Single cell gene expression analysis of bone marrow derived mesenchymal stem cells reveals a diminished subpopulation defined by vasculogenic markers

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Surgical Forum Abstracts

weeks (p⫽0.01, n⫽9/7). Average number of muscle fibers was lower, and fiber area was higher in MNC mice at all time points (p⬍0.05, n⫽2). CONCLUSIONS: High femoral ligation and excision is a reproducible model of limb ischemia in C57BL/6 mice that shows response to MNC injection. These studies suggest several parameters of human trials can be tested in a small animal, cost-effective manner, allowing optimization of human trial parameters.

Single cell gene expression analysis of bone marrow derived mesenchymal stem cells reveals a diminished subpopulation defined by vasculogenic markers Michael Sorkin MD, Jason P Glotzbach MD, Michael Januszyk MD, Jerry Chen MD, Victor W Wong MD, Kristine C Rustad, Michael T Longaker MD, MBA, FACS, Geoffrey C Gurtner MD, FACS Stanford University, Stanford, CA INTRODUCTION: Pathophysiologic changes associated with diabetes severely impair neovascularization and are accountable for complications affecting the cardiovascular system and wound healing. Bone marrow derived mesenchymal stem cells (BM-MSCs) are recruited to ischemic wounds and play a major role in vasculogenesis. In this study we hypothesized that the impaired neovascularization capacity in diabetic mice is reflected by transcriptional changes within BM-MSCs. METHODS: Total bone marrow cells were harvested from wildtype (C57BL/6) and diabetic (db/db) mice. BM-MSCs with the surface marker profile lin- CD45-Sca1⫹ were sorted as single cells using FACS. Microfluidic single cell transcriptional analysis was performed across an array of 48 gene targets. Mathematical clustering analysis was employed to identify subpopulations. RESULTS: Transcriptional analysis revealed multiple genes affected by diabetic metabolism. Especially genes associated with vasculogenesis (Flt1, Flt4, KDR and Vcam1) were found to be downregulated in diabetic mice. Furthermore, cells expressing the transcriptional factors associated with ‘stemness’ characteristics (Klf4, Id2, Notch1) were depleted in the diabetic group. Using cell clustering analysis, a subpopulation of cells that was defined by the expression of vasculogenic genes was found to be diminished in diabetic mice. CONCLUSIONS: Recruitment of bone marrow derived stem cells to peripheral tissues plays a crucial role in the recovery from an ischemic insult. Here we show that a subpopulation of BM-MSCs defined by the expression of vasculogenic genes is significantly reduced in diabetic bone marrow. These findings suggest a pathophysiologic mechanism underlying impaired diabetic vasculogenesis and may serve as a guide for future therapeutic approaches.

Role of Notch1 in NIKS cellular differentiation Madhuchhanda Roy MD, PhD, Sandy J Schlosser BSc, B Lynn Allen-Hoffman PhD, Herb Chen MD, FACS, Timothy W King MD, PhD University of Wisconsin, Madison, WI

J Am Coll Surg

INTRODUCTION: Cutaneous wound healing is a major health problem in the US. Following injury, keratinocytes must dedifferentiate to proliferate, migrate across the wound bed, and finally redifferentiate into a multi-layered tissue for the wound to heal. NIKS human keratinocyte progenitor cells were successfully evaluated as a component of StrataGraft skin substitute in a Phase I/IIa safety and early efficacy clinical trial for temporary management of traumatic skin wounds. However, the molecular mechanisms underlying NIKS wound healing properties remain unclear. Transmembrane receptor notch is critical in epidermal development. Studies using NIKS indicated that notch1 signaling is essential for cellular proliferation and migration. We hypothesize that, in addition to regulating proliferation and migration, Notch1 signaling is associated with cellular differentiation to promote healing. METHODS: To determine the role of Notch1 in differentiation, subconfluent NIKS monolayers were exposed to increasing concentrations (60-660␮M) of calcium. Notch1 protein and markers of keratinocyte differentiation were detected by Western Blot. RESULTS: A high basal Notch1 level was detected at a calcium concentration of 60␮M. In response to higher calcium, Notch1 was downregulated in 4 hours and was undetectable after 8 days with a concomitant increase in the differentiation markers, keratin 1, keratin 10 and involucrin. CONCLUSIONS: Our results suggest that following injury, Notch1 signaling is required for NIKS proliferation and migration. Furthermore, Notch 1 attenuation may be necessary to allow for keratinocyte differentiation. Our findings could improve clinical outcome of millions of patients with cutaneous wounds by modulating Notch1 signaling in StrataGraft skin substitutes or by using a novel bioactive dressing.

Fibroblast growth factor and Notch signaling are associated with hepatic progenitor cell expansion after chronic liver injury Christopher L Vendryes MD, MS, Sarah B Utley BSc, David M James BSc, Nirmala Mavila PhD, Jenn C Phan BSc, Kasper S Wang MD, FACS Childrens Hospital Los Angeles/Saban Research Institute, Los Angeles, CA INTRODUCTION: Chronic liver diseases are commonly characterized by impaired regeneration and ductular proliferation of hepatic progenitor cells (HPC). With liver tissue transplantation limited by donor availability, alternative therapies such as HPC transplantation become important. However, the mechanisms regulating HPC proliferation and differentiation are not completely understood. We previously described fibroblast growth factor (FGF)-10 as a key regulator of embryonic HPC proliferation and survival (Berg et al, 2007). Moreover, Notch signaling is critical in bile duct development. We hypothesized FGF and Notch signaling pathways are active in the ductular proliferation of HPC. METHODS: Ductular proliferation of periportal HPC was induced with a hepatotoxin (3,5-diethoxycarbonyl-dihydrocollidine, DDC) in C57BL/6 mice for up to 14 days. Expression of FGF and Notch