Expression of pluripotent stem cell markers in the human fetal ovary

BACKGROUND: Human primordial germ cells (PGCs) can give rise to pluripotentstem cells such as embryonal carcinoma cells (ECCs) and embryonicgerm cells (EGCs). METHODS: In order to determine whether PGCs express markers associatedwith pluripotency in EGCs and ECCs, the following study crossexamines the expression patterns of multiple pluripotent markersin the human fetal ovary, 5.5–15 weeks post-fertilizaton(pF) and relates this expression with the ability to derivepluripotent EGCs in vitro. RESULTS: Specific subpopulations were identified which included OCT4⁺/Nanog⁺/cKIT⁺/VASA⁺PGCs and oogonia. Interestingly, these cells also expressedSSEA1 and alkaline phosphatase (AP) and SSEA4 expression occurredthroughout the entire gonad. Isolation of SSEA1⁺ cells fromthe gonad resulted in AP⁺ EGC colony formation. The number ofOCT4⁺ or Nanog⁺ expressing cells peaked by week 8 and then diminishedafter week 9 pF, as oogonia enter meiosis. In addition, theefficiency of EGC derivation was associated with the numberof OCT4⁺ cells. TRA-1-60 and TRA-1-81 were only detected inthe lining of the mesonephric ducts and occasionally in thegonad. CONCLUSIONS: These results demonstrate that PGCs, a unipotent cell, expressmost, but not all, of the markers associated with pluripotentcells in the human fetal ovary.     

人诱导性多潜能干细胞系的建立

背景：诱导性多潜能干细胞与肿瘤干细胞的发生过程极其相似,而且具有的干细胞特性极其接近人胚胎干细胞。因此,研究诱导性多潜能干细胞有利于人们进一步认识并了解人类发育以及肿瘤的发生过程。 目的：掌握建立人诱导性多潜能干细胞系的技术,以便为特异性疾病细胞的重编程建立技术平台,从而利用重编程技术研究疾病的发病机制。 方法：将含有Oct4、Sox2、Klf-4和c-Myc 4个转录因子的反转录病毒感染人皮肤成纤维细胞(HS27细胞),在人胚胎干细胞培养条件下诱导产生人胚胎干细胞样的克隆。挑取并进一步扩增,通过克隆形态、碱性磷酸酶活性、免疫荧光检测是否有人胚胎干细胞标记物Oct4、Sox2、c-Myc、Klf-4的表达,悬滴法检测HS27细胞来源的克隆形成畸胎瘤的能力和验证向3个胚层的分化能力。 结果与结论：经病毒感染诱导产生的胚胎干细胞样克隆呈绿色荧光蛋白阴性,克隆在细胞形态方面与人胚胎干细胞克隆相似,进一步扩增经碱性磷酸酶检测克隆呈阳性,免疫荧光检测克隆表达Oct4、Sox2、c-Myc、Klf-4,并且HS27细胞来源的克隆注入免疫缺陷小鼠体内可以形成畸胎瘤并经苏木精-伊红染色显示具有向三胚层分化能力。实验成功构建了人诱导性多潜能干细胞系,为下一步开展疾病细胞特异性重编程研究奠定了良好的实验基础。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Novel germ cell markers characterize testicular seminoma and fetal testis

Seminomas are characterized by expression of several stem cell markers, supporting their origin from germ cells. The current study focuses on novel germ cell markers in normal testes compared to those in fetal testes and different progression stages of seminomas. Microarray data were followed by RT-PCRs and immunohistochemistry on pure seminomas (pT1 to pT3) compared to adult and fetal testis. An upregulation of known germ cell markers, KIT, OCT4 and NANOG, was confirmed in seminoma specimens. We also identified novel germ cell markers such as BOB1 (POU2AF1, OBF1) and prominin 1 (PROM1, CD133), which were significantly upregulated in seminoma specimens, compared to normal testes. Furthermore, two Sertoli cell markers, SCGF (SCF) and the newly identified neuronal stem cell factor, MCFD2 (SDNSF), were expressed in seminoma cells. While BOB1 was expressed in fetal testis of second and third trimester of gestation, MCFD2 and PROM1 were only present in gonocytes up to the second trimester. All marker genes investigated were not further regulated in progressing tumour stages between pT1 and pT3. In conclusion, the germ cell markers described here provide evidence for the origin of seminoma cells, which could be from the developmental stage of early gonocytes or from spermatogonia re-expressing markers of the developing germ cells.     

Environmental toxicant-induced germ cell apoptosis in the human fetal testis

BACKGROUND: Disorders of the male reproductive system are increasing in prevalence. The term testicular dysgenesis syndrome emphasizes the importance of developmental influences on the aetiology of conditions including cryptorchidism, testicular germ cell cancer and reduced spermatogenesis. Men whose mothers smoked during pregnancy have lower sperm production. Cigarette smoke contains agents acting on the aryl hydrocarbon receptor (AHR). We have investigated the presence of AHR in the developing human testis and the effects of functional activation. METHODS AND RESULTS: Immunohistochemistry determined AHR to be expressed by germ cells in the human testis between 7 and 19 week gestation, but not by other cells. Treatment of cultured fetal testis with an AHR ligand present in tobacco smoke increased markers of cell apoptosis, and this was prevented by an AHR receptor antagonist. Immunohistochemistry indicated that apoptosis was restricted to germ cells. CONCLUSIONS: Germ cells in the developing human testis are a target for regulation by AHR ligands. Activation of AHR by environmental toxicants and AHR-induced apoptotic pathways may be the mechanism of action underlying the epidemiological findings of reduced spermatogenesis in men exposed to cigarette smoke before birth, and may also be of importance in other conditions comprising the testicular dysgenesis syndrome.     

Substrates for human pluripotent stem cell cultures in conditioned medium of mesenchymal stem cells

We aimed to establish a culture system of human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), free from xenogeneic proteins, Matrigel(?) and conditioned medium of mouse embryonic fibroblasts. The conditioned culture medium consisted of mesenchymal stem cells derived from human bone marrow. We examined surface properties suitable for hPSC culture by using self-assembled monolayers (SAMs) of alkanethiols with four different functional groups: CH(3), OH, COOH and NH(2). hPSCs neither adhered nor proliferated on surfaces with a water contact angle higher than 40°. Based on this finding, the contact angle of a polystyrene (PSt) culture dish was reduced to less than 40°, and COOH and OH groups were introduced to its surface by oxygen plasma treatment, making the PSt dish suitable for hPSC culture. This combination of a PSt dish treated with oxygen plasma treatment and conditioned medium of mesenchymal stem cells achieved a long-term maintenance of hPSCs without differentiation.     

Carbon nanotube-based substrates for modulation of human pluripotent stem cell fate

We investigated the biological response of human pluripotent stem cells (hPSCs) cultured on a carbon nanotube (CNT) array-based substrate with the long term goal to direct hPSC germ layer specification for a wide variety of tissue engineering applications. CNT arrays were fabricated using a chemical vapor deposition system allowing for control over surface roughness and mechanical stiffness. Our results demonstrated that hPSCs readily attach to hydrophilized and extracellular matrix coated CNT arrays. hPSCs cultured as colonies in conditions supporting self-renewal demonstrated the morphology and marker expression of undifferentiated hPSCs. Conditions inducing spontaneous differentiation lead to hPSC commitment to all three embryonic germ layers as assessed by immunostaining and RT-PCR analysis. Strikingly, the physical characteristics of CNT arrays favored mesodermal specification of hPSCs. This is contradictory to the behavior of hPSCs on traditional tissue culture plastic which promotes the development of ectoderm. Altogether, these results demonstrate the potential of CNT arrays to be used in the generation of new platforms that allow for precise control of hPSC differentiation by tuning the characteristics of their physical microenvironment.     

Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation

Recent studies from our laboratory have shown that acellular substrates generated from human fibroblasts successfully maintained human pluripotent stem cells (hPSCs) in their undifferentiated state for extended periods. Aiming at better characterization, we conducted proteomic analyses to identify the extracellular matrix (ECM) proteins in mouse embryonic- and two human fibroblast-derived acellular substrates. Our studies identified heparan sulfate proteoglycan (HSPG) as a core component of these substrates and immunocytochemical analyses confirmed the presence of HSPG as well as other ECM proteins identified through proteomic analyses. In our attempt to develop surfaces that mimic fibroblast-deposited ECM and their self-renewal capabilities, substrates comprising HSPG and other core ECM proteins were formulated and assessed for the function of hPSC self-renewal. WA09 and BG01v hPSCs maintained on these substrates exhibit multiple characteristics of pluripotency, including (i) tight colony formation with typical stem cell morphology; (ii) positive expression of alkaline phosphatase, (iii) positive expression of SSEA3, SSEA4 and Oct4 based on immunocytochemical analyses; (iv) POU5F1, NANOG and SOX2 mRNA expression; and (v) in vitro differentiation and expression of germ-layer-specific markers. Our studies also reveal that although HSPG by itself-does not support hPSC self-renewal, a substrate that combines HSPG and fibronectin is sufficient for undifferentiated propagation of hPSCs. These studies form the basis for identification of appropriate ECM components in a substrate that synergistically promotes activation of adhesion and signaling pathways responsible for hPSC self-renewal.     

人多能干细胞的冷冻与保存

背景：人多能干细胞的出现与发展是近年来生物医学研究领域的重大突破。但其在基础/临床研究中的广泛应用还有诸多限制,建立安全有效标准化的冷冻保存方案是人多能干细胞广泛应用面临的重大挑战。 目的：回顾人多能干细胞冷冻领域的研究进展,探索造成冷冻损伤的原因和机制及改进方式,致力于促进新的更有效的冷冻方案形成。 方法：以“人多能干细胞、人胚胎干细胞、人诱导多能干细胞、玻璃化、程序化冷冻、慢冻法、冷冻保存”为中文检索词,以“human pluripotent stem cells,human embryonic stem cell,human introduced pluripotent stem cell,vitrification,programmed cryopreservation,slow-freezing,cryopreservation”为英文检索词,应用计算机检索中国知网全文数据库、万方全文数据库、维普(VIP)期刊全文数据库、PubMed数据库有关人多能干细胞冷冻保存技术的文献,排除与研究目的无关及重复文献,保留58篇文献进一步总结分析。 结果与结论：了解人多能干细胞冷冻过程中造成冷冻损伤的原因和机制,是寻找高效的冻存方案的关键。需要更清晰的了解冷冻过程中损伤的原理,改进和创新低温生物技术来避免各种冷冻损伤的发生并致力于探讨可重复的,高效的,符合GMP要求的,能大规模冻人多能干细胞的方案。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

诱导多能性干细胞技术的进展

将人类体细胞诱导成为多能性干细胞(induced pluripotent stem cells,iPS cells)的研究成果为生物医学研究提供了广泛的前景,建立了一种全新的体细胞核重编程的方法,这种方法相对容易操作,而且比较稳定、安全,点燃了再生医学应用的新希望,本文从体细胞重编程的研究历程、iPS细胞技术的诱导因子、转录因子、载体及其应用价值方面进行了综述.     

Differentiation markers of Sertoli cells and germ cells in fetal and early postnatal human testis

The definition of the temporal sequence of appearance of fetal markers during prenatal and early postnatal development in Sertoli and germ cells may be important for understanding the mechanisms underlying their reexpression in disorders of the adult testis. For this reason, we studied the expression of Sertoli and germ cell markers in 25 human testes spanning a period from 8 gestational weeks to 4 years. Well-characterized antibodies were employed to anti-Müllerian hormone (AMH), cytokeratin 18 (CK18), vimentin (VIM), M2A-antigen (M2A), germ cell alkaline phosphatase (GCAP), and somatic angiotensin-converting enzyme (sACE) on formalin-fixed and microwave-pretreated paraffin sections. In Sertoli cells, AMH and VIM were consistently present. While VIM and CK18 were coexpressed in embryonic testes, CK18 was progressively downregulated and completely absent from the 20th gestational week. M2A was absent or moderately expressed in fetal Sertoli cells but increased during further development. In germ cells, M2A was consistently found in primordial germ cells (PGCs) as well as in M- and T₁-prespermatogonia. In contrast, sACE and GCAP were absent from PGCs but were a distinct feature of late M- and early T₁-prespermatogonia and appeared predominantly between the 18th and the 22nd gestational weeks. Both T₂-prespermatogonia and postnatal prespermatogonia were devoid of any marker. While CK18 represents a differentiation marker for fetal Sertoli cells, M2A, GCAP, and sACE can be used as differentiation markers for the discrimination of different germ cell types during human prespermatogenesis. Because various immunophenotypes reflect distinct differentiation stages, this knowledge may be important for understanding adult testicular pathology.     

Induced pluripotent stem cell consensus genes: implication for the risk of tumorigenesis and cancers in induced pluripotent stem cell therapy

Induced pluripotent stem cells (iPSCs) have recently boomed enthusiasm in stem cell therapy, whereas high potential tumorigenesis of iPSCs has become the biggest obstacle for clinic application and the tumorigenic genes in iPSCs have not been well documented. In this investigation, using tools of bioinformatics, we analyzed the all available datasets regarded to iPSCs from 11 differentiated cell lines and revealed 593 iPSC consensus genes. Notably, of the 593 genes, 209 were expressed in human tumor cell lines and cancer tissues, and some of them were expressed in the iPSC-differentiated hepatocytes; remarkably, 5 oncogenes were overexpressed in the iPSCs and an oncogene RAB25 in the iPSC-differentiated cells, suggesting that these iPSC consensus genes are implicated with the risk of tumorigenesis and cancers. This investigation provides useful information for designing new strategies and methods to curtail the expression of oncogenic genes in iPSCs and produce safe iPSC derivatives for stem cell therapy.     

胎儿真皮间充质干细胞中的全能性相关转录因子的表达

背景：人真皮纤维母细胞或间充质干细胞可形成诱导性多能干细胞,但不同研究者所用的转录因子组合却并不相同。 目的：分离人胎儿真皮间充质干细胞,检测其全能性相关转录因子的表达。 方法：水囊引产5月龄胎儿,按照既往分离培养间充质干细胞的方法,得到胎儿真皮间充质干细胞。 结果与结论：胎儿真皮间充质干细胞高表达Oct4和C-myc、中度表达Sox2,是形成诱导性多能干细胞较好的体细胞。     

干细胞分化抗原和细胞增殖抗原在人胶质瘤细胞株中的表达

研究表明肿瘤组织中存在肿瘤干细胞(tumor stem cells,TSC).TSC具有自我更新、无限增殖和多向分化等干细胞的生物学特性,并且表达多种干细胞分化抗原[1].这些干细胞抗原在胶质瘤组织中也大量表达[2].通过建立在干细胞分化抗原基础上的肿瘤细胞亚群分离技术,已经在胶质瘤组织和胶质瘤细胞株中分离和鉴定出TSC[3-4],但各种干细胞分化抗原在胶质瘤细胞株中的表达状况尚不清楚.我们在本研究中检测人胶质母细胞瘤细胞株中干细胞分化抗原和细胞增殖抗原的表达,为进一步研究TSC打下基础.     

Alfa-inhibin and transferrin in human fetal testis

Aim of this research was to investigate the immunohistochemical localization of alfa-inhibin and transferrin in the human fetal testis. In addition, an attempt was made to identify the Sertoli cells in primary cell cultures of fetal testes by the demonstration of the mRNA for transferrin and the presence of the FSH receptor. 15 fetal testes (8-12 weeks) were utilized. The results of this study show: 1. In sections, immunoreactivity for alfa-inhibin was present in the interstitial cells, whereas transferrin was localized in Sertoli cells; 2. After 20 days of primary culture: a) the percentage of immunoreactive cells for both the substances was about 15%; b) the presence of FSH receptor and the expression of transferrin mRNA were observed in 12-15% of cells. These findings suggest: 1. alfa-inhibin and transferrin are present in the human fetal testis; 2. the expression of transferrin mRNA and the presence of the FSH receptor permit the identification of the Sertoli cells in primary cultures of fetal testes (8-12 weeks).