Stem Cell Research & Therapy

This arcticle has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s13287-016-0438-5.

Human induced pluripotent stem cells offer the possibility of generating unlimited quantities of cells for autologous transplantation. By correcting the genetic defect underlying Z-allele α1-antitrypsin deficiency, we recently provided the first proof of principle for application of human induced pluripotent stem cells in the treatment of inherited genetic disorders. Several important safety concerns will need to be addressed before this can be translated into clinical practice.

Xenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis. SSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L−1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis. Supplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice. In conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.

The ubiquitin–proteasome system plays important roles in maintaining the self-renewal and differentiation of stem and progenitor cells through highly ordered degradation of cellular proteins. Fbxw11, an E3 ligase, participates in many important biological processes by targeting a broad range of proteins. However, its roles in hematopoietic stem/progenitor cells (HSPCs) have not been established. In this study, the effects of Fbxw11 on HSPCs were studied in vitro and in vivo by an overexpression strategy. Real-time PCR was performed to detect the expression of Fbxw11 in hematopoietic subpopulations. Colony-forming assays were performed to evaluate the in vitro function of Fbxw11 on HSPCs. Hoechst 33342 and Ki67 staining was performed to determine the cell-cycle distribution of HSPCs. Competitive transplantation experiments were used to evaluate the effect of Fbxw11 on the reconstitution potential of HSPCs. Single-cell RNA sequencing (scRNA-seq) was employed to reveal the transcriptomic alterations in HSPCs. The expression of Fbxw11 was higher in Lin−c-Kit+Sca-1+ (LSK) cells and myeloid progenitors than in lymphoid progenitors. Fbxw11 played negative roles in colony-forming and quiescence maintenance of HSPCs in vitro. Furthermore, serial competitive transplantation experiments revealed that Fbxw11 impaired the repopulation capacity of HSPCs. The proportion of granulocytes (Gr-1+CD11b+) in the differentiated mature cells was significantly higher than that in the control group, T cells and B cells were lower. Moreover, scRNA-seq revealed seven cell clusters in HSPCs. In addition, Fbxw11 downregulated the expression of Cebpa, Myc and Arid5b, which are significant regulators of HSPC activity, in most cell clusters. Our data demonstrate that Fbxw11 plays a negative role in the maintenance of HSPCs in vitro and repopulation capacity in vivo. Our data also provide valuable transcriptome references for HSPCs in homeostasis.

Endometrial mesenchymal-like stromal/stem cells (eMSCs) have been proposed as adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b&CD146) has been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity, relationship to primary counterpart, remains largely unclear. In this study, we applied 10X genomics single-cell RNA sequencing (scRNA-seq) to cultured human CD140b+CD146+ endometrial perivascular cells (ePCs) from menstrual and secretory endometrium. We also analyzed publicly available scRNA-seq data of primary endometrium and performed transcriptome comparison between cultured ePCs and primary ePCs at single-cell level. Transcriptomic expression-based clustering revealed limited heterogeneity within cultured menstrual and secretory ePCs. A main subpopulation and a small stress-induced subpopulation were identified in secretory and menstrual ePCs. Cell identity analysis demonstrated the similar cellular composition in secretory and menstrual ePCs. Marker gene expression analysis showed that the main subpopulations identified from cultured secretory and menstrual ePCs simultaneously expressed genes marking mesenchymal stem cell (MSC), perivascular cell, smooth muscle cell, and stromal fibroblast. GO enrichment analysis revealed that genes upregulated in the main subpopulation enriched in actin filament organization, cellular division, etc., while genes upregulated in the small subpopulation enriched in extracellular matrix disassembly, stress response, etc. By comparing subpopulations of cultured ePCs to the publicly available primary endometrial cells, it was found that the main subpopulation identified from cultured ePCs was culture-unique which was unlike primary ePCs or primary endometrial stromal fibroblast cells. In summary, these data for the first time provides a single-cell atlas of the cultured human CD140b+CD146+ ePCs. The identification of culture-unique relatively homogenous cell population of CD140b+CD146+ ePCs underscores the importance of in vivo microenvironment in maintaining cellular identity.

The first derivation of human embryonic stem cells brought with it a clear understanding that animal models of human disease might be replaced by an unlimited supply of human cells for research, drug discovery, and drug development. With the advent of clinical trials using human pluripotent stem cell-based therapies, it is both timely and relevant to reflect on factors that will facilitate future translation of this technology. Human pluripotent cells are increasingly being used to investigate the molecular mechanisms that underpin normal and pathological human development. Their differentiated progeny are also being used to identify novel pharmaceuticals, to screen for toxic effects of known chemicals, and to investigate cell or tissue transplantation strategies. The intrinsic assumption of these research efforts is that the information gained from these studies will be more accurate, and therefore of greater relevance, than traditional investigations based on animal models of human disease and injury. This review will therefore evaluate how animals and animal-derived products are used for human pluripotent stem cell research, and will indicate how efforts to further reduce or remove animals and animal products from this research will increase the clinical translation of human pluripotent stem cell technologies through drug discovery, toxicology screening, and cell replacement therapies.

Successful regenerative medicine strategies need the manipulation and control of macrophages’ phenotypic switching. Our previous study indicated that rat and porcine adipose tissue-derived small extracellular vesicles could successfully promote soft tissue repair. However, whether human adipose tissue-derived small extracellular vesicles (h-sEV-AT) showed the same ability to promote soft tissue regeneration and whether adipose tissue-derived small extracellular vesicles (sEV-AT) contribute to modulating the polarization of macrophages were unknown. In this study, we, for the first time, isolated h-sEV-AT from liposuction adipose tissue and characterized the morphology, size distribution, and marker protein. In vitro, we treated adipose-derived stromal/stem cells (ASCs), endothelial cells (ECs), and M1 macrophages with h-sEV-AT. In vivo, the ability of h-sEV-AT to promote soft tissue regeneration and polarize macrophages was investigated. The results indicated that h-sEV-AT possessed the characteristics of small extracellular vesicles (sEVs). In vitro, an obvious increase in adipogenesis and angiogenesis was induced by h-sEV-AT. In vivo, h-sEV-AT successfully induced the regeneration of adipose tissue and effectively accelerated full-thickness skin wound healing. Besides, we found that h-sEV-AT showed the ability to increase the percentage of M2 macrophages both in vivo and in vitro, which had been reported to contribute to tissue repair and regeneration. Taken together, these results suggested that h-sEV-AT showed the ability to induce soft tissue repair supported by not only the differentiation of ASCs and ECs but also the polarization of macrophages. Considering the abundant sources, high yield, and guaranteed effectiveness, this study provided a cell-free strategy for soft tissue regeneration that directly isolated small extracellular vesicles from human liposuction adipose tissue.