Stem Cell Biology and Regenerative Medicine.
Currently we are studying the reprogramming of cells, nano-drug-carriers and small vesicles called exosomes produced by adult stem cells.The cells have the remarkable ability to home to injured tissues and repair them by a variety of mechanisms that include differentiation, immune modulation, suppression of inflammation, stimulation of tissue-endogenous stem/progenitor cells, and perhaps transfer of mitochondria. Recently we and others found that many of the beneficial effects of MSCs are explained by their secretion of exosomes. The aim of our research is to bridge the gap between basic science and clinical translation in the field of regenerative medicine and experimental cell therapeutics.We are hoping to use the vesicles to package small-molecule, protein, and RNA drugs or even use them as therapies themselves.We have designed a multifunctional nanoplatform for engineering and reprogramming vesicles and proved their potential to reach injured/diseased or cancer cells. Additionally, engineered and reprogrammed vesicles are highly versatile systems that can be tunable for a broader range of applications.

All Publications

  • Pulsed focused ultrasound enhances the therapeutic effect of mesenchymal stromal cell-derived extracellular vesicles in acute kidney injury. Stem cell research & therapy Ullah, M., Liu, D. D., Rai, S., Razavi, M., Concepcion, W., Thakor, A. S. 2020; 11 (1): 398


    BACKGROUND: Acute kidney injury (AKI) is characterized by rapid failure of renal function and has no curative therapies. Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) are known to carry therapeutic factors, which have shown promise in regenerative medicine applications, including AKI. However, there remains an unmet need to optimize their therapeutic effect. One potential avenue of optimization lies in pulsed focused ultrasound (pFUS), where tissues-of-interest are treated with sound waves. pFUS has been shown to enhance MSC therapy via increased cell homing, but its effects on cell-free EV therapy remain largely unexplored.METHODS: We combine pFUS pretreatment of the kidney with MSC-derived EV therapy in a mouse model of cisplatin-induced AKI.RESULTS: EVs significantly improved kidney function, reduced injury markers, mediated increased proliferation, and reduced inflammation and apoptosis. While pFUS did not enhance EV homing to the kidney, the combined treatment resulted in a superior therapeutic effect compared to either treatment alone. We identified several molecular mechanisms underlying this synergistic therapeutic effect, including upregulation of proliferative signaling (MAPK/ERK, PI3K/Akt) and regenerative pathways (eNOS, SIRT3), as well as suppression of inflammation.CONCLUSION: Taken together, pFUS may be a strategy for enhancing the therapeutic efficacy of MSC-derived EV treatment for the treatment of AKI.

    View details for DOI 10.1186/s13287-020-01922-1

    View details for PubMedID 32928310

  • HSP70-Mediated NLRP3 Inflammasome Suppression Underlies Reversal of Acute Kidney Injury Following Extracellular Vesicle and Focused Ultrasound Combination Therapy. International journal of molecular sciences Ullah, M., Liu, D. D., Rai, S., Concepcion, W., Thakor, A. S. 2020; 21 (11)


    Acute kidney injury (AKI) is the abrupt loss of renal function, for which only supportive therapies exist. Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have been shown to be therapeutically effective in treating AKI by spurring endogenous cell proliferation and survival while suppressing inflammation. Pre-treating kidneys with pulsed focused ultrasound (pFUS) has also been shown to enhance MSC therapy for AKI, but its role in MSC-derived EV therapy remains unexplored. Using a mouse model of cisplatin-induced AKI, we show that combination therapy with pFUS and EVs restores physiological and molecular markers of kidney function, more so than either alone. Both pFUS and EVs downregulate heat shock protein 70 (HSP70), the NLRP3 inflammasome, and its downstream pro-inflammatory cytokines IL-1beta and IL-18, all of which are highly upregulated in AKI. In vitro knockdown studies suggest that HSP70 is a positive regulator of the NLRP3 inflammasome. Our study therefore demonstrates the ability of pFUS to enhance EV therapy for AKI and provides further mechanistic understanding of their anti-inflammatory and regenerative effects.

    View details for DOI 10.3390/ijms21114085

    View details for PubMedID 32521623

  • A Novel Approach to Deliver Therapeutic Extracellular Vesicles Directly into the Mouse Kidney Cells Ullah, M., Liu, D. D., Rai, S., Razavi, M., Choi, J., Wang, J., Concepcion, W., Thakor, A. S. 2020; 9 (4): 937

    View details for DOI 10.3390/cells9040937

  • Emerging role of stem cell-derived extravesicular microRNAs in age-associated human diseases and in different therapies of longevity. Ageing Research Reviews Ullah, M., Ng, N. N., Concepcion, W., Thakor, A. S. 2020; 57: 1-14
  • Emerging role of stem cell-derived extravesicular microRNAs in age-associated human diseases and in different therapies of longevity. Ageing Research Reviews Ullah, M., Ng, N. N., Concepcion, W., Thakor, A. S. 2020
  • Stem cell-derived extracellular vesicles mitigate ageing-associated arterial stiffness and hypertension JOURNAL OF EXTRACELLULAR VESICLES Feng, R., Ullah, M., Chen, K., Ali, Q., Lin, Y., Sun, Z. 2020; 9 (1): 1783869


    The prevalence of arterial stiffness and hypertension increases with age. This study investigates the effect of induced pluripotent mesenchymal stem cell-derived extracellular vesicles (EVs) on ageing-associated arterial stiffness and hypertension. EVs were collected and purified from induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSCs). Young and old male C57BL/6 mice were used. Mice in the EVs group were injected via tail vein once a week for four weeks (18 x 106 EVs/mouse/injection). Blood pressure (BP) was measured using the tail-cuff method and validated by direct cannulation. Pulse wave velocity (PWV) was measured using a Doppler workstation. PWV and BP were increased significantly in the old mice, indicating arterial stiffness and hypertension. Intravenous administration of EVs significantly attenuated ageing-related arterial stiffness and hypertension, while enhancing endothelium-dependent vascular relaxation and arterial compliance in the old EVs mice. Elastin degradation and collagen I deposition (fibrosis) were increased in aortas of the old mice, but EVs substantially improved ageing-associated structural remodelling. Mechanistically, EVs abolished downregulation of sirtuin type 1 (SIRT1), and endothelial nitric oxide synthase (eNOS) protein expression in aortas of the older mice. In cultured human aortic endothelial cells, EVs promoted the expression of SIRT1, AMP-activated protein kinase alpha (AMPKα), and eNOS. In conclusion, iPS-MSC-derived EVs attenuated ageing-associated vascular endothelial dysfunction, arterial stiffness, and hypertension, likely via activation of the SIRT1-AMPKα-eNOS pathway and inhibition of MMPs and elastase. Thus, EVs mitigate arterial ageing. This finding also sheds light into the therapeutic potential of EVs for ageing-related vascular diseases.EV: Extracellular vesicles; iPS: induced pluripotent stem cell; MSC: mesenchymal stem cell; AMPKα: AMP activated protein kinase α; eNOS: endothelial nitric oxide synthase; Sirt1: sirtuin 1; JNC7: Seventh Report of the Joint National Committee; CVD: cardiovascular disease; PWV: pulse wave velocity; BP: blood pressure; SNP: sodium nitroprusside.

    View details for DOI 10.1080/20013078.2020.1783869

    View details for Web of Science ID 000544469200001

    View details for PubMedID 32939234

    View details for PubMedCentralID PMC7480600

  • Reversing Acute Kidney Injury Using Pulsed Focused Ultrasound and MSC Therapy: A Role for HSP-Mediated PI3K/AKT Signaling. Molecular therapy. Methods & clinical development Ullah, M. n., Liu, D. D., Rai, S. n., Dadhania, A. n., Jonnakuti, S. n., Concepcion, W. n., Thakor, A. S. 2020; 17: 683–94


    Acute kidney injury (AKI) is characterized by a sudden failure of renal function, but despite increasing worldwide prevalence, current treatments are largely supportive, with no curative therapies. Mesenchymal stromal cell (MSC) therapy has been shown to have a promising regenerative effect in AKI but is limited by the ability of cells to home to damaged tissue. Pulsed focused ultrasound (pFUS), wherein target tissues are sonicated by short bursts of sound waves, has been reported to enhance MSC homing by upregulating local homing signals. However, the exact mechanism by which pFUS enhances MSC therapy remains insufficiently explored. In this study, we studied the effect of bone marrow-derived MSCs (BM-MSCs), in conjunction with pFUS, in a mouse model of cisplatin-induced AKI. Here, BM-MSCs improved kidney function, reduced histological markers of kidney injury, decreased inflammation and apoptosis, and promoted cellular proliferation. Surprisingly, whereas pFUS did not upregulate local cytokine expression or improve BM-MSC homing, it did potentiate the effect of MSC treatment in AKI. Further analysis linked this effect to the upregulation of heat shock protein (HSP)20/HSP40 and subsequent phosphatidylinositol 3-kinase (PI3K)/Akt signaling. In summary, our results suggest that pFUS and BM-MSCs have independent as well as synergistic therapeutic effects in the context of AKI.

    View details for DOI 10.1016/j.omtm.2020.03.023

    View details for PubMedID 32346546

    View details for PubMedCentralID PMC7177168

  • Controlled Nutrient Delivery to Pancreatic Islets Using Polydopamine-Coated Mesoporous Silica Nanoparticles. Nano letters Razavi, M. n., Primavera, R. n., Kevadiya, B. D., Wang, J. n., Ullah, M. n., Buchwald, P. n., Thakor, A. S. 2020


    In the present study, we created a nanoscale platform that can deliver nutrients to pancreatic islets in a controlled manner. Our platform consists of a mesoporous silica nanoparticle (MSNP), which can be loaded with glutamine (G: an essential amino acid required for islet survival and function). To control the release of G, MSNPs were coated with a polydopamine (PD) layer. With the optimal parameters (0.5 mg/mL and 0.5 h), MSNPs were coated with a layer of PD, which resulted in a delay of G release from MSNPs over 14 d (57.4 ± 4.7% release). Following syngeneic renal subcapsule islet transplantation in diabetic mice, PDG-MSNPs improved the engraftment of islets (i.e., enhanced revascularization and reduced inflammation) as well as their function, resulting in re-establishment of glycemic control. Collectively, our data show that PDG-MSNPs can support transplanted islets by providing them with a controlled and sustained supply of nutrients.

    View details for DOI 10.1021/acs.nanolett.0c02576

    View details for PubMedID 32909757

  • Stem cell-derived extracellular vesicles: role in oncogenic processes, bioengineering potential, and technical challenges. Stem cell research & therapy Ullah, M., Qiao, Y., Concepcion, W., Thakor, A. S. 2019; 10 (1): 347


    Extracellular vesicles (EVs) are cellular-derived versatile transporters with a specialized property for trafficking a variety of cargo, including metabolites, growth factors, cytokines, proteins, lipids, and nucleic acids, throughout the microenvironment. EVs can act in a paracrine manner to facilitate communication between cells as well as modulate immune, inflammatory, regenerative, and remodeling processes. Of particular interest is the emerging association between EVs and stem cells, given their ability to integrate complex inputs for facilitating cellular migration to the sites of tissue injury. Additionally, stem cell-derived EVs can also act in an autocrine manner to influence stem cell proliferation, mobilization, differentiation, and self-renewal. Hence, it has been postulated that stem cells and EVs may work synergistically in the process of tissue repair and that dysregulation of EVs may cause a loss of homeostasis in the microenvironment leading to disease. By harnessing the property of EVs for delivery of small molecules, stem cell-derived EVs possess significant potential as a platform for developing bioengineering approaches for next-generation cancer therapies and targeted drug delivery methods. Although one of the main challenges of clinical cancer treatment remains a lack of specificity for the delivery of effective treatment options, EVs can be modified via genetic, biochemical, or synthetic methods for enhanced targeting ability of chemotherapeutic agents in promoting tumor regression. Here, we summarize recent research on the bioengineering potential of EV-based cancer therapies. A comprehensive understanding of EV modification may provide a novel strategy for cancer therapy and for the utilization of EVs in the targeting of oncogenic processes. Furthermore, innovative and emerging new technologies are shifting the paradigm and playing pivotal roles by continually expanding novel methods and materials for synthetic processes involved in the bioengineering of EVs for enhanced precision therapeutics.

    View details for DOI 10.1186/s13287-019-1468-6

    View details for PubMedID 31771657

  • A Study Comparing the Effects of Targeted Intra-Arterial and Systemic Chemotherapy in an Orthotopic Mouse Model of Pancreatic Cancer. Scientific reports Rezaee, M., Wang, J., Razavi, M., Ren, G., Zheng, F., Hussein, A., Ullah, M., Thakor, A. S. 2019; 9 (1): 15929


    Systemic chemotherapy is the first line treatment for patients with unresectable pancreatic cancer, however, insufficient drug delivery to the pancreas is a major problem resulting in poor outcomes. We evaluated the therapeutic effects of targeted intra-arterial (IA) delivery of gemcitabine directly into the pancreas in an orthotopic mouse model of pancreatic cancer. Nude mice with orthotopic pancreatic tumors were randomly assigned into 3 groups receiving gemcitabine: systemic intravenous (IV) injection (low: 0.3mg/kg and high: 100mg/kg) and direct IA injection (0.3mg/kg). Treatments were administered weekly for 2 weeks. IA treatment resulted in a significantly greater reduction in tumor growth compared to low IV treatment. To achieve a comparable reduction in tumor growth as seen with IA treatment, gemcitabine had to be given IV at over 300x the dose (high IV treatment) which was associated with some toxicity. After 2 weeks, tumor samples from animals treated with IA gemcitabine had significantly lower residual cancer cells, higher cellular necrosis and evidence of increased apoptosis when compared to animals treated with low IV gemcitabine. Our study shows targeted IA injection of gemcitabine directly into the pancreas, via its arterial blood supply, has a superior therapeutic effect in reducing tumor growth compared to the same concentration administered by conventional systemic injection.

    View details for DOI 10.1038/s41598-019-52490-1

    View details for PubMedID 31685925

  • Effect of Pulsed Focused Ultrasound on the Native Pancreas. Ultrasound in medicine & biology Razavi, M. n., Zheng, F. n., Telichko, A. n., Ullah, M. n., Dahl, J. n., Thakor, A. S. 2019


    Pulsed focused ultrasound (pFUS) utilizes short cycles of sound waves to mechanically shake cells within tissues which, in turn, causes transient local increases in cytokines, growth factors and cell adhesion molecules. Although the effect of pFUS has been investigated in several different organs including the kidney, muscle and heart, its effect on the pancreas has not been investigated. In the present work, we applied pFUS to the rodent pancreas with the following parameters: 1.1-MHz frequency, 5-Hz pulse repetition frequency, 5% duty cycle, 10-ms pulse length, 160-s duration. Low-intensity pFUS had a spatial average temporal average intensity of 11.5 W/cm2 and a negative peak pressure of 3 MPa; high-intensity pFUS had a spatial average temporal average intensity of 18.5 W/cm2 and negative peak pressure of 4 MPa. Here we found that pFUS changed the expression of several cytokines while having no effect on the underlying tissue histology or health of pancreatic cells (as reflected by no significant change in plasma levels of amylase and lipase). Furthermore, we found that this effect on cytokine expression in the pancreas was acoustic intensity dependent; while pFUS at low intensities turned off the expression of several cytokines, at high intensities it had the opposite effect and turned on the expression of these cytokines. The ability to non-invasively manipulate the microenvironment of the pancreas using sound waves could have profound implications for priming and modulating this organ for the application of cellular therapies in the context of both regenerative medicine (i.e., diabetes and pancreatitis) and oncology (i.e., pancreatic cancer).

    View details for DOI 10.1016/j.ultrasmedbio.2019.11.016

    View details for PubMedID 31882169

  • A Study Comparing the Effects of Targeted Intra-Arterial and Systemic Chemotherapy in an Orthotopic Mouse Model of Pancreatic Cancer A Study Comparing the Effects of Targeted Intra-Arterial and Systemic Chemotherapy in an Orthotopic Mouse Model of Pancreatic Cancer R, M. 2019
  • Stem cell-derived extracellular vesicles: role in oncogenic processes, bioengineering potential, and technical challenges Stem cell-derived extracellular vesicles: role in oncogenic processes, bioengineering potential, and technical challenges Ullah, M. 2019
  • An emerging role of CD9 in stemness and chemoresistance An emerging role of CD9 in stemness and chemoresistance Ullah, M. 2019
  • Mesenchymal stem cells confer chemoresistance in breast cancer via a CD9 dependent mechanism Oncotarget Ullah, M., Akbar, A., Ng, N. N., Concepcion, W., Thakor, A. S. 2019; 10 (37): 3435-3450
  • Mesenchymal Stromal Cell Homing: Mechanisms and Strategies for Improvement. iScience Ullah, M. n., Liu, D. D., Thakor, A. S. 2019; 15: 421–38


    Mesenchymal stromal cells (MSCs) have been widely investigated for their therapeutic potential in regenerative medicine, owing to their ability to home damaged tissue and serve as a reservoir of growth factors and regenerative molecules. As such, clinical applications of MSCs are reliant on these cells successfully migrating to the desired tissue following their administration. Unfortunately, MSC homing is inefficient, with only a small percentage of cells reaching the target tissue following systemic administration. This attrition represents a major bottleneck in realizing the full therapeutic potential of MSC-based therapies. Accordingly, a variety of strategies have been employed in the hope of improving this process. Here, we review the molecular mechanisms underlying MSC homing, based on a multistep model involving (1) initial tethering by selectins, (2) activation by cytokines, (3) arrest by integrins, (4) diapedesis or transmigration using matrix remodelers, and (5) extravascular migration toward chemokine gradients. We then review the various strategies that have been investigated for improving MSC homing, including genetic modification, cell surface engineering, in vitro priming of MSCs, and in particular, ultrasound techniques, which have recently gained significant interest. Contextualizing these strategies within the multistep homing model emphasizes that our ability to optimize this process hinges on our understanding of its molecular mechanisms. Moving forward, it is only with a combined effort of basic biology and translational work that the potential of MSC-based therapies can be realized.

    View details for DOI 10.1016/j.isci.2019.05.004

    View details for PubMedID 31121468

  • Klotho deficiency accelerates stem cells aging by impairing telomerase activity. The journals of gerontology. Series A, Biological sciences and medical sciences Ullah, M., Sun, Z. 2018


    Understanding the effect of molecular pathways involved in the age-dependent deterioration of stem cell function is critical for developing new therapies. The overexpression of Klotho (KL), an anti-aging protein, causes treated animal models to enjoy extended lifespans. Now, the question stands: Does KL-deficiency accelerate stem cell aging and telomere shortening? If so, what are the specific mechanisms by which it does this, and is cycloastragenol (CAG) treatment enough to restore telomerase activity in aged stem cells? We found that KL-deficiency diminished telomerase activity by altering the expression of TERF1 and TERT, causing impaired differentiation potential, pluripotency, cellular senescence, and apoptosis in stem cells. Telomerase activity decreased with KL-siRNA knockdown. This suggests that both KL and telomeres regulate the stem cell aging process through telomerase subunits TERF1, POT1 and TERT using the TGFbeta, Insulin, and Wnt signaling. These pathways can rejuvenate stem cell populations in a CD90-dependent mechanism. Stem cell dysfunctions were largely provoked by KL-deficiency and telomere shortening, owing to altered expression of TERF1, TGFbeta1, CD90, POT1, TERT, and bFGF. The CAG treatment partially rescued telomerase deterioration, suggesting that KL plays a critical role in life-extension by regulating telomere length and telomerase activity.

    View details for DOI 10.1093/gerona/gly261

    View details for PubMedID 30452555

  • Stem cells and anti-aging genes: double-edged sword-do the same job of life extension STEM CELL RESEARCH & THERAPY Ullah, M., Sun, Z. 2018; 9: 3


    Aging impacts diseases and lifespan. With current knowledge of stem cells, it is feasible to design and test interventions that delay aging and improve both health and lifespan. Stem cells, together with anti-aging genes such as Klotho, play a crucial role in delaying the aging process. Stem cells in combination with anti-aging genes make a complex and protective shield, which stand against the eroding effects of aging. Increased wear and tear of the stem cells, as well as Klotho deficiency, is expected to heavily increase cellular damage and accelerate the process of aging. Stem cells in conjugation with anti-aging genes probably receive and neutralize most of the devastating signaling effects which are known to cause premature aging. The shield of stem cells combined with anti-aging genes is a primary target for absorbing the shock of aging. If this shield neutralizes the shocks, it could lead to a youthful state, but if not it will accelerate the aging journey. In this review, we concisely discuss the neutralizing ability, operated and regulated by stem cells and other life-extension factors. We suggest that stem cell interventions that increase rejuvenation and keep in balance the expression of anti-aging genes could delay the aging phenotypes and result in prolonged lifespan.

    View details for DOI 10.1186/s13287-017-0746-4

    View details for Web of Science ID 000419961900001

    View details for PubMedID 29321045

    View details for PubMedCentralID PMC5763529

  • Mesenchymal stromal cell homing: mechanisms and strategies for improvement Mesenchymal stromal cell homing: mechanisms and strategies for improvement Ullah, M. 2018
  • iPS-derived MSCs from an expandable bank to deliver a prodrug-converting enzyme that limits growth and metastases of human breast cancers (vol 3, 16064, 2017) CELL DEATH DISCOVERY Ullah, M., Kuroda, Y., Bartosh, T. J., Liu, F., Zhao, Q., Gregory, C., Reger, R., Xu, J., Lee, R. H., Prockop, D. J. 2017; 3: 17029


    [This corrects the article DOI: 10.1038/cddiscovery.2016.64.].

    View details for DOI 10.1038/cddiscovery.2017.29

    View details for Web of Science ID 000463082900043

    View details for PubMedID 28845293

    View details for PubMedCentralID PMC5563522

  • Stem Cell-Derived Extracellular Vesicles Attenuates Aging-Associated Arterial Stiffness and Hypertension Stem Cell-Derived Extracellular Vesicles Attenuates Aging-Associated Arterial Stiffness and Hypertension Ullah, M. 2017
  • iPS-derived MSCs from an expandable bank to deliver a prodrug-converting enzyme that limits growth and metastases of human breast cancers CELL DEATH DISCOVERY Ullah, M., Kuroda, Y., Bartosh, T. J., Liu, F., Zhao, Q., Gregory, C., Reger, R., Xu, J., Lee, R. H., Prockop, D. J. 2017; 3: 16064


    One attractive strategy to treat cancers is to deliver an exogenous enzyme that will convert a non-toxic compound to a highly toxic derivative. The strategy was tested with viral vectors but was disappointing because the efficiency of transduction into tumor cells was too low. Recent reports demonstrated that the limitation can be addressed by using tissue-derived mesenchymal stromal cells (MSCs) to deliver enzyme/prodrug systems that kill adjacent cancer cells through bystander effects. Here we addressed the limitation that tissue-derived MSCs vary in their properties and are difficult to generate in the large numbers needed for clinical applications. We prepared a Feeder Stock of MSCs from induced pluripotent stem cells (iPSs) that provided an extensively expandable source of standardized cells. We then transduced the iPS-derived MSCs to express cytosine deaminase and injected them locally into a mouse xenogeneic model of human breast cancer. After administration of the prodrug (5-fluorocytosine), the transduced iPS-MSCs both limited growth of preformed tumors and decreased lung metastases.

    View details for DOI 10.1038/cddiscovery.2016.64

    View details for Web of Science ID 000463082900010

    View details for PubMedID 28179988

    View details for PubMedCentralID PMC5292869

  • Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bartosh, T. J., Ullah, M., Zeitouni, S., Beaver, J., Prockop, D. J. 2016; 113 (42): E6447–E6456


    Patients with breast cancer often develop malignant regrowth of residual drug-resistant dormant tumor cells years after primary treatment, a process defined as cancer relapse. Deciphering the causal basis of tumor dormancy therefore has obvious therapeutic significance. Because cancer cell behavior is strongly influenced by stromal cells, particularly the mesenchymal stem/stromal cells (MSCs) that are actively recruited into tumor-associated stroma, we assessed the impact of MSCs on breast cancer cell (BCC) dormancy. Using 3D cocultures to mimic the cellular interactions of an emerging tumor niche, we observed that MSCs sequentially surrounded the BCCs, promoted formation of cancer spheroids, and then were internalized/degraded through a process resembling the well-documented yet ill-defined clinical phenomenon of cancer cell cannibalism. This suspected feeding behavior was less appreciable in the presence of a rho kinase inhibitor and in 2D monolayer cocultures. Notably, cannibalism of MSCs enhanced survival of BCCs deprived of nutrients but suppressed their tumorigenicity, together suggesting the cancer cells entered dormancy. Transcriptome profiles revealed that the resulting BCCs acquired a unique molecular signature enriched in prosurvival factors and tumor suppressors, as well as inflammatory mediators that demarcate the secretome of senescent cells, also referred to as the senescence-associated secretory phenotype. Overall, our results provide intriguing evidence that cancer cells under duress enter dormancy after cannibalizing MSCs. Importantly, our practical 3D coculture model could provide a valuable tool to understand the antitumor activity of MSCs and cell cannibalism further, and therefore open new therapeutic avenues for the prevention of cancer recurrence.

    View details for DOI 10.1073/pnas.1612290113

    View details for Web of Science ID 000385610400018

    View details for PubMedID 27698134

    View details for PubMedCentralID PMC5081643

  • Bone morphogenetic protein 2/SMAD signalling in human ligamentocytes of degenerated and aged anterior cruciate ligaments OSTEOARTHRITIS AND CARTILAGE Ruschke, K., Meier, C., Ullah, M., Krebs, A., Silberreis, K., Kohl, B., Knaus, P., Jagielski, M., Arens, S., Schulze-Tanzil, G. 2016; 24 (10): 1816–25


    Anterior cruciate ligament (ACL) degeneration leads to knee instability and favors osteoarthritis (OA) progression. During ageing the growth factor sensitivity of ligaments changes but nothing is known about BMP2-signalling and -sensitivity in degenerated ACLs. This study addressed the question whether a dysregulated BMP2 signalling might contribute to age- and OA-dependent ACL degeneration.ACL samples from patients with/without OA of different ages (<60 and ≥60 years, males, females) were graded histopathologically (n = 45). After stimulation of cultured ACL fibroblasts with 5 nM BMP2 for different time points, phosphorylation of SMAD1/5/8 and gene expression of crucial BMP2 signalling proteins, ligamentogenic and chondrogenic transcription factors, scleraxis (SCX) and SOX9, were analyzed.ACL samples displayed different grades of degeneration, often associated with synovitis and calcium deposits. Degeneration correlated significantly with synovitis. ACL fibroblasts expressed BMP type I receptors ALK3 and ALK6 and the BMP type II receptor BMPRII. Donors could be divided into "responders" and "non responders" since their BMP2 mediated SMAD1/5/8 phosphorylation level differed. Basal ID1 expression was lower in cells derived from OA compared with non-OA patients and BMP2 led to an ID1 induction in both. Irrespective of BMP2 stimulation, the donor age significantly influenced the expression profile of BMP6 and SCX but not BMP signalling. The BMP2-mediated SMAD6 expression differed between OA and healthy ACL fibroblasts.Our data indicate that the expression level of BMP2/SMAD target genes such as ID1 and SMAD6 was reduced in ACL fibroblasts derived from OA compared with non OA patients.

    View details for DOI 10.1016/j.joca.2016.05.014

    View details for Web of Science ID 000384130400016

    View details for PubMedID 27208419

  • Effect of Pulsed Focused Ultrasound on the Native Pancreas Effect of Pulsed Focused Ultrasound on the Native Pancreas Ullah, M. 2016
  • In vivo Endothelial Cell-specific Expression of AMPKα Attenuates Cold-induced Pulmonary Vascular Dysfunction and Hypertension by Mitigating Inflammation In vivo Endothelial Cell-specific Expression of AMPKα Attenuates Cold-induced Pulmonary Vascular Dysfunction and Hypertension by Mitigating Inflammation Ullah, M. 2016
  • Klotho deficiency accelerates stem cells aging by impairing telomerase activity Klotho deficiency accelerates stem cells aging by impairing telomerase activity Ullah, M. 2015
  • Rapid Analysis of Cell Surface N-Glycosylation from Living Cells Using Mass Spectrometry JOURNAL OF PROTEOME RESEARCH Hamouda, H., Kaup, M., Ullah, M., Berger, M., Sandig, V., Tauber, R., Blanchard, V. 2014; 13 (12): 6144–51


    Cell surfaces are covered with a dense carbohydrate layer referred to as the glycocalyx. Because different cell types express different glycan signatures, it is of paramount importance to have robust methods to analyze the glycome of living cells. To achieve this, a common procedure involves cell lysis and extraction of membrane (glyco)proteins and yields a major proportion of high-mannose N-glycans that most likely stem from intracellular proteins derived from the ER. Using HEK 293 cells as a model system, we developed a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells. We directly released glycopeptides from cell surfaces through tryptic digestion of freshly harvested and vital cells, thereby improving the detection and quantification of complex-type N-glycans by increasing their relative amount from 14 to 85%. It was also possible to detect 25 additional structures in HEK 293, 48 in AGE1.HN, 42 in CHO-K1, and 51 in Hep G2 cells. The additional signals provided deeper insight into cell-type-specific N-glycan features such as antennarity, fucosylation, and sialylation. Thus, this protocol, which can potentially be applied to any cells, will be useful in the fields of glycobiotechnology and biomarker discovery.

    View details for DOI 10.1021/pr5003005

    View details for Web of Science ID 000346039400076

    View details for PubMedID 25348702

  • Continuous Cultivation of Human Hamstring Tenocytes on Microcarriers in a Spinner Flask Bioreactor System BIOTECHNOLOGY PROGRESS Stich, S., Ibold, Y., Abbas, A., Ullah, M., Sittinger, M., Ringe, J., Schulze-Tanzil, G., Mueller, C., Kohl, B., John, T. 2014; 30 (1): 142–51


    Tendon healing is a time consuming process leading to the formation of a functionally altered reparative tissue. Tissue engineering-based tendon reconstruction is attracting more and more interest. The aim of this study was to establish tenocyte expansion on microcarriers in continuous bioreactor cultures and to study tenocyte behavior during this new approach. Human hamstring tendon-derived tenocytes were expanded in monolayer culture before being seeded at two different seeding densities (2.00 and 4.00 3 106 cells/1000 cm2 surface) on CytodexTM type 3 microcarriers. Tenocytes’ vitality, growth kinetics and glucose/ lactic acid metabolism were determined dependent on the seeding densities and stirring velocities (20 or 40 rpm) in a spinner flask bioreactor over a period of 2 weeks. Gene expression profiles of tendon extracellular matrix (ECM) markers (type I/III collagen, decorin, cartilage oligomeric protein [COMP], aggrecan) and the tendon marker scleraxis were analyzed using real time detection polymerase chain reaction (RTD-PCR). Type I collagen and decorin deposition was demonstrated applying immunolabeling. Tenocytes adhered on the carriers, remained vital, proliferated and revealed an increasing glucose consumption and lactic acid formation under all culture conditions. “Bead-to-bead” transfer of cells from one microcarrier to another, a prerequisite for continuous tenocyte expansion, was demonstrated by scanning electron microscopy. Type I and type III collagen gene expression was mainly unaffected, whereas aggrecan and partly also decorin and COMP expression was significantly downregulated compared to monolayer cultures. Scleraxis gene expression revealed no significant regulation on the carriers. In conclusion, tenocytes could be successfully expanded on microcarriers. Therefore, bioreactors are promising tools for continuous tenocyte expansion.

    View details for DOI 10.1002/btpr.1815

    View details for Web of Science ID 000331385800015

    View details for PubMedID 24124166

  • Molecular characterization of human mesenchymal stem cell differentiation to identify biomarkers for quality assurance in stem cell therapy Molecular characterization of human mesenchymal stem cell differentiation to identify biomarkers for quality assurance in stem cell therapy Ullah, M. 2014
  • Transdifferentiation of adipogenically differentiated cells into osteogenically or chondrogenically differentiated cells: Phenotype switching via dedifferentiation INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY Ullah, M., Sittinger, M., Ringe, J. 2014; 46: 124–37


    Reprogramming is a new wave in cellular therapies to achieve the vital goals of regenerative medicine. Transdifferentiation, whereas the differentiated state of cells could be reprogrammed into other cell types, meaning cells are no more locked in their differentiated circle. Hence, cells of choice from abundant and easily available sources such as fibroblast and adipose tissue could be converted into cells of demand, to restore the diseased tissues. Before diverting this new approach into effective clinical use, transdifferentiation could not be simply overlooked, as it challenges the normal paradigms of biological laws, where mature cells transdifferentiate not only within same germ layers, but even across the lineage boundaries. How unipotent differentiated cells reprogram into another, and whether transdifferentiation proceeds via a direct cell-to-cell conversion or needs dedifferentiation. To address such questions, MSC were adipogenically differentiated followed by direct transdifferentiation, and subsequently examined by histology, immunohistochemistry, qPCR and single cell analysis. Direct cellular conversion of adipogenic lineage cells into osteogenic or chondrogenic resulted in mixed culture of both lineage cells (adipogenic and new acquiring osteogenic/chondrogenic phenotypes). On molecular level, such conversion was confirmed by significantly upregulated expression of PPARG, FABP4, SPP1 and RUNX2. Chondrogenic transdifferentiation was verified by significantly upregulated expression of PPARG, FABP4, SOX9 and COL2A1. Single cell analysis did not support the direct cell-to-cell conversion, rather described the involvement of dedifferentiation. Moreover, some differentiated single cells did not change their phenotype and were resistant to transdifferentiation, suggesting that differentiated cells behave differently during cellular conversion. An obvious characterization of differentiated cells could be helpful to understand the process of transdifferentiation.

    View details for DOI 10.1016/j.biocel.2013.11.010

    View details for Web of Science ID 000330546600014

    View details for PubMedID 24269783

  • N-Glycosylation Profile of Undifferentiated and Adipogenically Differentiated Human Bone Marrow Mesenchymal Stem Cells: Towards a Next Generation of Stem Cell Markers STEM CELLS AND DEVELOPMENT Hamouda, H., Ullah, M., Berger, M., Sittinger, M., Tauber, R., Ringe, J., Blanchard, V. 2013; 22 (23): 3100–3113


    Mesenchymal stem cells (MSCs) are multipotent cells that are easy to isolate and expand, develop into several tissues, including fat, migrate to diseased organs, have immunosuppressive properties and secrete regenerative factors. This makes MSCs ideal for regenerative medicine. For application and regulatory purposes, knowledge of (bio)markers characterizing MSCs and their development stages is of paramount importance. The cell surface is coated with glycans that possess lineage-specific nature, which makes glycans to be promising candidate markers. In the context of soft tissue generation, we aimed to identify glycans that could be markers for MSCs and their adipogenically differentiated progeny. MSCs were isolated from human bone marrow, adipogenically stimulated for 15 days and adipogenesis was verified by staining the lipid droplets and quantitative real time polymerase chain reaction of the marker genes peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid binding protein-4 (FABP4). Using matrix-assisted laser desorption-ionization-time of flight mass spectrometry combined with exoglycosidase digestions, we report for the first time the N-glycome of MSCs during adipogenic differentiation. We were able to detect more than 100 different N-glycans, including high-mannose, hybrid, and complex N-glycans, as well as poly-N-acetyllactosamine chains. Adipogenesis was accompanied by an increased amount of biantennary fucosylated structures, decreased amount of fucosylated, afucosylated tri- and tetraantennary structures and increased sialylation. N-glycans H6N5F1 and H7N6F1 were significantly overexpressed in undifferentiated MSCs while H3N4F1 and H5N4F3 were upregulated in adipogenically differentiated MSCs. These glycan structures are promising candidate markers to detect and distinguish MSCs and their adipogenic progeny.

    View details for DOI 10.1089/scd.2013.0108

    View details for Web of Science ID 000327305000006

    View details for PubMedID 23829188

    View details for PubMedCentralID PMC3856714

  • Extracellular matrix of adipogenically differentiated mesenchymal stem cells reveals a network of collagen filaments, mostly interwoven by hexagonal structural units MATRIX BIOLOGY Ullah, M., Sittinger, M., Ringe, J. 2013; 32 (7-8): 452–65


    Extracellular matrix (ECM) is the non-cellular component of tissues, which not only provides biological shelter but also takes part in the cellular decisions for diverse functions. Every tissue has an ECM with unique composition and topology that governs the process of determination, differentiation, proliferation, migration and regeneration of cells. Little is known about the structural organization of matrix especially of MSC-derived adipogenic ECM. Here, we particularly focus on the composition and architecture of the fat ECM to understand the cellular behavior on functional bases. Thus, mesenchymal stem cells (MSC) were adipogenically differentiated, then, were transferred to adipogenic propagation medium, whereas they started the release of lipid droplets leaving bare network of ECM. Microarray analysis was performed, to indentify the molecular machinery of matrix. Adipogenesis was verified by Oil Red O staining of lipid droplets and by qPCR of adipogenic marker genes PPARG and FABP4. Antibody staining demonstrated the presence of collagen type I, II and IV filaments, while alkaline phosphatase activity verified the ossified nature of these filaments. In the adipogenic matrix, the hexagonal structures were abundant followed by octagonal structures, whereas they interwoven in a crisscross manner. Regarding molecular machinery of adipogenic ECM, the bioinformatics analysis revealed the upregulated expression of COL4A1, ITGA7, ITGA7, SDC2, ICAM3, ADAMTS9, TIMP4, GPC1, GPC4 and downregulated expression of COL14A1, ADAMTS5, TIMP2, TIMP3, BGN, LAMA3, ITGA2, ITGA4, ITGB1, ITGB8, CLDN11. Moreover, genes associated with integrins, glycoproteins, laminins, fibronectins, cadherins, selectins and linked signaling pathways were found. Knowledge of the interactive-language between cells and matrix could be beneficial for the artificial designing of biomaterials and bioscaffolds.

    View details for DOI 10.1016/j.matbio.2013.07.001

    View details for Web of Science ID 000329423200012

    View details for PubMedID 23851162

  • Mesenchymal stem cells and their chondrogenic differentiated and dedifferentiated progeny express chemokine receptor CCR9 and chemotactically migrate toward CCL25 or serum STEM CELL RESEARCH & THERAPY Ullah, M., Eucker, J., Sittinger, M., Ringe, J. 2013; 4: 99


    Guided migration of chondrogenically differentiated cells has not been well studied, even though it may be critical for growth, repair, and regenerative processes. The chemokine CCL25 is believed to play a critical role in the directional migration of leukocytes and stem cells. To investigate the motility effect of serum- or CCL25-mediated chemotaxis on chondrogenically differentiated cells, mesenchymal stem cells (MSCs) were induced to chondrogenic lineage cells.MSC-derived chondrogenically differentiated cells were characterized for morphology, histology, immunohistochemistry, quantitative polymerase chain reaction (qPCR), surface profile, and serum- or CCL25-mediated cell migration. Additionally, the chemokine receptor, CCR9, was examined in different states of MSCs.The chondrogenic differentiated state of MSCs was positive for collagen type II and Alcian blue staining, and showed significantly upregulated expression of COL2A1and SOX9, and downregulated expression of CD44, CD73, CD90, CD105 and CD166, in contrast to the undifferentiated and dedifferentiated states of MSCs. For the chondrogenic differentiated, undifferentiated, and dedifferentiated states of MSCs, the serum-mediated chemotaxis was in a percentage ratio of 33%:84%:85%, and CCL25-mediated chemotaxis was in percentage ratio of 12%:14%:13%, respectively. On the protein level, CCR9, receptor of CCL25, was expressed in the form of extracellular and intracellular domains. On the gene level, qPCR confirmed the expression of CCR9 in different states of MSCs.CCL25 is an effective cue to guide migration in a directional way. In CCL25-mediated chemotaxis, the cell-migration rate was almost the same for different states of MSCs. In serum-mediated chemotaxis, the cell-migration rate of chondrogenically differentiated cells was significantly lower than that in undifferentiated or dedifferentiated cells. Current knowledge of the surface CD profile and cell migration could be beneficial for regenerative cellular therapies.

    View details for DOI 10.1186/scrt310

    View details for Web of Science ID 000323904500001

    View details for PubMedID 23958031

    View details for PubMedCentralID PMC3854782

  • Reverse Differentiation as a Gene Filtering Tool in Genome Expression Profiling of Adipogenesis for Fat Marker Gene Selection and Their Analysis PLOS ONE Ullah, M., Stich, S., Haeupl, T., Eucker, J., Sittinger, M., Ringe, J. 2013; 8 (7): e69754


    During mesenchymal stem cell (MSC) conversion into adipocytes, the adipogenic cocktail consisting of insulin, dexamethasone, indomethacin and 3-isobutyl-1-methylxanthine not only induces adipogenic-specific but also genes for non-adipogenic processes. Therefore, not all significantly expressed genes represent adipogenic-specific marker genes. So, our aim was to filter only adipogenic-specific out of all expressed genes. We hypothesize that exclusively adipogenic-specific genes change their expression during adipogenesis, and reverse during dedifferentiation. Thus, MSC were adipogenic differentiated and dedifferentiated.Adipogenesis and reverse adipogenesis was verified by Oil Red O staining and expression of PPARG and FABP4. Based on GeneChips, 991 genes were differentially expressed during adipogenesis and grouped in 4 clusters. According to bioinformatic analysis the relevance of genes with adipogenic-linked biological annotations, expression sites, molecular functions, signaling pathways and transcription factor binding sites was high in cluster 1, including all prominent adipogenic genes like ADIPOQ, C/EBPA, LPL, PPARG and FABP4, moderate in clusters 2-3, and negligible in cluster 4. During reversed adipogenesis, only 782 expressed genes (clusters 1-3) were reverted, including 597 genes not reported for adipogenesis before. We identified APCDD1, CHI3L1, RARRES1 and SEMA3G as potential adipogenic-specific genes.The model system of adipogenesis linked to reverse adipogenesis allowed the filtration of 782 adipogenic-specific genes out of total 991 significantly expressed genes. Database analysis of adipogenic-specific biological annotations, transcription factors and signaling pathways further validated and valued our concept, because most of the filtered 782 genes showed affiliation to adipogenesis. Based on this approach, the selected and filtered genes would be potentially important for characterization of adipogenesis and monitoring of clinical translation for soft-tissue regeneration. Moreover, we report 4 new marker genes.

    View details for DOI 10.1371/journal.pone.0069754

    View details for Web of Science ID 000322838900077

    View details for PubMedID 23922792

    View details for PubMedCentralID PMC3724870

  • Transdifferentiation of mesenchymal stem cells-derived adipogenic-differentiated cells into osteogenic- or chondrogenic-differentiated cells proceeds via dedifferentiation and have a correlation with cell cycle arresting and driving genes DIFFERENTIATION Ullah, M., Stich, S., Notter, M., Eucker, J., Sittinger, M., Ringe, J. 2013; 85 (3): 78–90


    It is generally accepted that after differentiation bone marrow mesenchymal stem cells (MSC) become lineage restricted and unipotent in an irreversible manner. However, current results imply that even terminally differentiated cells transdifferentiate across lineage boundaries and therefore act as a progenitor cells for other lineages. This leads to the questions that whether transdifferentiation occurs via direct cell-to-cell conversion or dedifferentiation to a progenitor cells and subsequent differentiation, and whether MSC potency decreases or increases during differentiation. To address these questions, MSC were differentiated into adipogenic lineage cells, followed by dedifferentiation. The process of dedifferentiation was also confirmed by single cell clonal analysis. Finally the dedifferentiated cells were used for adipogenesis, osteogenesis and chondrogenesis. Histology, FACS, qPCR and GeneChip analyses of undifferentiated MSC, adipogenic-differentiated and dedifferentiated cells were performed. Interestingly, gene profiling and bioinformatics demonstrated that upregulation (DHCR24, G0S2, MAP2K6, SESN3) and downregulation (DST, KAT2, MLL5, RB1, SMAD3, ZAK) of distinct genes have an association with cell cycle arrest in adipogenic-differentiated cells and perhaps narrow down the lineage potency. However, the upregulation (CCND1, CHEK, HGF, HMGA2, SMAD3) and downregulation (CCPG1, RASSF4, RGS2) of these genes have an association with cell cycle progression and maybe motivate dedifferentiation of adipogenic-differentiated cells. We found that dedifferentiated cells have a multilineage potency comparable to MSC, and also observed the associative role of proliferation genes with cell cycle arrest and progression. Concluded, our results indicate that transdifferentiation of adipogenic-differentiated cells into osteogenic- or chondrogenic-differentiated cells proceeds via dedifferentiation and correlates with cell cycle arresting and deriving genes. Regarding clinical use, the knowledge of potency and underlying mechanisms are prerequisites.

    View details for DOI 10.1016/j.diff.2013.02.001

    View details for Web of Science ID 000320766300002

    View details for PubMedID 23644554

  • Migration and Differentiation Capacity of Mesenchymal Stem Cells from Patients with Osteoarthritis-Towards In Situ Joint Cartilage Tissue Engineering Migration and Differentiation Capacity of Mesenchymal Stem Cells from Patients with Osteoarthritis-Towards In Situ Joint Cartilage Tissue Engineering Ullah, M. 2013
  • Differentiation, Dedifferentiation and Transdifferentiation Potential and Mechanisms of Human Bone Marrow-Derived Mesenchymal Stem Cells. Differentiation, Dedifferentiation and Transdifferentiation Potential and Mechanisms of Human Bone Marrow-Derived Mesenchymal Stem Cells. ULLAH, M. 2013
  • A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures BIORESEARCH OPEN ACCESS Ullah, M., Hamouda, H., Stich, S., Sittinger, M., Ringe, J. 2012; 1 (6): 297–305


    Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering.

    View details for DOI 10.1089/biores.2012.0279

    View details for Web of Science ID 000215142400005

    View details for PubMedID 23514965

    View details for PubMedCentralID PMC3559221

  • Changes in N-Glycosylation Profile of Human MSC During Adipogenic Development-Towards a Next Generation of Markers for Regenerative Medicine Changes in N-Glycosylation Profile of Human MSC During Adipogenic Development-Towards a Next Generation of Markers for Regenerative Medicine Ullah, M. 2012
  • Differentiation, Dedifferentiation and Transdifferentiation Potential and Mechanisms of Human MSC Differentiation, Dedifferentiation and Transdifferentiation Potential and Mechanisms of Human MSC Ullah, M. 2012