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 reproduc-ible model of limb ischemia in C57BL/6 mice that shows response toMNC injection. These studies suggest several parameters of humantrials can be tested in a small animal, cost-effective manner, allowingoptimization of human trial parameters.

Single cell gene expression analysis of bone marrowderived mesenchymal stem cells reveals a diminishedsubpopulation defined by vasculogenic markersMichael 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, FACSStanford University, Stanford, CA

INTRODUCTION: Pathophysiologic changes associated with diabe-tes severely impair neovascularization and are accountable for com-plications affecting the cardiovascular system and wound healing.Bone marrow derived mesenchymal stem cells (BM-MSCs) are re-cruited to ischemic wounds and play a major role in vasculogenesis.In this study we hypothesized that the impaired neovascularizationcapacity in diabetic mice is reflected by transcriptional changeswithin BM-MSCs.

METHODS: Total bone marrow cells were harvested from wildtype(C57BL/6) and diabetic (db/db) mice. BM-MSCs with the surfacemarker profile lin- CD45-Sca1� were sorted as single cells usingFACS. Microfluidic single cell transcriptional analysis was performedacross an array of 48 gene targets. Mathematical clustering analysiswas employed to identify subpopulations.

RESULTS: Transcriptional analysis revealed multiple genes affectedby diabetic metabolism. Especially genes associated with vasculogen-esis (Flt1, Flt4, KDR and Vcam1) were found to be downregulated indiabetic mice. Furthermore, cells expressing the transcriptional fac-tors associated with ‘stemness’ characteristics (Klf4, Id2, Notch1)were depleted in the diabetic group. Using cell clustering analysis, asubpopulation of cells that was defined by the expression of vasculo-genic genes was found to be diminished in diabetic mice.

CONCLUSIONS: Recruitment of bone marrow derived stem cellsto peripheral tissues plays a crucial role in the recovery from anischemic insult. Here we show that a subpopulation of BM-MSCsdefined by the expression of vasculogenic genes is significantly re-duced in diabetic bone marrow. These findings suggest a pathophys-iologic mechanism underlying impaired diabetic vasculogenesis andmay serve as a guide for future therapeutic approaches.

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

INTRODUCTION: Cutaneous wound healing is a major healthproblem in the US. Following injury, keratinocytes must dedifferen-tiate to proliferate, migrate across the wound bed, and finally redif-ferentiate into a multi-layered tissue for the wound to heal. NIKShuman keratinocyte progenitor cells were successfully evaluated as acomponent of StrataGraft skin substitute in a Phase I/IIa safety andearly efficacy clinical trial for temporary management of traumaticskin wounds. However, the molecular mechanisms underlying NIKSwound healing properties remain unclear. Transmembrane receptornotch is critical in epidermal development. Studies using NIKS in-dicated that notch1 signaling is essential for cellular proliferation andmigration. We hypothesize that, in addition to regulating prolifera-tion and migration, Notch1 signaling is associated with cellular dif-ferentiation to promote healing.

METHODS: To determine the role of Notch1 in differentiation,subconfluent NIKS monolayers were exposed to increasing concen-trations (60-660�M) of calcium. Notch1 protein and markers ofkeratinocyte differentiation were detected by Western Blot.

RESULTS: A high basal Notch1 level was detected at a calciumconcentration of 60�M. In response to higher calcium, Notch1 wasdownregulated in 4 hours and was undetectable after 8 days with aconcomitant increase in the differentiation markers, keratin 1, kera-tin 10 and involucrin.

CONCLUSIONS: Our results suggest that following injury, Notch1signaling is required for NIKS proliferation and migration. Further-more, Notch 1 attenuation may be necessary to allow for keratino-cyte differentiation. Our findings could improve clinical outcome ofmillions of patients with cutaneous wounds by modulating Notch1signaling in StrataGraft skin substitutes or by using a novel bioactivedressing.

Fibroblast growth factor and Notch signaling areassociated with hepatic progenitor cell expansion afterchronic liver injuryChristopher L Vendryes MD, MS, Sarah B Utley BSc,David M James BSc, Nirmala Mavila PhD, Jenn C Phan BSc,Kasper S Wang MD, FACSChildrens Hospital Los Angeles/Saban Research Institute, LosAngeles, CA

INTRODUCTION: Chronic liver diseases are commonly character-ized by impaired regeneration and ductular proliferation of hepaticprogenitor cells (HPC). With liver tissue transplantation limited bydonor availability, alternative therapies such as HPC transplantationbecome important. However, the mechanisms regulating HPC pro-liferation and differentiation are not completely understood. We pre-viously described fibroblast growth factor (FGF)-10 as a key regula-tor of embryonic HPC proliferation and survival (Berg et al, 2007).Moreover, Notch signaling is critical in bile duct development. Wehypothesized FGF and Notch signaling pathways are active in theductular proliferation of HPC.

METHODS: Ductular proliferation of periportal HPC was inducedwith a hepatotoxin (3,5-diethoxycarbonyl-dihydrocollidine, DDC)in C57BL/6 mice for up to 14 days. Expression of FGF and Notch

S102 Surgical Forum Abstracts J Am Coll Surg