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Single Cell Transcriptomic Analysis of Human Fetal Bone Marrow-Derived Mesenchymal Stem Cells in Wound Healing
Sacha M.L. Khong, PhD, Dominik Duscher, MD, Michael Januszyk, MD, Peter A. Than, MD, Zeshaan Maan, MD, Geoffrey C. Gurtner, MD.
Stanford University, Stanford, CA, USA.
Purpose: Chronic wounds are a source of significant morbidity for the 6.5 million afflicted Americans, at an annual cost of \ billion. Stem cell-based therapies have recently emerged as a promising new treatment for chronic wounds. They are thought to function through a combination of: 1) secretion of progenitor-recruiting trophic factors, 2) modulation of the local immune response, 3) enhancement of angiogenesis and 4) improvement of extracellular matrix production. These effects represent a singular collection of the various therapeutic approaches to wound healing. Current strategies in cell-based wound therapy employ whole stem cell populations. With increasing evidence indicating substantial heterogeneity within seemingly homogenous stem cell populations, we aim to characterize functionally distinct subsets of fetal bone marrow derived mesenchymal stem cells (BM-MSCs), and determine the mechanisms that underlie their efficacy.
Methods: BM-MSCs were expanded in culture to passage 3. Wound healing function was assessed using a murine stented excisional wound model previously developed and validated by our laboratory. Fluorescently-labeled BM-MSCs and fibroblasts (control) were seeded into a pullulan-collagen hydrogel which was then applied to the wounds on the day of wounding. Wound closure was assessed by daily photographs with histologic and immunohistochemical analysis of the tissue after wound closure.
High throughput single cell multiplex qPCR was performed on Fluorescent Activated Cell Sorted (FACS) BM-MSCs plated in a tissue culture dish. A panel of 96 genes were selected through a comprehensive literature search on key known functional genes pertaining to wound healing, with a further streamlining of genes obtained by advanced bioinformatics analysis using Ingenuity Pathway Analysis (IPA). Data was analyzed using a novel computational methodology developed in our laboratory utilizing principles of information theory and fuzzy logic to group cells based on their transcriptional signatures.
Results: BM-MSCs trilineage differentiation as well as accelerated wound closure (BM-MSC13 ± 0.0 days vs fibroblast 15 ± 0.25, p=0.0004) in C57BL/6 mice was confirmed. Single cell transcriptional analysis on tissue culture plated BM-MSCs revealed 2 distinct subpopulations. Cluster 1 was defined by the high expression of thrombospondin (TSP-1), vascular endothelial growth factor (VEGF), and focal adhesion kinase (FAK), suggesting a more angiogenic function. Cluster 2 was defined by the high expression of stromal cell derived factor 1 (SDF-1), tumor necrosis factor stimulated gene 6 (TNFAIP 6), and transforming growth factor beta 1 (TGFβ1), suggesting a more immunoregulatory function. After introduction to the wound environment, the transcriptional profile of BM-MSCs switch to more abundantly express platelet endothelial cell adhesion molecule (PECAM), stanniocalcin (STC-1), bone morphogenic protein (BMP2) and cluster of differentiation 9 (CD9), suggesting a role in signaling cellular differentiation.
Conclusions: A previously described homogenous population of BM-MSCs were characterized for wound healing potential into two functionally distinct populations. Our single cell transcriptomic analysis revealed a subcluster of BM-MSCs with upregulation of key angiogenic factors and a separate subcluster upregulating key immunoregulatory factors. A better understanding of functionally relevant subpopulations and the critical proteins necessary for improved wound healing may permit the development of an advanced cell-free biologic dressing.
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