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Porcine embryonic fibroblasts (PEFs) can be directly reprogrammed into porcine induced pluripotent stem cells (piPSCs). However, the reprogramming process is generally lengthy and inefficient. Here, we established a fast and efficient induction system of piPSCs from porcine Sertoli cells (SCs) via forced expression of pig Yamanaka factors. The alkaline phosphatase (AP)-positive colonies from SCs developed on Day 3 after lentivirus infection, and were expanded and then picked up on Day 7, whereas reprogramming process from PEFs did not show any colonies in the same period. The picked piPSCs strongly expressed pluripotent genes, had the differentiation capacity to three germ layers, and could be also induced into primordial germ cell-like cells. Screening for transcription factor combinations showed that POU class 5 homeobox 1 (OCT4) is the core factor for AP-positive colony formation, and two factors (OCT4 and c-MYC) could successfully reprogram SCs into piPSCs. We then compared the RNA-sequencing data of piPSCs derived from SCs and PEFs, and found that the most significant difference was the activation of Transforming Growth Factor β signaling pathway. We also compared the RNA levels of SCs and PEFs, and found that SCs exhibited higher Wnt signaling activity and Bone Morphogenetic Protein 4 expression than PEFs, which might be correlated with higher cell proliferation rate and reprogramming efficiency. In summary, the data demonstrated that starting cell sources of piPSCs significantly affect reprogramming dynamics and SCs could serve as cell sources for efficient reprogramming., (© 2022 Wiley Periodicals LLC.)

Orchitis is one of the leading causes of male animal infertility and is associated with inflammatory reactions caused by the bacterium. It has been reported that there is a mutual coupling effect between endoplasmic reticulum stress (ERS) and inflammatory response. Our studies showed that lipopolysaccharide (LPS) could cause testicular damages, apoptosis, ERS, and inflammatory responses in spermatogonial stem cells (SSCs); ERS-related apoptosis proteins were activated and the expression of ERS genes was significantly upregulated; meanwhile, the expression of Toll-like receptor 4 and inflammation factors was apparently increased with LPS treatment. Moreover, melatonin (MEL) could rescue testicular damage, and significantly inhibited the expression of ERS-related apoptosis genes, ERS markers, and inflammatory factors in SSCs and MEL played repairing and anti-infection roles in LPS-induced testicular damage. Therefore, MEL may be used as a drug to prevent and control bacterial infections in male reproductive systems. However, the specific molecular mechanism of MEL to resist ERS and inflammatory response remains to be further studied., (© 2020 Wiley Periodicals LLC.)

Spermatogenesis is a complex process that originates from and depends on the spermatogonial stem cells (SSCs). The number of SSCs is rare, which makes the separation and enrichment of SSCs difficult and inefficient. The transcription factor PAX7 maintains fertility in normal spermatogenesis in mice. However, for large animals, much less is known about the SSCs' self-renewal regulation, especially in dairy goats. We isolated and enriched the CD49f-positive and negative dairy goat testicular cells by magnetic-activated cell sorting strategies. The RNA- sequencing and experimental data revealed that cells with a high CD49f and PAX7 expression are undifferentiated spermatogonia in goat testis. Our findings indicated that ZBTB16 (PLZF), PAX7, LIN28A, BMPR1B, FGFR1, and FOXO1 were expressed higher in CD49f-positive cells as compared to negative cells and goat fibroblasts cells. The expression and distribution of PAX7 in dairy goat also have been detected, which gradually decreased in testis tissue along with the increasing age. When the PAX7 gene was overexpressed in dairy goat immortal mGSCs-I-SB germ cell lines, the expression of PLZF, GFRα1, ID4, and OCT4 was upregulated. Together, our data demonstrated that there is a subset of spermatogonial stem cells with a high expression of PAX7 among the CD49f + spermatogonia, and PAX7 can maintain the self-renewal of CD49f-positive SSCs., (© 2020 Wiley Periodicals LLC.)

LIN28A serves as a crucial marker of dairy goat male germline stem cells (GmGSCs). In our previous study, we demonstrated that LIN28A promotes proliferation, self-renewal, and maintains the stemness of GmGSCs. Here, we found that LIN28A could activate the transcription of NANOG in a let-7g independent manner. We cloned the 5' upstream of two NANOG genes which were located on chromosome 15 ( NANOG-ch15) and chromosome 5 ( NANOG-ch5), respectively, and then examined their promoter activities and promoter methylation levels. Results showed that NANOG-ch15 is a pseudogene whereas NANOG-ch5 is active in Capra hircus. Bioinformatics analysis indicated that the 5' upstream region of NANOG-ch5 does not have typical CpG islands but contains several CG enrichment regions and several LIN28A binding sites. Deletion analysis suggested that NANOG-ch5 promoter can be activated by LIN28A directly binding to the site -210 but not by the indirect effect from the inhibition of let-7g, which is known to be downregulated by LIN28A. Mechanistically, LIN28A recruits and interacts with 5-methylcytosine-dioxygenase Ten-Eleven translocation 1 (TET1) to NANOG-ch5 gene promoter binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. These results revealed the role of LIN28A in NANOG transcriptional regulation via epigenetic DNA modifications to maintain the stemness of GmGSC., (© 2018 Wiley Periodicals, Inc.)

Reelin plays important roles in brain development. Reeler mutant mice that lack the protein reelin (RELN) suffer from cell type- and region-dependent changes in their neocortical layers, and adult reeler mutant mice have dilated seminiferous tubules. Meanwhile, the mechanism by which Reelin regulates the spermatogenic cell development in mice and their reproductive abilities remains unclear. In the present study, we used reeler mutant mice to investigate the effects of Reelin on reproduction in mice. The results indicated variations in sex hormone expression among the reeler mice, indicating that they produce few offspring and their spermatogenic cells are irregularly developed. Moreover, glial cell line-derived neurotrophic factor (GDNF)/GDNF family receptor alpha 1, Ras/extracellular regulated protein kinases (ERK), and promyelocytic leukemia zinc finger (PLZF)/chemokine (C-X-C motif) receptor 4 (CXCR4) serve as potential regulatory pathways that respond to the changes in sertoli cells and the niche of male germ cells. Our findings provided valuable insights into the role of reeler in the reproductive abilities of male mice and development of their spermatogonia stem cells., (© 2018 Wiley Periodicals, Inc.)

Promyelocytic leukemia zinc finger PLZF, known as ZBTB16 or ZFP145 is a critical zinc finger protein of male germline stem cells (mGSCs), it's an essential transcriptional factor for goat testis development and spermatogenesis. Loss of PLZF results in progressive depletion of SSCs after the first wave of spermatogenesis leading to eventual spermatogenic arrest, apparently the result of a shift in the balance in SSC fate away from self-renewal and toward differentiation. Cumulating evidences have demonstrated that microRNAs are expressed in a cell-specific or stage-specific manner during spermatogenesis and acts as regulators on specific makers such as Stra8, ETV5, and PLZF. However, the post transcriptional function of PLZF still poorly elucidate in mGSCs. Bioinformatic analysis and dual luciferase reporter assay showed that miR-19b-3p binds the 3'UTR of PLZF, suggesting that PLZF is a direct target of miR-19b-3p. The profile of miR-19b-3p and PLZF analyzed in dairy goat testis at different age showed that miR-19b-3p was significantly up-regulated in goat testis at 1, 3, 6 months and downregulated at 12, 18, and 24 months which was inversely correlated with PLZF in the same testis. Focusing on the role of miR-19b-3p, we found that miR-19b-3p changes c-KIT and mTOR signaling through PLZF to promote proliferation in goat nGSCs and infertile mice testes. Over-expression of PLZF significantly reversed miR-19b-3p-mediated proliferation in mice testes. We found also that miR-19b-3p reduced heterochromatin-mediated senescence through PLZF localized on HP1α. Taken together, our findings indicate that miR-19b-3p promotes proliferation and reduces heterochromatin-mediated senescence through PLZF in mGSCs., (© 2017 Wiley Periodicals, Inc.)

Adipose-derived mesenchymal stem cells (ADSCs) are proven to provide good effects in numerous tissue engineering application and other cell-based therapies. However, the difficulty in the proliferation of ADSCs, known as the "Hayflick limit" in vitro, limits their clinical application. Here, we immortalized canine ADSCs (cADSCs) with SV40 gene and transplanted them into busulfan-induced seminiferous tubules of infertile mice. The proliferation of these immortalized cells was improved significantly. Then, cellular differentiation assays showed that the immortalized cADSCs could differentiate into three-germ-layer cells, osteogenesis, chondrogenesis, adipogenesis phenotypes, and primordial germ cell-like cells (PGCLCs). In addition, the immortalized cADSCs can proliferate in the busulfan-induced seminiferous tubules of infertile mice. These findings confirmed that the immortalized cADSCs maintain the criteria of cADSCs., (© 2017 Wiley Periodicals, Inc.)

The protein encoded by double sex and mab-3 related transcription factor 1 (Dmrt1) gene contains a double sex/mab-3 domain, which was considered as one of the most conservative structures in sex determination. However, its effect on spermatogenesis of dairy goat spermatogonial stem cells (SSCs) remains to be clarified. For the first time, the roles of Dmrt1 in spermatogenesis of livestock are highlighted. Here, we investigated the expression pattern of Dmrt1 in the testes of dairy goats. Dmrt1 primarily located in undifferentiated SSCs. Moreover, Dmrt1 enhanced differentiation and proliferation of mGSCs. On the contrary, the level of meiosis was down-regulated, as Dmrt1 determines whether SSCs undergo mitosis and spermatogonial differentiation or meiosis. In the busulfan-treated mice testes, Dmrt1 repair germ cell damage was emphasized as well. Our results exposed that Dmrt1 maintenance mGSCs in two ways: facilitating proliferation and self-renewal of SSCs; and reducing the inflammatory response caused by reproductive injury. These findings identify a central role for Dmrt1 in controlling population stability and injury restoring of SSCs., (© 2017 Wiley Periodicals, Inc.)

Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function for continual spermatogenesis. Studies of in vitro culture of germline stem cells are important and meaningful for basic biological research and practical application. Growth factors, such as GDNF, bFGF, CSF1, and EGF, could maintain the self-renewal of mGSCs. Insulin-like growth factor 1 (IGF-1), an important growth factor, and its pathway have been reported to maintain the survival of several types of stem cells and play important roles in male reproduction. However, the mechanism through which the IGF-1 pathway acts to regulate the self-renewal of mGSCs remains unclear. We analyzed the effect of IGF-1 on the proliferation and apoptosis of bovine mGSCs. We evaluated the expression profile of long noncoding RNA (LncRNA) H19 in bovine and mouse tissues. Moreover, we investigated whether LncRNA H19 could regulate the IGF-1 pathway. Results showed that IGF-1 could activate the phosphorylation of AKT and ERK signaling pathways, and the IGF-1 pathway played an important role in regulating the proliferation and apoptosis of bovine mGSCs. The proliferation rate of mGSCs decreased, whereas the apoptosis rate of mGSCs increased when the IGF-1 receptor (IGF-1R) was blocked using the IGF-1R-specific inhibitor (picropodophyllin). LncRNA H19 could regulate the IGF-1 signaling pathway and, consequently, the proliferation and apoptosis of mGSCs. The number of cells in the seminiferous tubule decreased when H19 was interfered by injecting a virus-containing supernatant. Hence, LncRNA H19 participated in the regulation of the proliferation and apoptosis of mGSCs via the IGF-1 signaling pathway., (© 2018 Wiley Periodicals, Inc.)

Resveratrol (RSV) has been shown to affect the differentiation of several types of stem cells, while the detailed mechanism is elusive. Here, we aim to investigate the function of RSV in self-renewal of mouse embryonic stem cells (ESCs) and the related mechanisms. In contrast with its reported roles, we found unexpectedly that differentiated ESCs or iPSCs treated by RSV would not show further differentiation, but regained a naïve pluripotency state with higher expressions of core transcriptional factors and with the ability to differentiate into all three germ layers when transplanted in vivo. In accordance with these findings, RSV also enhanced cell cycle progression of ESCs via regulating cell cycle-related proteins. Finally, enhanced activation of JAK/STAT3 signaling pathway and suppressed activation of mTOR were found essential in enhancing the self-renewal of ESCs by RSV. Our finding discovered a novel function of RSV in enhancing the self-renewal of ESCs, and suggested that the timing of treatment and concentration of RSV determined the final effect of it. Our work may contribute to understanding of RSV in the self-renewal maintenance of pluripotent stem cells, and may also provide help to the generation and maintenance of iPSCs in vitro. J. Cell. Biochem. 118: 1928-1935, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Previous studies have shown that promyelocytic leukemia zinc finger (PLZF), chemokine (C-X-C motif) receptor 4 (CXCR4) and mir146a were associated with the self-renewal of mouse spermatogonial stem cells (SSCs); however, there is little information on their effects on the fate of livestock SSCs. Here, we have identified a regulatory pathway in dairy goat mGSCs, involving PLZF, mir146a and the SDF-1 receptor CXCR4. PLZF overexpression downregulated mir146a and simultaneously upregulated the expression of CXCR4 protein, whereas PLZF knockdown (siPLZF) induced the specifically opposite effects. The in vitro assays demonstrated that PLZF specifically interacts with and suppresses the mir146a promoter, and mir146a targets CXCR4 to impede its translation. The levels of ERK1/2 phosphorylation in the mGSCs overexpressed CXCR4 and PLZF were upregulated, respectively, whereas mir146a expression was decreased and CXCR4 protein was increased. Mir146a overexpression and siPLZF impaired mGSC proliferation and differentiation, however, Mir146a knockdown induced the opposite effects. The effects of PLZF and mir146a were mediated regulation by mir146a and CXCR4, respectively. Overexpression of CXCR4 or addition of CXCL12 in cultures of dairy goat mGSCs resulted in the upregulation of their signaling, and the phosphorylation of ERK1/2 was increased. Collectively, these findings indicate that PLZF is an important transcription factor in the regulation of the expression of CXCR4 to promote dairy goat mGSC proliferation by targeting mir146a., (© 2015 Wiley Periodicals, Inc.)

The balance between the self-renewal and differentiation of male germline stem cells (mGSCs) is critical for the initiation and maintenance of mammalian spermatogenesis. The promyelocytic leukemia zinc finger (PLZF), a zinc finger protein, is a critical factor for maintaining the self-renewal of mGSCs, so, evaluation of the PLZF pathway in mGSCs may provide a deeper insight into mammalian spermatogenesis. miRNA was also an important regulating factor for the self-renewal and differentiation of mGSCs; however, there is currently no data indicating that which miRNA regulate the self-renewal and differentiation of mGSCs via PLZF. Here, we predicted the prospective miRNA targeting to PLZF using the online Bioinformatics database-Targetscan, and performed an analysis of the dual-luciferase recombinant vector, psiCHCEKTM-2-PLZF-3'UTR. miR-544 mimics (miR-544m), miR-544 inhibitors (miR-544i), Control (NC, scrambled oligonucleotides transfection), pPLZF-IRES2-EGFP or PLZF siRNA were transfected into mGSCs; the cells proliferation was evaluated by BRDU incorporation assay and flow cytometry, and the mGSC marker, GFRa1, PLZF, KIT, DAZL, and VASA expression were analyzed by RT-qPCR, immunofluorescence and Western blot. The results showed that miR-544 regulates dairy goat male germline stem cell self-renewal via targeting PLZF. Our study identifies a new regulatory pathway for PLZF and expands upon the PLZF regulatory network in mGSCs., (© 2015 Wiley Periodicals, Inc.)

miRNAs, a type of small RNA, play critical roles in mammalian spermatogenesis. Spermatogonia are the foundation of spermatogenesis and are valuable for the study of spermatogenesis. However, the expression profiling of the miRNAs in spermatogonia of dairy goats remains unclear. CD49f has been one of the surface markers used for spermatogonia enrichment by magnetic activated cell sorting (MACS). Therefore, we used a CD49f microbead antibody to purify CD49f-positive and -negative cells of dairy goat testicular cells by MACS and then analysed the miRNA expression in these cells in depth using Illumina sequencing technology. The results of miRNA expression profiling in purified CD49f-positive and -negative testicular cells showed that 933 miRNAs were upregulated in CD49f-positive cells and 916 miRNAs were upregulated in CD49f-negative cells with a twofold increase, respectively; several miRNAs and marker genes specific for spermatogonial stem cells (SSCs) in testis had a higher expression level in CD49f-positive testicular cells, including miR-221, miR-23a, miR-29b, miR-24, miR-29a, miR-199b, miR-199a, miR-27a, and miR-21 and CD90, Gfra1, and Plzf. The bioinformatics analysis of differently expressed miRNAs indicated that the target genes of these miRNAs in CD49f-positive cells were involved in cell-cycle biological processes and the cell-cycle KEGG pathway. In conclusion, our comparative miRNAome data provide useful miRNA profiling data of dairy goat spermatogonia cells and suggest that CD49f could be used to enrich dairy goat spermatogonia-like cells, including SSCs., (© 2014 Wiley Periodicals, Inc.)

Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro. Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on goat mGSCs., (© 2014 Wiley Periodicals, Inc.)

miRNAs are expressed in many mammalian cells, acting specific roles in regulating gene expression or mediating special mRNAs cleavage by targeting their 3'-untranslated region (3'UTR). Some miRNAs are essential and important for animal development. However, it is still unclear what the relationship is between miR-34c and mammalian spermatogonial stem cells (SSCs). We found that a conserved microRNA-34c through its target-Nanos2, regulating SSCs' differentiation in mouse. Immunohistochemistry analysis of Nanos2 and miR-34c FISH results revealed the opposite expression trends between them. Seven bioinformatics websites and programs predicted that miR-34c has interaction sites in Nanos2's 3'UTR. Dual-luciferase reporter vector and mutated dual-luciferase reporter vector analysis validated that they are interacted. After transfection miR-34c mimics into mouse SSCs, or miR-34c lentiviral vector in vitro co-cultivation with seminiferous tubules, and Western blot analysis demonstrated that miR-34c over-expression could suppress Nanos2 expression in post-transcription level. Our experiments identified that miR-34c may promote meiosis process by interacting with Nanos2 leading up-regulation of Stra8 in mouse spermatogonial stem cells., (© 2013 Wiley Periodicals, Inc.)

Boule is a conserved gene in meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Deletion of Boule was found to block meiosis in spermatogenesis, which contributes to infertility. Up to date, the expression and function of Boule in the goat testis are not known. The objectives of this study were to investigate the expression pattern of Boule in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Boule in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. Over-expression of Boule promoted the expression of meiosis-related genes in dairy goat mGSCs. The expression of Stra8 was up-regulated by over-expression of Boule analyzed by Western blotting and Luciferase reporter assay. While, Cdc25a, the downstream regulator of Boule, was found not to affect the expression of Stra8, and our data illustrated that Cdc25a did not regulate meiosis via Stra8. The expression of Stra8 and Boule was up-regulated by RA induction. Taken together, results suggest the Boule plays an important role in dairy goat spermatogenesis and that over-expression of Boule may promote spermatogenesis and meiosis in dairy goat., (Copyright © 2012 Wiley Periodicals, Inc.)

A small molecular chemical-Reversine has been shown to promote cell reprogramming and induce dedifferentiation of multiple terminally differentiated mesodermal origin cells, and then differentiate into other cell types within mesodermal lineages as well as neuroectodermal. However, the possibilities of these cells to give rise to germ cell lineages have not been examined. The objective of the current study was to detect the effect of Reversine on PMDSCs differentiation into germ cells. PMDSCs from fetal porcine skeletal muscle and their potential of differentiation into germ cells in vitro were investigated. The phenotype, proliferation potential, characteristic markers of the first adhesion cells (pp1), and the purified 2 times cells (pp3) were analyzed by growth curve, FACS, and RT-PCR, respectively. Then, the purified cells were induced with 10% or 20% bovine follicular fluid (FF), the results showed that some of the induced pp3 cells were similar as porcine oocyte, and expressed germ cell and oocyte markers analyzed by semi-quantitative RT-PCR and immunofluorescent staining. Reversine clearly increased the potentiality of PMDSCs differentiation into large round germ-like cells in FF induction medium analyzed by morpholgogy, QRT-PCR and immunofluoresce. The BrdU labeled PMDSCs might differentiate into female germ-like cells in recipient's kidney capsule, which were positive for germ cell and meiotic markers (Dazl, Vasa, Figla, Stra8, Scp3) and oocyte markers (Zp2, Zp3). These findings provided an efficient model to study the mechanism of cell proliferation and germ cell differentiation in livestock promoted by Reversine., (Copyright © 2012 Wiley Periodicals, Inc.)

Previous studies have demonstrated that germ cells can be derived from mouse embryonic stem cells (ESCs). However, there is still no efficient system, which can visualize the stage of germ cell specification in vitro, and further to identify and enrich germ cells derived from ESCs. Figla (factor in the germline, alpha) gene encodes a germ cell specific transcription factor that coordinates the expression of the oocyte-specific zona pellucida (Zp) genes and is essential for folliculogenesis in mouse. Here, we first constructed a pFigla-EGFP recombinant plasmid that expressed enhanced green fluorescent protein (EGFP) under the control of Figla promoter, and generated and characterized an ESC line stably carrying this pFigla-EGFP reporter construct. Then the ESCs were induced to differentiate into female germ-like cells by culturing adherent embryoid bodies (EBs) in retinoic acid (RA) induction medium or transplanting ESCs under the kidney capsule with ovarian cells. A population of differentiated ESCs expressed GFP, and these cells were analyzed by RT-PCR and immunofluorescence. The GFP positive cells showed the expression of germ cell markers Vasa, meiotic specific gene Stra8, Scp3, oocyte markers Gdf9, Zp3 and Figla, indicating that this method could be used for the purification and selection of female germ cells. Our study establishes a new selective system of female germ-like cell derivation and offers an approach for further research on the development and the differentiation of germ cells derived from stem cells., (© 2011 Wiley Periodicals, Inc.)

Studies have shown that male germline stem cells (mGSCs), which are responsible for maintaining spermatogenesis in the male, could be obtained from mouse and human testis. However, the traditional cultural methods were mostly dependent on serum and feeder, and the initial mGSCs were either obtained from neonatal mice or the detailed description of its potency and origin was not provided. Here we reported a novel (retinol (RE) serum-free and feeder-free) system for the successful culture of adult germline stem cells from adult Kunming mice (8-24 weeks) testis. The isolated mGSCs cultured in RE serum-free and feeder-free medium maintained the typical morphology of undifferentiated embryonic stem cells (ESCs), and they proliferated well in RE medium analyzed by proliferation assay, RT-PCR, microarray, and Western blotting. These cells also showed typical properties of ESCs (alkaline phosphatase (AP) positive, expressions of Oct4, Sox2, Nanog, and SSEA1, with the capacity to form teratomas and differentiate into various types of cells within three germ layers). Taken together, we conclude that RE promotes the self-renewal of mGSCs and maintains the pluripotency of mGSCs, the RE serum-free and feeder-free system may be useful for the culture of pluripotent stem cell lines from adult testis tissues, which provides a new resource for tissue engineering and therapy for infertility., (Copyright © 2011 Wiley-Liss, Inc.)

Here we report the isolation and characterization of pluripotent stem cells from adult goat skin. We found that these primary cells have the properties of embryonic stem cells (ESC), including the expression of appropriate immunological markers and the capability of forming embryoid bodies. The subcultured cells also show the characteristics of stem cells, such as the expression of CK19, beta(1-)integrin, P63, and formation of holo-clones in culture. Therefore, we termed these cells epidermal adult stem cells (EpiASC), although their origin was not identified. We have shown that clones of individual EpiASC proliferate and differentiate in culture to produce neurons, cardiomyocytes, osteoblasts, and occytes. Further, we cultivated EpiASC on bioengineered dermis and denuded human amniotic membrane (HAM), to reconstruct artificial skin and corneal epithelium. We successfully transplanted those artificial tissues in goats with acute full-thickness skin defect (AFTSD) and limbal stem cell deficiency (LSCD), respectively. Our results showed that indeed EpiASC reconstructed the skin (hair was observed in restored areas), and repaired the damaged cornea of goats with total LSCD. These data confirm that EpiASC can differentiate into different functional cell types in vivo or in vitro. Due to their high degree of inherent plasticity, and to their easy accessibility for collection from the skin, EpiASC are excellent candidate sources for diverse cell therapies., ((c) 2006 Wiley-Liss, Inc.)