Differentiation of mesenchymal stem cells to germ‐like cells under induction of Sertoli cell‐conditioned medium and retinoic acid

The aim of this research was to find a way to differentiate germ cells from umbilical cord Wharton's jelly mesenchymal stem cells (MSCs) to support in vitro spermatogenesis. A small piece of Wharton's jelly was cultured in high‐glucose Dulbecco's modified Eagle's medium in present of 10% foetal calf serum. After the fourth passage, the cells were isolated and cultured in Sertoli cell‐conditioned medium under induction of two different doses of retinoic acid (10^?5, 10^?6 m ). The differentiation of MSC to germ‐like cells was evaluated by expression of Oct4, Nanog, Plzf, Stra8 and Prm1 genes during different days of culture through qPCR. The results showed that there were downregulation of Oct4 and Nanog and upregulation of pre‐meiotic germ cell marker (stra8) and haploid cell marker (Prm1) when MSCs are differentiated over time. The expression of Bax gene (an apoptotic marker) was significantly observed in high dosage of retinoic acid (RA). As a result, RA has positive effects on proliferation and differentiation of MSCs, but its effects are related to dosage. The success of this method can introduce umbilical cord MSC as a source of germ cells for treatment of infertility in future.     

诱导小鼠骨髓间充质干细胞向雄性生殖细胞的定向分化

目的探讨小鼠骨髓间充质干细胞是否能够在体外被诱导发生向雄性生殖细胞方向的分化。方法从雄性小鼠骨髓中分离能够长期贴壁生长的细胞,并鉴定其是否为间充质干细胞。对分离的细胞进行生殖细胞特异性报告基因标记(stra-8-GFP)。采用视黄酸诱导标记的细胞发生向生殖细胞方向的分化。通过观察报告基因表达和生殖细胞相关基因mRNA表达情况确定是否发生了分化。结果从小鼠骨髓中分离到的贴壁生长的细胞表达间充质干细胞的表面标志CD90、CD44、CD105和Sca-1;细胞在体外可以被诱导分化为成骨、成软骨及成脂肪细胞。报告基因标记的间充质干细胞在被视黄酸诱导2d后开始表达绿色荧光蛋白和生殖细胞相关基因Mvh、Fragilis和Stella的mRNA。未经视黄酸诱导的细胞不表达绿色荧光蛋白和生殖细胞相关基因。结论小鼠骨髓间充质干细胞在体外可以被视黄酸诱导发生向雄性生殖细胞方向的分化。     

全反式视黄酸影响小鼠胚胎干细胞向原始生殖细胞分化的相关基因表达

目的 探讨全反式视黄酸(all-trans retinoic acid,atRA)诱导小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)向原始生殖细胞(Primordial germ cellS,PGCs)分化的相关基因表达改变及其机制.方法 mESCs分化形成拟胚体(embryoid bodies,Ebs),不同浓度atRA(1 μM,2 μM,5 μM)持续诱导Ebs 16h、2d和5d,观察Ebs形态变化,实时PCR和Western blot检测PGCs分化相关基因和蛋白表达变化.分析基因肩动子中RA反应元件,以Stra8(Stimulated by Retinoic Acid Gene 8,Stra8)基因为阳性对照,确定各基因对atRA刺激的原发性反应和继发性反应.结果 不同浓度atRA诱导Ebs 16h和2d后形态无明显变化.诱导后Ebs的PGCs分化相天基因mRNA表达分4种情况:16h内(Stra8)表达量达最大,然后下降;2d内(Scp3)表达量达最大,然后下降;5d达到最大量(Mvh);表达量无明显变化(Fragilis,Blimp-1和Prm1).Mvh表达符合继发反应特点;Stra8和Scp3表达符合原发反应特点;Blimp1,Prm1和Fragilis表达可能与atRA调节无关.Stra8蛋白变化与mRNA变化相一致,而Mvh则不一致.结论 atRA诱导mESCs向PGCs分化,各阶段标志基因表达变化均在相对短的时间内完成(2～5d),atRA诱导浓度可选用1 μM.根据基因表达模式初步得出,atRA调控的靶基因为Mvh,具备继发反应特点,与特异性分化有关,而Blimp-1、Fragilis和Prm1调控PGCs分化可能与atRA无关.     

Retinoic acid and 17β-estradiol improve male germ cell differentiation from mouse-induced pluripotent stem cells

This research aimed to explore the impacts of retinoic acid (RA)/17β-estradiol (E) induction and embryoid body formation to enhance differentiation of mouse-induced pluripotent stem cells (miPSCs) into male germ cells in vitro. Flow cytometry and qPCR were conducted to describe miPSCs differentiation process. Various temporal expression profiles of germ cell-related genes were traced. Stra8 gene expression increased in the RA group on the 4th day compared to other groups. The RA group experienced a more significant increase than E group. The expression of Sycp3 increased in RA + E group on 4th day compared with other groups. Expression of AKAP3 enhanced in the RA + E group than other groups on day 4. Moreover, miPSCs showed that this gene expression in the RA + E group was increased in comparison to RA and E groups on day 7. AKAP3 gene expression on day 7 of miPSCs decreased in RA and E groups. Flow cytometry data indicated that 3%–8% of the cells in sub-G1 stage were haploid after RA and E induction compared to other groups on day 4. This study showed that miPSCs possess the power for differentiating into male germ cells in vitro via formation of embryoid body by RA with/or E induction.     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

Insights into bone marrow‐derived mesenchymal stem cells safety for cutaneous repair and regeneration

Wound healing involves the orchestration of a complex process of interactions between numerous types of cell, components of extracellular matrix and signalling molecules following injury, which is usually a highly successful biological course to reconstruct the integrity of the skin. Nevertheless, when skin is severely damaged, the injured skin is limited in its ability to repair itself and possibly results in the hypertrophic scars or so‐called keloids, and non healing wound or ulcer. Bone marrow‐derived mesenchymal stem cells (BM‐MSCs) are being clinically explored as a promising therapy in the field of tissue repair and regeneration. However, potential risks associated with these cell‐based therapies remain uncertain. The aim of this review is to summarise the safety issues accompanying the administration of BM‐MSCs for acute or chronic skin repair and regeneration. More importantly, this review highlights the requirement for fundamental research to improve future clinical application of these strategies, as well as for regulatory authorities to establish clinical criteria to identify the qualitative requirements for the manufacture process of cells products, which will ensure the manufacture process of the best benefit‐to‐risk ratio of cell‐based therapy for the patients.     

Isolation and characterization of turkey bone marrow‐derived mesenchymal stem cells

Flexor tendon injury is often associated with suboptimal outcomes and results in substantial digit dysfunction. Stem cells have been isolated from several experimental animals for the growing interest and needs of utilizing cell‐based therapies. Recently, turkey has been developed as a new large animal model for flexor tendon research. In the present study, we reported the isolation and characterization of bone marrow‐derived mesenchymal stem cells (BMSCs) from 8‐ to 12‐month‐old heritage‐breed turkeys. The isolated cells demonstrated fibroblast‐like morphology, clonogenic capacity, and high proliferation rate. These cells were positive for surface antigens CD90, CD105, and CD44, but were negative for CD45. The multipotency of turkey BMSCs was determined by differentiating cells into osteogenic, adipogenic, chondrogenic, and tenogenic lineages. There was upregulated gene expression of tenogenic markers, including mohawk, tenomodulin, and EGR1 as well as increased collagen synthesis in BMP12 induced cells. The successful isolation and verification of bone marrow‐derived MSCs from turkey would provide opportunities of studying cell‐based therapies and developing new treatments for tendon injuries using this novel preclinical large animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1419–1428, 2019.     

诱导多能干细胞体外向生精细胞自发分化潜能的研究

目的:探讨诱导多能干细胞(iPS)体外培养自发分化过程中生精细胞相关基因的表达,评估iPS体外向生精细胞自发分化的潜能。方法:经类胚体(EB)形成,体外诱导iPS向生精细胞分化,实时定量PCR和PCR检测生精细胞相关基因的表达。结果:实时定量PCR和PCR结果显示iPS经EB形成诱导分化后生精细胞不同时期的相关基因均有不同程度表达。iPS体外培养自发分化后生精细胞相关基因出现4种时间表达特征:Oct4基因表达量呈波浪状上升;Dppa3和Stra8基因表达量随诱导时间延长而下降;Dazl基因表达量呈波浪状下降;减数分裂前期基因Tex14、Msy2,减数分裂期基因Scp1、Scp3以及单倍体基因Akap3随着诱导时间延长先表达增加,而后表达下降。结论:iPS在经EB自发分化过程中表达生精细胞不同时期的相关基因,并且表达雄性配子单倍体基因,具有向雄性配子的分化潜能。     

Evaluation of adipose‐derived stromal vascular fraction or bone marrow‐derived mesenchymal stem cells for treatment of osteoarthritis

The purpose of this study was the assessment of clinical, biochemical, and histologic effects of intraarticular administered adipose‐derived stromal vascular fraction or bone marrow‐derived mesenchymal stem cells for treatment of osteoarthritis. Osteoarthritis was induced arthroscopically in the middle carpal joint of all horses, the contralateral joint being sham‐operated. All horses received treatment on Day 14. Eight horses received placebo treatment and eight horses received adipose‐derived stromal vascular fraction in their osteoarthritis‐affected joint. The final eight horses were treated the in osteoarthritis‐affected joint with bone marrow‐derived mesenchymal stem cells. Evaluations included clinical, radiographic, synovial fluid analysis, gross, histologic, histochemical, and biochemical evaluations. No adverse treatment‐related events were observed. The model induced a significant change in all but two parameters, no significant treatment effects were demonstrated, with the exception of improvement in synovial fluid effusion PGE2 levels with bone marrow‐derived mesenchymal stem cells when compared to placebo. A greater improvement was seen with bone marrow‐derived mesenchymal stem cells when compared to adipose‐derived stromal vascular fraction and placebo treatment. Overall, the findings of this study were not significant enough to recommend the use of stem cells for the treatment of osteoarthritis represented in this model. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1675–1680, 2009     

Comparison of the osteogenic capacity of minipig and human bone marrow‐derived mesenchymal stem cells

Minipigs are a recommended large animal model for preclinical testing of human orthopedic implants. Mesenchymal stem cells (MSCs) are the key repair cells in bone healing and implant osseointegration, but the osteogenic capacity of minipig MSCs is incompletely known. The aim of this study was to isolate and characterize minipig bone marrow (BM) and peripheral blood (PB) MSCs in comparison to human BM‐MSCs. BM sample was aspirated from posterior iliac crest of five male Göttingen minipigs (age 15 ± 1 months). PB sample was drawn for isolation of circulating MSCs. MSCs were selected by plastic‐adherence as originally described by Friedenstein. Cell morphology, colony formation, proliferation, surface marker expression, and differentiation were examined. Human BM‐MSCs were isolated and cultured from adult fracture patients (n = 13, age 19–60 years) using identical techniques. MSCs were found in all minipig BM samples, but no circulating MSCs could be detected. Minipig BM‐MSCs had similar morphology, proliferation, and colony formation capacities as human BM‐MSCs. Unexpectedly, minipig BM‐MSCs had a significantly lower ability than human BM‐MSCs to form differentiated and functional osteoblasts. This observation emphasizes the need for species‐specific optimization of MSC culture protocol before direct systematic comparison of MSCs between human and various preclinical large animal models can be made. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1019–1025, 2012     

The fate of transplanted xenogeneic bone marrow‐derived stem cells in rat intervertebral discs

Intervertebral disc degeneration is a major cause and a risk factor for chronic low back pain. The potential of using stem cells to treat disc degeneration has been raised. The aims of our study were to assess whether xenogeneic bone‐marrow derived stem cells could survive in a rat disc degeneration model and to determine which cell types, if any, survived and differentiated into disc‐like cells. Human bone‐marrow derived CD34⁺ (hematopoietic progenitor cells) and CD34^? (nonhematopoietic progenitor cells, including mesenchymal stem cells) cells were isolated, fluorescent‐labeled, and injected into rat coccygeal discs. The rats were sacrificed at day 1, 10, 21, and 42. Treated discs were examined by histological and immunostaining techniques and compared to control discs. The survival of transplanted cells was further confirmed with a human nuclear specific marker. Fluorescent labeled CD34^? cells were detected until day 42 in the nucleus pulposus of the injected discs. After 3 weeks these cells had differentiated into cells expressing chondrocytic phenotype (Collagen II and Sox‐9). In contrast, the fluorescent labeled CD34⁺ cells could not be detected after day 21. No fluorescence‐positive cells were detected in the noninjected control discs. Further, no inflammatory cells infiltrated the nucleus pulposus, even though these animals had not received immunosuppressive treatment. Our data provide evidence that transplanted human BM CD34^? cells survived and differentiated within the relative immune privileged nucleus pulposus of intervertebral disc degeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:374–379, 2009     

干细胞向雄性配子诱导分化研究进展

人类雄性配子的重要功能之一是完整地将父代遗传信息传递给子代。配子发生,尤其雄性配子发生是个极其复杂的细胞分化过程,它包括有丝分裂与减数分裂两大过程。然而由于缺乏可重复、高效率研究雄性配子发生的体外培养体系,生殖细胞发生、发育机制研究进展缓慢。干细胞经体外诱导向雄性生殖细胞分化的研究,将推进生殖细胞发育研究,甚至生殖生物学的发展。本文综述近年原始生殖细胞体外培养及干细胞向雄性配子诱导分化的研究进展。     

In vivo bioluminescence imaging of magnetically targeted bone marrow‐derived mesenchymal stem cells in skeletal muscle injury model

The purpose of this study is to clarify the kinetics of transplanted mesenchymal stem cells (MSCs) in rat skeletal muscle injury model and the contribution of the magnetic cell delivery system to muscle injury repair. A magnetic field generator was used to apply an external magnetic force to the injury site of the tibia anterior muscle, and 1 × 10⁶ MSCs labeled with ferucarbotran–protamine complexes, which were isolated from luciferase transgenic rats, were injected into the injury site. MSCs were injected with and without an external magnetic force (MSC M+ and MSC M? groups, respectively), and phosphate‐buffered saline was injected into injury sites as a control. In vivo bioluminescence imaging was performed immediately after the transplantation and, at 12, 24, and 72 h, and 1 and 4 weeks post‐transplantation. Also, muscle regeneration and function were histologically and electromechanically evaluated. In vivo bioluminescence imaging showed that the photon of the MSC M+ group was significantly higher than that of the MSC M? group throughout the observation period. In addition, muscle regeneration and function in the MSC M+ group was histologically and functionally better than that of the MSC M? group. The results of our study indicated that magnetic cell delivery system may be of use in directing the transplanted MSCs to the injury site to promote skeletal muscle regeneration. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 754–759, 2013     

Cross‐linking affects cellular condensation and chondrogenesis in type II collagen‐GAG scaffolds seeded with bone marrow‐derived mesenchymal stem cells

The formation of cartilaginous tissue by chondroprogenitor cells, whether in vivo or in vitro, appears to require a critical initial stage of “condensation” in which intercellular space is reduced through an aggregation of cells, leading to development of cell‐to‐cell junctions followed by chondrocytic differentiation. The objective of this study was to investigate the association of aggregation (condensation) of mesenchymal stem cell (MSCs) and chondrogenesis in vitro. Previous work with chondrocytes indicated that the cross‐link density and related cell‐mediated contraction of collagen scaffolds significantly affects cartilaginous tissue formation within the cell‐seeded construct. Based on this finding, we hypothesized that the cell‐aggregating effect of the contraction of MSC‐seeded collagen scaffolds of lower cross‐link density favors chondrogenesis; scaffolds of higher cross‐link density, which resist cell‐mediated contraction, would demonstrate a lower cell number density (i.e., subcritical packing density) and less cartilage formation. Type II collagen–GAG scaffolds, chemically cross‐linked to achieve a range of cross‐link densities, were seeded with caprine MSCs and cultured for 4 weeks. Constructs with low cross‐link densities experienced cell‐mediated contraction, increased cell number densities, and a greater degree of chondrogenesis (indicated by the chondrocytic morphology of cells, and synthesis of GAG and type II collagen) compared to more highly cross‐linked scaffolds that resisted cellular contraction. These results provide a foundation for further investigation of the mechanisms by which condensation of mesenchymal cells induces chondrogenesis in this in vitro model, and may inform cross‐linking protocols for collagen scaffolds for use in cartilage tissue engineering. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1184–1192, 2010     

脐带间充质干细胞增强无精子症模型睾丸特异性基因表达

在生殖男科领域，探索人脐带间充质干细胞（HUCMSCs）对无精子症的治疗作用。HUCMSCs具有潜在的免疫抑制功能，并能够分泌多种细胞因子和生长因子，因此具有潜在的临床应用价值。作为探索，我们移植HUCMSCs进入无精子症小鼠睾丸间质，检测是否能够促进精子发生过程。从不同来源的脐带中分离HUCMSCs，移植进入白消安处理的小鼠睾丸间质中，采用注射生理盐水和HEK293细胞作为对照，对侧睾丸不注射。三周之后，RT-PCR检测10个生殖细胞特异性表达的基因，并检N3个特异性蛋白的表达。结果表明，注射人脐带间充质干细胞之后的表达明显高于对照组，证实生殖细胞特异性基因的表达上调，从而表明HUCMSCs对睾丸生精功能的恢复具有促进作用，为治疗无精子症探索一条新的途径。     

Mechanical activation of β‐catenin regulates phenotype in adult murine marrow‐derived mesenchymal stem cells

Regulation of skeletal remodeling appears to influence the differentiation of multipotent mesenchymal stem cells (MSC) resident in the bone marrow. As murine marrow cultures are contaminated with hematopoietic cells, they are problematic for studying direct effects of mechanical input. Here we use a modified technique to isolate marrow‐derived MSC (mdMSC) from adult mice, yielding a population able to differentiate into adipogenic and osteogenic phenotypes that is devoid of hematopoietic cells. In pure mdMSC populations, a daily strain regimen inhibited adipogenic differentiation, suppressing expression of PPARγ and adiponectin. Strain increased β‐catenin and inhibition of adipogenesis required this effect. Under osteogenic conditions, strain activated β‐catenin signaling and increased expression of WISP1 and COX2. mdMSC were also generated from mice lacking caveolin‐1, a protein known to sequester β‐catenin: caveolin‐1^(?/?) mdMSC exhibited retarded differentiation along both adipogenic and osteogenic lineages but retained mechanical responses that involved β‐catenin activation. Interestingly, caveolin‐1^(?/?) mdMSC failed to express bone sialoprotein and did not form mineralized nodules. In summary, mdMSC from adult mice respond to both soluble factors and mechanical input, with mechanical activation of β‐catenin influencing phenotype. As such, these cells offer a useful model for studies of direct mechanical regulation of MSC differentiation and function. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1531–1538, 2010