Human umbilical cord blood (CB)-derived unrestricted somatic stem cells (USSCs) have previously been demonstrated to have a broad differentiation potential and regenerative beneficial effects when administered in animal models of multiple degenerative diseases. Here we demonstrated that USSCs could be induced to express genes that hallmark keratinocyte differentiation. We also demonstrated that USSCs express type VII collagen (C7), a protein that is absent or defective in patients with an inherited skin disease, recessive dystrophic epidermolysis bullosa (RDEB). In mice with full-thickness excisional wounds, a single intradermal injection of USSCs at a 1-cm distance to the wound edge resulted in significantly accelerated wound healing. USSC-treated wounds displayed a higher density of CD31(+) cells, and the wounds healed with a significant increase in skin appendages. These beneficial effects were demonstrated without apparent differentiation of the injected USSCs into keratinocytes or endothelial cells. In vivo bioluminescent imaging (BLI) revealed specific migration of USSCs modified with a luciferase reporter gene, from a distant intradermal injection site to the wound, as well as following systemic injection of USSCs. These data suggest that CB-derived USSCs could significantly contribute to wound repair and be potentially used in cell therapy for patients with RDEB.
Delayed healing of skin wounds is a major morbidity. Repeated wounding is characteristic in patients with recessive dystrophic epidermolysis bullosa (RDEB), caused by mutations in COL7A1 gene. Stem cell therapy offers an option in treating this disease (Kiru/Cairo et al. PNAS, 2011). Recently, cord blood (CB) derived pluripotent stem cells, USSCs, have been applied in several animal models of degenerative diseases with beneficial outcomes. To determine the potential of USSCs in the treatment of RDEB and its associated wounding phenotype. CB-USSCs were characterized for genetic and functional properties. Their in vivo functions were evaluated in a murine full-thickness excisional wound healing model and by bioluminescent imaging (BLI), using USSCs modified with a luciferase reporter gene. CB-USSCs share several embryonic stem cell properties and could be induced to express hallmark genes of keratinocyte differentiation. USSCs constitutively express Col7A1, supporting their therapeutic potential in the treatment of patients with RDEB. In the wounding model, a single USSC intradermal injection promoted epithelialization and facilitated formation and remodeling of epidermis, accompanied by a significantly accelerated rate of wound healing on days 6-10 post wounding (F(1,168)=50.8 P < .01). In vivo BLI revealed specific migration of USSCs from a distant intradermal injection site toward the wound, as well as following systemic injection. Temporal quantification on the total bioluminescence indicated an overall 59.9% signal loss over 3 days followed by a 95.06% loss at 1 week. The bioluminescence in the area of wound was then maintained at ∼0.5-1% level till the end of the experiment (3 month). USSCs express several chemokine receptors that may mediate their migration to the wound, including CXCR4 (for SDF1), CCR7 (for CCL21) and PDGFRα (for HMGB1). In vitro chemotaxis assays indicated that SDF-1 significantly enhanced USSC migration at a concentration of 100ng/ml, while neither CCL21 nor HMGB1 showed significance even at a concentration of 10μg/ml. The effects of such chemokine/receptor interactions on USSC recruitment in vivo are now being investigated. These results suggest significant beneficial effects of CB-USSCs on wound healing and raised the possibility of USSC's therapeutic beneifit in the treatment of patients with RDEB.
Recessive Dystrophic Epidermolysis bullosa (RDEB) is a severe inherited skin-blistering disease caused by mutations in the Col7A1 gene. COL7A1 is synthesized by keratinocytes and fibroblasts and is a major component of anchoring fibrils that extend from basement membrane to papillary dermis. A recent animal study suggested that the hematopoietic enriched cells ameliorated the blistering phenotype in the RDEB mice. However, only limited donor epithelial engraftment and COL7A1 protein were observed and the possibility of rare stem cells co-purifying with enriched cells cannot be excluded. As multiple populations of primitive stem cells with multi-lineage differentiation potential have been identified from HUCB, including unrestricted somatic stem cells (USSCs), they may offer potential for the treatment of RDEB.GoalTo isolate USSCs from HUCB and determine the potential in treating RDEB and promoting wound healing.MethodsUSSCs were initiated from HUCB in 30% FBS and 10−7M dexamethasone. Q-RT-PCR was utilized to assess gene expression of Nanog, Oct4, Sox2 and Col7A1. DNA methylation at the enhancer and promoter of both Oct4 and Nanog genes was analyzed by bisulfate sequencing. The expression of CCR2 was analyzed by immunocytochemistry.ResultsHUCB-USSCs are lineage negative and share overlapping but distinct surface markers with MSCs. They express a low but consistent level of Nanog, Oct4 and Sox2. Significantly, the Nanog and Oct4 expression in USSCs is about 20- and 400- fold higher than that in human fibroblasts. Their level was further increased 10 fold following the treatment with DNA methylation inhibitor, 5-azacytidine. An average of 65% and 47% of the CpGs were unmethylated in the enhancer and promoter of the Nanog gene respectively, while 56% and 80% were unmethylated at those of the Oct4 gene. We also showed that USSCs express Col7A1, at a level comparable to human keratinocytes, suggesting that USSCs, once engraft in the skin, could rescue the defective anchoring fiber formation by secreting COL7A1. In addition, we demonstrated that USSCs express the CCR2, a receptor for several chemoattractant proteins, such as MCP-1, that have been shown to be highly expressed in the dermal wounds, implying a migratory ability of USSCs under wound conditions. We are now investigating the role of USSCs in promoting wound healing in immunodeficient mice with excisional wounds and comparing the effects of routes of injection on their long-term engraftment. Recessive Dystrophic Epidermolysis bullosa (RDEB) is a severe inherited skin-blistering disease caused by mutations in the Col7A1 gene. COL7A1 is synthesized by keratinocytes and fibroblasts and is a major component of anchoring fibrils that extend from basement membrane to papillary dermis. A recent animal study suggested that the hematopoietic enriched cells ameliorated the blistering phenotype in the RDEB mice. However, only limited donor epithelial engraftment and COL7A1 protein were observed and the possibility of rare stem cells co-purifying with enriched cells cannot be excluded. As multiple populations of primitive stem cells with multi-lineage differentiation potential have been identified from HUCB, including unrestricted somatic stem cells (USSCs), they may offer potential for the treatment of RDEB. GoalTo isolate USSCs from HUCB and determine the potential in treating RDEB and promoting wound healing. To isolate USSCs from HUCB and determine the potential in treating RDEB and promoting wound healing. MethodsUSSCs were initiated from HUCB in 30% FBS and 10−7M dexamethasone. Q-RT-PCR was utilized to assess gene expression of Nanog, Oct4, Sox2 and Col7A1. DNA methylation at the enhancer and promoter of both Oct4 and Nanog genes was analyzed by bisulfate sequencing. The expression of CCR2 was analyzed by immunocytochemistry. USSCs were initiated from HUCB in 30% FBS and 10−7M dexamethasone. Q-RT-PCR was utilized to assess gene expression of Nanog, Oct4, Sox2 and Col7A1. DNA methylation at the enhancer and promoter of both Oct4 and Nanog genes was analyzed by bisulfate sequencing. The expression of CCR2 was analyzed by immunocytochemistry. ResultsHUCB-USSCs are lineage negative and share overlapping but distinct surface markers with MSCs. They express a low but consistent level of Nanog, Oct4 and Sox2. Significantly, the Nanog and Oct4 expression in USSCs is about 20- and 400- fold higher than that in human fibroblasts. Their level was further increased 10 fold following the treatment with DNA methylation inhibitor, 5-azacytidine. An average of 65% and 47% of the CpGs were unmethylated in the enhancer and promoter of the Nanog gene respectively, while 56% and 80% were unmethylated at those of the Oct4 gene. We also showed that USSCs express Col7A1, at a level comparable to human keratinocytes, suggesting that USSCs, once engraft in the skin, could rescue the defective anchoring fiber formation by secreting COL7A1. In addition, we demonstrated that USSCs express the CCR2, a receptor for several chemoattractant proteins, such as MCP-1, that have been shown to be highly expressed in the dermal wounds, implying a migratory ability of USSCs under wound conditions. We are now investigating the role of USSCs in promoting wound healing in immunodeficient mice with excisional wounds and comparing the effects of routes of injection on their long-term engraftment. HUCB-USSCs are lineage negative and share overlapping but distinct surface markers with MSCs. They express a low but consistent level of Nanog, Oct4 and Sox2. Significantly, the Nanog and Oct4 expression in USSCs is about 20- and 400- fold higher than that in human fibroblasts. Their level was further increased 10 fold following the treatment with DNA methylation inhibitor, 5-azacytidine. An average of 65% and 47% of the CpGs were unmethylated in the enhancer and promoter of the Nanog gene respectively, while 56% and 80% were unmethylated at those of the Oct4 gene. We also showed that USSCs express Col7A1, at a level comparable to human keratinocytes, suggesting that USSCs, once engraft in the skin, could rescue the defective anchoring fiber formation by secreting COL7A1. In addition, we demonstrated that USSCs express the CCR2, a receptor for several chemoattractant proteins, such as MCP-1, that have been shown to be highly expressed in the dermal wounds, implying a migratory ability of USSCs under wound conditions. We are now investigating the role of USSCs in promoting wound healing in immunodeficient mice with excisional wounds and comparing the effects of routes of injection on their long-term engraftment.
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin blistering disease caused by mutations in Col7a1gene, which encodes a major component in anchoring fibrils (Christiano et.al. Nat Gen 1993). An initial report has shown promises of Allogeneic (Allo) -SCT for the treatment of RDEB (Wagner et.al. N Engl J Med 2010). However no distinct anchoring fibrils were observed in the recipient skin and the functional cell populations are to be characterized. Stem cells with pluripotent properties, including unrestricted somatic stem cells (USSCs) have been isolated from HUCB and may represent novel stem cells for the RDEB regenerative therapy (Liao/Cairo et al, Exp. Hem, 2011). To determine the potential of USSCs in promoting wound healing and treating RDEB. USSCs were isolated from HUCB and characterized by flow cytometry, RT-PCR, bisulfate sequencing and immunocytochemistry. A 1cm2 full-thickness excisional wound was created at the dorsal of NSG mice, followed by intradermal injection of 1 million USSCs or PBS at a 1cm- distance from the margin of the wound. USSCs transcribe a low level of ES factors and have a mosaic DNA methylation pattern at the regulatory regions of Nanog and Oct4. USSCs express Col7a1 at a level that is comparable to human keratinocytes and fibroblasts. Through an initial treatment of ascorbic acid, EGF and BMP4 for 7 days followed by culture in defined K-SFM medium, USSCs were able to differentiate and express keratinocyte- specific genes. In the mouse wounding model, the wounds closed at a faster rate in USSC-treated mice as compared to PBS and a significant difference was observed between the two groups on days 6-10 post wounding (F(1,168) = 50.8, P<0.01). USSCs promoted epithelialization and facilitated formation and remodeling of epidermis. More host-derived endothelial cells (PECAM+) were recruited to the USSC-treated wounds as early as three days post wounding. Furthermore, the USSC-treated skin healed with skin appendages in the center of the wounded area. However human cells could be sporadically detected only in the basal membrane in skin sections without apparent differentiation. This suggests that the improved murine wound healing is due to a paracrine effect of USSCs. HUCB- USSCs showed beneficial effects in cutaneous regeneration. The ability of USSCs to express Col7A1 and promote wound healing suggests their potential application in the RDEB therapy.
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