Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.     

Early-outgrowth of endothelial progenitor cells can function as antigen-presenting cells

Endothelial progenitor cells (EPCs) have been recently found to exist circulating in peripheral blood of adults, and home to sites of neovascularization in peripheral tissues. They can also be differentiated from peripheral blood mononuclear cells (PBMNCs). In tumor tissues, EPCs are found in highly vascularized lesions. Few reports exist in the literature concerning the characteristics of EPCs, especially related to their surface antigen expressions, except for endothelial markers. Here, we aimed to investigate the surface expression of differentiation markers, and the functional activities of early-outgrowth of EPCs (EO-EPCs), especially focusing on their antigen-presenting ability. EO-EPCs were generated from PBMNCs, by culture in the presence of angiogenic factors. These EO-EPCs had the morphological and functional features of endothelial cells and, additionally, they shared antigen-presenting ability. They induced the proliferation of allogeneic lymphocytes in a mixed-lymphocyte reaction, and could generate cytotoxic lymphocytes, with the ability to lyze tumor cells in an antigen-specific manner. The antigen-presenting ability of EO-EPCs, however, was weaker than that of monocyte-derived dendritic cells, but stronger than peripheral blood monocytes. Since EO-EPCs play an important role in the development of tumor angiogenesis, targeting EPCs would be an effective anti-angiogenic strategy. Alternatively, due to their antigen-presenting ability, EO-EPCs can be used as the effectors of anti-tumor immunotherapy. Since they share endothelial antigens, the activation of a cellular immunity against angiogenic vessels can be expected. In conclusion, EO-EPCs should be an interesting alternative for the development of new therapeutic strategies to combat cancer, either as the effectors or as the targets of cancer immunotherapy.     

Lysis of endothelial cells by autologous lymphokine-activated killer cells

The mechanisms of lysis of endothelial cells derived from human umbilical vein (HUVEC) by autologous lymphokine-activated killer (LAK) cells, generated from cord blood lymphocytes of the same donor, were investigated. Freshly isolated HUVEC as well as HUVEC cultured for several passages were efficiently lysed by autologous LAK cells, and their susceptibility to the LAK cells was almost the some as that of allogenic HUVEC. Complement-depletion experiments revealed that the lysis was mainly dependent on CD16-natural killer (NK) LAK cells. Pretreatment of HUVEC with recombinant interferon (rIFN) for 24 h made them resistant to lysis by autologous LAK cells, while pretreatment with either rIL-1. rTNF, or acidic or basic fibroblast growth factor did not alter the lytic sensitivity of HUVEC. The resistance of rIFN-treated HUVEC was specific to lysis by CD16⁺ NK LAK cells, and their lysis by CD3⁺ T-LAK cells was not significantly altered. Moreover, in comparison with control HUVEC or rIL-1-treated HUVEC, rIFN-treated HUVEC had a significantly less potent inhibitory effect on the lysis of untreated HUVEC, when used as an unlabeled target. This suggests that rIFN treatment may down-regulate the recognition of some molecules on HUVEC by rIL-2-activated NK cells. These data suggest that damage of the endothelium during LAK therapy is mainly dependent on LAK cells with a NK phenotype that can specifically recognize a certain molecule on autologous endothelial cells.     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.     

神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

干细胞概述

干细胞是存在于胚胎和成体中的一类特殊细胞,它能长期地自我更新,在特定的条件下具有分化形成多种终末细胞的能力,不同来源的干细胞分化潜能各异。从早期胚胎内细胞团分离的胚胎干细胞能分化形成个体所有的细胞类型,并具有在体外无限增殖的能力,是最具有临床应用前景和研究价值的干细胞之一。在成体各种组织和器官中也存在成体干细胞,用于维持机体结构和功能的稳态。近期有关成体干细胞可塑性的研究和成体组织中多能干细胞存在的证据扩大了人们对成体干细胞分化潜能的认识。干细胞具有的多向分化潜能和自我更新能力使其成为未来再生医学的重要种子细胞,并成为研究人类早期胚层特化和器官形成、药物筛选以及基因治疗的最佳工具。     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

骨髓间充质干细胞向神经细胞分化的研究

无论是在体外实验、还是在体内实验,MSCs都可以向中枢神经系统(CNS)神经细胞分化,但争议颇多。因为功能性神经元不仅要具有典型神经元的形态、特异性标记,还要求具有可兴奋性、能和其他神经元形成突触联系、产生突触电位等,所以对于骨髓间充质干细胞是否能诱导出真正具有功能的神经元存在很大分歧。在此对MSCs向神经细胞诱导分化研究的现况、存在的问题及发展前景给以综述。     

Transdifferentiation of mesenchymal stem cells derived from human fetal lung to hepatocyte-like cells

The great shortage of human hepatic cells makes it desirable to generate extrahepatic stem or precursor cells. In recent years, it has been reported that human multipotential mesenchymal stem cells (hMSCs) differentiate into hepatocyte-like cells. The fetal lung is one of the largest organs containing many MSCs that can be easily obtained. Whether MSCs from fetal lung can differentiate into hepatocytes or bile duct cells is an important issue in basic medicine and clinical application. We isolated fetal lung cells, and expanded and analyzed them. At passage 4, their morphologic, immunophenotyping and cytokine secretions were similar to adult bone marrow-derived MSCs. We conclude that these cells from fetal lung are MSCs, indicating that human fetal lung is an ideal source of hMSCs. hMSCs from fetal lung induced in special differentiation medium showed homogeneous and small polygonal endothelial-like morphology, expressing weak mRNA, as well as Alb and AFP. This implies that hMSCs from fetal lung can differentiate into hepatocyte-like cells.     

Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.     

Conversion of female germline stem cells from neonatal and prepubertal mice into pluripotent stern cells

Pluripotent stem cells derived from neonatal or adult testes are a useful tool to examine the mechanisms of pluripotency and a resource for cell-based therapies. However, therapies usingthese cells will only benefit males but not females. Recently, female germline stem cells （FGSCs） were discovered in ovaries. Whether FGSCs can be converted into pluripotent stem cells, similar to spermatogonial stem cells, is unknown. Here, we demonstrate that female embryonic stem-like cells （fESLCs） can be generated within 1 month from the stably proliferating FGSCs cultured in embryonic stem cell （ESC） medium, fESLCs exhibit properties similar to those of ESCs in terms of marker expression and differentiation potential. Thus, our findings suggest that generation of patient-specific fESLCs is feasible and provides a foundation for personalized regenerative applications.     

Th17细胞和Treg细胞与动脉粥样硬化

动脉粥样硬化从脂质条纹的形成到更复杂的病变和斑块破裂的进程是由多种不同类型的细胞和细胞因子网络共同参与作用的,其中最主要的是Th17细胞和Treg细胞及它们分泌的细胞因子。大量研究显示,Th17细胞对动脉粥样硬化的作用仍存在争议,但大部分研究仍认为其具有促动脉粥样硬化的作用。Treg细胞具有抗动脉粥样硬化的作用,Th17／Treg平衡对动脉粥样硬化的发生和发展具有重要的调节作用。本文将对Th17细胞、Treg细胞的生物学特性以及Th17细胞、Treg细胞和Th17／Treg平衡对动脉粥样硬化影响的最新研究进展做一综述。     

Response of the human testis to long-term estrogen treatment: Morphology of Sertoli cells,Leydig cells and spermatogonial stem cells

Summary The present investigation is concerned with the morphological changes observed in human testicular tissue following prolonged estrogen administration. Testicular material obtained from 11 transsexual patients who had been submitted to long-term estrogen treatment prior to sex-reversal surgery was studied by means of light- and electron microscopy.The testes of all patients examined present a more or less uniform appearance: There are narrow seminiferous cords surrounded by an extensively thickened lamina propria. They contain Sertoli cells and spermatogonia exclusively. There is no evidence of typical Leydig cells.The persisting spermatogonia show the characteristic features of pale type-A spermatogonia, whereas dark type-A spermatogonia are almost completely eliminated from the epithelium. In view of the fact that spermatogonia that survived radiotherapy and treatment with various noxious agents have recently been regarded as the stem cells of the human testis, it is suggested that also the majority of those spermatogonial types that are less sensitive to disturbances of the endocrine balance may consist of stem cells. The present results, therefore, corroborate the concept that the stem cells of the human testis may be derived from pale type-A spermatogonia or the variants of this cell type.Sertoli cells display two types of ovoid nuclei. In contrast to untreated material the nuclei lie adjacent to the basal lamina, and organelles and telolysosomes are confined to the apical cytoplasm. The apico-basal differentiation of mature cells, therefore, is not observed. Moreover, typical organelles and inclusions of mature cells are absent, as are the junctional specializations. Thus, Sertoli cells have transformed into immature cells, resembling precursors prior to puberty.Fibroblast-like cells in the interstitial tissue, which display strongly lobulated nuclei, a well-developed smooth endoplasmic reticulum, lipid droplets, and numerous inclusions are assumed to represent dedifferentiated Leydig cells.Since after estrogen treatment serum testosterone and gonadotropin levels are known to be reduced, it appears that the morphological changes correlate well with the endocrine status.     

Efficient propagation of single cells Accutase-dissociated human embryonic stem cells

Human embryonic stem cells (hESCs) hold great promise for cell-based therapies and drug screening applications. However, growing and processing large quantities of undifferentiated hESCs is a challenging task. Conventionally, hESCs are passaged as clusters, which can limit their growth efficiency and use in downstream applications. This study demonstrates that hESCs can be passaged as single cells using Accutase, a formulated mixture of digestive enzymes. In contrast to trypsin treatment, Accutase treatment does not significantly affect the viability and proliferation rate of hESC dissociation into single cells. Accutase-dissociated single cells can be separated by FACS and proliferate as fully pluripotent hESCs. An Oct4-eGFP reporter construct engineered into hESCs was used to monitor the pluripotency of hESCs passaged with Accutase. Compared to collagenase-passaged hESCs, Accutase-treated cultures contained a larger proportion of undifferentiated (Oct4-positive) cells. Additionally, Accutase-passaged undifferentiated hESCs could be grown as monolayers without the need for monitoring and/or selection for quality hESC colonies.     

干细胞向甲状旁腺细胞的分化及在甲状旁腺功能减退中的研究进展

干细胞是一类能够自我复制、自我更新,具有多向分化潜能,能产生多种分化细胞类型的细胞,由于它的自我复制和多向分化潜能,干细胞技术已经被广泛的应用于细胞移植治疗中。甲状旁腺功能减退作为一种内分泌疾病,目前的治疗方案都不能从根本上治疗该疾病。因此应用干细胞来源的甲状旁腺细胞移植治疗甲状旁腺功能减退越来越受到医学界的重视。目前,已有科研团队报道了应用胚胎干细胞（ESCs）、胸腺上皮细胞和扁桃体间充质细胞向甲状旁腺细胞分化及其在甲状旁腺功能减退中的治疗。本文将干细胞向甲状旁腺细胞的分化及在甲状旁腺功能减退治疗中的研究进展进行综述。