Increasing evidence supports the cancer stem cell hypothesis, which postulates that cancer stem cells are responsible for tumor initiation, metastasis, and resistance to treatments. Therefore, they ...are the cells to target to cure a cancer. To study the behavior of cancer stem cells, markers for prospective isolation of cancer stem cells are crucial. Recently, CD133 has been used extensively as a marker for the identification of stem cells from normal and cancerous tissues. Several more recent studies, however, indicate that CD133 are expressed in differentiated epithelial cells in various organs, and CD133-negative cancer cells can also initiate tumors. The findings suggest that CD133 is not restricted to somatic stem cells and cancer stem cells. However, in many cases CD133 may be used in combination with other markers or methods to acquire stem cells. In this review, we summarize findings in CD133 expression in various tissues and critically discuss its applications in stem cell isolation.
Three‐dimensional (3D) culture has been shown to improve pluripotent gene expression in mesenchymal stem cells (MSCs), but the underlining mechanisms were poorly understood. Here, we found that the ...relaxation of cytoskeleton tension of MSCs in 3D culture was critically associated with the expressional up‐regulation of Nanog. Cultured in spheroids, MSCs showed decreased integrin‐based cell–matrix adhesion but increased cadherin‐based cell–cell interaction. Different from that in 2D culture, where MSCs exhibited branched and multiple‐directed F‐actin stress bundles at the cell edge and strengthened stress fibres transversing the cell body, MSCs cultured in spheroids showed compact cell body, relaxed cytoskeleton tension with very thin cortical actin filament outlining the cell, and increased expression of Nanog along with reduced levels of Suv39h1 (H3K9 methyltransferase) and H3K9me3. Notably, pharmaceutical inhibition of actin polymerization with cytochalasin D or silencing Suv39h1 expression with siRNA in 2D‐cultured MSCs elevated the expression of Nanog via H3K9 demethylation. Thus, our data suggest that 3D culture increases the expression of Nanog through the relaxation of actin cytoskeleton, which mediates reduced Suv39h1 and H3K9me3 levels.
The mouse excisional wound healing model has been used extensively to study wound healing and cutaneous regeneration. However, as mouse skin is mobile, contraction accounts for a large part of wound ...closure. In the mouse excisional wound splinting model, a splinting ring tightly adheres to the skin around the wound, preventing local skin contraction. The wound therefore heals through granulation and re-epithelialization, a process similar to that occurring in humans. The model, which takes 2-4 weeks to carry out, can be used to study the effects of stem cells on cutaneous repair or regeneration. In this protocol, we also describe how to implant stem cells onto the wound bed in Matrigel and/or into the surrounding tissue through injection. Serial wound tissue samples at different time points can be harvested to monitor the engraftment and the effects of stem cells in angiogenesis and wound healing.
Mesenchymal stem cells (MSCs) hold profound promise in tissue repair/regeneration. However, MSCs undergo remarkable spontaneous differentiation and aging during monolayer culture expansion. In this ...study, we found that 2–3 days of three‐dimensional (3D) spheroid culture of human MSCs (hMSCs) that had been expanded in monolayer for six passages increased their clonogenicity and differentiation potency to neuronal cells. Moreover, in accordance with these changes, the expression levels of miRNA which were involved in stem cell potency were changed and levels of histone H3 acetylation in K9 in promoter regions of Oct4, Sox2 and Nanog were elevated. Our results indicate that spheroid culture increases their multi‐potency and changes the epigenetic status of pluripotent genes in hMSCs.
Although chronic wounds are common, treatment for these disabling conditions remains limited and largely ineffective. In this study, we examined the benefit of bone marrow-derived mesenchymal stem ...cells (BM-MSCs) in wound healing. Using an excisional wound splinting model, we showed that injection around the wound and application to the wound bed of green fluorescence protein (GFP)(+) allogeneic BM-MSCs significantly enhanced wound healing in normal and diabetic mice compared with that of allogeneic neonatal dermal fibroblasts or vehicle control medium. Fluorescence-activated cell sorting analysis of cells derived from the wound for GFP-expressing BM-MSCs indicated engraftments of 27% at 7 days, 7.6% at 14 days, and 2.5% at 28 days of total BM-MSCs administered. BM-MSC-treated wounds exhibited significantly accelerated wound closure, with increased re-epithelialization, cellularity, and angiogenesis. Notably, BM-MSCs, but not CD34(+) bone marrow cells in the wound, expressed the keratinocyte-specific protein keratin and formed glandular structures, suggesting a direct contribution of BM-MSCs to cutaneous regeneration. Moreover, BM-MSC-conditioned medium promoted endothelial cell tube formation. Real-time polymerase chain reaction and Western blot analysis revealed high levels of vascular endothelial growth factor and angiopoietin-1 in BM-MSCs and significantly greater amounts of the proteins in BM-MSC-treated wounds. Thus, our data suggest that BM-MSCs promote wound healing through differentiation and release of proangiogenic factors. Disclosure of potential conflicts of interest is found at the end of this article.
Bone marrow derived mesenchymal stem cells (BM-MSCs) have been shown to enhance wound healing; however, the mechanisms involved are barely understood. In this study, we examined paracrine factors ...released by BM-MSCs and their effects on the cells participating in wound healing compared to those released by dermal fibroblasts. Analyses of BM-MSCs with Real-Time PCR and of BM-MSC-conditioned medium by antibody-based protein array and ELISA indicated that BM-MSCs secreted distinctively different cytokines and chemokines, such as greater amounts of VEGF-alpha, IGF-1, EGF, keratinocyte growth factor, angiopoietin-1, stromal derived factor-1, macrophage inflammatory protein-1alpha and beta and erythropoietin, compared to dermal fibroblasts. These molecules are known to be important in normal wound healing. BM-MSC-conditioned medium significantly enhanced migration of macrophages, keratinocytes and endothelial cells and proliferation of keratinocytes and endothelial cells compared to fibroblast-conditioned medium. Moreover, in a mouse model of excisional wound healing, where concentrated BM-MSC-conditioned medium was applied, accelerated wound healing occurred compared to administration of pre-conditioned or fibroblast-conditioned medium. Analysis of cell suspensions derived from the wound by FACS showed that wounds treated with BM-MSC-conditioned medium had increased proportions of CD4/80-positive macrophages and Flk-1-, CD34- or c-kit-positive endothelial (progenitor) cells compared to wounds treated with pre-conditioned medium or fibroblast-conditioned medium. Consistent with the above findings, immunohistochemical analysis of wound sections showed that wounds treated with BM-MSC-conditioned medium had increased abundance of macrophages. Our results suggest that factors released by BM-MSCs recruit macrophages and endothelial lineage cells into the wound thus enhancing wound healing.
Our understanding of the role of bone marrow (BM)‐derived cells in cutaneous homeostasis and wound healing had long been limited to the contribution of inflammatory cells. Recent studies, however, ...suggest that the BM contributes a significant proportion of noninflammatory cells to the skin, which are present primarily in the dermis in fibroblast‐like morphology and in the epidermis in a keratinocyte phenotype; and the number of these BM‐derived cells increases markedly after wounding. More recently, several studies indicate that mesenchymal stem cells derived from the BM could significantly impact wound healing in diabetic and nondiabetic animals, through cell differentiation and the release of paracrine factors, implying a profound therapeutic potential. This review discusses the most recent understanding of the contribution of BM‐derived noninflammatory cells to cutaneous homeostasis and wound healing. STEM CELLS 2010;28:905–915
Skin stem cells can regenerate epidermal appendages; however, hair follicles (HF) lost as a result of injury are barely regenerated. Here we show that macrophages in wounds activate HF stem cells, ...leading to telogen-anagen transition (TAT) around the wound and de novo HF regeneration, mostly through TNF signalling. Both TNF knockout and overexpression attenuate HF neogenesis in wounds, suggesting dose-dependent induction of HF neogenesis by TNF, which is consistent with TNF-induced AKT signalling in epidermal stem cells in vitro. TNF-induced β-catenin accumulation is dependent on AKT but not Wnt signalling. Inhibition of PI3K/AKT blocks depilation-induced HF TAT. Notably, Pten loss in Lgr5
HF stem cells results in HF TAT independent of injury and promotes HF neogenesis after wounding. Thus, our results suggest that macrophage-TNF-induced AKT/β-catenin signalling in Lgr5
HF stem cells has a crucial role in promoting HF cycling and neogenesis after wounding.
The genesis of the hair follicle relies on signals derived from mesenchymal cells in the dermis during skin morphogenesis and regeneration. Multipotent skin-derived precursors (SKPs), which exhibit ...long term proliferation potential when being cultured in spheroids, have been shown to induce hair genesis and hair follicle regeneration in mice, implying a therapeutic potential of SKPs in hair follicle regeneration and bioengineering. However, the hair-inductive property of SKPs declines progressively upon ex vivo culture expansion, suggesting that the expressions of the genes responsible for hair induction are epigenetically unstable. In this study, we found that TSA markedly alleviated culture expansion induced SKP senescence, increased the expression and activity of alkaline phosphatase (AP) in the cells and importantly restored the hair inductive capacity of SKPs. TSA increased the acetylation level of histone H3, including the K19/14 sites in the promoter regions of bone morphogenetic proteins (BMPs) genes, which were associated with elevated gene expression and BMP signaling activity, suggesting a potential attribution of BMP pathway in TSA induced recovery of the hair inductive capacity of SKPs.
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