Wnt/β-catenin signaling is critical for dedifferentiation of aged epidermal cells in vivo and in vitro

Aged epidermal cells have the capacity to dedifferentiate into stem cell-like cells. However, the signals that regulate the dedifferentiation of aged epidermal cells remain unclear. Here, we provide evidence that Wnt/β-catenin is critical for aged epidermal cell dedifferentiation in vivo and in vitro. Some aged epidermal cells in human ultrathin epidermal sheets lacking basal stem cells transplanted onto wounds dedifferentiated into stem cell-like cells that were positive for CK19 and β1 integrin but negative for CK10. In addition, Wnt/β-catenin pathway was activated during this process. There was increased expression of Wnt-1, Wnt-4, Wnt-7a, β-catenin, cyclin D1, and c-myc. Secreted frizzled-related protein 1, a Wnt/β-catenin pathway inhibitor, blocked dedifferentiation in vivo. Then, the activator, a highly specific glycogen synthase kinase (GSK)-3β inhibitor, of Wnt/β-catenin pathway was added to the culture medium of aged epidermal cells. Surprisingly, we found that the activator induced higher expression of CK19, β1 integrin, Oct4, and Nanog proteins. The induced aged epidermal cells exhibited high colony-forming efficiency, long-term proliferative potential and could regenerate a skin equivalent (as do epidermal stem cells). These results suggested that activation of Wnt/β-catenin pathway induced the dedifferentiation of aged epidermal cells, which suggest a new approach to generate epidermal stem cell-like cells.     

Beta-catenin can induce hair follicle stem cell differentiation into transit-amplifying cells through c-myc activation

Hair follicle stem cells play important roles in maintaining homeostasis and skin tissue self-renewal. Transit-amplifying cells represent the transition of cells from hair follicle stem cells into differentiated epidermal cells. Thus far, the signaling pathway and the molecular biological mechanism that regulate the proliferation and differentiation of hair follicle stem cells remain unclear. In this paper, we studied the relationship between β-catenin and c-myc during the process of the differentiation of hair follicle stem cells into transit-amplifying cells. Based on our results, the expression of β-catenin can activate the nuclear gene c-myc and regulate the expression of transit-amplifying cell markers K15, K19, a6-integrin and β1-integrin, indicating that β-catenin is involved in the transformation process from hair follicle stem cells to transit-amplifying cells and suggesting that β-catenin plays an important biological role in the induction of this differentiation process.     

Ring1 promotes the transformation of hepatic progenitor cells into cancer stem cells through the Wnt/β-catenin signaling pathway

The proliferation of hepatic progenitor cells (HPCs) is observed in reactive conditions of the liver and primary liver cancers. Ring1 as a member of polycomb-group proteins which play vital roles in carcinogenesis and stem cell self-renewal was increased in HCC patients and promoted proliferation and survival of cancer cell by degrading p53. However, the mechanisms of Ring1 driving the progression of hepatocarcinogenesis have not been elucidated. In this study, forced expression Ring1 and Ring1 siRNA lentiviral vectors were utilized to stably overexpression and silence Ring1 in HPC cell line (WB-F344), respectively. Our finding indicated that overexpression of Ring1 in HPCs promoted colony formation, cell multiplication, and invasion in vitro, conversely depletion of Ring1 repressed the biological functions of HPCs relative to controls. The expression of β-catenin was upregulated in the HPCs with overexpression of Ring1, and the correlation analysis also showed that β-catenin and Ring1 had a significant correlation in the liver cancer tissues and adjacent tissues. The activation of the Wnt/β-catenin signaling pathway significantly increased the expression of liver cancer stem cells related (LCSCs)-related molecular markers CD90 and EpCAM, which led to the transformation of HPCs into LCSCs. Most importantly, the injection of HPCs with overexpressed Ring1 into the subcutaneous of nude mice leads to the formation of poorly differentiated HCC neoplasm. Our findings elucidate that overexpression of Ring1 the activated Wnt/β-catenin signaling pathway and drove the transformation of HPCs into cancer stem cell-like cells, suggesting Ring1 has extraordinary potential in early diagnosis of HCC.     

Effects of Wnt3a on proliferation and differentiation of human epidermal stem cells

Epidermal stem cells maintain development and homeostasis of mammalian epidermis throughout life. However, the molecular mechanisms involved in the proliferation and differentiation of epidermal stem cells are far from clear. In this study, we investigated the effects of Wnt3a and Wnt/β-catenin signaling on proliferation and differentiation of human fetal epidermal stem cells. We found both Wnt3a and active β-catenin, two key members of the Wnt/β-catenin signaling, were expressed in human fetal epidermis and epidermal stem cells. In addition, Wnt3a protein can promote proliferation and inhibit differentiation of epidermal stem cells in vitro culture. Our results suggest that Wnt/β-catenin signaling plays important roles in human fetal skin development and homeostasis, which also provide new insights on the molecular mechanisms of oncogenesis in human epidermis.     

Dedifferentiation derived cells exhibit phenotypic and functional characteristics of epidermal stem cells

Differentiated epidermal cells can dedifferentiate into stem cells or stem cell‐like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation‐derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c‐nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and β₁‐integrin emerged at the wound‐neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and β₁‐integrin⁺ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10⁺ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation‐derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, β₁‐integrin, Oct4 and Nanog in dedifferentiation‐derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation‐derived cells demonstrated: (1) the numbers of colonies consisting of 5–10 cells and greater than 10 cells were increased 5.9‐fold and 6.7‐fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 × 10⁵ cells versus 1 × 10¹² cells) were all significantly increased and (4) dedifferentiation‐derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation‐derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.     

Pluripotent stem cells induced from mouse neural stem cells and small intestinal epithelial cells by small molecule compounds

Recently, we reported a chemical approach to generate pluripotent stem cells from mouse fibroblasts. However, whether chemically induced pluripotent stem cells (CiPSCs) can be derived from other cell types remains to be demonstrated. Here, using lineage tracing, we first verify the generation of CiPSCs from fibroblasts. Next, we demonstrate that neural stem cells (NSCs) from the ectoderm and small intestinal epithelial cells (IECs) from the endoderm can be chemically reprogrammed into pluripotent stem cells. CiPSCs derived from NSCs and IECs resemble mouse embryonic stem cells in proliferation rate, global gene expression profile, epigenetic status, self-renewal and differentiation capacity, and germline transmission competency. Interestingly, the pluripotency gene Sall4 is expressed at the initial stage in the chemical reprogramming process from different cell types, and the same core small molecules are required for the reprogramming, suggesting conservation in the molecular mechanism underlying chemical reprogramming from these diverse cell types. Our analysis also shows that the use of these small molecules should be fine-tuned to meet the requirement of reprogramming from different cell types. Together, these findings demonstrate that full chemical reprogramming approach can be applied in cells of different tissue origins and suggest that chemical reprogramming is a promising strategy with the potential to be extended to more initial types.     

High glucose-induced hyperosmolarity impacts proliferation,cytoskeleton remodeling and migration of human induced pluripotent stem cells via aquaporin-1

Background and objective: Hyperglycemia leads to adaptive cell responses in part due to hyperosmolarity. In endothelial and epithelial cells, hyperosmolarity induces aquaporin-1 (AQP1) which plays a role in cytoskeletal remodeling, cell proliferation and migration. Whether such impairments also occur in human induced pluripotent stem cells (iPS) is not known. We therefore investigated whether high glucose-induced hyperosmolarity impacts proliferation, migration, expression of pluripotency markers and actin skeleton remodeling in iPS cells in an AQP1-dependent manner. Methods and results: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, Sox2, Klf4, c-Myc). After reprogramming, iPS cells were characterized by their adaptive responses to high glucose-induced hyperosmolarity by incubation with 5.5 mmol/L glucose, high glucose (HG) at 30.5 mM, or with the hyperosmolar control mannitol (HM). Exposure to either HG or HM increased the expression of AQP1. AQP1 co-immunoprecipitated with β-catenin. HG and HM induced the expression of β-catenin. Under these conditions, iPS cells showed increased ratios of F-actin to G-actin and formed increased tubing networks. Inhibition of AQP1 with small interfering RNA (siRNA) reverted the inducing effects of HG and HM. Conclusions: High glucose enhances human iPS cell proliferation and cytoskeletal remodeling due to hyperosmolarity-induced upregulation of AQP1.     

The β-catenin/Tcf4/survivin signaling maintains a less differentiated phenotype and high proliferative capacity of human corneal epithelial progenitor cells

It is clear that the microenvironment or niche plays an important role in determining the fate of stem cells: being stem cells or differentiated. However, the intrinsic pathways controlling the fate of adult stem cells in different niches are largely unknown. This study was to explore the role of β-catenin/Tcf4/survivin signaling in determining the fate of human corneal epithelial stem cells in different media. We observed that the low calcium serum-free media, especially CnT-20, promoted proliferative capacity, colony forming efficiency and stem cell-like phenotype of human corneal epithelial cells (HCECs) when compared with the cells cultured in a high calcium serum-containing medium SHEM. Three key factors in Wnt signaling, β-catenin, Tcf4 and survivin, were found to be expressed higher by HCECs grown in CnT-20 than those cultured in SHEM, as evaluated by real-time PCR, Western blotting and immunostaining. Transfection of siRNA-Tcf4 at 10-50nM knocked down Tcf4, and also significantly suppressed its down stream molecule survivin at both mRNA and protein levels in HCECs. Furthermore, Tcf4 silencing significantly suppressed the proliferative capacity of HCECs, measured by WST-1 assay, compared with the control groups, untreated or transfected with non-coding sequence siRNA-fluorescein. These findings demonstrate that low calcium serum free media promote ex vivo expansion of corneal epithelial progenitor cells that retain a less differentiated phenotype and high proliferative capacity via β-catenin/Tcf4/survivin signaling, a novel intrinsic pathway. This study may have high impact and clinic implication on the expansion of corneal epithelial stem cells in regenerative medicine, especially for ocular surface reconstruction.     

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

We describe derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension culture-reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor-expressing cells based on their differential survival and proliferation in suspension culture. Seamless integration of SiPSC reprogramming and directed differentiation enabled scalable production of beating cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step toward the development of robust PSC generation, expansion and differentiation technology.     

Vitamin D and calcium signaling in epidermal stem cells and their regeneration

Epidermal stem cells (SCs) residing in the skin play an essential role for epidermal regeneration during cutaneous wound healing. Upon injury, distinct epidermal SCs residing in the interfollicular epidermis and/or hair follicles are activated to proliferate. Subsequently, SCs and progeny migrate, differentiate and restore the epidermis. We review a role of the vitamin D signaling through its receptor of vitamin D receptor (Vdr) in these processes. Vdr conditional knockout (cKO) mouse skin experiences a delay in wound re-epithelialization under low dietary calcium conditions, stimulating our efforts to examine a cooperative role of Vdr with calcium signaling through the calcium sensing receptor in the epidermis. We review the role of vitamin D and calcium signaling in different processes essential for injury induced epidermal regeneration during cutaneous wound repair. First, we discuss their roles in self-renewal of epidermal SCs through β-catenin signaling. Then, we describe epidermal remodeling, in which SCs and progeny migrate and differentiate to restore the epidermis, events controlled by the E-cadherin mediated adherens junction signaling. Finally, we discuss the potential mechanisms for vitamin D and calcium signaling to regulate injury induced epidermal regeneration mutually and interdependently.