Transcription and imprinting dynamics in developing postnatal male germline stem cells

Postnatal spermatogonial stem cells (SSCs) progress through proliferative and developmental stages to populate the testicular niche prior to productive spermatogenesis. To better understand, we conducted extensive genomic profiling at multiple postnatal stages on subpopulations enriched for particular markers (THY1, KIT, OCT4, ID4, or GFRa1). Overall, our profiles suggest three broad populations of spermatogonia in juveniles: (1) epithelial-like spermatogonia (THY1⁺; high OCT4, ID4, and GFRa1), (2) more abundant mesenchymal-like spermatogonia (THY1⁺; moderate OCT4 and ID4; high mesenchymal markers), and (3) (in older juveniles) abundant spermatogonia committing to gametogenesis (high KIT⁺). Epithelial-like spermatogonia displayed the expected imprinting patterns, but, surprisingly, mesenchymal-like spermatogonia lacked imprinting specifically at paternally imprinted loci but fully restored imprinting prior to puberty. Furthermore, mesenchymal-like spermatogonia also displayed developmentally linked DNA demethylation at meiotic genes and also at certain monoallelic neural genes (e.g., protocadherins and olfactory receptors). We also reveal novel candidate receptor–ligand networks involving SSCs and the developing niche. Taken together, neonates/juveniles contain heterogeneous epithelial-like or mesenchymal-like spermatogonial populations, with the latter displaying extensive DNA methylation/chromatin dynamics. We speculate that this plasticity helps SSCs proliferate and migrate within the developing seminiferous tubule, with proper niche interaction and membrane attachment reverting mesenchymal-like spermatogonial subtype cells back to an epithelial-like state with normal imprinting profiles.     

人胚胎生殖干细胞的分离和体外培养

目的：体外培养人胚胎生殖干细胞(EG),在不添加细胞因子的培养条件下,观察细胞生长情况。方法：取5～10周人胚胎的生殖腺嵴和肠背系膜,进行组织块培养,采用组织化学及免疫细胞化学技术对培养的细胞进行鉴定。结果：培养4d后,在成纤维细胞的上面出现EG细胞集落;培养2周后,显示细胞呈圆形,胞质染成深蓝色;细胞染色体均为正常的二倍体核型;碱性磷酸酶活性强阳性;并检测到SSEA-1。结论：体外培养人胚胎生殖腺嵴,利用源于自身胚胎组织的成纤维细胞作为饲养层,可观察到EG细胞集落的形成;培养的细胞初步鉴定为人胚胎干细胞。     

Homeodomain interacting protein kinase (HPK‐1) is required in the soma for robust germline proliferation in C. elegans

Background: Tightly regulated pathways maintain the balance between proliferation and differentiation within stem cell populations. In Caenorhabditis elegans, the germline is the only tissue that is maintained by stem‐like cells into adulthood. In the current study, we investigated the role played by a member of the Homeodomain interacting protein kinase (HIPK) family of serine/threonine kinases, HPK‐1, in the development and maintenance of the C. elegans germline. Results: We report that HPK‐1 is required for promotion of germline proliferation during development and into adulthood. Additionally, we show that HPK‐1 is required in the soma for regulation of germline proliferation. We also show that HPK‐1 is a predominantly nuclear protein expressed in several somatic tissues including germline‐interacting somatic cells. Conclusions: Our observations are consistent with a conserved role for HIPKs in the control of cellular proliferation and identify a new context for such control in germ cell proliferation. Developmental Dynamics 242:1250–1261, 2013. © 2013 Wiley Periodicals, Inc.     

Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro

BACKGROUND: The purpose of this study was to establish the culture conditions required to isolate, identify and expand male germ stem cell-like cells (GSC-LC) from the testicular tissue of patients with non-obstructive azoospermia (NOA). METHODS AND RESULTS: Testicular tissues obtained from patients (two with maturation arrest (MA, n = 2) and Sertoli cell-only syndrome (SCOS, n = 11) were dissociated and plated into gelatin-coated dishes. After 2-4 weeks, cultures from both MA patients (100%) and four SCOS patients (36.3%) exhibited multicellular colonies, which proliferated successfully until passage 10. GSC-LC in the colonies displayed alkaline phosphatase activity, as well as Oct-4 and integrin b1 expression after every passage. After the fifth passage, GSC-LC were differentiated by encapsulation in calcium alginate and further cultivation. At 2 and 6 weeks, cells expressed c-Kit, Scp3, testis-specific histone protein 2B (TH2B), and transition protein (TP)-1. Fluorescence in situ hybridization additionally disclosed a few tetraploid and haploid cells at 6 weeks. Human oocytes were activated in the absence of artificial activation and cleaved after the injection of presumptive spermatids. CONCLUSIONS: Our novel culture system may be useful for diagnosing the existence of germ cells and facilitating the treatment of NOA patients.     

恒河猴骨髓间充质干细胞的体外培养及其端粒酶活性

目的：建立骨髓间充质干细胞(MSCs)的培养方法,并检测其细胞周期及端粒酶活性。方法：无菌抽取恒河猴胫骨骨髓,采用贴壁筛选法分离、培养骨髓MSCs;原位杂交方法检测其端粒酶活性;流式细胞仪检测细胞周期。结果：培养的骨髓MSCs呈梭形,平行排列或成集落;细胞端粒酶活性呈阳性反应;细胞周期中G1、S、G2期的细胞所占百分比分别为89．5％、7．8％、7．7％。结论：培养的骨髓间MSCs处于未分化阶段,具有较强的增殖能力。     

端粒酶与组织工程种子细胞衰老的研究

种子细胞是组织工程基本要素之一,目前种子细胞主要来源是自体的同源细胞,存在容易衰老,不易大量扩增的缺陷。端粒及端粒酶在细胞的衰老过程中扮演着重要角色。通过回顾有关研究,探讨端粒酶在延缓组织工程种子细胞衰老中的作用。     

Potency of germ cells and its relevance for regenerative medicine

Germline stem cells, which can self-renew and generate gametes, are unique stem cells in that they are solely dedicated to transmit genetic information from generation to generation. The germ cells have a special place in the life cycle because they must be able to retain the ability to recreate the organism, a property known as developmental totipotency. Several lines of evidence have suggested the extensive proliferation activity and pluripotency of prenatal, neonatal and adult germline stem cells. We showed that adult male germline stem cells, spermatogonial stem cells, can be converted into embryonic stem cell-like cells, which can differentiate into the somatic stem cells of three germ layers. Different cell types such as vascular, heart, liver, pancreatic and blood cells could also be obtained from these stem cells. Understanding how spermatogonial stem cells can give rise to pluripotent stem cells and how somatic stem cells differentiate into germ cells could give significant insight into the regulation of developmental totipotency as well as having important implications for male fertility and regenerative medicine.     

人胚胎干细胞向间充质干细胞分化的低血清诱导法的建立

目的建立人胚胎干细胞向间充质干细胞分化的低血清诱导方法。方法用低血清培养体系培养人胚胎干细胞,每3 d半量换液,7~10 d后消化成单细胞用扩增培养基培养,每3 d传代1次。结果人胚胎干细胞呈克隆样增殖,边缘呈锯齿状,表达多能干细胞标志OCT4、SOX2和NANOG。诱导3 d后,克隆间隙出现体积较小的成纤维样细胞,增殖能力强,经两次传代后可获得形态一致的长梭形细胞。这些细胞表达间质细胞标志VIMENTIN,细胞均表达CD105、CD90、CD73、CD44和HLA-ABC,不表达CD34、CD45和HLA-DR,且具有成脂和成骨分化能力。结论用低血清诱导法能快速获得较高纯度的人胚胎干细胞源性间充质干细胞。     

骨髓间充质干细胞对衰老大鼠肠损伤修复作用的研究

 目的 探讨骨髓间充质干细胞（MSCs）在D-半乳糖制备的衰老大鼠小肠损伤中的作用。方法SD大鼠30只,随机均分为3组：对照组、衰老模型组和MSCs防治组。给衰老模型组大鼠每日皮下注射D-半乳糖400mg/kg,连续4个月。MSCs防治组在衰老模型制备成功后,给予尾静脉输注3×106 个MSCs。分别采用硫代巴比妥酸和黄嘌呤氧化法检测小肠组织中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性;观察3组大鼠小肠组织结构的差异,并用表达绿色荧光蛋白（GFP）的慢病毒载体标记MSCs,以确定MSCs的植入情况。结果 GFP标记的MSCs移植给大鼠后,能向小肠组织迁移并存活。与衰老模型组相比,MSCs防治组SOD 的活性明显升高,MDA含量明显降低,差异有显著性意义,分别为：（133.7±3.6）,（105.1±4.3）U/ml,P <0.01;（5.9±0.1）,（6.9±0.1）(nmol/ml),P <0.01。模型组大鼠肠道粘膜损伤严重,而MSCs防治组大鼠的小肠损伤有明显修复。结论 骨髓间充质干细胞能够一定程度上减轻D-半乳糖致衰老大鼠的小肠损伤。     

Human amniotic fluid stem cells support undifferentiated propagation and pluripotency of human embryonic stem cell without b-FGF in a density dependent manner

Human embryonic stem cells (hESCs) are pluripotent cells which can give rise to almost all adult cell lineages. Culture system of hESCs is complex, requiring exogenous b-FGF and feeder cell layer. Human mesenchymal stem cells (MSCs) not only synthesize soluble cytokines or factors such as b-FGF, but also provide other mechanism which might play positive role on sustaining hESCs propagation and pluripotency. Human amniotic fluid stem (AFS) cells, which share characteristics of both embryonic and adult stem cells, have been regarded as promising cells for regenerative medicine. Taking advantage by AFS cells, we studied the ability of AFS cells in supporting undifferentiated propagation and pluripotency of Chinese population derived X-01 hESCs. Human AF-type amniotic fluid stem cells (hAF-AFSCs) transcribed genes including Activin A, TGF-β1, Noggin and b-FGF, which involved in maintaining pluripotency and self-renewal of hESCs. Compared to mouse embryonic fibroblasts (MEFs), hAF-AFSCs secreted higher concentration of b-FGF which was important in hESCs culture (P < 0.05). The hESCs were propagated more than 30 passages on hAF-AFSCs layer with exogenous b-FGF supplementation, keeping undifferentiated status. While exogenous b-FGF was obviated, propagation of hESCs with undifferentiated status was dependent on density of hAF-AFSC feeder layer. Lower density of hAF-AFSCs resulted in rapid decline in undifferentiated clone number, while higher ones hindered the growth of colonies. The most appropriate hAF-AFSCs feeder density to maintain the X-01 hESC line without exogenous b-FGF was 15-20×10⁴/well. To the best of our knowledge, this is the first study demonstrating that hAF-AFSCs could support undifferentiated propagation and pluripotency of Chinese population derived hESCs without exogenous b-FGF supplementation.     

Sept4/ARTS is required for stem cell apoptosis and tumor suppression

Inhibitor of Apoptosis Proteins (IAPs) are frequently overexpressed in tumors and have become promising targets for developing anti-cancer drugs. IAPs can be inhibited by natural antagonists, but a physiological requirement of mammalian IAP antagonists remains to be established. Here we show that deletion of the mouse Sept4 gene, which encodes the IAP antagonist ARTS, promotes tumor development. Sept4-null mice have increased numbers of hematopoietic stem and progenitor cells, elevated XIAP protein, increased resistance to cell death, and accelerated tumor development in an Eμ-Myc background. These phenotypes are partially suppressed by inactivation of XIAP. Our results suggest that apoptosis plays an important role as a frontline defense against cancer by restricting the number of normal stem cells.     

Co-culture with Schwann cells is an effective way for adipose-derived stem cells neural transdifferentiation

Introduction

Adipose-derived stem cells (ADSCs) could accomplish neural transdifferentiation with the presence of certain growth factors in vitro. It has been proved that bone marrow stromal cells (BMSCs) can realize neural transdifferentiation only by being co-cultured with Schwann cells (SCs), and in our former studies we have confirmed that ADSCs could do so too. This paper aims to investigate whether the neural induction efficiency of co-culture is as high as that of other strategies using chemicals or chemicals combined with some growth factors.

Material and methods

We isolated and multiplied ADSCs from adult Sprague-Dawley rats, and SCs from sciatic nerves of 1-to-2-day-old Sprague-Dawley rat pups, then induced ADSCs neural transdifferentiation through 2% dimethyl sulphoxide (DMSO) and DMSO combined with growth factors. Meanwhile we co-cultured ADSCs and SCs in Transwell culture dishes without intercellular contacts. Immunostaining and RT-PCR were adopted to investigate the neural transdifferentiation of ADSCs. Then we compared the expression differences for genes S100, nestin and GFAP of the above three protocols by real-time PCR.

Results

Both immunostaining and RT-PCR proved that ADSCs could accomplish neural transdifferentiation through each of the above three protocols. And real-time PCR further shows that the gene expression relative quantities for the above three genes are not statistically different between co-culture and induction through DMSO combined with growth factors (p > 0.05), but both of them are statistically different from induction only by DMSO (p < 0.05).

Conclusions

Co-culturing ADSCs and SCs may be a simple, effective and practical way for ADSCs neural transdifferentiation.     

MPTP诱导人神经干细胞衰老模型的建立

目的:建立MPTP诱导的人神经干细胞(hN SCs)衰老模型,探讨其衰老的相关生物学特点,从而为人神经干细胞衰老的研究提供细胞模型工具。方法:人胚胎干细胞系(H9)经体外诱导分化得到hN SCs,将第3代细胞接种到培养皿。在完全培养基基础上加入终浓度400μmol/L的MPTP处理24 h,处理结束后通过CCK8和Ki67染色、β-半乳糖苷酶染色、活性氧水平检测验证神经干细胞衰老模型的建立,应用Western Blot法检测相关基因SOD2,p21和p53的表达变化。结果:400μmol/L MPTP作用24 h后,hN SCs提前衰老,表现为CCK8光密度值显著下降,ki67阳性细胞比例显著下降,β-半乳糖苷酶阳性细胞百分比显著升高,细胞活性氧水平显著升高。进一步在分子水平上表现为SOD2蛋白表达水平明显下降,p21,p53蛋白表达水平显著升高。结论:本研究证实400μmol/L MPTP作用24 h可建立人神经干细胞体外衰老模型,该模型的生物学变化可能是由SOD2,p21,p53的表达水平引起的。     

类胚体中残留胚胎干细胞数量与其致瘤性相关性的初步研究

目的 探讨类胚体(EBs)中残留未分化胚胎干细胞(ESCs)的数量与其致瘤性的相关性.方法 小鼠R1胚胎干细胞株,体外类胚体诱导分化10d,流式细胞仪检测残留未分化ESCs表面标志SSEA-1阳性率.将第10天EBs消化打散后重新给予ESCs常规培养体系培养,观察EBs中残留未分化ESCs形态,流式细胞仪检测残留细胞表面标志物;第10天EBs消化打散后以104～2×106细胞量分别注射至裸鼠四肢肌肉内,观察不同细胞数量与畸胎瘤形成的相关性.结果 ESCs分化为EBs 10 d后有(13.5±0.75)%的细胞表达SSEA-1,提示存在残留未分化ESCs.残留未分化细胞生长形态呈克隆样,高表达SSEA-1等未分化ESCs标志.EBs消化打散后仅在注射2×l06个细胞的部位形成畸胎瘤,瘤体组织中存在成熟的内胚层、中胚层和外胚层组织,其余各组均未见畸胎瘤的形成.结论 胚胎干细胞分化为类胚体后仍存在残留未分化胚胎干细胞,并需要一定细胞数量才具有致瘤性.     

Immunology of stem cells and cancer stem cells

The capacity of pluri-potent stem cells to repair the tissues in which stem cells reside holds great promise in development of novel cell replacement therapeutics for treating chronic and degenerative diseases. However, numerous reports show that stem cell therapy, even in an autologous setting, triggers lymphocyte infiltration and inflammation. Therefore, an important question to be answered is how the host immune system responds to engrafted autologous stem cells or allogeneous stem cells. In this brief review, we summarize the progress in several related areas in this field, including some of our data, in four sections： （1） immunogenicity of stem cells; （2） strategies to inhibit immune rejection to allograft stem cells; （3） immune responses to cancer stem cells; and （4） mesenchymal stem cells in immune regulation. Improvement of our understanding on these and other aspects of immune system-stem cell interplay would greatly facilitate the development of stem cell-based therapeutics for regenerative purposes. Cellular ＆ Molecular Immunology.     

Medullary thymic epithelial stem cells: role in thymic epithelial cell maintenance and thymic involution

The thymus consists of two distinct anatomical regions, the cortex and the medulla; medullary thymic epithelial cells (mTECs) play a crucial role in establishing central T-cell tolerance for self-antigens. Although the understanding of mTEC development in thymic organogenesis as well as the regulation of their differentiation and maturation has improved, the mechanisms of postnatal maintenance remain poorly understood. This issue has a central importance in immune homeostasis and physiological thymic involution as well as autoimmune disorders in various clinicopathological settings. Recently, several reports have demonstrated the existence of TEC stem or progenitor cells in the postnatal thymus, which are either bipotent or unipotent. We identified stem cells specified for mTEC-lineage that are generated in the thymic ontogeny and may sustain mTEC regeneration and lifelong central T-cell self-tolerance. This finding suggested that the thymic medulla is maintained autonomously by its own stem cells. Although several issues, including the relationship with other putative TEC stem/progenitors, remain unclear, further examination of mTEC stem cells (mTECSCs) and their regulatory mechanisms may contribute to the understanding of postnatal immune homeostasis. Possible relationships between decline of mTECSC activity and early thymic involution as well as various autoimmune disorders are discussed.