Professional Education

  • Doctor of Philosophy, Stanford University, BIOE-PHD (2017)
  • Master of Science, Harvard University, Engineering Sciences (BioE) (2012)
  • Bachelor of Arts, Harvard University, Engineering Sciences (BioE) (2012)

All Publications

  • Bioengineering strategies to accelerate stem cell therapeutics NATURE Madl, C. M., Heilshorn, S. C., Blau, H. M. 2018; 557 (7705): 335–42


    Although only a few stem cell-based therapies are currently available to patients, stem cells hold tremendous regenerative potential, and several exciting clinical applications are on the horizon. Biomaterials with tuneable mechanical and biochemical properties can preserve stem cell function in culture, enhance survival of transplanted cells and guide tissue regeneration. Rapid progress with three-dimensional hydrogel culture platforms provides the opportunity to grow patient-specific organoids, and has led to the discovery of drugs that stimulate endogenous tissue-specific stem cells and enabled screens for drugs to treat disease. Therefore, bioengineering technologies are poised to overcome current bottlenecks and revolutionize the field of regenerative medicine.

    View details for DOI 10.1038/s41586-018-0089-z

    View details for Web of Science ID 000432242000045

    View details for PubMedID 29769665

  • Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling. Nature materials Madl, C. M., LeSavage, B. L., Dewi, R. E., Dinh, C. B., Stowers, R. S., Khariton, M., Lampe, K. J., Nguyen, D., Chaudhuri, O., Enejder, A., Heilshorn, S. C. 2017; 16 (12): 1233–42


    Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ∼0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell-cell contact and promote β-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.

    View details for DOI 10.1038/nmat5020

    View details for PubMedID 29115291

  • Bio-Orthogonally Crosslinked, Engineered Protein Hydrogels with Tunable Mechanics and Biochemistry for Cell Encapsulation ADVANCED FUNCTIONAL MATERIALS Madl, C. M., Katz, L. M., Heilshorn, S. C. 2016; 26 (21): 3612-3620
  • Tuning Bulk Hydrogel Degradation by Simultaneous Control of Proteolytic Cleavage Kinetics and Hydrogel Network Architecture ACS MACRO LETTERS Madl, C. M., Katz, L. M., Heilshorn, S. C. 2018; 7 (11): 1302–7
  • Investigating the interplay between substrate stiffness and ligand chemistry in directing mesenchymal stem cell differentiation within 3D macro-porous substrates. Biomaterials Haugh, M. G., Vaughan, T. J., Madl, C. M., Raftery, R. M., McNamara, L. M., O'Brien, F. J., Heilshorn, S. C. 2018; 171: 23–33


    Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior.

    View details for DOI 10.1016/j.biomaterials.2018.04.026

    View details for PubMedID 29677521

  • Engineering Hydrogel Microenvironments to Recapitulate the Stem Cell Niche. Annual review of biomedical engineering Madl, C. M., Heilshorn, S. C. 2018; 20: 21–47


    Stem cells are a powerful resource for many applications including regenerative medicine, patient-specific disease modeling, and toxicology screening. However, eliciting the desired behavior from stem cells, such as expansion in a naive state or differentiation into a particular mature lineage, remains challenging. Drawing inspiration from the native stem cell niche, hydrogel platforms have been developed to regulate stem cell fate by controlling microenvironmental parameters including matrix mechanics, degradability, cell-adhesive ligand presentation, local microstructure, and cell-cell interactions. We survey techniques for modulating hydrogel properties and review the effects of microenvironmental parameters on maintaining stemness and controlling differentiation for a variety of stem cell types. Looking forward, we envision future hydrogel designs spanning a spectrum of complexity, ranging from simple, fully defined materials for industrial expansion of stem cells to complex, biomimetic systems for organotypic cell culture models.

    View details for DOI 10.1146/annurev-bioeng-062117-120954

    View details for PubMedID 29220201

  • Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D. Journal of visualized experiments : JoVE LeSavage, B. L., Suhar, N. A., Madl, C. M., Heilshorn, S. C. 2018


    Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

    View details for DOI 10.3791/57739

    View details for PubMedID 29863669

  • Bioorthogonal Strategies for Engineering Extracellular Matrices ADVANCED FUNCTIONAL MATERIALS Madl, C. M., Heilshorn, S. C. 2018; 28 (11)
  • In-situ tissue regeneration through SDF-1 alpha driven cell recruitment and stiffness-mediated bone regeneration in a critical-sized segmental femoral defect ACTA BIOMATERIALIA Cipitria, A., Boettcher, K., Schoenhals, S., Garske, D. S., Schmidt-Bleek, K., Ellinghaus, A., Dienelt, A., Peters, A., Mehta, M., Madl, C. M., Huebsch, N., Mooney, D. J., Duda, G. N. 2017; 60: 50–63


    In-situ tissue regeneration aims to utilize the body's endogenous healing capacity through the recruitment of host stem or progenitor cells to an injury site. Stromal cell-derived factor-1α (SDF-1α) is widely discussed as a potent chemoattractant. Here we use a cell-free biomaterial-based approach to (i) deliver SDF-1α for the recruitment of endogenous bone marrow-derived stromal cells (BMSC) into a critical-sized segmental femoral defect in rats and to (ii) induce hydrogel stiffness-mediated osteogenic differentiation in-vivo. Ionically crosslinked alginate hydrogels with a stiffness optimized for osteogenic differentiation were used. Fast-degrading porogens were incorporated to impart a macroporous architecture that facilitates host cell invasion. Endogenous cell recruitment to the defect site was successfully triggered through the controlled release of SDF-1α. A trend for increased bone volume fraction (BV/TV) and a significantly higher bone mineral density (BMD) were observed for gels loaded with SDF-1α, compared to empty gels at two weeks. A trend was also observed, albeit not statistically significant, towards matrix stiffness influencing BV/TV and BMD at two weeks. However, over a six week time-frame, these effects were insufficient for bone bridging of a segmental femoral defect. While mechanical cues combined with ex-vivo cell encapsulation have been shown to have an effect in the regeneration of less demanding in-vivo models, such as cranial defects of nude rats, they are not sufficient for a SDF-1α mediated in-situ regeneration approach in segmental femoral defects of immunocompetent rats, suggesting that additional osteogenic cues may also be required.Stromal cell-derived factor-1α (SDF-1α) is a chemoattractant used to recruit host cells for tissue regeneration. The concept that matrix stiffness can direct mesenchymal stromal cell (MSC) differentiation into various lineages was described a decade ago using in-vitro experiments. Recently, alginate hydrogels with an optimized stiffness and ex-vivo encapsulated MSCs were shown to have an effect in the regeneration of skull defects of nude rats. Here, we apply this material system, loaded with SDF-1α and without encapsulated MSCs, to (i) recruit endogenous cells and (ii) induce stiffness-mediated osteogenic differentiation in-vivo, using as model system a load-bearing femoral defect in immunocompetent rats. While a cell-free approach is of great interest from a translational perspective, the current limitations are described.

    View details for DOI 10.1016/j.actbio.2017.07.032

    View details for Web of Science ID 000411421400005

    View details for PubMedID 28739546

  • Tyrosine-Selective Functionalization for Bio-Orthogonal Cross-Linking of Engineered Protein Hydrogels. Bioconjugate chemistry Madl, C. M., Heilshorn, S. C. 2017


    Engineered protein hydrogels have shown promise as artificial extracellular matrix materials for the 3D culture of stem cells due to the ability to decouple hydrogel biochemistry and mechanics. The modular design of these proteins allows for incorporation of various bioactive sequences to regulate cellular behavior. However, the chemistry used to cross-link the proteins into hydrogels can limit what bioactive sequences can be incorporated, in order to prevent nonspecific cross-linking within the bioactive region. Bio-orthogonal cross-linking chemistries may allow for the incorporation of any arbitrary bioactive sequence, but site-selective and scalable incorporation of bio-orthogonal reactive groups such as azides that do not rely on commonly used amine-reactive chemistry is often challenging. In response, we have optimized the reaction of an azide-bearing 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) with engineered elastin-like proteins (ELPs) to selectively azide-functionalize tyrosine residues within the proteins. The PTAD-azide functionalized ELPs cross-link with bicyclononyne (BCN) functionalized ELPs via the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to form hydrogels. Human mesenchymal stem cells and murine neural progenitor cells encapsulated within these hydrogels remain highly viable and maintain their phenotypes in culture. Tyrosine-specific modification may expand the number of bioactive sequences that can be designed into protein-engineered materials by permitting incorporation of lysine-containing sequences without concern for nonspecific cross-linking.

    View details for DOI 10.1021/acs.bioconjchem.6b00720

    View details for PubMedID 28151642

  • YAP-dependent mechanotransduction is required for proliferation and migration on native-like substrate topography BIOMATERIALS Mascharak, S., Benitez, P. L., Proctor, A. C., Madl, C. M., Hu, K. H., Dewi, R. E., Butte, M. J., Heilshorn, S. C. 2017; 115: 155-166


    Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials.

    View details for DOI 10.1016/j.biomaterials.2016.11.019

    View details for Web of Science ID 000390642100014

    View details for PubMedID 27889666

  • Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation NATURE MATERIALS Huebsch, N., Lippens, E., Lee, K., Mehta, M., Koshy, S. T., Darnell, M. C., Desai, R. M., Madl, C. M., Xu, M., Zhao, X., Chaudhuri, O., Verbeke, C., Kim, W. S., Alim, K., Mammoto, A., Ingber, D. E., Duda, G. N., Mooney, D. J. 2015; 14 (12): 1269-1277


    The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.

    View details for DOI 10.1038/NMAT4407

    View details for Web of Science ID 000365839000025

    View details for PubMedID 26366848

    View details for PubMedCentralID PMC4654683

  • Matrix interactions modulate neurotrophin-mediated neurite outgrowth and pathfinding NEURAL REGENERATION RESEARCH Madl, C. M., Heilshorn, S. C. 2015; 10 (4): 514-517


    Both matrix biochemistry and neurotrophic factors are known to modulate neurite outgrowth and pathfinding; however, the interplay between these two factors is less studied. While previous work has shown that the biochemical identity of the matrix can alter the outgrowth of neurites in response to neurotrophins, the importance of the concentration of cell-adhesive ligands is unknown. Using engineered elastin-like protein matrices, we recently demonstrated a synergistic effect between matrix-bound cell-adhesive ligand density and soluble nerve growth factor treatment on neurite outgrowth from dorsal root ganglia. This synergism was mediated by Schwann cell-neurite contact through L1CAM. Cell-adhesive ligand density was also shown to alter the pathfinding behavior of dorsal root ganglion neurites in response to a gradient of nerve growth factor. While more cell-adhesive matrices promoted neurite outgrowth, less cell-adhesive matrices promoted more faithful neurite pathfinding. These studies emphasize the importance of considering both matrix biochemistry and neurotrophic factors when designing biomaterials for peripheral nerve regeneration.

    View details for DOI 10.4103/1673-5374.155426

    View details for Web of Science ID 000354156200002

    View details for PubMedID 26170800

    View details for PubMedCentralID PMC4424732

  • Matrix RGD ligand density and L1CAM-mediated Schwann cell interactions synergistically enhance neurite outgrowth. Acta biomaterialia Romano, N. H., Madl, C. M., Heilshorn, S. C. 2015; 11: 48-57


    The innate biological response to peripheral nerve injury involves a complex interplay of multiple molecular cues to guide neurites across the injury gap. Many current strategies to stimulate regeneration take inspiration from this biological response. However, little is known about the balance of cell-matrix and Schwann cell-neurite dynamics required for regeneration of neural architectures. We present an engineered extracellular matrix (eECM) microenvironment with tailored cell-matrix and cell-cell interactions to study their individual and combined effects on neurite outgrowth. This eECM regulates cell-matrix interactions by presenting integrin-binding RGD (Arg-Gly-Asp) ligands at specified densities. Simultaneously, the addition or exclusion of nerve growth factor (NGF) is used to modulate L1CAM-mediated Schwann cell-neurite interactions. Individually, increasing the RGD ligand density from 0.16 to 3.2mM resulted in increasing neurite lengths. In matrices presenting higher RGD ligand densities, neurite outgrowth was synergistically enhanced in the presence of soluble NGF. Analysis of Schwann cell migration and co-localization with neurites revealed that NGF enhanced cooperative outgrowth between the two cell types. Interestingly, neurites in NGF-supplemented conditions were unable to extend on the surrounding eECM without the assistance of Schwann cells. Blocking studies revealed that L1CAM is primarily responsible for these Schwann cell-neurite interactions. Without NGF supplementation, neurite outgrowth was unaffected by L1CAM blocking or the depletion of Schwann cells. These results underscore the synergistic interplay between cell-matrix and cell-cell interactions in enhancing neurite outgrowth for peripheral nerve regeneration.

    View details for DOI 10.1016/j.actbio.2014.10.008

    View details for PubMedID 25308870

    View details for PubMedCentralID PMC4528982

  • Co-Release of Cells and Polymeric Nanoparticles from Sacrificial Microfibers Enhances Nonviral Gene Delivery Inside 3D Hydrogels TISSUE ENGINEERING PART C-METHODS Madl, C. M., Keeney, M., Li, X., Han, L., Yang, F. 2014; 20 (10): 798-805
  • Co-release of cells and polymeric nanoparticles from sacrificial microfibers enhances nonviral gene delivery inside 3D hydrogels. Tissue engineering. Part C, Methods Madl, C. M., Keeney, M., Li, X., Han, L., Yang, F. 2014; 20 (10): 798-805


    Hydrogels can promote desirable cellular phenotype by mimicking tissue-like stiffness or serving as a gene delivery depot. However, nonviral gene delivery inside three-dimensional (3D) hydrogels remains a great challenge, and increasing hydrogel stiffness generally results in further decrease in gene delivery efficiency. Here we report a method to enhance nonviral gene delivery efficiency inside 3D hydrogels across a broad range of stiffness using sacrificial microfibers for co-releasing cells and polymeric nanoparticles (NPs). We fabricated hydrolytically degradable alginate as sacrificial microfibers, and optimized the degradation profile of alginate by varying the degree of oxidization. Scanning electron microscopy confirmed degradation of alginate microfibers inside hydrogels, leaving behind microchannel-like structures within 3D hydrogels. Sacrificial microfibers also serve as a delivery vehicle for co-releasing encapsulated cells and NPs, allowing cell attachment and spreading within the microchannel surface upon microfiber degradation. To examine the effects of sacrificial microfibers on nonviral gene delivery inside 3D hydrogels, alginate microfibers containing human embryonic kidney 293 cells and polymeric NPs were encapsulated within 3D hydrogel scaffolds with varying stiffness (9, 58, and 197 kPa). Compared with cells encapsulated in bulk hydrogels, we observed up to 15-fold increase in gene delivery efficiency using sacrificial microfibers, and gene delivery efficiency increased as hydrogel stiffness increased. The platform reported herein provides a strategy for enhancing nonviral gene delivery inside 3D hydrogels across a broad range of stiffness, and may aid tissue regeneration by engaging both mechanotransduction and nonviral gene delivery.

    View details for DOI 10.1089/ten.TEC.2013.0669

    View details for PubMedID 24483329

  • Presentation of BMP-2 Mimicking Peptides in 3D Hydrogels Directs Cell Fate Commitment in Osteoblasts and Mesenchymal Stem Cells BIOMACROMOLECULES Madl, C. M., Mehta, M., Duda, G. N., Heilshorn, S. C., Mooney, D. J. 2014; 15 (2): 445-455


    Many strategies for controlling the fate of transplanted stem cells rely on the concurrent delivery of soluble growth factors that have the potential to produce undesirable secondary effects in surrounding tissue. Such off target effects could be eliminated by locally presenting growth factor peptide mimics from biomaterial scaffolds to control stem cell fate. Peptide mimics of bone morphogenetic protein 2 (BMP-2) were synthesized by solid phase Fmoc-peptide synthesis and covalently bound to alginate hydrogels via either carbodiimide or sulfhydryl-based coupling strategies. Successful peptide conjugation was confirmed by (1)H NMR spectroscopy and quantified by fluorescently labeling the peptides. Peptides derived from the knuckle epitope of BMP-2, presented from both 2D surfaces and 3D alginate hydrogels, were shown to increase alkaline phosphatase activity in clonally derived murine osteoblasts. Furthermore, when presented in 3D hydrogels, these peptides were shown to initiate Smad signaling, upregulate osteopontin production, and increase mineral deposition with clonally derived murine mesenchymal stem cells. These data suggest that these peptide-conjugated hydrogels may be effective alternatives to local BMP-2 release in directly and spatially eliciting osteogenesis from transplanted or host osteoprogenitors in the future.

    View details for DOI 10.1021/bm401726u

    View details for Web of Science ID 000331342200001

    View details for PubMedID 24400664

    View details for PubMedCentralID PMC3930060

  • Synthesis and bonding of trans-dichlorobis(1,3-diaminopropane)ruthenium(III) chloride to a self assembled monolayer and its effect on conducting polymer sheet conductivity INORGANICA CHIMICA ACTA Kariuki, P. N., Madl, C. M., Martin, J. J., Upreti, S., Jones, W. E. 2012; 383: 320-326
  • Vapor phase polymerization of poly (3,4-ethylenedioxythiophene) on flexible substrates for enhanced transparent electrodes SYNTHETIC METALS Madl, C. M., Kariuki, P. N., Gendron, J., Piper, L. F., Jones, W. E. 2011; 161 (13-14): 1159-1165