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Debrided Skin as a Source of Autologous Stem Cells for Wound Repair

From the August Edition of Stem Cells
Paper commentary by Stuart P. Atkinson

Tissue resident adult stem cells, such as mesenchymal stem cells (MSCs) or adipose-derived stem cells (ASCs), have previously demonstrated a capacity to repair extensively injured tissues (Picinich et al, Horwitz and Dominici). However, major traumatic injuries such as large surface area burns, which constitute 5%–10% of military casualties, limit the availability of autologous stem cell populations for wound repair and such injuries also require extensive reconstruction. The process of wound debridement; the medical removal of a patient's dead, damaged, or infected tissue to improve the healing potential of the remaining healthy tissue, typically involves the removal of subcutaneous layers and associated tissue structures, including portions of intact hypodermal adipose tissue. This led the group of Robert J. Christy at the United States Army Institute of Surgical Research, Fort Sam Houston, Texas, USA to investigate the potential of debrided skin to be a source of viable autologous stem cells for use in wound treatments. Their report (Natesan et al) is published in the August Edition of Stem Cells.

Analysis of debrided skin samples indicated 40%–80% tissue viability and complete loss of both the epidermis (the outer layer of the skin) and reticular dermis (the underlying dense irregular connective tissue which gives the skin its strength and elasticity, housing glands, hair follicles, blood vessels and nerves). Underneath these layers lies the hypodermis, containing subcutaneous fat and connective tissue and larger blood vessels and nerves. Although some hypodermal areas showed complete necrosis of both fat cells and dermis other regions of the tissue contained intact adipocytes and completely intact blood vessels. Intact microvessels marked by CD31 (PECAM1) were associated with PDGFRB+ cells. From these hypodermal samples, a floating fraction and a pellet fraction of cells were isolated. Cells from the floating fraction deemed to originate from the perivascular space of blood vessels surrounding adipocytes and which adhered after plating overnight were considered P0 and named debrided skin adipose-derived stem cells, or dsASCs, and were further characterised. Analysis showed that these cells exhibited characteristic fibroblast-like morphology and stained positive for stem cell-associated surface markers including CD54 (ICAM1), CD71 (TFRC), CD90 (THY1), CD105 (ENG), STRO-1, and PDGFRB, but were negative for endothelial (CD31) and leukocytic (CD45 (PTPRC) markers, indicating the selective adherence of stem cells. It has been demonstrated across numerous tissues that the stem cell niche is localized to perivascular regions and that resident stem cells within the niche express markers of both MSCs (CD105, STRO-1, and CD34) and pericytic (NG2 (CSPG4) and PDGFRB) lineages (Crisan et al, Katz et al, Shi and Gronthos, Traktuev et al, Amos et al). Perivascular cells are in direct contact with the inner endothelium and aid the formation of stable vessel network and this interaction is required to restore an appropriate microenvironment for tissue remodeling during skin regeneration (Rubina et al). Stemness of dsASCs was assessed in P0 and P1 cells by FACS and showed that a two-fold higher percentage of CD54+CD71+CD90+CD105+PDGFRb+STRO-1+ cells was present within the P1 cells, while the P1 cells were also CD19-CD44+ providing evidence for preferential expansion of stem cells. Further expansion of the dsASCs in culture led to an apparent increase in the expression levels of CD71, CD90, CD105 and PDGFRb transcripts and showed a four to five fold increase in cell number every 48 hours up to P4, after which the proliferation rate marginally decreased to three- to four fold every 48 hours up to P8.

Further, analysis of P2 dsASCs demonstrated that these cells exhibited multilineage differentiation potential. Culture under conditions conducive to adipogenic differentiation led to the cells accumulating lipid (as detected by oil red 0 staining) and a large increase in PPARG transcript expression compared to control ASCs. An increase in adiponectin transcript levels demonstrated the ability of dsASCs to undergo adipogenesis through the activation of a glucose-regulated signaling pathway, while GLUT4 (SLC2A4) and leptin levels were also induced. Osteogenic differentiation led to a matrix mineralization by day 8 post-induction and increased over time, with the cells positively staining for calcium by day 21. Terminal differentiation was confirmed by the expression of RUNX2, a master osteogenic transcription factor responsible for mature osteoblast formation. Vascular differentiation of dsASCs was also demonstrated, as when seeded over a three-dimensional matrix of Matrigel and induced with endothelial cell-specific media the dsASCs spontaneously joined, forming tubular structures which covered the entire surface, creating a dense cellular network within 10 days which expressed both CD31 and PDGFRB in high levels. The engraftment efficiency of P2 dsASCs was also assessed using an excision wound splinting model in immunodeficient rats (Chen et al). After 8 days dsASC-Matrigel composites integrated within the healing wound bed and re-epithelialisation was evident at the epidermal region while by day 12 the wound bed was vascularised. dsASCs integrated within the healing wound bed were identified with human-specific anti-mitochondrial protein antibody.

Overall, this suggests that appropriate treatment of debrided tissue from injured patients can yield useful stem cells which can be amplified in culture, exhibit multilineage differentiation potential and show wound healing capabilities. Although patient-specific induced pluripotent stem cells (iPSC) technology promises to provide a source of cells for cell replacement therapy, this technology is still in its relative infancy, and so research such as this into the amplification of resident adult stem cell populations for therapeutic means represents an important and useful avenue for regenerative medicine.


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