Ngan F. Huang

All Publications

• A mouse model of volumetric muscle loss and therapeutic scaffold implantation. Nature protocols Hu, C., Chiang, G., Chan, A. H., Alcazar, C., Nakayama, K. H., Quarta, M., Rando, T. A., Huang, N. F. 2024

  Abstract

  Skeletal myofibers naturally regenerate after damage; however, impaired muscle function can result in cases when a prominent portion of skeletal muscle mass is lost, for example, following traumatic muscle injury. Volumetric muscle loss can be modeled in mice using a surgical model of muscle ablation to study the pathology of volumetric muscle loss and to test experimental treatments, such as the implantation of acellular scaffolds, which promote de novo myogenesis and angiogenesis. Here we provide step-by-step instructions to perform full-thickness surgical ablation, using biopsy punches, and to remove a large volume of the tibialis anterior muscle of the lower limb in mice. This procedure results in a reduction in muscle mass and limited regeneration capacity; the approach is easy to reproduce and can also be applied to larger animal models. For therapeutic applications, we further explain how to implant bioscaffolds into the ablated muscle site. With adequate training and practice, the surgical procedure can be performed within 30 min.

  View details for DOI 10.1038/s41596-024-01059-y

  View details for PubMedID 39424992

• Editorial: Methods in cardiovascular biologics and regenerative medicine. Frontiers in cardiovascular medicine Gurusamy, N., Bergmann, O., Castaldo, C., Huang, N. F., Lien, C. L., Tan, J. J., Engel, F. B. 2024; 11: 1477927

  View details for DOI 10.3389/fcvm.2024.1477927

  View details for PubMedID 39350966

  View details for PubMedCentralID PMC11440514

• Overcoming big bottlenecks in vascular regeneration. Communications biology Fantini, D. A., Yang, G., Khanna, A., Subramanian, D., Phillippi, J. A., Huang, N. F. 2024; 7 (1): 876

  Abstract

  Bioengineering and regenerative medicine strategies are promising for the treatment of vascular diseases. However, current limitations inhibit the ability of these approaches to be translated to clinical practice. Here we summarize some of the big bottlenecks that inhibit vascular regeneration in the disease applications of aortic aneurysms, stroke, and peripheral artery disease. We also describe the bottlenecks preventing three-dimensional bioprinting of vascular networks for tissue engineering applications. Finally, we describe emerging technologies and opportunities to overcome these challenges to advance vascular regeneration.

  View details for DOI 10.1038/s42003-024-06567-x

  View details for PubMedID 39020071

• Skeletal muscle-on-a-chip in microgravity as a platform for regeneration modeling and drug screening. Stem cell reports Kim, S., Ayan, B., Shayan, M., Rando, T. A., Huang, N. F. 2024

  Abstract

  Microgravity has been shown to lead to both muscle atrophy and impaired muscle regeneration. The purpose was to study the efficacy of microgravity to model impaired muscle regeneration in an engineered muscle platform and then to demonstrate the feasibility of performing drug screening in this model. Engineered human muscle was launched to the International Space Station National Laboratory, where the effect of microgravity exposure for 7 days was examined by transcriptomics and proteomics approaches. Gene set enrichment analysis of engineered muscle cultured in microgravity, compared to normal gravity conditions, highlighted a metabolic shift toward lipid and fatty acid metabolism, along with increased apoptotic gene expression. The addition of pro-regenerative drugs, insulin-like growth factor-1 (IGF-1) and a 15-hydroxyprostaglandin dehydrogenase inhibitor (15-PGDH-i), partially inhibited the effects of microgravity. In summary, microgravity mimics aspects of impaired myogenesis, and the addition of these drugs could partially inhibit the effects induced by microgravity.

  View details for DOI 10.1016/j.stemcr.2024.06.010

  View details for PubMedID 39059375

• Institutional Support for the Career Advancement of Women Faculty in Science and Academic Medicine: Successes, Challenges, and Future Directions. Arteriosclerosis, thrombosis, and vascular biology Chen, Z. B., Aikawa, E., Alfaidi, M., Ali, K., Clift, C. L., Erbay, E., Fredman, G., Gomez, D., Huang, N. F., Lu, H. S., Nguyen, P. K., Darc Oliveira, S., Rodriguez-Miguelez, P., SenthilKumar, G., Zhang, H., by the ATVB WLC 2024

  Abstract

  Institutional support is crucial for the successful career advancement of all faculty but in particular those who are women. Evolving from the past, in which gender disparities were prevalent in many institutions, recent decades have witnessed significant progress in supporting the career advancement of women faculty in science and academic medicine. However, continued advancement is necessary as previously unrecognized needs and new opportunities for improvement emerge. To identify the needs, opportunities, and potential challenges encountered by women faculty, the Women's Leadership Committee of the Arteriosclerosis, Thrombosis, and Vascular Biology Council developed an initiative termed GROWTH (Generating Resources and Opportunities for Women in Technology and Health). The committee designed a survey questionnaire and interviewed 19 leaders with roles and responsibilities in faculty development from a total of 12 institutions across various regions of the United States. The results were compiled, analyzed, and discussed. Based on our interviews and analyses, we present the current status of these representative institutions in supporting faculty development, highlighting efforts specific to women faculty. Through the experiences, insights, and vision of these leaders, we identified success stories, challenges, and future priorities. Our article provides a primer and a snapshot of institutional efforts to support the advancement of women faculty. Importantly, this article can serve as a reference and resource for academic entities seeking ideas to gauge their commitment level to women faculty and to implement new initiatives. Additionally, this article can provide guidance and strategies for women faculty as they seek support and resources from their current or prospective institutions when pursuing new career opportunities.

  View details for DOI 10.1161/ATVBAHA.124.320910

  View details for PubMedID 38957985

• Bioengineering Cell Therapy for Treatment of Peripheral Artery Disease. Arteriosclerosis, thrombosis, and vascular biology Huang, N. F., Stern, B., Oropeza, B. P., Zaitseva, T. S., Paukshto, M. V., Zoldan, J. 2024

  Abstract

  Peripheral artery disease is an atherosclerotic disease associated with limb ischemia that necessitates limb amputation in severe cases. Cell therapies comprised of adult mononuclear or stromal cells have been clinically tested and show moderate benefits. Bioengineering strategies can be applied to modify cell behavior and function in a controllable fashion. Using mechanically tunable or spatially controllable biomaterials, we highlight examples in which biomaterials can increase the survival and function of the transplanted cells to improve their revascularization efficacy in preclinical models. Biomaterials can be used in conjunction with soluble factors or genetic approaches to further modulate the behavior of transplanted cells and the locally implanted tissue environment in vivo. We critically assess the advances in bioengineering strategies such as 3-dimensional bioprinting and immunomodulatory biomaterials that can be applied to the treatment of peripheral artery disease and then discuss the current challenges and future directions in the implementation of bioengineering strategies.

  View details for DOI 10.1161/ATVBAHA.123.318126

  View details for PubMedID 38174560

• Special issue: Frontiers in biomaterials for cardiovascular health. Journal of biomedical materials research. Part A Bashur, C. A., Huang, N. F. 2023

  View details for DOI 10.1002/jbm.a.37658

  View details for PubMedID 38126535

• Editorial: Insights in cardiovascular biologics and regenerative medicine: 2022. Frontiers in cardiovascular medicine Madeddu, P., Huang, N. F. 2023; 10: 1333866

  View details for DOI 10.3389/fcvm.2023.1333866

  View details for PubMedID 38045911

  View details for PubMedCentralID PMC10691737

• Cardiovascular human organ-on-a-chip platform for disease modeling, drug development, and personalized therapy. Journal of biomedical materials research. Part A Khanna, A., Oropeza, B. P., Huang, N. F. 2023

  Abstract

  Cardiovascular organ-on-a-chip (OoC) devices are composed of engineered or native functional tissues that are cultured under controlled microenvironments inside microchips. These systems employ microfabrication and tissue engineering techniques to recapitulate human physiology. This review focuses on human OoC systems to model cardiovascular diseases, to perform drug screening, and to advance personalized medicine. We also address the challenges in the generation of organ chips that can revolutionize the large-scale application of these systems for drug development and personalized therapy.

  View details for DOI 10.1002/jbm.a.37602

  View details for PubMedID 37668192

• Combinatorial extracellular matrix cues with mechanical strain induce differential effects on myogenesis in vitro. Biomaterials science Chan, A. H., Jain, I., Oropeza, B. P., Zhou, T., Nelsen, B., Geisse, N. A., Huang, N. F. 2023

  Abstract

  Skeletal muscle regeneration remains a clinical unmet need for volumetric muscle loss and atrophy where muscle function cannot be restored to prior capacity. Current experimental approaches do not account for the complex microenvironmental factors that modulate myogenesis. In this study we developed a biomimetic tissue chip platform to systematically study the combined effects of the extracellular matrix (ECM) microenvironment and mechanical strain on myogenesis of murine myoblasts. Using stretchable tissue chips composed of collagen I (C), fibronectin (F) and laminin (L), as well as their combinations thereof, we tested the addition of mechanical strain regimens on myogenesis at the transcriptomic and translational levels. Our results show that ECMs have a significant effect on myotube formation in C2C12 murine myoblasts. Under static conditions, laminin substrates induced the longest myotubes, whereas fibronectin produced the widest myotubes. Combinatorial ECMs showed non-intuitive effects on myotube formation. Genome-wide analysis revealed the upregulation in actin cytoskeletal related genes that are suggestive of myogenesis. When mechanical strain was introduced to C + F + L combinatorial ECM substrates in the form of constant or intermittent uniaxial strain at low (5%) and high (15%) levels, we observed synergistic enhancements in myotube width, along with transcriptomic upregulation in myosin heavy chain genes. Together, these studies highlight the complex role of microenvironmental factors such as ECM interactions and strain on myotube formation and the underlying signaling pathways.

  View details for DOI 10.1039/d3bm00448a

  View details for PubMedID 37477446

• Development of a rat model of lymphedema and the implantation of a collagen-based medical device for therapeutic intervention. Frontiers in cardiovascular medicine Nguyen, D., Dionyssiou, D., Zaitseva, T. S., Zhou, A. T., Sue, G., Deptula, P., Moroz, M. A., Tabada, P., Rockson, S. G., Paukshto, M. V., Cheng, M. H., Huang, N. F. 2023; 10: 1214116

  Abstract

  Secondary lymphedema is a common condition among cancer survivors, and treatment strategies to prevent or treat lymphedema are in high demand. The development of novel strategies to diagnose or treat lymphedema would benefit from a robust experimental animal model of secondary lymphedema. The purpose of this methods paper is to describe and summarize our experience in developing and characterizing a rat hindlimb model of lymphedema. Here we describe a protocol to induce secondary lymphedema that takes advantage of micro computed tomography imaging for limb volume measurements and visualization of lymph drainage with near infrared imaging. To demonstrate the utility of this preclinical model for studying the therapeutic benefit of novel devices, we apply this animal model to test the efficacy of a biomaterials-based implantable medical device.

  View details for DOI 10.3389/fcvm.2023.1214116

  View details for PubMedID 37469481

  View details for PubMedCentralID PMC10353614

• Multiomics Analyses of Peripheral Artery Disease Muscle Biopsies. Circulation research Jain, I., Oropeza, B. P., Huang, N. F. 2023; 132 (11): 1444-1446

  View details for DOI 10.1161/CIRCRESAHA.123.322913

  View details for PubMedID 37228238

• Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function. Journal of biomedical materials research. Part A Shayan, M., Huang, M. S., Navarro, R., Chiang, G., Hu, C., Oropeza, B. P., Johansson, P. K., Suhar, R. A., Foster, A. A., LeSavage, B. L., Zamani, M., Enejder, A., Roth, J. G., Heilshorn, S. C., Huang, N. F. 2023

  Abstract

  Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

  View details for DOI 10.1002/jbm.a.37520

  View details for PubMedID 36861665

• Biomedical Applications of Collagen. Bioengineering (Basel, Switzerland) Huang, N. F., Zaitseva, T. S., Paukshto, M. V. 2023; 10 (1)

  Abstract

  Extracellular matrix proteins (ECMs) provide structural support and dynamic signaling cues that regulate cell behavior and tissue morphogenesis [...].

  View details for DOI 10.3390/bioengineering10010090

  View details for PubMedID 36671662

• Chronic nicotine impairs the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells in a murine model of peripheral arterial disease. JVS-vascular science Chan, A. H., Hu, C., Chiang, G. C., Ekweume, C., Huang, N. F. 2023; 4: 100115

  Abstract

  Objective: Lifestyle choices such as tobacco and e-cigarette use are a risk factor for peripheral arterial disease (PAD) and may influence therapeutic outcomes. The effect of chronic nicotine exposure on the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) was assessed in a murine model of PAD.Methods: Mice were exposed to nicotine or phosphate-buffered saline (PBS) for 28days, followed by induction of limb ischemia and iPSC-EC transplantation. Cells were injected into the ischemic limb immediately after induction of hindlimb ischemia and again 7days later. Limb perfusion was assessed by laser Doppler spectroscopy, and transplant cell survival was monitored for 14days afterward using bioluminescence imaging, followed by histological analysis of angiogenesis.Results: Transplant cell retention progressively decreased over time after implantation based on bioluminescence imaging, and there were no significant differences in cell survival between mice with chronic exposure to nicotine or PBS. However, compared with mice without nicotine exposure, mice with prior nicotine exposure had had an impaired therapeutic response to iPSC-EC therapy based on decreased vascular perfusion recovery. Mice with nicotine exposure, followed by cell transplantation, had significantly lower mean perfusion ratio after 14days (0.47± 0.07) compared with mice undergoing cell transplantation without prior nicotine exposure (0.79± 0.11). This finding was further supported by histological analysis of capillary density, in which animals with prior nicotine exposure had a lower capillary density (45.9± 4.7 per mm2) compared with mice without nicotine exposure (66.5± 8.1 per mm2). Importantly, the ischemic limbs mice exposed to nicotine without cell therapy also showed significant impairment in perfusion recovery after 14days, compared with mice that received PBS+ iPSC-EC treatment. This result suggested that mice without chronic nicotine exposure could respond to iPSC-EC implantation into the ischemic limb by inducing perfusion recovery, whereas mice with chronic nicotine exposure did not respond to iPSC-EC therapy.Conclusions: Together, these findings show that chronic nicotine exposure adversely affects the ability of iPSC-EC therapy to promote vascular perfusion recovery and angiogenesis in a murine PAD model.

  View details for DOI 10.1016/j.jvssci.2023.100115

  View details for PubMedID 37519333

• Single-Cell Transcriptomic Census of Endothelial Changes Induced by Matrix Stiffness and the Association with Atherosclerosis. Advanced functional materials Zamani, M., Cheng, Y. H., Charbonier, F., Gupta, V. K., Mayer, A. T., Trevino, A. E., Quertermous, T., Chaudhuri, O., Cahan, P., Huang, N. F. 2022; 32 (47)

  Abstract

  Vascular endothelial cell (EC) plasticity plays a critical role in the progression of atherosclerosis by giving rise to mesenchymal phenotypes in the plaque lesion. Despite the evidence for arterial stiffening as a major contributor to atherosclerosis, the complex interplay among atherogenic stimuli in vivo has hindered attempts to determine the effects of extracellular matrix (ECM) stiffness on endothelial-mesenchymal transition (EndMT). To study the regulatory effects of ECM stiffness on EndMT, an in vitro model is developed in which human coronary artery ECs are cultured on physiological or pathological stiffness substrates. Leveraging single-cell RNA sequencing, cell clusters with mesenchymal transcriptional features are identified to be more prevalent on pathological substrates than physiological substrates. Trajectory inference analyses reveal a novel mesenchymal-to-endothelial reverse transition, which is blocked by pathological stiffness substrates, in addition to the expected EndMT trajectory. ECs pushed to a mesenchymal character by pathological stiffness substrates are enriched in transcriptional signatures of atherosclerotic ECs from human and murine plaques. This study characterizes at single-cell resolution the transcriptional programs that underpin EC plasticity in both physiological or pathological milieus, and thus serves as a valuable resource for more precisely defining EndMT and the transcriptional programs contributing to atherosclerosis.

  View details for DOI 10.1002/adfm.202203069

  View details for PubMedID 36816792

  View details for PubMedCentralID PMC9937733

• Engineering Spatiotemporal Control in Vascularized Tissues. Bioengineering (Basel, Switzerland) Khanna, A., Oropeza, B. P., Huang, N. F. 2022; 9 (10)

  Abstract

  A major challenge in engineering scalable three-dimensional tissues is the generation of a functional and developed microvascular network for adequate perfusion of oxygen and growth factors. Current biological approaches to creating vascularized tissues include the use of vascular cells, soluble factors, and instructive biomaterials. Angiogenesis and the subsequent generation of a functional vascular bed within engineered tissues has gained attention and is actively being studied through combinations of physical and chemical signals, specifically through the presentation of topographical growth factor signals. The spatiotemporal control of angiogenic signals can generate vascular networks in large and dense engineered tissues. This review highlights the developments and studies in the spatiotemporal control of these biological approaches through the coordinated orchestration of angiogenic factors, differentiation of vascular cells, and microfabrication of complex vascular networks. Fabrication strategies to achieve spatiotemporal control of vascularization involves the incorporation or encapsulation of growth factors, topographical engineering approaches, and 3D bioprinting techniques. In this article, we highlight the vascularization of engineered tissues, with a focus on vascularized cardiac patches that are clinically scalable for myocardial repair. Finally, we discuss the present challenges for successful clinical translation of engineered tissues and biomaterials.

  View details for DOI 10.3390/bioengineering9100555

  View details for PubMedID 36290523

• Scalable macroporous hydrogels enhance stem cell treatment of volumetric muscle loss. Biomaterials Eugenis, I., Wu, D., Hu, C., Chiang, G., Huang, N. F., Rando, T. A. 2022; 290: 121818

  Abstract

  Volumetric muscle loss (VML), characterized by an irreversible loss of skeletal muscle due to trauma or surgery, is accompanied by severe functional impairment and long-term disability. Tissue engineering strategies combining stem cells and biomaterials hold great promise for skeletal muscle regeneration. However, scaffolds, including decellularized extracellular matrix (dECM), hydrogels, and electrospun fibers, used for VML applications generally lack macroporosity. As a result, the scaffolds used typically delay host cell infiltration, transplanted cell proliferation, and new tissue formation. To overcome these limitations, we engineered a macroporous dECM-methacrylate (dECM-MA) hydrogel, which we will refer to as a dECM-MA sponge, and investigated its therapeutic potential in vivo. Our results demonstrate that dECM-MA sponges promoted early cellularization, endothelialization, and establishment of a pro-regenerative immune microenvironment in a mouse VML model. In addition, dECM-MA sponges enhanced the proliferation of transplanted primary muscle stem cells, muscle tissue regeneration, and functional recovery four weeks after implantation. Finally, we investigated the scale-up potential of our scaffolds using a rat VML model and found that dECM-MA sponges significantly improved transplanted cell proliferation and muscle regeneration compared to conventional dECM scaffolds. Together, these results validate macroporous hydrogels as novel scaffolds for VML treatment and skeletal muscle regeneration.

  View details for DOI 10.1016/j.biomaterials.2022.121818

  View details for PubMedID 36209578

• Single-Cell Transcriptomic Census of Endothelial Changes Induced by Matrix Stiffness and the Association with Atherosclerosis ADVANCED FUNCTIONAL MATERIALS Zamani, M., Cheng, Y., Charbonier, F., Gupta, V., Mayer, A. T., Trevino, A. E., Quertermous, T., Chaudhuri, O., Cahan, P., Huang, N. F. 2022

  View details for DOI 10.1002/adfm.202203069

  View details for Web of Science ID 000852765400001

• Editorial: Frontiers in Cardiovascular Medicine: Rising Stars 2021. Frontiers in cardiovascular medicine Wang, L., Xu, G. E., Pan, L., Aikawa, E., Aikawa, M., Xiao, J., Huang, N. F. 2022; 9: 928981

  View details for DOI 10.3389/fcvm.2022.928981

  View details for PubMedID 35757336

  View details for PubMedCentralID PMC9218798

• Advances in three-dimensional bioprinted stem cell-based tissue engineering for cardiovascular regeneration. Journal of molecular and cellular cardiology Khanna, A., Ayan, B., Undieh, A. A., Yang, Y. P., Huang, N. F. 2022; 169: 13-27

  Abstract

  Three-dimensional (3D) bioprinting of cellular or biological components are an emerging field to develop tissue structures that mimic the spatial, mechanochemical and temporal characteristics of cardiovascular tissues. 3D multi-cellular and multi-domain organotypic biological constructs can better recapitulate in vivo physiology and can be utilized in a variety of applications. Such applications include in vitro cellular studies, high-throughput drug screening, disease modeling, biocompatibility analysis, drug testing and regenerative medicine. A major challenge of 3D bioprinting strategies is the inability of matrix molecules to reconstitute the complexity of the extracellular matrix and the intrinsic cellular morphologies and functions. An important factor is the inclusion of a vascular network to facilitate oxygen and nutrient perfusion in scalable and patterned 3D bioprinted tissues to promote cell viability and functionality. In this review, we summarize the new generation of 3D bioprinting techniques, the kinds of bioinks and printing materials employed for 3D bioprinting, along with the current state-of-the-art in engineered cardiovascular tissue models. We also highlight the translational applications of 3D bioprinting in engineering the myocardium cardiac valves, and vascular grafts. Finally, we discuss current challenges and perspectives of designing effective 3D bioprinted constructs with native vasculature, architecture and functionality for clinical translation and cardiovascular regeneration.

  View details for DOI 10.1016/j.yjmcc.2022.04.017

  View details for PubMedID 35569213

• INSULIN-LIKE GROWTH FACTOR-1 LADEN SCAFFOLDS WITH REHABILITATIVE EXERCISE IMPROVES MUSCLE REGENERATION Huang, N. F., Nakayam, K., Alcazar, C., Hu, C., Rando, T. MARY ANN LIEBERT, INC. 2022: S584

  View details for Web of Science ID 000821187303171

• Dual delivery of BMP-2 and IGF-1 through injectable hydrogel promotes cranial bone defect healing. Tissue engineering. Part A Park, Y., Lin, S., Bai, Y., Moeinzadeh, S., Kim, S., Huang, J., Lee, U., Huang, N. F., Yang, Y. P. 2022

  Abstract

  Critical size cranial bone defect remains a great clinical challenge. With advantages in regenerative medicine, injectable hydrogels incorporated with bioactive molecules show great potential in promoting cranial bone repair. Recently, we developed a dual delivery system by sequential release of BMP2 followed by IGF1 in microparticles (MPs), and an injectable alginate/collagen (alg/col)-based hydrogel. In this study we aim to evaluate the effect of dual delivery of BMP2 and IGF1 in MPs through the injectable hydrogel in critical-size cranial bone defect healing. The gelatin MPs (gMPs) loaded with BMP2 and poly(lactic-co-glycolic acid)-poly(ethylene glycol)-carboxyl (PLGA-PEG-COOH) MPs (pMPs) loaded with IGF1 were prepared, respectively. The encapsulation efficiency and release profile of growth factors in MPs were measured. A cranial defect model was applied to evaluate the efficacy of the dual delivery system in bone regeneration. Adult SD rats were subjected to osteotomy to make an ⌀8-mm cranial defect. The injectable hydrogel contained MPs loading with BMP2 (2 µg), IGF1 (2 µg), or a combination of BMP2 (1 µg) and IGF1 (1 µg) were injected to the defect site. New bone formation was evaluated by micro-CT, histological analysis, and immunohistochemistry after 4 or 8 weeks. Data showed that dual delivery of the low-dose BMP2 and IGF1 in MPs through alg/col-based hydrogel successfully restored cranial bone as early as 4 weeks after implantation, whose effect was comparable to the single delivery of high-dose BMP2 in MPs. In conclusion, this study suggests that dual delivery of BMP2 and IGF1 in MPs in alg/col-based hydrogel achieves early bone regeneration in critical size bone defect, with advantage in reducing the dose of BMP2.

  View details for DOI 10.1089/ten.TEA.2022.0002

  View details for PubMedID 35357948

• Comparative Effects of Basic Fibroblast Growth Factor Delivery or Voluntary Exercise on Muscle Regeneration after Volumetric Muscle Loss. Bioengineering (Basel, Switzerland) Hu, C., Ayan, B., Chiang, G., Chan, A. H., Rando, T. A., Huang, N. F. 1800; 9 (1)

  Abstract

  Volumetric muscle loss (VML) is associated with irreversibly impaired muscle function due to traumatic injury. Experimental approaches to treat VML include the delivery of basic fibroblast growth factor (bFGF) or rehabilitative exercise. The objective of this study was to compare the effects of spatially nanopatterned collagen scaffold implants with either bFGF delivery or in conjunction with voluntary exercise. Aligned nanofibrillar collagen scaffold bundles were adsorbed with bFGF, and the bioactivity of bFGF-laden scaffolds was examined by skeletal myoblast or endothelial cell proliferation. The therapeutic efficacy of scaffold implants with either bFGF release or exercise was examined in a murine VML model. Our results show an initial burst release of bFGF from the scaffolds, followed by a slower release over 21 days. The released bFGF induced myoblast and endothelial cell proliferation in vitro. After 3 weeks of implantation in a mouse VML model, twitch force generation was significantly higher in mice treated with bFGF-laden scaffolds compared to bFGF-laden scaffolds with exercise. However, myofiber density was not significantly improved with bFGF scaffolds or voluntary exercise. In contrast, the scaffold implant with exercise induced more re-innervation than all other groups. These results highlight the differential effects of bFGF and exercise on muscle regeneration.

  View details for DOI 10.3390/bioengineering9010037

  View details for PubMedID 35049746

• Extracellular Matrix-Based Biomaterials for Cardiovascular Tissue Engineering. Journal of cardiovascular development and disease Khanna, A., Zamani, M., Huang, N. F. 2021; 8 (11)

  Abstract

  Regenerative medicine and tissue engineering strategies have made remarkable progress in remodeling, replacing, and regenerating damaged cardiovascular tissues. The design of three-dimensional (3D) scaffolds with appropriate biochemical and mechanical characteristics is critical for engineering tissue-engineered replacements. The extracellular matrix (ECM) is a dynamic scaffolding structure characterized by tissue-specific biochemical, biophysical, and mechanical properties that modulates cellular behavior and activates highly regulated signaling pathways. In light of technological advancements, biomaterial-based scaffolds have been developed that better mimic physiological ECM properties, provide signaling cues that modulate cellular behavior, and form functional tissues and organs. In this review, we summarize the in vitro, pre-clinical, and clinical research models that have been employed in the design of ECM-based biomaterials for cardiovascular regenerative medicine. We highlight the research advancements in the incorporation of ECM components into biomaterial-based scaffolds, the engineering of increasingly complex structures using biofabrication and spatial patterning techniques, the regulation of ECMs on vascular differentiation and function, and the translation of ECM-based scaffolds for vascular graft applications. Finally, we discuss the challenges, future perspectives, and directions in the design of next-generation ECM-based biomaterials for cardiovascular tissue engineering and clinical translation.

  View details for DOI 10.3390/jcdd8110137

  View details for PubMedID 34821690

• What Makes a Great Mentor: Interviews With Recipients of the ATVB Mentor of Women Award. Arteriosclerosis, thrombosis, and vascular biology Zhang, H., Chen, Z. B., Fredman, G., Gomez, D., Grumbach, I. M., Huang, N. F., Nguyen, P., Ouimet, M., Sutton, N. R., Aikawa, E. 2021: ATVBAHA121316558

  View details for DOI 10.1161/ATVBAHA.121.316558

  View details for PubMedID 34547925

• peri-Adventitial delivery of smooth muscle cells in porous collagen scaffolds for treatment of experimental abdominal aortic aneurysm. Biomaterials science Mulorz, J., Shayan, M., Hu, C., Alcazar, C., Chan, A. H., Briggs, M., Wen, Y., Walvekar, A. P., Ramasubramanian, A. K., Spin, J. M., Chen, B., Tsao, P. S., Huang, N. F. 2021

  Abstract

  Abdominal aortic aneurysm (AAA) is associated with the loss of vascular smooth muscle cells (SMCs) within the vessel wall. Direct delivery of therapeutic cells is challenging due to impaired mechanical integrity of the vessel wall. We hypothesized that porous collagen scaffolds can be an effective vehicle for the delivery of human-derived SMCs to the site of AAA. The purpose was to evaluate if the delivery of cell-seeded scaffolds can abrogate progressive expansion in a mouse model of AAA. Collagen scaffolds seeded with either primary human aortic SMCs or induced pluripotent stem cell derived-smooth muscle progenitor cells (iPSC-SMPs) had >80% in vitro cell viability and >75% cell penetrance through the scaffold's depth, while preserving smooth muscle phenotype. The cell-seeded scaffolds were successfully transplanted onto the murine aneurysm peri-adventitia on day 7 following AAA induction using pancreatic porcine elastase infusion. Ultrasound imaging revealed that SMC-seeded scaffolds significantly reduced the aortic diameter by 28 days, compared to scaffolds seeded with iPSC-SMPs or without cells (acellular scaffold), respectively. Bioluminescence imaging demonstrated that both cell-seeded scaffold groups had cellular localization to the aneurysm but a decline in survival with time. Histological analysis revealed that both cell-seeded scaffold groups had more SMC retention and less macrophage invasion into the medial layer of AAA lesions, when compared to the acellular scaffold treatment group. Our data suggest that scaffold-based SMC delivery is feasible and may constitute a platform for cell-based AAA therapy.

  View details for DOI 10.1039/d1bm00685a

  View details for PubMedID 34522940

• Modest Gains After an 8-Week Exercise Program Correlate With Reductions in Non-traditional Markers of Cardiovascular Risk FRONTIERS IN CARDIOVASCULAR MEDICINE Liang, G., Huang, X., Hirsch, J., Mehmi, S., Fonda, H., Chan, K., Huang, N. F., Aalami, O., Froelicher, V. F., Lee, D. P., Myers, J., Lee, A. S., Nguyen, P. K. 2021; 8: 669110

  Abstract

  Background: Although engaging in physical exercise has been shown to reduce the incidence of cardiovascular events, the molecular mechanisms by which exercise mediates these benefits remain unclear. Based on epidemiological evidence, reductions in traditional risk factors only accounts for 50% of the protective effects of exercise, leaving the remaining mechanisms unexplained. The objective of this study was to determine whether engaging in a regular exercise program in a real world clinical setting mediates cardiovascular protection via modulation of non-traditional risk factors, such as those involved in coagulation, inflammation and metabolic regulation. Methods and Results: We performed a prospective, cohort study in 52 sedentary patients with cardiovascular disease or cardiovascular risk factors at two tertiary medical centers between January 1, 2016 and December 31, 2019. Prior to and at the completion of an 8-week exercise program, we collected information on traditional cardiovascular risk factors, exercise capacity, and physical activity and performed plasma analysis to measure levels of fibrinolytic, inflammatory and metabolic biomarkers to assess changes in non-traditional cardiovascular risk factors. The median weight change, improvement in physical fitness, and change in physical activity for the entire cohort were: -4.6 pounds (IQR: +2 pounds, -11.8 pounds), 0.37 METs (IQR: -0.076 METs, 1.06 METs), and 252.7 kcals/week (IQR: -119, 921.2 kcals/week). In addition to improvement in blood pressure and cholesterol, patients who lost at least 5 pounds, expended at least 1,000 additional kcals/week, and/or achieved ≥0.5 MET increase in fitness had a significant reduction in plasminogen activator inhibitor-1 [9.07 ng/mL (95% CI: 2.78-15.35 ng/mL); P = 0.026], platelet derived growth factor beta [376.077 pg/mL (95% CI: 44.69-707.46 pg/mL); P = 0.026); and angiopoietin-1 [(1104.11 pg/mL (95% CI: 2.92-2205.30 pg/mL); P = 0.049)]. Conclusion: Modest improvements in physical fitness, physical activity, and/or weight loss through a short-term exercise program was associated with decreased plasma levels of plasminogen activator inhibitor, platelet derived growth factor beta, and angiopoietin, which have been associated with impaired fibrinolysis and inflammation.

  View details for DOI 10.3389/fcvm.2021.669110

  View details for Web of Science ID 000668338500001

  View details for PubMedID 34222367

  View details for PubMedCentralID PMC8245677

• Recent advances in bioprinting technologies for engineering cardiac tissue. Materials science & engineering. C, Materials for biological applications Agarwal, T., Fortunato, G. M., Hann, S. Y., Ayan, B., Vajanthri, K. Y., Presutti, D., Cui, H., Chan, A. H., Costantini, M., Onesto, V., Di Natale, C., Huang, N. F., Makvandi, P., Shabani, M., Maiti, T. K., Zhang, L. G., De Maria, C. 2021; 124: 112057

  Abstract

  Annually increasing incidence of cardiac-related disorders and cardiac tissue's minimal regenerative capacity have motivated the researchers to explore effective therapeutic strategies. In the recent years, bioprinting technologies have witnessed a great wave of enthusiasm and have undergone steady advancements over a short period, opening the possibilities for recreating engineered functional cardiac tissue models for regenerative and diagnostic applications. With this perspective, the current review delineates recent developments in the sphere of engineered cardiac tissue fabrication, using traditional and advanced bioprinting strategies. The review also highlights different printing ink formulations, available cellular opportunities, and aspects of personalized medicines in the context of cardiac tissue engineering and bioprinting. On a concluding note, current challenges and prospects for further advancements are also discussed.

  View details for DOI 10.1016/j.msec.2021.112057

  View details for PubMedID 33947551

• Engineering Cardiovascular Tissue Chips for Disease Modeling and Drug Screening Applications. Frontiers in bioengineering and biotechnology Chan, A. H., Huang, N. F. 2021; 9: 673212

  Abstract

  In recent years, the cost of drug discovery and development have been progressively increasing, but the number of drugs approved for treatment of cardiovascular diseases (CVDs) has been limited. Current in vitro models for drug development do not sufficiently ensure safety and efficacy, owing to their lack of physiological relevance. On the other hand, preclinical animal models are extremely costly and present problems of inaccuracy due to species differences. To address these limitations, tissue chips offer the opportunity to emulate physiological and pathological tissue processes in a biomimetic in vitro platform. Tissue chips enable in vitro modeling of CVDs to give mechanistic insights, and they can also be a powerful approach for drug screening applications. Here, we review recent advances in CVD modeling using tissue chips and their applications in drug screening.

  View details for DOI 10.3389/fbioe.2021.673212

  View details for PubMedID 33959600

• Transplantation of insulin-like growth factor-1 laden scaffolds combined with exercise promotes neuroregeneration and angiogenesis in a preclinical muscle injury model BIOMATERIALS SCIENCE Alcazar, C. A., Hu, C., Rando, T. A., Huang, N. F., Nakayama, K. H. 2020; 8 (19): 5376–89

  Abstract

  The regeneration of skeletal muscle can be permanently impaired by traumatic injuries, despite the high regenerative capacity of native muscle. An attractive therapeutic approach for treating severe muscle inuries is the implantation of off-the-shelf engineered biomimetic scaffolds into the site of tissue damage to enhance muscle regeneration. Anisotropic nanofibrillar scaffolds provide spatial patterning cues to create organized myofibers, and growth factors such as insulin-like growth factor-1 (IGF-1) are potent inducers of both muscle regeneration as well as angiogenesis. The aim of this study was to test the therapeutic efficacy of anisotropic IGF-1-releasing collagen scaffolds combined with voluntary exercise for the treatment of acute volumetric muscle loss, with a focus on histomorphological effects. To enhance the angiogenic and regenerative potential of injured murine skeletal muscle, IGF-1-laden nanofibrillar scaffolds with aligned topography were fabricated using a shear-mediated extrusion approach, followed by growth factor adsorption. Individual scaffolds released a cumulative total of 1244 ng ± 153 ng of IGF-1 over the course of 21 days in vitro. To test the bioactivity of IGF-1-releasing scaffolds, the myotube formation capacity of murine myoblasts was quantified. On IGF-1-releasing scaffolds seeded with myoblasts, the resulting myotubes formed were 1.5-fold longer in length and contained 2-fold greater nuclei per myotube, when compared to scaffolds without IGF-1. When implanted into the ablated murine tibialis anterior muscle, the IGF-1-laden scaffolds, in conjunction with voluntary wheel running, significantly increased the density of perfused microvessels by greater than 3-fold, in comparison to treatment with scaffolds without IGF-1. Enhanced myogenesis was also observed in animals treated with the IGF-1-laden scaffolds combined with exercise, compared to control scaffolds transplanted into mice that did not receive exercise. Furthermore, the abundance of mature neuromuscular junctions was greater by approximately 2-fold in muscles treated with IGF-1-laden scaffolds, when paired with exercise, in comparison to the same treatment without exercise. These findings demonstrate that voluntary exercise improves the regenerative effect of growth factor-laden scaffolds by augmenting neurovascular regeneration, and have important translational implications in the design of off-the-shelf therapeutics for the treatment of traumatic muscle injury.

  View details for DOI 10.1039/d0bm00990c

  View details for Web of Science ID 000573820100008

  View details for PubMedID 32996916

  View details for PubMedCentralID PMC7531607

• Pre-Clinical Cell Therapeutic Approaches for Repair of Volumetric Muscle Loss. Bioengineering (Basel, Switzerland) Shayan, M., Huang, N. F. 2020; 7 (3)

  Abstract

  Extensive damage to skeletal muscle tissue due to volumetric muscle loss (VML) is beyond the inherent regenerative capacity of the body, and results in permanent functional debilitation. Current clinical treatments fail to fully restore native muscle function. Recently, cell-based therapies have emerged as a promising approach to promote skeletal muscle regeneration following injury and/or disease. Stem cell populations, such as muscle stem cells, mesenchymal stem cells and induced pluripotent stem cells (iPSCs), have shown a promising capacity for muscle differentiation. Support cells, such as endothelial cells, nerve cells or immune cells, play a pivotal role in providing paracrine signaling cues for myogenesis, along with modulating the processes of inflammation, angiogenesis and innervation. The efficacy of cell therapies relies on the provision of instructive microenvironmental cues and appropriate intercellular interactions. This review describes the recent developments of cell-based therapies for the treatment of VML, with a focus on preclinical testing and future trends in the field.

  View details for DOI 10.3390/bioengineering7030097

  View details for PubMedID 32825213

• Delivery of hepatocyte growth factor mRNA from nanofibrillar scaffolds in a pig model of peripheral arterial disease. Regenerative medicine S Zaitseva, T., Yang, G., Dionyssiou, D., Zamani, M., Sawamura, S., Yakubov, E., Ferguson, J., Hallett, R. L., Fleischmann, D., Paukshto, M. V., Huang, N. F. 2020

  Abstract

  Background: Chemical modification of mRNA (mmRNA) substantially improves their stability and translational efficiency within cells. Nanofibrillar collagen scaffolds were previously shown to enable the spatially localized delivery and temporally controlled release of mmRNA encoding HGF both in vitro and in vivo. Materials &methods: Herein we developed an improved slow-releasing HGF mmRNA scaffold and tested its therapeutic efficacy in a porcine model of peripheral arterial disease. Results & conclusion: The HGF mmRNA wasreleased from scaffolds in a temporally controlled fashion in vitro with preserved transfection activity. The mmRNA scaffolds improved vascular regeneration when sutured to the ligated porcine femoral artery. These studies validate the therapeutic potential of HGF mmRNA delivery from nanofibrillar scaffolds for treatment of peripheral arterial disease.

  View details for DOI 10.2217/rme-2020-0023

  View details for PubMedID 32772903

• Effects of nicotine on the translation of stem cell therapy. Regenerative medicine Chan, A. H., Huang, N. F. 2020

  Abstract

  Although stem cell therapy has tremendous therapeutic potential, clinical translation of stem cell therapy has yet to be fully realized. Recently, patient comorbidities and lifestyle choices have emerged to be important factors in the efficacy of stem cell therapy. Tobacco usage is an important risk factor for numerous diseases, and nicotine exposure specifically has become increasing more prevalent with the rising use of electronic cigarettes. This review describes the effects of nicotine exposure on the function of various stem cells. We place emphasis on the differential effects of nicotine exposure in vitro and as well as in preclinical models. Further research on the effects of nicotine on stem cells will deepen our understanding of how lifestyle choices can impact the outcome of stem cell therapies.

  View details for DOI 10.2217/rme-2020-0032

  View details for PubMedID 32618492

• Delivery of Human Stromal Vascular Fraction Cells on Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease. Frontiers in bioengineering and biotechnology Hu, C. n., Zaitseva, T. S., Alcazar, C. n., Tabada, P. n., Sawamura, S. n., Yang, G. n., Borrelli, M. R., Wan, D. C., Nguyen, D. H., Paukshto, M. V., Huang, N. F. 2020; 8: 689

  Abstract

  Cell therapy for treatment of peripheral arterial disease (PAD) is a promising approach but is limited by poor cell survival when cells are delivered using saline. The objective of this study was to examine the feasibility of aligned nanofibrillar scaffolds as a vehicle for the delivery of human stromal vascular fraction (SVF), and then to assess the efficacy of the cell-seeded scaffolds in a murine model of PAD. Flow cytometric analysis was performed to characterize the phenotype of SVF cells from freshly isolated lipoaspirate, as well as after attachment onto aligned nanofibrillar scaffolds. Flow cytometry results demonstrated that the SVF consisted of 33.1 ± 9.6% CD45+ cells, a small fraction of CD45-/CD31+ (4.5 ± 3.1%) and 45.4 ± 20.0% of CD45-/CD31-/CD34+ cells. Although the subpopulations of SVF did not change significantly after attachment to the aligned nanofibrillar scaffolds, protein secretion of vascular endothelial growth factor (VEGF) significantly increased by six-fold, compared to SVF cultured in suspension. Importantly, when SVF-seeded scaffolds were transplanted into immunodeficient mice with induced hindlimb ischemia, the cell-seeded scaffolds induced a significant higher mean perfusion ratio after 14 days, compared to cells delivered using saline. Together, these results show that aligned nanofibrillar scaffolds promoted cellular attachment, enhanced the secretion of VEGF from attached SVF cells, and their implantation with attached SVF cells stimulated blood perfusion recovery. These findings have important therapeutic implications for the treatment of PAD using SVF.

  View details for DOI 10.3389/fbioe.2020.00689

  View details for PubMedID 32766213

  View details for PubMedCentralID PMC7380169

• Multi-scale cellular engineering: From molecules to organ-on-a-chip. APL bioengineering Huang, N. F., Chaudhuri, O. n., Cahan, P. n., Wang, A. n., Engler, A. J., Wang, Y. n., Kumar, S. n., Khademhosseini, A. n., Li, S. n. 2020; 4 (1): 010906

  Abstract

  Recent technological advances in cellular and molecular engineering have provided new insights into biology and enabled the design, manufacturing, and manipulation of complex living systems. Here, we summarize the state of advances at the molecular, cellular, and multi-cellular levels using experimental and computational tools. The areas of focus include intrinsically disordered proteins, synthetic proteins, spatiotemporally dynamic extracellular matrices, organ-on-a-chip approaches, and computational modeling, which all have tremendous potential for advancing fundamental and translational science. Perspectives on the current limitations and future directions are also described, with the goal of stimulating interest to overcome these hurdles using multi-disciplinary approaches.

  View details for DOI 10.1063/1.5129788

  View details for PubMedID 32161833

  View details for PubMedCentralID PMC7054123

• Vascularization of Engineered Spatially Patterned Myocardial Tissue Derived From Human Pluripotent Stem Cells in vivo FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY Wanjare, M., Kawamura, M., Hu, C., Alcazar, C., Wang, H., Woo, Y., Huang, N. F. 2019; 7

  View details for DOI 10.3389/fbioe.2019.00208

  View details for Web of Science ID 000483596200001

• Vascularization of Engineered Spatially Patterned Myocardial Tissue Derived From Human Pluripotent Stem Cells in vivo. Frontiers in bioengineering and biotechnology Wanjare, M., Kawamura, M., Hu, C., Alcazar, C., Wang, H., Woo, Y. J., Huang, N. F. 2019; 7: 208

  Abstract

  Tissue engineering approaches to regenerate myocardial tissue after disease or injury is promising. Integration with the host vasculature is critical to the survival and therapeutic efficacy of engineered myocardial tissues. To create more physiologically oriented engineered myocardial tissue with organized cellular arrangements and endothelial interactions, randomly oriented or parallel-aligned microfibrous polycaprolactone scaffolds were seeded with human pluripotent stem cell-derived cardiomyocytes (iCMs) and/or endothelial cells (iECs). The resultant engineered myocardial tissues were assessed in a subcutaneous transplantation model and in a myocardial injury model to evaluate the effect of scaffold anisotropy and endothelial interactions on vascular integration of the engineered myocardial tissue. Here we demonstrated that engineered myocardial tissue composed of randomly oriented scaffolds seeded with iECs promoted the survival of iECs for up to 14 days. However, engineered myocardial tissue composed of aligned scaffolds preferentially guided the organization of host capillaries along the direction of the microfibers. In a myocardial injury model, epicardially transplanted engineered myocardial tissues composed of randomly oriented scaffolds seeded with iCMs augmented microvessel formation leading to a significantly higher arteriole density after 4 weeks, compared to engineered tissues derived from aligned scaffolds. These findings that the scaffold microtopography imparts differential effect on revascularization, in which randomly oriented scaffolds promote pro-survival and pro-angiogenic effects, and aligned scaffolds direct the formation of anisotropic vessels. These findings suggest a dominant role of scaffold topography over endothelial co-culture in modulating cellular survival, vascularization, and microvessel architecture.

  View details for DOI 10.3389/fbioe.2019.00208

  View details for PubMedID 31552234

  View details for PubMedCentralID PMC6733921

• Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration COMMUNICATIONS BIOLOGY Nakayama, K. H., Quarta, M., Paine, P., Alcazar, C., Karakikes, I., Garcia, V., Abilez, O. J., Calvo, N. S., Simmons, C. S., Rando, T. A., Huang, N. F. 2019; 2

  View details for DOI 10.1038/s42003-019-0416-4

  View details for Web of Science ID 000467214900002

• Engineering Biomimetic Materials for Skeletal Muscle Repair and Regeneration ADVANCED HEALTHCARE MATERIALS Nakayama, K. H., Shayan, M., Huang, N. F. 2019; 8 (5)

  View details for DOI 10.1002/adhm.201801168

  View details for Web of Science ID 000461575200004

• Endothelial Cell Mechanotransduction in the Dynamic Vascular Environment ADVANCED BIOSYSTEMS Charbonier, F. W., Zamani, M., Huang, N. F. 2019; 3 (2)

  View details for DOI 10.1002/adbi.201800252

  View details for Web of Science ID 000458425000013

• Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration. Communications biology Nakayama, K. H., Quarta, M. n., Paine, P. n., Alcazar, C. n., Karakikes, I. n., Garcia, V. n., Abilez, O. J., Calvo, N. S., Simmons, C. S., Rando, T. A., Huang, N. F. 2019; 2 (1): 170

  Abstract

  Traumatic skeletal muscle injuries cause irreversible tissue damage and impaired revascularization. Engineered muscle is promising for enhancing tissue revascularization and regeneration in injured muscle. Here we fabricated engineered skeletal muscle composed of myotubes interspersed with vascular endothelial cells using spatially patterned scaffolds that induce aligned cellular organization, and then assessed their therapeutic benefit for treatment of murine volumetric muscle loss. Murine skeletal myoblasts co-cultured with endothelial cells in aligned nanofibrillar scaffolds form endothelialized and aligned muscle with longer myotubes, more synchronized contractility, and more abundant secretion of angiogenic cytokines, compared to endothelialized engineered muscle formed from randomly-oriented scaffolds. Treatment of traumatically injured muscle with endothelialized and aligned skeletal muscle promotes the formation of highly organized myofibers and microvasculature, along with greater vascular perfusion, compared to treatment of muscle derived from randomly-oriented scaffolds. This work demonstrates the potential of endothelialized and aligned engineered skeletal muscle to promote vascular regeneration following transplantation.

  View details for DOI 10.1038/s42003-019-0416-4

  View details for PubMedID 31924993

• Small Molecule Derived From Carboxyethylpyrrole Protein Adducts Promotes Angiogenesis in a Mouse Model of Peripheral Arterial Disease. Journal of the American Heart Association Hou, L., Yang, G., Tang, S., Alcazar, C., Joshi, P., Strassberg, Z., Kim, M., Kawamura, M., Woo, Y. J., Shrager, J., Ding, S., Huang, N. F. 2018; 7 (18): e009234

  Abstract

  Background CEP (omega-[2-carboxyethyl]pyrrole) protein adducts are the end products of lipid oxidation associated with inflammation and have been implicated in the induction of angiogenesis in pathological conditions such as tissue ischemia. We synthesized small molecules derived from CEP protein adducts and evaluated the angiogenic effect of the CEP analog CEP 03 in the setting of peripheral arterial disease. Methods and Results The angiogenic effect of CEP 03 was assessed by invitro analysis of primary human microvascular endothelial cell proliferation and tubelike formation in Matrigel (Corning). In the presence of CEP 03, proliferation of endothelial cells invitro increased by 27±18% under hypoxic (1% O2) conditions, reaching similar levels to that of VEGF A (vascular endothelial growth factor A) stimulation (22±10%), relative to the vehicle control treatment. A similar effect of CEP 03 was demonstrated in the increased number of tubelike branches in Matrigel, reaching >70% induction in hypoxia, compared with the vehicle control. The therapeutic potential of CEP 03 was further evaluated in a mouse model of peripheral arterial disease by quantification of blood perfusion recovery and capillary density. In the ischemic hind limb, treatment of CEP 03 encapsulated within Matrigel significantly enhanced blood perfusion by 2-fold after 14days compared with those treated with Matrigel alone. Moreover, these results concurred with histological finding that treatment of CEP 03 in Matrigel resulted in a significant increase in microvessel density compared with Matrigel alone. Conclusions Our data suggest that CEP 03 has a profound positive effect on angiogenesis and neovessel formation and thus has therapeutic potential for treatment of peripheral arterial disease.

  View details for PubMedID 30371212

• Rehabilitative exercise and spatially patterned nanofibrillar scaffolds enhance vascularization and innervation following volumetric muscle loss NPJ REGENERATIVE MEDICINE Nakayama, K. H., Alcazar, C., Yang, G., Quarta, M., Paine, P., Doan, L., Davies, A., Rando, T. A., Huang, N. F. 2018; 3: 16

  Abstract

  Muscle regeneration can be permanently impaired by traumatic injuries, despite the high regenerative capacity of skeletal muscle. Implantation of engineered biomimetic scaffolds to the site of muscle ablation may serve as an attractive off-the-shelf therapeutic approach. The objective of the study was to histologically assess the therapeutic benefit of a three-dimensional spatially patterned collagen scaffold, in conjunction with rehabilitative exercise, for treatment of volumetric muscle loss. To mimic the physiologic organization of skeletal muscle, which is generally composed of myofibers aligned in parallel, three-dimensional parallel-aligned nanofibrillar collagen scaffolds were fabricated. When implanted into the ablated murine tibialis anterior muscle, the aligned nanofibrillar scaffolds, in conjunction with voluntary caged wheel exercise, significantly improved the density of perfused microvessels, in comparison to treatments of the randomly oriented nanofibrillar scaffold, decellularized scaffold, or in the untreated control group. The abundance of neuromuscular junctions was 19-fold higher when treated with aligned nanofibrillar scaffolds in conjunction with exercise, in comparison to treatment of aligned scaffold without exercise. Although, the density of de novo myofibers was not significantly improved by aligned scaffolds, regardless of exercise activity, the cross-sectional area of regenerating myofibers was increased by > 60% when treated with either aligned and randomly oriented scaffolds, in comparison to treatment of decellularized scaffold or untreated controls. These findings demonstrate that voluntary exercise improved the regenerative effect of aligned scaffolds by augmenting neurovascularization, and have important implications in the design of engineered biomimetic scaffolds for treatment of traumatic muscle injury.

  View details for PubMedID 30245849

• Near-Infrared IIb Fluorescence Imaging of Vascular Regeneration with Dynamic Tissue Perfusion Measurement and High Spatial Resolution ADVANCED FUNCTIONAL MATERIALS Ma, Z., Zhang, M., Yue, J., Alcazar, C., Zhong, Y., Doyle, T. C., Dai, H., Huang, N. F. 2018; 28 (36)

  View details for DOI 10.1002/adfm.201803417

  View details for Web of Science ID 000443375900026

• Protein-engineered hydrogels enhance the survival of induced pluripotent stem cell-derived endothelial cells for treatment of peripheral arterial disease BIOMATERIALS SCIENCE Foster, A. A., Dewi, R. E., Cai, L., Hou, L., Strassberg, Z., Alcazar, C. A., Heilshorn, S. C., Huang, N. F. 2018; 6 (3): 614–22

  Abstract

  A key feature of peripheral arterial disease (PAD) is damage to endothelial cells (ECs), resulting in lower limb pain and restricted blood flow. Recent preclinical studies demonstrate that the transplantation of ECs via direct injection into the affected limb can result in significantly improved blood circulation. Unfortunately, the clinical application of this therapy has been limited by low cell viability and poor cell function. To address these limitations we have developed an injectable, recombinant hydrogel, termed SHIELD (Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery) for cell transplantation. SHIELD provides mechanical protection from cell membrane damage during syringe flow. Additionally, secondary in situ crosslinking provides a reinforcing network to improve cell retention, thereby augmenting the therapeutic benefit of cell therapy. In this study, we demonstrate the improved acute viability of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following syringe injection delivery in SHIELD, compared to saline. Using a murine hind limb ischemia model of PAD, we demonstrate enhanced iPSC-EC retention in vivo and improved neovascularization of the ischemic limb based on arteriogenesis following transplantation of iPSC-ECs delivered in SHIELD.

  View details for PubMedID 29406542

  View details for PubMedCentralID PMC5829050

• Aligned Nanofibrillar Scaffolds for Controlled Delivery of Modified mRNA. Tissue engineering. Part A Zaitseva, T. n., Alcazar, C. n., Zamani, M. n., Hou, L. n., Sawamura, S. n., Yakubov, E. n., Hopkins, M. n., Woo, Y. J., Paukshto, M. n., Huang, N. F. 2018

  Abstract

  RNA-based vector delivery is a promising gene therapy approach. Recent advances in chemical modification of mRNA structure to form modified mRNA (mmRNA or cmRNA or modRNA) have substantially improved their stability and translational efficiency within cells. However, mmRNA conventionally delivered in solution can be taken up non-specifically or become cleared away prematurely, which markedly limits the potential benefit of mmRNA therapy. To address this limitation, we developed mmRNA-incorporated nanofibrillar scaffolds that could target spatially localized delivery and temporally controlled release of the mmRNA both in vitro and in vivo. To establish the efficacy of mmRNA therapy, mmRNA encoding reporter proteins such as green fluorescence protein (GFP) or firefly luciferase (Fluc) was loaded into aligned nanofibrillar collagen scaffolds. The mmRNA was released from mmRNA-loaded scaffolds in a transient and temporally controlled fashion and induced transfection in human fibroblasts in a dose-dependent manner. In vitro transfection was further verified using mmRNA encoding the angiogenic growth factor, hepatocyte growth factor (HGF). Finally, scaffold-based delivery of HGF mmRNA to the site of surgically induced muscle injury in mice resulted in significantly higher vascular regeneration after 14 days, compared to implantation of Fluc mmRNA-releasing scaffolds. After transfection with Fluc mmRNA-releasing scaffold in vivo, Fluc activity was detectable and localized to the muscle region, based on non-invasive bioluminescence imaging. Scaffold-based local mmRNA delivery as an off-the-shelf form of gene therapy has broad translatability for treating a broad range of diseases or injuries.

  View details for PubMedID 29717619

• Multicellular Interactions in 3D Engineered Myocardial Tissue. Frontiers in cardiovascular medicine Zamani, M., Karaca, E., Huang, N. F. 2018; 5: 147

  Abstract

  Cardiovascular disease is a leading cause of death in the US and many countries worldwide. Current cell-based clinical trials to restore cardiomyocyte (CM) health by local delivery of cells have shown only moderate benefit in improving cardiac pumping capacity. CMs have highly organized physiological structure and interact dynamically with non-CM populations, including endothelial cells and fibroblasts. Within engineered myocardial tissue, non-CM populations play an important role in CM survival and function, in part by secreting paracrine factors and cell-cell interactions. In this review, we summarize the progress of engineering myocardial tissue with pre-formed physiological multicellular organization, and present the challenges toward clinical translation.

  View details for PubMedID 30406114

• Big bottlenecks in cardiovascular tissue engineering COMMUNICATIONS BIOLOGY Huang, N. F., Serpooshan, V., Morris, V. B., Sayed, N., Pardon, G., Abilez, O. J., Nakayama, K. H., Pruitt, B. L., Wu, S. M., Yoon, Y., Zhang, J., Wu, J. C. 2018; 1

  View details for DOI 10.1038/s42003-018-0202-8

  View details for Web of Science ID 000461126500237

• Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells CELLULAR AND MOLECULAR BIOENGINEERING Kim, J. J., Hou, L., Yang, G., Mezak, N. P., Wanjare, M., Joubert, L. M., Huang, N. F. 2017; 10 (5): 417–32

  View details for PubMedID 28936269

• Delivery of Hepatocyte Growth Factor mRNA From Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease Huang, N. F., Hou, L., Alcazar, C., Strassberg, Z., Hopkins, M., Zaitseva, T., Yakubov, E., Paukshto, M. V. LIPPINCOTT WILLIAMS & WILKINS. 2017

  View details for Web of Science ID 000408316600193

• Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells Hou, L., Kim, J. J., Wanjare, M. J., Patlolla, B., Coller, J., Natu, V., Hastie, T., Huang, N. F. FEDERATION AMER SOC EXP BIOL. 2017

  View details for Web of Science ID 000405986501003

• Regulation of the microenvironment for cardiac tissue engineering. Regenerative medicine Wanjare, M., Huang, N. F. 2017; 12 (2): 187-201

  Abstract

  The microenvironment of myocardium plays an important role in the fate and function of cardiomyocytes (CMs). Cardiovascular tissue engineering strategies commonly utilize stem cell sources in conjunction with microenvironmental cues that often include biochemical, electrical, spatial and biomechanical factors. Microenvironmental stimulation of CMs, in addition to the incorporation of intercellular interactions from non-CMs, results in the generation of engineered cardiac constructs. Current studies suggest that use of these factors when engineering cardiac constructs improve cardiac function when implanted in vivo. In this review, we summarize the approaches to modulate biochemical, electrical, biomechanical and spatial factors to induce CM differentiation and their subsequent organization for cardiac tissue engineering application.

  View details for DOI 10.2217/rme-2016-0132

  View details for PubMedID 28244821

  View details for PubMedCentralID PMC5348721

• A comparison of the pro-angiogenic potential of human induced pluripotent stem cell derived endothelial cells and induced endothelial cells in a murine model of peripheral arterial disease. International journal of cardiology Clayton, Z. E., Yuen, G. S., Sadeghipour, S., Hywood, J. D., Wong, J. W., Huang, N. F., Ng, M. K., Cooke, J. P., Patel, S. 2017

  Abstract

  Endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) promote angiogenesis, and more recently induced endothelial cells (iECs) have been generated via fibroblast trans-differentiation. These cell types have potential as treatments for peripheral arterial disease (PAD). However, it is unknown whether different reprogramming methods produce cells that are equivalent in terms of their pro-angiogenic capabilities.We aimed to directly compare iPSC-ECs and iECs in an animal model of PAD, in order to identify which cell type, if any, displays superior therapeutic potential.IPSC-ECs and iECs were generated from human fibroblasts, and transduced with a reporter construct encoding GFP and firefly luciferase for bioluminescence imaging (BLI). Endothelial phenotype was confirmed using in vitro assays. NOD-SCID mice underwent hindlimb ischaemia surgery and received an intramuscular injection of either 1×10(6) iPSC-ECs, 1×10(6) iECs or control vehicle only. Perfusion recovery was measured by laser Doppler. Hindlimb muscle samples were taken for histological analyses.Perfusion recovery was enhanced in iPSC-EC treated mice on day 14 (Control vs. iPSC-EC; 0.35±0.04 vs. 0.54±0.08, p<0.05) and in iEC treated mice on days 7 (Control vs. iEC; 0.23±0.02 vs. 0.44±0.06, p<0.05), 10 (0.31±0.04 vs. 0.64±0.07, p<0.001) and 14 (0.35±0.04 vs. 0.68±0.07, p<0.001) post-treatment. IEC-treated mice also had greater capillary density in the ischaemic gastrocnemius muscle (Control vs. iEC; 125±10 vs. 179±11 capillaries/image; p<0.05). BLI detected iPSC-EC and iEC presence in vivo for two weeks post-treatment.IPSC-ECs and iECs exhibit similar, but not identical, endothelial functionality and both cell types enhance perfusion recovery after hindlimb ischaemia.

  View details for DOI 10.1016/j.ijcard.2017.01.125

  View details for PubMedID 28209385

• Boosting the down-shifting luminescence of rare-earth nanocrystals for biological imaging beyond 1500 nm. Nature communications Zhong, Y. n., Ma, Z. n., Zhu, S. n., Yue, J. n., Zhang, M. n., Antaris, A. L., Yuan, J. n., Cui, R. n., Wan, H. n., Zhou, Y. n., Wang, W. n., Huang, N. F., Luo, J. n., Hu, Z. n., Dai, H. n. 2017; 8 (1): 737

  Abstract

  In vivo fluorescence imaging in the near-infrared region between 1500-1700 nm (NIR-IIb window) affords high spatial resolution, deep-tissue penetration, and diminished auto-fluorescence due to the suppressed scattering of long-wavelength photons and large fluorophore Stokes shifts. However, very few NIR-IIb fluorescent probes exist currently. Here, we report the synthesis of a down-conversion luminescent rare-earth nanocrystal with cerium doping (Er/Ce co-doped NaYbF4 nanocrystal core with an inert NaYF4 shell). Ce doping is found to suppress the up-conversion pathway while boosting down-conversion by ~9-fold to produce bright 1550 nm luminescence under 980 nm excitation. Optimization of the inert shell coating surrounding the core and hydrophilic surface functionalization minimize the luminescence quenching effect by water. The resulting biocompatible, bright 1550 nm emitting nanoparticles enable fast in vivo imaging of blood vasculature in the mouse brain and hindlimb in the NIR-IIb window with short exposure time of 20 ms for rare-earth based probes.Fluorescence imaging in the near-infrared window between 1500-1700 nm (NIR-IIb window) offers superior spatial resolution and tissue penetration depth, but few NIR-IIb probes exist. Here, the authors synthesize rare earth down-converting nanocrystals as promising fluorescent probes for in vivo imaging in this spectral region.

  View details for PubMedID 28963467

  View details for PubMedCentralID PMC5622117

• Anisotropic microfibrous scaffolds enhance the organization and function of cardiomyocytes derived from induced pluripotent stem cells. Biomaterials science Wanjare, M. n., Hou, L. n., Nakayama, K. H., Kim, J. J., Mezak, N. P., Abilez, O. J., Tzatzalos, E. n., Wu, J. C., Huang, N. F. 2017; 5 (8): 1567–78

  Abstract

  Engineering of myocardial tissue constructs is a promising approach for treatment of coronary heart disease. To engineer myocardial tissues that better mimic the highly ordered physiological arrangement and function of native cardiomyocytes, we generated electrospun microfibrous polycaprolactone scaffolds with either randomly oriented (14 μm fiber diameter) or parallel-aligned (7 μm fiber diameter) microfiber arrangement and co-seeded the scaffolds with human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and endothelial cells (iECs) for up to 12 days after iCM seeding. Here we demonstrated that aligned microfibrous scaffolds induced iCM alignment along the direction of the aligned microfibers after 2 days of iCM seeding, as well as promoted greater iCM maturation by increasing the sarcomeric length and gene expression of myosin heavy chain adult isoform (MYH7), in comparison to randomly oriented scaffolds. Furthermore, the benefit of scaffold anisotropy was evident in the significantly higher maximum contraction velocity of iCMs on the aligned scaffolds, compared to randomly oriented scaffolds, at 12 days of culture. Co-seeding of iCMs with iECs led to reduced contractility, compared to when iCMs were seeded alone. These findings demonstrate a dominant role of scaffold anisotropy in engineering cardiovascular tissues that maintain iCM organization and contractile function.

  View details for PubMedID 28715029

  View details for PubMedCentralID PMC5567776

• Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome. Arteriosclerosis, thrombosis, and vascular biology Carcamo-Orive, I. n., Huang, N. F., Quertermous, T. n., Knowles, J. W. 2017; 37 (11): 2038–42

  Abstract

  Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations.

  View details for PubMedID 28729365

  View details for PubMedCentralID PMC5669062

• In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse. Journal of visualized experiments : JoVE Yuan, K., Orcholski, M. E., Huang, N. F., de Jesus Perez, V. A. 2016

  Abstract

  Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

  View details for DOI 10.3791/54617

  View details for PubMedID 28060266

• Combinatorial extracellular matrix microenvironments promote survival and phenotype of human induced pluripotent stem cell-derived endothelial cells in hypoxia ACTA BIOMATERIALIA Hou, L., CoIler, J., Natu, V., Hastie, T. J., Huang, N. F. 2016; 44: 188-199

  Abstract

  Recent developments in cell therapy using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) hold great promise for treating ischemic cardiovascular tissues. However, poor post-transplantation viability largely limits the potential of stem cell therapy. Although the extracellular matrix (ECM) has become increasingly recognized as an important cell survival factor, conventional approaches primarily rely on single ECMs for in vivo co-delivery with cells, even though the endothelial basement membrane is comprised of a milieu of different ECMs. To address this limitation, we developed a combinatorial ECM microarray platform to simultaneously interrogate hundreds of micro-scale multi-component chemical compositions of ECMs on iPSC-EC response. After seeding iPSC-ECs onto ECM microarrays, we performed high-throughput analysis of the effects of combinatorial ECMs on iPSC-EC survival, endothelial phenotype, and nitric oxide production under conditions of hypoxia (1% O2) and reduced nutrients (1% fetal bovine serum), as is present in ischemic injury sites. Using automated image acquisition and analysis, we identified combinatorial ECMs such as collagen IV+gelatin+heparan sulfate+laminin and collagen IV+fibronectin+gelatin+heparan sulfate+laminin that significantly improved cell survival, nitric oxide production, and CD31 phenotypic expression, in comparison to single-component ECMs. These results were further validated in conventional cell culture platforms and within three-dimensional scaffolds. Furthermore, this approach revealed complex ECM interactions and non-intuitive cell behavior that otherwise could not be easily determined using conventional cell culture platforms. Together these data suggested that iPSC-EC delivery within optimal combinatorial ECMs may improve their survival and function under the condition of hypoxia with reduced nutrients.Human endothelial cells (ECs) derived from induced pluripotent stem cells (iPSC-ECs) are promising for treating diseases associated with reduced nutrient and oxygen supply like heart failure. However, diminished iPSC-EC survival after implantation into diseased environments limits their therapeutic potential. Since native ECs interact with numerous extracellular matrix (ECM) proteins for functional maintenance, we hypothesized that combinatorial ECMs may improve cell survival and function under conditions of reduced oxygen and nutrients. We developed a high-throughput system for simultaneous screening of iPSC-ECs cultured on multi-component ECM combinations under the condition of hypoxia and reduced serum. Using automated image acquisition and analytical algorithms, we identified combinatorial ECMs that significantly improved cell survival and function, in comparison to single ECMs. Furthermore, this approach revealed complex ECM interactions and non-intuitive cell behavior that otherwise could not be easily determined.

  View details for DOI 10.1016/j.actbio.2016.08.003

  View details for Web of Science ID 000385594700017

  View details for PubMedCentralID PMC5045796

• Aligned nanofibrillar collagen scaffolds - Guiding lymphangiogenesis for treatment of acquired lymphedema. Biomaterials Hadamitzky, C., Zaitseva, T. S., Bazalova-Carter, M., Paukshto, M. V., Hou, L., Strassberg, Z., Ferguson, J., Matsuura, Y., Dash, R., Yang, P. C., Kretchetov, S., Vogt, P. M., Rockson, S. G., Cooke, J. P., Huang, N. F. 2016; 102: 259-267

  Abstract

  Secondary lymphedema is a common disorder associated with acquired functional impairment of the lymphatic system. The goal of this study was to evaluate the therapeutic efficacy of aligned nanofibrillar collagen scaffolds (BioBridge) positioned across the area of lymphatic obstruction in guiding lymphatic regeneration. In a porcine model of acquired lymphedema, animals were treated with BioBridge scaffolds, alone or in conjunction with autologous lymph node transfer as a source of endogenous lymphatic growth factor. They were compared with a surgical control group and a second control group in which the implanted BioBridge was supplemented with exogenous vascular endothelial growth factor-C (VEGF-C). Three months after implantation, immunofluorescence staining of lymphatic vessels demonstrated a significant increase in lymphatic collectors within close proximity to the scaffolds. To quantify the functional impact of scaffold implantation, bioimpedance was used as an early indicator of extracellular fluid accumulation. In comparison to the levels prior to implantation, the bioimpedance ratio was significantly improved only in the experimental BioBridge recipients with or without lymph node transfer, suggesting restoration of functional lymphatic drainage. These results further correlated with quantifiable lymphatic collectors, as visualized by contrast-enhanced computed tomography. They demonstrate the therapeutic potential of BioBridge scaffolds in secondary lymphedema.

  View details for DOI 10.1016/j.biomaterials.2016.05.040

  View details for PubMedID 27348849

• Vascularization of three-dimensional engineered tissues for regenerative medicine applications. Acta biomaterialia Kim, J. J., Hou, L., Huang, N. F. 2016; 41: 17-26

  Abstract

  Engineering of three-dimensional (3D) tissues is a promising approach for restoring diseased or dysfunctional myocardium with a functional replacement. However, a major bottleneck in this field is the lack of efficient vascularization strategies, because tissue constructs produced in vitro require a constant flow of oxygen and nutrients to maintain viability and functionality. Compared to angiogenic cell therapy and growth factor treatment, bioengineering approaches such as spatial micropatterning, integration of sacrificial materials, tissue decellularization, and 3D bioprinting enable the generation of more precisely controllable neovessel formation. In this review, we summarize the state-of-the-art approaches to develop 3D tissue engineered constructs with vasculature, and demonstrate how some of these techniques have been applied towards regenerative medicine for treatment of heart failure.Tissue engineering is a promising approach to replace or restore dysfunctional tissues/organs, but a major bottleneck in realizing its potential is the challenge of creating scalable 3D tissues. Since most 3D engineered tissues require a constant supply of nutrients, it is necessary to integrate functional vasculature within the tissues in order to facilitate the transport of nutrients. To address these needs, researchers are employing biomaterial engineering and design strategies to foster vessel formation within 3D tissues. This review highlights the state-of-the-art bioengineering tools and technologies to create vascularized 3D tissues for clinical applications in regenerative medicine, highlighting the application of these technologies to engineer vascularized cardiac patches for treatment of heart failure.

  View details for DOI 10.1016/j.actbio.2016.06.001

  View details for PubMedID 27262741

• Distilling complexity to advance cardiac tissue engineering SCIENCE TRANSLATIONAL MEDICINE Ogle, B. M., Bursac, N., Domian, I., Huang, N. F., Menasche, P., Murry, C. E., Pruitt, B., Radisic, M., Wu, J. C., Wu, S. M., Zhang, J., Zimmermann, W., Vunjak-Novakovic, G. 2016; 8 (342)

  Abstract

  The promise of cardiac tissue engineering is in the ability to recapitulate in vitro the functional aspects of a healthy heart and disease pathology as well as to design replacement muscle for clinical therapy. Parts of this promise have been realized; others have not. In a meeting of scientists in this field, five central challenges or "big questions" were articulated that, if addressed, could substantially advance the current state of the art in modeling heart disease and realizing heart repair.

  View details for DOI 10.1126/scitranslmed.aad2304

  View details for Web of Science ID 000377443800001

  View details for PubMedID 27280684

• Targeted delivery of human iPS-ECs overexpressing IL-8 receptors inhibits neointimal and inflammatory responses to vascular injury in the rat. American journal of physiology. Heart and circulatory physiology Giordano, S., Zhao, X., Xing, D., Hage, F., Oparil, S., Cooke, J. P., Lee, J., Nakayama, K. H., Huang, N. F., Chen, Y. 2016; 310 (6): H705-15

  Abstract

  Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 10(6) HiPS-ECs, 1.5 × 10(6) HiPS-Null-ECs, or 1.5 × 10(6) HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury.

  View details for DOI 10.1152/ajpheart.00587.2015

  View details for PubMedID 26801304

• Stem cell-based therapies to promote angiogenesis in ischemic cardiovascular disease AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Hou, L., Kim, J. J., Woo, Y. J., Huang, N. F. 2016; 310 (4): H455-H465

  Abstract

  Stem cell therapy is a promising approach for treatment of tissue ischemia associated with myocardial infarction and peripheral arterial disease. Stem and progenitor cells derived from bone marrow or from pluripotent stem cells have shown therapeutic benefit in boosting angiogenesis as well as restoring tissue function. Notably, adult stem and progenitor cells including mononuclear cells, endothelial progenitor cells, and mesenchymal stem cells have progressed into clinical trials and have shown positive benefits. In this review, we overview the major classes of stem and progenitor cells, including pluripotent stem cells, and summarize the state-of-the-art in applying these cell types for treating myocardial infarction and peripheral arterial disease.

  View details for DOI 10.1152/ajpheart.00726.2015

  View details for PubMedID 26683902

• Polymer-DNA Nanoparticle-Induced CXCR4 Overexpression Improves Stem Cell Engraftment and Tissue Regeneration in a Mouse Hindlimb Ischemia Model THERANOSTICS Deveza, L., Choi, J., Lee, J., Huang, N., Cooke, J., Yang, F. 2016; 6 (8): 1176-1189

  Abstract

  Peripheral arterial disease affects nearly 202 million individuals worldwide, sometimes leading to non-healing ulcers or limb amputations in severe cases. Genetically modified stem cells offer potential advantages for therapeutically inducing angiogenesis via augmented paracrine release mechanisms and tuned dynamic responses to environmental stimuli at disease sites. Here, we report the application of nanoparticle-induced CXCR4-overexpressing stem cells in a mouse hindlimb ischemia model. We found that CXCR4 overexpression improved stem cell survival, modulated inflammation in situ, and accelerated blood reperfusion. These effects, unexpectedly, led to complete limb salvage and skeletal muscle repair, markedly outperforming the efficacy of the conventional angiogenic factor control, VEGF. Importantly, assessment of CXCR4-overexpressing stem cells in vitro revealed that CXCR4 overexpression induced changes in paracrine signaling of stem cells, promoting a therapeutically desirable pro-angiogenic and anti-inflammatory phenotype. These results suggest that nanoparticle-induced CXCR4 overexpression may promote favorable phenotypic changes and therapeutic efficacy of stem cells in response to the ischemic environment.

  View details for DOI 10.7150/thno.12866

  View details for Web of Science ID 000378529400009

  View details for PubMedID 27279910

  View details for PubMedCentralID PMC4893644

• Nanoscale Patterning of Extracellular Matrix Alters Endothelial Function under Shear Stress NANO LETTERS Nakayama, K. H., Surya, V. N., Gole, M., Walker, T. W., Yang, W., Lai, E. S., Ostrowski, M. A., Fuller, G. G., Dunn, A. R., Huang, N. F. 2016; 16 (1): 410-419

  Abstract

  The role of nanotopographical extracellular matrix (ECM) cues in vascular endothelial cell (EC) organization and function is not well-understood, despite the composition of nano- to microscale fibrillar ECMs within blood vessels. Instead, the predominant modulator of EC organization and function is traditionally thought to be hemodynamic shear stress, in which uniform shear stress induces parallel-alignment of ECs with anti-inflammatory function, whereas disturbed flow induces a disorganized configuration with pro-inflammatory function. Since shear stress acts on ECs by applying a mechanical force concomitant with inducing spatial patterning of the cells, we sought to decouple the effects of shear stress using parallel-aligned nanofibrillar collagen films that induce parallel EC alignment prior to stimulation with disturbed flow resulting from spatial wall shear stress gradients. Using real time live-cell imaging, we tracked the alignment, migration trajectories, proliferation, and anti-inflammatory behavior of ECs when they were cultured on parallel-aligned or randomly oriented nanofibrillar films. Intriguingly, ECs cultured on aligned nanofibrillar films remained well-aligned and migrated predominantly along the direction of aligned nanofibrils, despite exposure to shear stress orthogonal to the direction of the aligned nanofibrils. Furthermore, in stark contrast to ECs cultured on randomly oriented films, ECs on aligned nanofibrillar films exposed to disturbed flow had significantly reduced inflammation and proliferation, while maintaining intact intercellular junctions. This work reveals fundamental insights into the importance of nanoscale ECM interactions in the maintenance of endothelial function. Importantly, it provides new insight into how ECs respond to opposing cues derived from nanotopography and mechanical shear force and has strong implications in the design of polymeric conduits and bioengineered tissues.

  View details for DOI 10.1021/acs.nanolett.5b04028

  View details for Web of Science ID 000368322700064

  View details for PubMedCentralID PMC4758680

• Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances Arteriogenesis. ACS nano Nakayama, K. H., Hong, G., Lee, J. C., Patel, J., Edwards, B., Zaitseva, T. S., Paukshto, M. V., Dai, H., Cooke, J. P., Woo, Y. J., Huang, N. F. 2015; 9 (7): 6900-6908

  Abstract

  The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nanoscale signaling cues from aligned nanofibrillar scaffolds in the setting of tissue ischemia. Thread-like nanofibrillar scaffolds with porous structure were fabricated from aligned-braided membranes generated under shear from liquid crystal collagen solution. Human ECs showed greater outgrowth from aligned scaffolds than from nonpatterned scaffolds. Integrin α1 was in part responsible for the enhanced cellular outgrowth on aligned nanofibrillar scaffolds, as the effect was abrogated by integrin α1 inhibition. To test the efficacy of EC-seeded aligned nanofibrillar scaffolds in improving neovascularization in vivo, the ischemic limbs of mice were treated with EC-seeded aligned nanofibrillar scaffold; EC-seeded nonpatterned scaffold; ECs in saline; aligned nanofibrillar scaffold alone; or no treatment. After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion recovery when treated with EC-seeded aligned nanofibrillar scaffolds, in comparison to ECs in saline or no treatment. In ischemic hindlimbs treated with scaffolds seeded with human ECs derived from induced pluripotent stem cells (iPSC-ECs), single-walled carbon nanotube (SWNT) fluorophores were systemically delivered to quantify microvascular density after 28 days. Near infrared-II (NIR-II, 1000-1700 nm) imaging of SWNT fluorophores demonstrated that iPSC-EC-seeded aligned scaffolds group showed significantly higher microvascular density than the saline or cells groups. These data suggest that treatment with EC-seeded aligned nanofibrillar scaffolds improved blood perfusion and arteriogenesis, when compared to treatment with cells alone or scaffold alone, and have important implications in the design of therapeutic cell delivery strategies.

  View details for DOI 10.1021/acsnano.5b00545

  View details for PubMedID 26061869

• Activation of the Wnt/Planar Cell Polarity Pathway Is Required for Pericyte Recruitment during Pulmonary Angiogenesis. American journal of pathology Yuan, K., Orcholski, M. E., Panaroni, C., Shuffle, E. M., Huang, N. F., Jiang, X., Tian, W., Vladar, E. K., Wang, L., Nicolls, M. R., Wu, J. Y., de Jesus Perez, V. A. 2015; 185 (1): 69-84

  Abstract

  Pericytes are perivascular cells localized to capillaries that promote vessel maturation, and their absence can contribute to vessel loss. Whether impaired endothelial-pericyte interaction contributes to small vessel loss in pulmonary arterial hypertension (PAH) is unclear. Using 3G5-specific, immunoglobulin G-coated magnetic beads, we isolated pericytes from the lungs of healthy subjects and PAH patients, followed by lineage validation. PAH pericytes seeded with healthy pulmonary microvascular endothelial cells failed to associate with endothelial tubes, resulting in smaller vascular networks compared to those with healthy pericytes. After the demonstration of abnormal polarization toward endothelium via live-imaging and wound-healing studies, we screened PAH pericytes for abnormalities in the Wnt/planar cell polarity (PCP) pathway, which has been shown to regulate cell motility and polarity in the pulmonary vasculature. PAH pericytes had reduced expression of frizzled 7 (Fzd7) and cdc42, genes crucial for Wnt/PCP activation. With simultaneous knockdown of Fzd7 and cdc42 in healthy pericytes in vitro and in a murine model of angiogenesis, motility and polarization toward pulmonary microvascular endothelial cells were reduced, whereas with restoration of both genes in PAH pericytes, endothelial-pericyte association was improved, with larger vascular networks. These studies suggest that the motility and polarity of pericytes during pulmonary angiogenesis are regulated by Wnt/PCP activation, which can be targeted to prevent vessel loss in PAH.

  View details for DOI 10.1016/j.ajpath.2014.09.013

  View details for PubMedID 25447046

• Bilayered vascular graft derived from human induced pluripotent stem cells with biomimetic structure and function REGENERATIVE MEDICINE Nakayama, K. H., Joshi, P. A., Lai, E. S., Gujar, P., Joubert, L., Chen, B., Huang, N. F. 2015; 10 (6): 745-755

  Abstract

  We developed an aligned bi-layered vascular graft derived from human induced pluripotent stem cells (iPSCs) that recapitulates the cellular composition, orientation, and anti-inflammatory function of blood vessels.The luminal layer consisted of longitudinal-aligned nanofibrillar collagen containing primary endothelial cells (ECs) or iPSC-derived ECs (iPSC-ECs). The outer layer contained circumferentially oriented nanofibrillar collagen with primary smooth muscle cells (SMCs) or iPSC-derived SMCs(iPSC-SMCs).On the aligned scaffolds, cells organized F-actin assembly within 8º from the direction of nanofibrils. When compared to randomly-oriented scaffolds, EC-seeded aligned scaffolds had significant reduced inflammatory response, based on adhesivity to monocytes.This study highlights the importance of anisotropic scaffolds in directing cell form and function, and has therapeutic significance as physiologically relevant blood vessels.

  View details for DOI 10.2217/rme.15.45

  View details for Web of Science ID 000364574000005

  View details for PubMedID 26440211

• Targeted Delivery of Rat Aortic Endothelial Cells (RAECs) Overexpressing Interleukin-8 (IL8) Receptors Inhibits Neointimal and Inflammatory Responses to Endoluminal Injury of Carotid Artery and Acute Pulmonary Hypertension (PAH) in Rats Giordano, S., Zhao, X., Xing, D., Hage, F., Oparil, S., Townes, T., Sun, C., Wu, L. N., Cooke, J. P., Lee, J., Nakayama, K., Huang, N. F., Chen, Y. ELSEVIER SCIENCE INC. 2014: S44–S45

  View details for DOI 10.1016/j.freeradbiomed.2014.10.397

  View details for Web of Science ID 000359525800102

• Avidity-controlled hydrogels for injectable co-delivery of induced pluripotent stem cell-derived endothelial cells and growth factors. Journal of controlled release Mulyasasmita, W., Cai, L., Dewi, R. E., Jha, A., Ullmann, S. D., Luong, R. H., Huang, N. F., Heilshorn, S. C. 2014; 191: 71-81

  Abstract

  To translate recent advances in induced pluripotent stem cell biology to clinical regenerative medicine therapies, new strategies to control the co-delivery of cells and growth factors are needed. Building on our previous work designing Mixing-Induced Two-Component Hydrogels (MITCHs) from engineered proteins, here we develop protein-polyethylene glycol (PEG) hybrid hydrogels, MITCH-PEG, which form physical gels upon mixing for cell and growth factor co-delivery. MITCH-PEG is a mixture of C7, which is a linear, engineered protein containing seven repeats of the CC43 WW peptide domain (C), and 8-arm star-shaped PEG conjugated with either one or two repeats of a proline-rich peptide to each arm (P1 or P2, respectively). Both 20kDa and 40kDa star-shaped PEG variants were investigated, and all four PEG-peptide variants were able to undergo a sol-gel phase transition when mixed with the linear C7 protein at constant physiological conditions due to noncovalent hetero-dimerization between the C and P domains. Due to the dynamic nature of the C-P physical crosslinks, all four gels were observed to be reversibly shear-thinning and self-healing. The P2 variants exhibited higher storage moduli than the P1 variants, demonstrating the ability to tune the hydrogel bulk properties through a biomimetic peptide-avidity strategy. The 20kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40kDa variants. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) adopted a well-spread morphology within three-dimensional MITCH-PEG cultures, and MITCH-PEG provided significant protection from cell damage during ejection through a fine-gauge syringe needle. In a mouse hindlimb ischemia model of peripheral arterial disease, MITCH-PEG co-delivery of hiPSC-ECs and VEGF was found to reduce inflammation and promote muscle tissue regeneration compared to a saline control.

  View details for DOI 10.1016/j.jconrel.2014.05.015

  View details for PubMedID 24848744

• Role of extracellular matrix signaling cues in modulating cell fate commitment for cardiovascular tissue engineering. Advanced healthcare materials Nakayama, K. H., Hou, L., Huang, N. F. 2014; 3 (5): 628-641

  Abstract

  It is generally agreed that engineered cardiovascular tissues require cellular interactions with the local milieu. Within the microenvironment, the extracellular matrix (ECM) is an important support structure that provides dynamic signaling cues in part through its chemical, physical, and mechanical properties. In response to ECM factors, cells activate biochemical and mechanotransduction pathways that modulate their survival, growth, migration, differentiation, and function. This Review describes the role of ECM chemical composition, spatial patterning, and mechanical stimulation in the specification of cardiovascular lineages, with a focus on stem cell differentiation, direct transdifferentiation, and endothelial-to-mesenchymal transition. The translational application of ECMs will be discussed in the context of cardiovascular tissue engineering and regenerative medicine.

  View details for DOI 10.1002/adhm.201300620

  View details for PubMedID 24443420

• Near-infrared II fluorescence for imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement. Circulation. Cardiovascular imaging Hong, G., Lee, J. C., Jha, A., Diao, S., Nakayama, K. H., Hou, L., Doyle, T. C., Robinson, J. T., Antaris, A. L., Dai, H., Cooke, J. P., Huang, N. F. 2014; 7 (3): 517-525

  Abstract

  Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here, we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000-1400 nm) of photon wavelengths.Because of the reduced photon scattering of NIR-II fluorescence compared with traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microscopic computed tomography. Furthermore, imaging during 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P<0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy.The penetration depth of millimeters, high spatial resolution, and fast acquisition rate of NIR-II imaging make it a useful imaging tool for murine models of vascular disease.

  View details for DOI 10.1161/CIRCIMAGING.113.000305

  View details for PubMedID 24657826

• Extracellular matrix-mediated endothelial differentiation of human induced pluripotent stem cells Hou, L., Huang, N. FEDERATION AMER SOC EXP BIOL. 2014

  View details for Web of Science ID 000346651002118

• Microvascular Endothelial Cells Migrate Upstream and Align Against the Shear Stress Field Created by Impinging Flow BIOPHYSICAL JOURNAL Ostrowski, M. A., Huang, N. F., Walker, T. W., Verwijlen, T., Poplawski, C., Khoo, A. S., Cooke, J. P., Fuller, G. G., Dunn, A. R. 2014; 106 (2): 366-374

  Abstract

  At present, little is known about how endothelial cells respond to spatial variations in fluid shear stress such as those that occur locally during embryonic development, at heart valve leaflets, and at sites of aneurysm formation. We built an impinging flow device that exposes endothelial cells to gradients of shear stress. Using this device, we investigated the response of microvascular endothelial cells to shear-stress gradients that ranged from 0 to a peak shear stress of 9-210 dyn/cm(2). We observe that at high confluency, these cells migrate against the direction of fluid flow and concentrate in the region of maximum wall shear stress, whereas low-density microvascular endothelial cells that lack cell-cell contacts migrate in the flow direction. In addition, the cells align parallel to the flow at low wall shear stresses but orient perpendicularly to the flow direction above a critical threshold in local wall shear stress. Our observations suggest that endothelial cells are exquisitely sensitive to both magnitude and spatial gradients in wall shear stress. The impinging flow device provides a, to our knowledge, novel means to study endothelial cell migration and polarization in response to gradients in physical forces such as wall shear stress.

  View details for DOI 10.1016/j.bpj.2013.11.4502

  View details for Web of Science ID 000330132500005

  View details for PubMedID 24461011

  View details for PubMedCentralID PMC3907231

• Characterization of a Fluorescent Probe for Imaging Nitric Oxide JOURNAL OF VASCULAR RESEARCH Ghebremariam, Y. T., Huang, N. F., Karnbharnpati, S., Volz, K. S., Joshi, G. G., Anslyn, E. V., Cooke, J. P. 2014; 51 (1): 68-79

  Abstract

  Nitric oxide (NO), a potent vasodilator and anti-atherogenic molecule, is synthesized in various cell types, including vascular endothelial cells (ECs). The biological importance of NO enforces the need to develop and characterize specific and sensitive probes. To date, several fluorophores, chromophores and colorimetric techniques have been developed to detect NO or its metabolites (NO(2) and NO(3)) in biological fluids, viable cells or cell lysates.Recently, a novel probe (NO(550)) has been developed and reported to detect NO in solutions and in primary astrocytes and neuronal cells with a fluorescence signal arising from a nonfluorescent background.Here, we report further characterization of this probe by optimizing conditions for the detection and imaging of NO products in primary vascular ECs, fibroblasts, and embryonic stem cell- and induced pluripotent stem cell-derived ECs in the absence and presence of pharmacological agents that modulate NO levels. In addition, we studied the stability of this probe in cells over time and evaluated its compartmentalization in reference to organelle-labeling dyes. Finally, we synthesized an inherently fluorescent diazo ring compound (AZO(550)) that is expected to form when the nonfluorescent NO(550) reacts with cellular NO, and compared its cellular distribution with that of NO(550).NO(550) is a promising agent for imaging NO at baseline and in response to pharmacological agents that modulate its levels.

  View details for DOI 10.1159/000356445

  View details for Web of Science ID 000329771700007

  View details for PubMedID 24335468

  View details for PubMedCentralID PMC3927988

• Hindlimb ischemia MANUAL OF RESEARCH TECHNIQUES IN CARDIOVASCULAR MEDICINE Lee, J. C., Huang, N. F., Cooke, J. P., Ardehali, H., Bolli, R., Losordo, D. W. 2014: 177–86

  View details for Web of Science ID 000360465900023

• Multi-cellular interactions sustain long-term contractility of human pluripotent stem cell-derived cardiomyocytes. American journal of translational research Burridge, P. W., Metzler, S. A., Nakayama, K. H., Abilez, O. J., Simmons, C. S., Bruce, M. A., Matsuura, Y., Kim, P., Wu, J. C., Butte, M., Huang, N. F., Yang, P. C. 2014; 6 (6): 724-735

  Abstract

  Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca(2+) ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases.

  View details for PubMedID 25628783

• Effects of Dimethylarginine Dimethylaminohydrolase-1 Overexpression on the Response of the Pulmonary Vasculature to Hypoxia AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Bakr, A., Pak, O., Taye, A., Hamada, F., Hemeida, R., Janssen, W., Gierhardt, M., Ghofrani, H. A., Seeger, W., Grimminger, F., Schermuly, R. T., Witzenrath, M., Brandes, R. P., Huang, N., Cooke, J. P., Weissmann, N., Sommer, N. 2013; 49 (3): 491-500

  Abstract

  Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1(tg)) on HPV and chronic hypoxia-induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 3',5'-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1(tg) mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1(tg). The administration of either Nω-nitro-l-arginine or 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1(tg) mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia-induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO-cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia-induced PH.

  View details for DOI 10.1165/rcmb.2012-0330OC

  View details for Web of Science ID 000324195400018

  View details for PubMedID 23642043

• Conversion of Human Fibroblasts to Functional Endothelial Cells by Defined Factors ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Li, J., Huang, N. F., Zou, J., Laurent, T. J., Lee, J. C., Okogbaa, J., Cooke, J. P., Ding, S. 2013; 33 (6): 1366-?

  Abstract

  Transdifferentiation of fibroblasts to endothelial cells (ECs) may provide a novel therapeutic avenue for diseases, including ischemia and fibrosis. Here, we demonstrate that human fibroblasts can be transdifferentiated into functional ECs by using only 2 factors, Oct4 and Klf4, under inductive signaling conditions.To determine whether human fibroblasts could be converted into ECs by transient expression of pluripotency factors, human neonatal fibroblasts were transduced with lentiviruses encoding Oct4 and Klf4 in the presence of soluble factors that promote the induction of an endothelial program. After 28 days, clusters of induced endothelial (iEnd) cells seemed and were isolated for further propagation and subsequent characterization. The iEnd cells resembled primary human ECs in their transcriptional signature by expressing endothelial phenotypic markers, such as CD31, vascular endothelial-cadherin, and von Willebrand Factor. Furthermore, the iEnd cells could incorporate acetylated low-density lipoprotein and form vascular structures in vitro and in vivo. When injected into the ischemic limb of mice, the iEnd cells engrafted, increased capillary density, and enhanced tissue perfusion. During the transdifferentiation process, the endogenous pluripotency network was not activated, suggesting that this process bypassed a pluripotent intermediate step.Pluripotent factor-induced transdifferentiation can be successfully applied for generating functional autologous ECs for therapeutic applications.

  View details for DOI 10.1161/ATVBAHA.112.301167

  View details for Web of Science ID 000319119500038

  View details for PubMedID 23520160

• The modulation of endothelial cell morphology, function, and survival using anisotropic nanofibrillar collagen scaffolds BIOMATERIALS Huang, N. F., Okogbaa, J., Lee, J. C., Jha, A., Zaitseva, T. S., Paukshto, M. V., Sun, J. S., Punjya, N., Fuller, G. G., Cooke, J. P. 2013; 34 (16): 4038-4047

  Abstract

  Endothelial cells (ECs) are aligned longitudinally under laminar flow, whereas they are polygonal and poorly aligned in regions of disturbed flow. The unaligned ECs in disturbed flow fields manifest altered function and reduced survival that promote lesion formation. We demonstrate that the alignment of the ECs may directly influence their biology, independent of fluid flow. We developed aligned nanofibrillar collagen scaffolds that mimic the structure of collagen bundles in blood vessels, and examined the effects of these materials on EC alignment, function, and in vivo survival. ECs cultured on 30-nm diameter aligned fibrils re-organized their F-actin along the nanofibril direction, and were 50% less adhesive for monocytes than the ECs grown on randomly oriented fibrils. After EC transplantation into both subcutaneous tissue and the ischemic hindlimb, EC viability was enhanced when ECs were cultured and implanted on aligned nanofibrillar scaffolds, in contrast to non-patterned scaffolds. ECs derived from human induced pluripotent stem cells and cultured on aligned scaffolds also persisted for over 28 days, as assessed by bioluminescence imaging, when implanted in ischemic tissue. By contrast, ECs implanted on scaffolds without nanopatterning generated no detectable bioluminescent signal by day 4 in either normal or ischemic tissues. We demonstrate that 30-nm aligned nanofibrillar collagen scaffolds guide cellular organization, modulate endothelial inflammatory response, and enhance cell survival after implantation in normal and ischemic tissues.

  View details for DOI 10.1016/j.biomaterials.2013.02.036

  View details for PubMedID 23480958

• Spatial patterning of endothelium modulates cell morphology, adhesiveness and transcriptional signature BIOMATERIALS Huang, N. F., Lai, E. S., Ribeiro, A. J., Pan, S., Pruitt, B. L., Fuller, G. G., Cooke, J. P. 2013; 34 (12): 2928-2937

  Abstract

  Microscale and nanoscale structures can spatially pattern endothelial cells (ECs) into parallel-aligned organization, mimicking their cellular alignment in blood vessels exposed to laminar shear stress. However, the effects of spatial patterning on the function and global transcriptome of ECs are incompletely characterized. We used both parallel-aligned micropatterned and nanopatterned biomaterials to evaluate the effects of spatial patterning on the phenotype of ECs, based on gene expression profiling, functional characterization of monocyte adhesion, and quantification of cellular morphology. We demonstrate that both micropatterned and aligned nanofibrillar biomaterials could effectively guide EC organization along the direction of the micropatterned channels or nanofibrils, respectively. The ability of ECs to sense spatial patterning cues were abrogated in the presence of cytoskeletal disruption agents. Moreover, both micropatterned and aligned nanofibrillar substrates promoted an athero-resistant EC phenotype by reducing endothelial adhesiveness for monocytes and platelets, as well as by downregulating the expression of adhesion proteins and chemokines. We further found that micropatterned ECs have a transcriptional signature that is unique from non-patterned ECs, as well as from ECs aligned by shear stress. These findings highlight the importance of spatial patterning cues in guiding EC organization and function, which may have clinical relevance in the development of vascular grafts that promote patency.

  View details for DOI 10.1016/j.biomaterials.2013.01.017

  View details for Web of Science ID 000316038900008

  View details for PubMedID 23357369

  View details for PubMedCentralID PMC3581686

• Human induced pluripotent stem cell-derived endothelial cells exhibit functional heterogeneity. American journal of translational research Rufaihah, A. J., Huang, N. F., Kim, J., Herold, J., Volz, K. S., Park, T. S., Lee, J. C., Zambidis, E. T., Reijo-Pera, R., Cooke, J. P. 2013; 5 (1): 21-35

  Abstract

  Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) are promising for treatment of vascular diseases. However, hiPSC-ECs purified based on CD31 expression are comprised of arterial, venous, and lymphatic subtypes. It is unclear whether hiPSC-ECs are heterogeneous in nature, and whether there may be functional benefits of enriching for specific subtypes. Therefore, we sought to characterize the hiPSC-ECs and enrich for each subtype, and demonstrate whether such enrichment would have functional significance. The hiPSC-ECs were generated from differentiation of hiPSCs using vascular endothelial growth factor (VEGF)-A and bone morphogenetic protein-4. The hiPSC-ECs were purified based on positive expression of CD31. Subsequently, we sought to enrich for each subtype. Arterial hiPSC-ECs were induced using higher concentrations of VEGF-A and 8-bromoadenosine-3':5'-cyclic monophosphate in the media, whereas lower concentrations of VEGF-A favored venous subtype. VEGF-C and angiopoietin-1 promoted the expression of lymphatic phenotype. Upon FACS purification based on CD31+ expression, the hiPSC-EC population was observed to display typical endothelial surface markers and functions. However, the hiPSC-EC population was heterogeneous in that they displayed arterial, venous, and to a lesser degree, lymphatic lineage markers. Upon comparing vascular formation in matrigel plugs in vivo, we observed that arterial enriched hiPSC-ECs formed a more extensive capillary network in this model, by comparison to a heterogeneous population of hiPSC-ECs. This study demonstrates that FACS purification of CD31+ hiPSC-ECs produces a diverse population of ECs. Refining the differentiation methods can enrich for subtype-specific hiPSC-ECs with functional benefits of enhancing neovascularization.

  View details for PubMedID 23390563

• Chemotaxis of human induced pluripotent stem cell-derived endothelial cells. American journal of translational research Huang, N. F., Dewi, R. E., Okogbaa, J., Lee, J. C., Jalilrufaihah, A., Heilshorn, S. C., Cooke, J. P. 2013; 5 (5): 510-520

  Abstract

  This study examined the homing capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and their response to chemotactic gradients of stromal derived factor-1α (SDF). We have previously shown that EC derived from murine pluripotent stem cells can home to the ischemic hindlimb of the mouse. In the current study, we were interested to understand if ECs derived from human induced pluripotent stem cells are capable of homing. The homing capacity of iPSC-ECs was assessed after systemic delivery into immunodeficient mice with unilateral hindlimb ischemia. Furthermore, the iPSC-ECs were evaluated for their expression of CXCR4 and their ability to respond to SDF chemotactic gradients in vitro. Upon systemic delivery, the iPSC-ECs transiently localized to the lungs but did not home to the ischemic limb over the course of 14 days. To understand the mechanism of the lack of homing, the expression levels of the homing receptor, CXCR4, was examined at the transcriptional and protein levels. Furthermore, their ability to migrate in response to chemokines was assessed using microfluidic and scratch assays. Unlike ECs derived from syngeneic mouse pluripotent stem cells, human iPSC-ECs do not home to the ischemic mouse hindlimb. This lack of functional homing may represent an impairment of interspecies cellular communication or a difference in the differentiation state of the human iPSC-ECs. These results may have important implications in therapeutic delivery of iPSC-ECs.

  View details for PubMedID 23977410

• Multifunctional in vivo vascular imaging using near-infrared II fluorescence NATURE MEDICINE Hong, G., Lee, J. C., Robinson, J. T., Raaz, U., Xie, L., Huang, N. F., Cooke, J. P., Dai, H. 2012; 18 (12): 1841-?

  Abstract

  In vivo real-time epifluorescence imaging of mouse hind limb vasculatures in the second near-infrared region (NIR-II) is performed using single-walled carbon nanotubes as fluorophores. Both high spatial (∼30 μm) and temporal (<200 ms per frame) resolution for small-vessel imaging are achieved at 1-3 mm deep in the hind limb owing to the beneficial NIR-II optical window that affords deep anatomical penetration and low scattering. This spatial resolution is unattainable by traditional NIR imaging (NIR-I) or microscopic computed tomography, and the temporal resolution far exceeds scanning microscopic imaging techniques. Arterial and venous vessels are unambiguously differentiated using a dynamic contrast-enhanced NIR-II imaging technique on the basis of their distinct hemodynamics. Further, the deep tissue penetration and high spatial and temporal resolution of NIR-II imaging allow for precise quantifications of blood velocity in both normal and ischemic femoral arteries, which are beyond the capabilities of ultrasonography at lower blood velocities.

  View details for DOI 10.1038/nm.2995

  View details for PubMedID 23160236

• Endothelial Cells Derived From Nuclear Reprogramming CIRCULATION RESEARCH Wong, W. T., Huang, N. F., Botham, C. M., Sayed, N., Cooke, J. P. 2012; 111 (10): 1363-1375

  Abstract

  The endothelium plays a pivotal role in vascular homeostasis, regulating the tone of the vascular wall, and its interaction with circulating blood elements. Alterations in endothelial functions facilitate the infiltration of inflammatory cells and permit vascular smooth muscle proliferation and platelet aggregation. Therefore, endothelial dysfunction is an early event in disease processes including atherosclerosis, and because of its critical role in vascular health, the endothelium is worthy of the intense focus it has received. However, there are limitations to studying human endothelial function in vivo, or human vascular segments ex vivo. Thus, methods for endothelial cell (EC) culture have been developed and refined. Recently, methods to derive ECs from pluripotent cells have extended the scientific range of human EC studies. Pluripotent stem cells may be generated, expanded, and then differentiated into ECs for in vitro studies. Constructs for molecular imaging can also be employed to facilitate tracking these cells in vivo. Furthermore, one can generate patient-specific ECs to study the effects of genetic or epigenetic alterations on endothelial behavior. Finally, there is the opportunity to apply these cells for vascular therapy. This review focuses on the generation of ECs from stem cells; their characterization by genetic, histological, and functional studies; and their translational applications.

  View details for DOI 10.1161/CIRCRESAHA.111.247213

  View details for Web of Science ID 000310501300017

  View details for PubMedID 23104878

  View details for PubMedCentralID PMC3526979

• Aligned nanofibrillar collagen regulates endothelial organization and migration REGENERATIVE MEDICINE Lai, E. S., Huang, N. F., Cooke, J. P., Fuller, G. G. 2012; 7 (5): 649-661

  Abstract

  Modulating endothelial cell (EC) morphology and motility, with the aim to influence their biology, might be beneficial for the treatment of vascular disease. We examined the effect of nanoscale matrix anisotropy on EC organization and migration for vascular tissue engineering applications.We developed a flow processing technique to generate anisotropic nanofibrillar collagen. Human ECs were cultured on aligned or on randomly oriented collagen, and their cellular alignment and cytoskeletal organization were characterized by immunofluorescence staining and time-lapse microscopy.ECs were elongated along the direction of aligned collagen nanofibrils and had organized focal adhesions. Cellular protrusion migrated with greater directionality and higher velocity along the anisotropic nanofibrils compared with cells on random nanofibrils. The flow technique can be adapted to fabricate vascular grafts that support the endothelial phenotype.Aligned nanofibrillar collagen regulates EC organization and migration, which can significantly contribute to the development of vascular grafts.

  View details for DOI 10.2217/RME.12.48

  View details for Web of Science ID 000308387900011

  View details for PubMedID 22954436

  View details for PubMedCentralID PMC3589994

• Bioluminescence Imaging of Stem Cell-Based Therapeutics for Vascular Regeneration THERANOSTICS Huang, N. F., Okogbaa, J., Babakhanyan, A., Cooke, J. P. 2012; 2 (4): 346-354

  Abstract

  Stem cell-based therapeutics show promise for treatment of vascular diseases. However, the survival of the cells after in vivo injection into diseased tissues remains a concern. In the advent of non-invasive optical imaging techniques such as bioluminescence imaging (BLI), cell localization and survival can be easily monitored over time. This approach has recently been applied towards monitoring stem cell treatments for vascular regeneration of the coronary or peripheral arteries. In this review, we will describe the application of BLI for tracking transplanted stem cells and associating their viability with therapeutic efficacy, in preclinical disease models of vascular disease.

  View details for DOI 10.7150/thno.3694

  View details for Web of Science ID 000304031200003

  View details for PubMedID 22509198

  View details for PubMedCentralID PMC3326722

• Endothelial Cells Derived From Human iPSCS Increase Capillary Density and Improve Perfusion in a Mouse Model of Peripheral Arterial Disease ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Rufaihah, A. J., Huang, N. F., Jame, S., Lee, J. C., Nguyen, H. N., Byers, B., De, A., Okogbaa, J., Rollins, M., Reijo-Pera, R., Gambhir, S. S., Cooke, J. P. 2011; 31 (11): E72-U44

  Abstract

  Stem cell therapy for angiogenesis and vascular regeneration has been investigated using adult or embryonic stem cells. In the present study, we investigated the potential of endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) to promote the perfusion of ischemic tissue in a murine model of peripheral arterial disease.Endothelial differentiation was initiated by culturing hiPSCs for 14 days in differentiation media supplemented with BMP-4 and vascular endothelial growth factor. The hiPSC-ECs exhibited endothelial characteristics by forming capillary-like structures in matrigel and incorporating acetylated-LDL. They stained positively for EC markers such as KDR, CD31, CD144, and eNOS. In vitro exposure of hiPSC-ECs to hypoxia resulted in increased expression of various angiogenic related cytokines and growth factors. hiPSC-ECs were stably transduced with a double fusion construct encoded by the ubiquitin promoter, firefly luciferase for bioluminescence imaging and green fluorescence protein for fluorescent detection. The hiPSC-ECs (5×10(5)) were delivered by intramuscular injection into the ischemic hindlimb of SCID mice at day 0 and again on day 7 after femoral artery ligation (n=8). Bioluminescence imaging showed that hiPSC-ECs survived in the ischemic limb for at least 2 weeks. In addition, laser Doppler imaging showed that the ratio of blood perfusion was increased by hiPSC-EC treatment by comparison to the saline-treated group (0.58±0.12 versus 0.44±0.04; P=0.005). The total number of capillaries in the ischemic limb of mice receiving hiPSC-EC injections was greater than those in the saline-treated group (1284±155 versus 797±206 capillaries/mm(2)) (P<0.002).This study is a first step toward development of a regenerative strategy for peripheral arterial disease based on the use of ECs derived from hiPSCs.

  View details for DOI 10.1161/ATVBAHA.111.230938

  View details for PubMedID 21836062

• Regulation of the Matrix Microenvironment for Stem Cell Engineering and Regenerative Medicine ANNALS OF BIOMEDICAL ENGINEERING Huang, N. F., Li, S. 2011; 39 (4): 1201-1214

  Abstract

  The extracellular matrix (ECM) microenvironment consists of structural and functional molecules. The ECM relays both biochemical and biophysical cues to and from the cells to modulate cell behavior and function. The biophysical cues can be engineered and applied to cells by means of spatial patterning, matrix rigidity, and matrix actuation. Tissue engineering strategies that utilize ECMs to direct stem cell organization and lineage specification show tremendous potential. This review describes the technologies for modulating ECM spatial patterning, matrix rigidity, chemical composition, and matrix actuation. The role of ECMs in vascular tissue engineering is then discussed as a model of tissue engineering and regenerative medicine.

  View details for DOI 10.1007/s10439-011-0297-2

  View details for Web of Science ID 000289103500006

  View details for PubMedID 21424849

  View details for PubMedCentralID PMC3568678

• Proteomic identification of biomarkers of vascular injury. American journal of translational research Huang, N. F., Kurpinski, K., fang, q., Lee, R. J., Li, S. 2011; 3 (2): 139-148

  Abstract

  Predictive biomarkers may be beneficial for detecting, diagnosing, and assessing the risk of restenosis and vascular injury. We utilized proteomic profiling to identify protein markers in the blood following vascular injury, and corroborated the differential protein expression with immunological approaches. Rats underwent carotid artery injury, and plasma was collected after 2 or 5 weeks. Proteomic profiling was carried out by two-dimensional differential in-gel electrophoresis. The differentially expressed plasma proteins were identified by mass spectroscopy and confirmed by immunoblotting. Proteomic profiling by two-dimensional differential in-gel electrophoresis and mass spectroscopy revealed plasma proteins that were differentially expressed at 2 weeks after injury. Among the proteins identified included vitamin D binding protein (VDBP), aldolase A (aldo A), and apolipoproteinE (apoE). Immunoblotting results validated a significant reduction in these proteins in the plasma at 2 or 5 weeks after vascular injury, in comparison to control animals without vascular injury. These findings suggest that VDBP, aldo A, and apoE may be biomarkers for vascular injury, which will have important prognostic and diagnostic implications.

  View details for PubMedID 21416056

  View details for PubMedCentralID PMC3056560

• A matrix micropatterning platform for cell localization and stem cell fate determination ACTA BIOMATERIALIA Huang, N. F., Patlolla, B., Abilez, O., Sharma, H., Rajadas, J., Beygui, R. E., Zarins, C. K., Cooke, J. P. 2010; 6 (12): 4614-4621

  Abstract

  To study the role of cell-extracellular matrix (ECM) interactions, microscale approaches provide the potential to perform high throughput assessment of the effect of the ECM microenvironment on cellular function and phenotype. Using a microscale direct writing (MDW) technique, we characterized the generation of multicomponent ECM microarrays for cellular micropatterning, localization and stem cell fate determination. ECMs and other biomolecules of various geometries and sizes were printed onto epoxide-modified glass substrates to evaluate cell attachment by human endothelial cells. The endothelial cells displayed strong preferential attachment to the ECM patterned regions and aligned their cytoskeleton along the direction of the micropatterns. We next generated ECM microarrays that contained one or more ECM components (namely gelatin, collagen IV and fibronectin) and then cultured murine embryonic stem cell (ESCs) on the microarrays. The ESCs selectively attached to the micropatterned features and expressed markers associated with a pluripotent phenotype, such as E-cadherin and alkaline phosphatase, when maintained in growth medium containing leukemia inhibitory factor. In the presence of the soluble factors retinoic acid and bone morphogenetic protein-4 the ESCs differentiated towards the ectodermal lineage on the ECM microarray with differential ECM effects. The ESCs cultured on gelatin showed significantly higher levels of pan cytokeratin expression, when compared with cells cultured on collagen IV or fibronectin, suggesting that gelatin preferentially promotes ectodermal differentiation. In summary, our results demonstrate that MDW is a versatile approach to print ECMs of diverse geometries and compositions onto surfaces, and it is amenable to the generation of multicomponent ECM microarrays for stem cell fate determination.

  View details for DOI 10.1016/j.actbio.2010.06.033

  View details for Web of Science ID 000284385300018

  View details for PubMedID 20601236

  View details for PubMedCentralID PMC2957527

• Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells STEM CELLS AND DEVELOPMENT Huang, N. F., Fleissner, F., Sun, J., Cooke, J. P. 2010; 19 (10): 1617-1625

  Abstract

  Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester (L-NAME) for up to 11 days were not significantly different from vehicle-treated cells in EC markers. However, by 14 days, L-NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of L-NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of L-NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by L-NAME, confirming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function.

  View details for DOI 10.1089/scd.2009.0417

  View details for Web of Science ID 000282394400014

  View details for PubMedID 20064011

  View details for PubMedCentralID PMC3121801

• Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Huang, N. F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M. D., Wu, J. C., Gambhir, S. S., Cooke, J. P. 2010; 30 (5): 984-U224

  Abstract

  We examined the effect of delivery modality on the survival, localization, and functional effects of exogenously administered embryonic stem cells (ESCs) or endothelial cells derived from them (ESC-ECs) in the ischemic hindlimb.Murine ESCs or ESC-ECs were stably transduced with a construct for bioluminescence imaging (BLI) and fluorescent detection. In a syngeneic murine model of limb ischemia, ESCs or ESC-ECs were delivered by intramuscular (IM), intrafemoral artery (IA), or intrafemoral vein injections (n=5 in each group). For 2 weeks, cell survival and localization were tracked by BLI and confirmed by immunohistochemistry, and functional improvement was assessed by laser Doppler perfusion. BLI showed that ESCs localized to the ischemic limb after IM or IA, but not after intrafemoral vein administration. Regardless of the route of administration, ESCs were detected outside the hindlimb circulation in the spleen or lungs. ESCs did not improve limb perfusion and generated teratomas. In contrast, ESC-ECs delivered by all 3 modalities localized to the ischemic limb, as assessed by BLI. Most surprisingly, ESC-EC injected intrafemoral vein eventually localized to the ischemic limb after initially lodging in the pulmonary circulation. Immunohistochemical studies confirmed the engraftment of ESC-ECs into the limb vasculature after 2 weeks. Notably, ESC-ECs were not detected in the spleen or lungs after 2 weeks, regardless of route of administration. Furthermore, ESC-ECs significantly improved limb perfusion and neovascularization compared with the parental ESCs or the vehicle control group.In contrast to parental ESCs, ESC-ECs preferentially localized in the ischemic hindlimb by IA, IM, and intrafemoral vein delivery. ESC-ECs engrafted into the ischemic microvasculature, enhanced neovascularization, and improved limb perfusion.

  View details for DOI 10.1161/ATVBAHA.110.202796

  View details for PubMedID 20167654

• Engineering of aligned skeletal muscle by micropatterning AMERICAN JOURNAL OF TRANSLATIONAL RESEARCH Huang, N. F., Lee, R. J., Li, S. 2010; 2 (1): 43-55

  Abstract

  Tissue engineered skeletal muscle has tremendous potential for the treatment of muscular injury or muscular dysfunction. However, in vitro methods to generate skeletal muscle with physiologically aligned myofiber structure remains limited. To develop a robust in vitro model that resembles the physiologically aligned structure of muscle fibers, we fabricated micropatterned polymer membranes of poly(dimethylsiloxane) (PDMS) with parallel microgrooves, and then examined the effect of micropatterning on myoblast cellular organization and the cell fusion process. In comparison to the myoblasts on non-patterned PDMS films, myoblasts on micropatterned PDMS films had well-organized F-actin assembly in close proximity to the direction of microgrooves, along with enhanced levels of myotube formation at early time points. The increase of cell cycle regulator p21(WAF/Cip1) and the organized interactions of N-cadherin in myoblasts on micropatterned surfaces may contribute to the enhanced formation of myotubes. Similar results of cellular alignment was observed when myoblasts were cultured on microfluidically patterned poly(2-hydroxyethyl methacrylate) (pHEMA) microgrooves, and the micropatterns were found to detach from the Petri dish over time. To apply this technology for generating aligned tissue-like muscle constructs, we developed a methodology to transfer the aligned myotubes to biodegradable collagen gels. Histological analysis revealed the persistence of aligned cellular organization in the collagen gels. Together, these results demonstrate that micropatterned PDMS or pHEMA can promote cell alignment and fusion along the direction of the microgrooves, and this platform can be utilized to transfer aligned myotubes on biodegradable hydrogels. This study highlights the importance of spatial cues in creating aligned skeletal muscle for tissue engineering and muscular regeneration applications.

  View details for Web of Science ID 000208694000003

  View details for PubMedCentralID PMC2826821

• Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion 82nd National Conference and Exhibitions and Scientific Sessions of the American-Heart-Association Huang, N. F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M. D., Wu, J. C., Gambhir, S. S., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 2009: S1152–S1152

  View details for Web of Science ID 000271831504251

• Human Induced Pluripotent Stem Cell-derived Endothelial Cells Exhibit Heterogeneity Jalil, R. A., Nguyen, H. N., Huang, N. F., Hunter, A. L., Pera, R. R., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 2009: S1092

  View details for Web of Science ID 000271831503797

• Bone marrow-derived mesenchymal stem cells in fibrin augment angiogenesis in the chronically infarcted myocardium REGENERATIVE MEDICINE Huang, N. F., Lam, A., fang, q., Sievers, R. E., Li, S., Lee, R. J. 2009; 4 (4): 527-538

  Abstract

  Current efforts to treat myocardial infarction include the delivery of cells and matrix scaffolds. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells that secrete angiogenic growth factors, and fibrin has been shown to be a biomaterial that provides structural support to cells and tissues. The objective of this study was to characterize the attachment and viability of BM-MSCs in fibrin in vitro, and then to assess the efficacy of treatment with BM-MSCs in fibrin for promoting neovascularization in the chronically infarcted myocardium.BM-MSCs were cultured in fibrin and assessed for cell attachment and viability by using immunofluorescence staining for actin filaments and Live/Dead((R)) viability assays, respectively. To determine the efficacy of BM-MSCs in fibrin in vivo, chronically infarcted rat hearts were treated with either cells, cells in fibrin, fibrin or saline (n = 9). After 5 weeks, the infarct scar tissues were assessed for neovascularization.BM-MSCs exhibited robust cell attachment and viability when cultured in fibrin in vitro. Furthermore, when injected together into the infarcted tissue, BM-MSCs in fibrin could enhance neovasculature formation by increasing capillary density, in comparison to treatment by cells or fibrin separately. Concomitant to significant improvement in capillary density was an increase in the levels of VEGF in the infarct scar.This study demonstrates the angiogenic potential of the combined delivery of BM-MSCs and fibrin, and highlights the advantage of stem cell-matrix approaches for myocardial repair.

  View details for DOI 10.2217/RME.09.32

  View details for Web of Science ID 000268122400010

  View details for PubMedID 19580402

  View details for PubMedCentralID PMC2778008

• Embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia. Journal of visualized experiments : JoVE Huang, N. F., Niiyama, H., De, A., Gambhir, S. S., Cooke, J. P. 2009

  Abstract

  Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD. In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.

  View details for DOI 10.3791/1034

  View details for PubMedID 19229180

  View details for PubMedCentralID PMC2781824

• Murine model of hindlimb ischemia. Journal of visualized experiments : JoVE Niiyama, H., Huang, N. F., Rollins, M. D., Cooke, J. P. 2009

  Abstract

  In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.

  View details for DOI 10.3791/1035

  View details for PubMedID 19229179

  View details for PubMedCentralID PMC2763292

• Mesenchymal stem cells for vascular regeneration REGENERATIVE MEDICINE Huang, N. F., Li, S. 2008; 3 (6): 877-892

  Abstract

  Mesenchymal stem cells (MSCs) have tremendous potential for regenerative medicine, and have been researched for the treatment of cardiovascular diseases. MSCs are a promising cell type because of their ease of isolation and expansion, their multipotency and their low immunogenicity. However, in order to fully utilize the therapeutic potential of MSCs, it is important to understand the intrinsic property of MSCs and the role of the microenvironment in modulating MSC behavior and function. Microenvironmental factors such as mechanical cues, soluble factors and matrix properties not only regulate MSC differentiation, but also modulate MSC signaling to the surrounding environment. Understanding the properties of MSCs and the role of the microenvironment will be beneficial for developing in vivo therapies for the construction of tissue-engineered vascular grafts and the treatment of ischemic cardiac tissues.

  View details for DOI 10.2217/17460751.3.6.877

  View details for Web of Science ID 000261008000015

  View details for PubMedID 18947310

  View details for PubMedCentralID PMC2596657

• Mechanobiology of mesenchymal stem cells and their use in cardiovascular repair FRONTIERS IN BIOSCIENCE-LANDMARK Park, J. S., Huang, N. F., Kurpinski, K. T., Patel, S., Hsu, S., Li, S. 2007; 12: 5098-5116

  Abstract

  Mesenchymal stem cells (MSCs) derived from bone marrow have shown great promise in tissue repair. While these cells induce little immune response, they show marked self-renewal properties and can differentiate into many cell types. Recent evidence shows that mechanical factors such as fluid shear stress, mechanical strain and the rigidity of extracellular matrix can regulate the proliferation and differentiation of MSCs through various signaling pathways. Transplanted MSCs enhance angiogenesis and contribute to remodeling of the vasculature. In this review, we will focus on the responses of vascular cells and MSCs to shear stress, strain and matrix rigidity and will discuss the use of MSCs in myocardial repair and vascular tissue engineering.

  View details for DOI 10.2741/2551

  View details for Web of Science ID 000253943900025

  View details for PubMedID 17569633

• Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering TISSUE ENGINEERING Huang, N. F., Lee, R. J., Li, S. 2007; 13 (8): 1809-1823

  Abstract

  The field of cardiovascular tissue engineering has experienced tremendous advances in the past several decades, but the clinical reality of engineered heart tissue and vascular conduits remains immature. Stem cells and progenitor cells are promising cell sources for engineering functional cardiovascular tissues. To realize the therapeutic potential of stem cells and progenitor cells, we need to understand how microenvironmental cues modulate and guide stem cell differentiation and organization. This review describes the current understanding of the chemical and physical regulation of embryonic and adult stem cells for potential applications in cardiovascular repair, focusing on cardiac therapies after myocardial infarction and the engineering of vascular conduits.

  View details for DOI 10.1089/ten.2006.0096

  View details for Web of Science ID 000248742200003

  View details for PubMedID 17518703

• Antibody targeting of stem cells to infarcted myocardium STEM CELLS Lee, R. J., fang, q., Davol, P. A., Gu, Y., Sievers, R. E., Grabert, R. C., Gall, J. M., Tsang, E., Yee, M. S., Fok, H., Huang, N. F., Padbury, J. F., Larrick, J. W., Lum, L. G. 2007; 25 (3): 712-717

  Abstract

  Hematopoietic stem cell (HSC) therapy for myocardial repair is limited by the number of stem cells that migrate to, engraft in, and proliferate at sites of injured myocardium. To alleviate this limitation, we studied whether a strategy using a bispecific antibody (BiAb) could target human stem cells specifically to injured myocardium and preserve myocardial function. Using a xenogeneic rat model whereby ischemic injury was induced by transient ligation of the left anterior descending artery (LAD), we determined the ability of a bispecific antibody to target human CD34+ cells to specific antigens expressed in ischemic injured myocardium. A bispecific antibody comprising an anti-CD45 antibody recognizing the common leukocyte antigen found on HSCs and an antibody recognizing myosin light chain, an organ-specific injury antigen expressed by infarcted myocardium, was prepared by chemical conjugation. CD34+ cells armed and unarmed with this BiAb were injected intravenously in rats 2 days postmyocardial injury. Immunohistochemistry studies showed that the armed CD34+ cells specifically localized to the infarcted region of the heart, colocalized with troponin T-stained cells, and colocalization with vascular structures. Compared to unarmed CD34+ cells, the bispecific antibody improved delivery of the stem cells to injured myocardium, and such targeted delivery was correlated with improved myocardial function 5 weeks after infarction (p < .01). Bispecific antibody targeting offers a unique means to improve the delivery of stem cells to facilitate organ repair and a tool to study stem cell biology.

  View details for DOI 10.1634/stemcells.2005-0602

  View details for Web of Science ID 000244847100020

  View details for PubMedID 17138964

• Myotube assembly on nanofibrous and micropatterned polymers NANO LETTERS HUANG, N. F., Patel, S., Thakar, R. G., Wu, J., Hsiao, B. S., Chu, B., Lee, R. J., Li, S. 2006; 6 (3): 537-542

  Abstract

  Skeletal muscle consists of parallel bundles of myotubes formed by the fusion of myoblasts. We fabricated nanofibrous and micropatterned polymers as cell culture substrates to guide the morphogenesis of muscular tissue. The nanoscale and microscale topographic features regulate cell and cytoskeleton alignment, myotube assembly, myotube striation, and myoblast proliferation. This bottom-up approach from nanoscale to tissue level demonstrates the potential of nanofibrous polymers for engineering the assembly of cell and tissue structure.

  View details for DOI 10.1021/nl060060o

  View details for Web of Science ID 000236049800040

  View details for PubMedID 16522058

• A rodent model of myocardial infarction for testing the efficacy of cells and polymers for myocardial reconstruction NATURE PROTOCOLS Huang, N. F., Sievers, R. E., Park, J. S., fang, q., Li, S., Lee, R. J. 2006; 1 (3): 1596-1609

  Abstract

  We have developed a robust rat model of myocardial infarction (MI). Here we describe the step-by-step protocol for creating an ischemia-reperfusion rat model of MI. We also describe how to deliver therapeutic injections of mesenchymal stem cells (MSCs) together with fibrin, to show an application of this model. In addition, to confirm the presence of fibrin and cells in the infarct, visualization of MSCs and fibrin by histological techniques are also described. The ischemia-reperfusion MI model can be modified and generalized for use with various injectable polymers, cell types, drugs, DNA and combinations thereof. The model can be created in 7 days or less, depending on the timing of therapeutic intervention.

  View details for DOI 10.1038/nprot.2006.188

  View details for Web of Science ID 000251155400065

  View details for PubMedID 17406452

• Mechanotransduction in endothelial cell migration JOURNAL OF CELLULAR BIOCHEMISTRY Li, S., HUANG, N. F., HSU, S. 2005; 96 (6): 1110-1126

  Abstract

  The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. EC migration can be regulated by different mechanisms such as chemotaxis, haptotaxis, and mechanotaxis. This review will focus on fluid shear stress-induced mechanotransduction during EC migration. EC migration and mechanotransduction can be modulated by cytoskeleton, cell surface receptors such as integrins and proteoglycans, the chemical and physical properties of extracellular matrix (ECM) and cell-cell adhesions. The shear stress applied on the luminal surface of ECs can be sensed by cell membrane and associated receptor and transmitted throughout the cell to cell-ECM adhesions and cell-cell adhesions. As a result, shear stress induces directional migration of ECs by promoting lamellipodial protrusion and the formation of focal adhesions (FAs) at the front in the flow direction and the disassembly of FAs at the rear. Persistent EC migration in the flow direction can be driven by polarized activation of signaling molecules and the positive feedback loops constituted by Rho GTPases, cytoskeleton, and FAs at the leading edge. Furthermore, shear stress-induced EC migration can overcome the haptotaxis of ECs. Given the hemodynamic environment of the vascular system, mechanotransduction during EC migration has a significant impact on vascular development, angiogenesis, and vascular wound healing.

  View details for DOI 10.1002/jcb.20614

  View details for Web of Science ID 000233353000002

  View details for PubMedID 16167340

• Injectable biopolymers enhance angiogenesis after myocardial infarction TISSUE ENGINEERING HUANG, N. F., Yu, J. S., Sievers, R., Li, S., Lee, R. J. 2005; 11 (11-12): 1860-1866

  Abstract

  Novel strategies by which to repair ischemic myocardium after myocardial infarction include the use of three-dimensional polymer scaffolds. A comparative study was carried out to assess the therapeutic potential of fibrin, collagen I, and Matrigel as injectable biopolymers for repair after myocardial infarction. Using a rat model of left coronary artery occlusion followed by reperfusion, local injection of the biopolymers into the infarct zone yielded significantly higher levels of capillary formation, when compared with the saline control group, at 5 weeks posttreatment. However, the degree of angiogenesis was not significantly different among the biopolymers. In addition, the collagen biopolymer significantly enhanced infiltration of myofibroblasts into the infarct area when compared with the control group. The results of this study highlight the potential clinical benefit of these biopolymers as injectable scaffolds or cell delivery vehicles to the infarct zone after infarction.

  View details for Web of Science ID 000234829500025

  View details for PubMedID 16411832

• Base hydrolysis of phosphodiester bonds in pneumococcal polysaccharides BIOPOLYMERS Pujar, N. S., HUANG, N. F., Daniels, C. L., Dieter, L., Dayton, M. G., Lee, A. L. 2004; 75 (1): 71-84

  Abstract

  A comprehensive study of the base hydrolysis of all phosphodiester bond-containing capsular polysaccharides of the 23-valent pneumococcal vaccine is described here. Capsular polysaccharides from serotypes 6B, 10A, 17F, 19A, 19F, and 20 contain a phosphodiester bond that connects the repeating units in these polysaccharides (also referred to as backbone phosphodiester bonds), and polysaccharides from serotypes 11A, 15B, 18C, and 23F contain a phosphodiester bond that links a side chain to their repeating units. Molecular weight measurements of the polysaccharides, using high performance size exclusion chromatography with tandem multiangle laser light scattering and refractive index detection, was used to evaluate the kinetics of hydrolysis. The measurement of molecular weight provides a high degree of sensitivity in the case of small extents of reaction, thus allowing reliable measurements of the kinetics over short times. Pseudo-first-order rate constants for these polysaccharides were estimated using a simple model that accounts for the polydispersity of the starting sample. It was found that the relative order of backbone phosphodiester bond instability due to base hydrolysis was 19A > 10A > 19F > 6B > 17F, 20. Degradation of side-chain phosphodiester bonds was not observed, although the high degree of sensitivity in measurements is lost in this case, due to the low contribution of the side chains to the total polysaccharide molecular weight. In comparison with literature data on pneumococcal polysaccharide 6A, 19A was found to be the more labile, and hence appears to be the most labile pneumococcal polysaccharide studied to date. The rate of hydrolysis increased at higher pH and in the presence of divalent cation, but the extent was lower than expected based on similar data on RNA. Finally, the differences in the phosphodiester bond stabilities were analyzed by considering stereochemical factors in these polysaccharides. These results also provide a framework for evaluation of molecular integrity of phosphodiester-bond-containing polysaccharides in different solution conditions.

  View details for DOI 10.1002/bip.20087

  View details for Web of Science ID 000223580500005

  View details for PubMedID 15307199

• Tissue engineering of muscle on micropatterned polymer films. Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference Huang, N. F., Thakar, R. G., Wong, M., Kim, D., Lee, R. J., Li, S. 2004; 7: 4966-4969

  Abstract

  Tissue engineered skeletal muscle has potential physiologically relevant environments to study myogenesis and investigate the organization, differentiation and proliferation to be used for the therapy of muscular dysfunction. In order to engineer skeletal muscle that better resemble the structured architecture in vivo, we cultured myoblasts on topographically micropatterned elastic polymer films with 10-mum wide microgrooves. The organization and differentiation of myoblasts on nonpatterned and micropatterned PDMS films were characterized. In comparison to the myoblasts on nonpatterned PDMS films, myoblasts on micropatterned PDMS films aligned themselves along the direction of the microgrooves. The myoblasts on micropatterned films formed long and unbranched myotubes that had uniform diameter and aligned in the microgroove direction, suggesting that microgrooves promote end-to end fusion of myoblasts; in contrast, myotubes formed on nonpatterned surface were short and less uniform in diameter, and oriented in various directions. This study demonstrates a new approach to engineer muscular tissues on flexible substrate, and highlights the importance of topographical cues for creating more engineer skeletal muscle.

  View details for PubMedID 17271429

• Skin substitutes and wound healing: Current status and challenges WOUNDS-A COMPENDIUM OF CLINICAL RESEARCH AND PRACTICE Eisenbud, D., HUANG, N. F., Luke, S., Silberklang, M. 2004; 16 (1): 2-17

  View details for Web of Science ID 000188459400002

• Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Levenberg, S., HUANG, N. F., Lavik, E., Rogers, A. B., Itskovitz-Eldor, J., Langer, R. 2003; 100 (22): 12741-12746

  Abstract

  Human embryonic stem (hES) cells hold promise as an unlimited source of cells for transplantation therapies. However, control of their proliferation and differentiation into complex, viable 3D tissues is challenging. Here we examine the use of biodegradable polymer scaffolds for promoting hES cell growth and differentiation and formation of 3D structures. We show that complex structures with features of various committed embryonic tissues can be generated, in vitro, by using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment such as poly(lactic-co-glycolic acid)/poly(L-lactic acid) polymer scaffolds. We found that hES cell differentiation and organization can be influenced by the scaffold and directed by growth factors such as retinoic acid, transforming growth factor beta, activin-A, or insulin-like growth factor. These growth factors induced differentiation into 3D structures with characteristics of developing neural tissues, cartilage, or liver, respectively. In addition, formation of a 3D vessel-like network was observed. When transplanted into severe combined immunodeficient mice, the constructs continue to express specific human proteins in defined differentiated structures and appear to recruit and anastamose with the host vasculature. This approach provides a unique culture system for addressing questions in cell and developmental biology, and provides a potential mechanism for creating viable human tissue structures for therapeutic applications.

  View details for DOI 10.1073/pnas.1735463100

  View details for Web of Science ID 000186301100043

  View details for PubMedID 14561891

  View details for PubMedCentralID PMC240688

• Regulation of vascular smooth muscle cells by micropatterning BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Thakar, R. G., Ho, F., HUANG, N. F., Liepmann, D., Li, S. 2003; 307 (4): 883-890

  Abstract

  Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.

  View details for DOI 10.1016/S0006-291X(03)01285-3

  View details for Web of Science ID 000184510100021

  View details for PubMedID 12878194

• Apoptosis in skin wound healing WOUNDS-A COMPENDIUM OF CLINICAL RESEARCH AND PRACTICE Huang, N. F., Zac-Varghese, S., Luke, S. 2003; 15 (6): 182-194

  View details for Web of Science ID 000183521400003

• Detection of numerical chromosomal abnormalities in epithelial ovarian neoplasms by fluorescence in situ hybridization (Fish) and a review of the current literature APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Huang, N. F., Gupta, M., Varghese, S., Rao, S., Luke, S. 2002; 10 (2): 187-193

  Abstract

  Preliminary retrospective chromosomal analysis was performed using fluorescence in situ hybridization (FISH) with alphoid DNA probes for chromosomes 1, 3, 6, 8, 12, 17, and X. Twenty-four epithelial ovarian tumors were examined in this pilot study, including 8 borderline (LMP) serous tumors, 9 serous carcinoma, and 7 mucinous carcinoma. Hybridization signals were counted to demonstrate the frequency of aneusomy, trace chromosomal progression, and identify the predominance of chromosome copy number abnormalities that are specific to a particular histotype. The preliminary results revealed almost an equal number of mean aneusomies in serous (58.13 +/- 13%) and mucinous (64.33 +/- 10%) carcinoma, both of which were slightly higher than borderline serous tumors (50.57 +/- 17%). Hyposomies 3 and X were significantly higher in mucinous than in serous ovarian carcinomas, and lowest in borderline serous tumors (P<0.05 and P<0.01). Signal losses were a more frequent abnormality in all three histologic subtypes. Mucinous carcinomas showed a loss of chromosomes 8 (45.00 +/- 28%) and 3 (43.14 +/- 16%), in addition to a loss of chromosome X (56.29 +/- 12%). Serous carcinomas showed a gain of chromosome 1 (39.44 +/- 32%), followed by losses of chromosomes 6 (37.00 +/- 20%), 17 (36.44 +/- 19%), and 8 (36.89 +/- 19%). In borderline serous tumors, the most frequent findings were losses of chromosomes 6 (38.00 +/- 17%), 12 (36.88 +/- 17%), and 3 (36.13 +/- 21%). However, further research is necessary to substantiate these preliminary results and elucidate their clinical significance. A brief review of the literature pertaining to interphase cytogenetics in ovarian epithelial tumors is discussed also.

  View details for Web of Science ID 000175774500016

  View details for PubMedID 12051640

• Optimization of fluorescence in situ hybridization (FISH) in cytological specimens. Cell vision : the journal of analytical morphology Luke, S., HUANG, N. F., Goyal, N., Varkey, J. A., Alsay-Oculam, A. A., Gupta, M., Ladoulis, C. T. 1998; 5 (1): 85-86

  View details for PubMedID 9660738