Human placenta feeder layers support undifferentiated growth of primate embryonic stem cells

Various undifferentiated embryonic stem (ES) cells can grow on mouse embryonic fibroblast (MEF) feeders. However, the risk of zoonosis from animal feeders to human ES cells generally excludes the clinical use of these human ES cells. We have found that human placenta is a useful source of feeder cells for the undifferentiated growth of primate ES cells. As on MEF feeders, primate ES cells cultured on human amniotic epithelial (HAE) feeder cells and human chorionic plate (HCP) cells had undifferentiated growth. The cultured primate ES cells expressed Oct-4, alkaline phosphatase, and SSEA-4. The primate ES cells on HAE feeder cells produced typical immature teratomas in vivo after injection into severe combined immunodeficient mice. Human placenta is quite novel and important because it would provide a relatively available source of feeders for the growth of human ES cells for therapeutic purposes that are also free of ethical complications.     

Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells

Human embryonic stem (hES) cells are typically derived and serially propagated on inactivated murine embryonic fibroblast (MEF) feeders. The use of MEFs and other components of animal origin in the culture media for hES cell support substantially elevates the risk of contaminating these cell lines with infectious agents of animal origin thereby severely limiting their potential for clinical application. We have previously shown that it is possible to derive and establish new hES cell lines in a xeno-free culture system using human fetal muscle fibroblast feeders. In this report, we have comparatively evaluated a panel of 11 different human adult, fetal, and neonatal feeders for hES cell support and have ranked them as supportive and non-supportive. We report that two adult skin fibroblast cell lines established in-house from abdominal skin biopsies supported prolonged undifferentiated hES cell growth for over 30 weekly passages in culture. Furthermore, hES cell lines cultured on adult skin fibroblast feeders retain hES cell morphology and remain pluripotent. Also, differences in feeder support exist between human cell types and sources. The use of human adult skin feeders is convenient for hES cell support given the ease of obtaining skin biopsies.     

Enhancement of NF-kappaB expression and activity upon differentiation of human embryonic stem cell line SNUhES3

NF-kappaB is involved in many biological processes including proliferation, survival, and differentiation. Because human embryonic stem (ES) cells have the potential to differentiate to various lineages, understanding mechanisms involved in stemness and lineage differentiation is an important issue. We investigated expression of NF-kappaB in the human ES cell lines SNUhES3 and MizhES4 and found that expression of NF-kappaB mRNA and protein in these two cell lines was significantly lower compared to those of other adult cell lines. However, when SNUhES3 cells were induced to differentiate by retinoic acid, expression levels of NF-kappaB significantly increased compared to undifferentiated SNUhES3 cells. As the components of tumor necrosis factor-alpha (TNF-alpha) signaling are expressed comparably in undifferentiated and differentiated SNUhES3 cells, we examined the responsiveness of SNUhES3 cells to treatment with TNF-alpha, an agonist of NF-kappaB signaling. Nuclear localization of NF-kappaB in response to TNF-alpha was evident in differentiated, but not undifferentiated, SNUhES3 cells. In agreement with this observation, induction of interleukin-8 (IL-8) in response to TNF-alpha was seen only in differentiated SNUhES3 cells. On the basis of an IkappaB kinase (IKK) inhibitor study, expression of IL-8 induced by TNF-alpha was dependent on NF-kappaB activity. Taken together, our results suggest that expression and activity of NF-kappaB is comparatively low in undifferentiated human ES cells, but increases during differentiation of the ES cells.     

Hematopoietic differentiation of embryoid bodies derived from the human embryonic stem cell line SNUhES3 in co-culture with human bone marrow stromal cells

Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic precursor cells via two methods: the formation of embryoid bodies (EBs) and co-culture with mouse bone marrow (BM) stromal cells. In this study, the above two methods have been combined by co-culture of human ES-cell-derived EBs with human BM stromal cells. The efficacy of this method was compared with that using EB formation alone. The undifferentiated human ES cell line SNUhES3 was allowed to form EBs for two days, then EBs were induced to differentiate in the presence of a different serum concentration (EB and EB/high FBS group), or co- cultured with human BM stromal cells (EB/BM co-culture group). Flow cytometry and hematopoietic colony-forming assays were used to assess hematopoietic differentiation in the three groups. While no significant increase of CD34+/CD45- or CD34+/CD38- cells was noted in the three groups on days 3 and 5, the percentage of CD34+/CD45- cells and CD34+/ CD38- cells was significantly higher in the EB/BM co-culture group than in the EB and EB/high FBS groups on day 10. The number of colony-forming cells (CFCs) was increased in the EB/BM co-culture group on days 7 and 10, implying a possible role for human BM stromal cells in supporting hematopoietic differentiation from human ES cell-derived EBs. These results demonstrate that co-culture of human ES-cell-derived EBs with human BM stromal cells might lead to more efficient hematopoietic differentiation from human ES cells cultured alone. Further study is warranted to evaluate the underlying mechanism.     

The TRA-1-60 and TRA-1-81 human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma

We have previously identified the cell adhesion protein podocalyxin expressed in a human pluripotent stem cell, embryonal carcinoma (EC), which is a malignant germ cell. Podocalyxin is a heavily glycosylated membrane protein with amino acid sequence homology to the hematopoietic stem cell marker CD34. Since the initial discovery of podocalyxin in a cancerous stem cell, numerous new studies have identified podocalyxin in many different human cancers and in embryonic stem cells lines (ES) derived from human embryos. Embryonal carcinoma, as do all human pluripotent stem cells, expresses TRA-1-60 and TRA-1-81 antigens, and although their molecular identities are unknown, they are commonly used as markers of undifferentiated pluripotent human stem cells. We report here that purified podocalyxin from embryonal carcinoma has binding activity with the TRA-1-60 and TRA-1-81 antibodies. Embryonal carcinoma cells treated with retinoic acid undergo differentiation and lose the TRA-1-60/TRA-1-81 markers from their plasma membrane surface. We show that podocalyxin is modified in the retinoic acid-treated cells and has an apparent molecular mass of 170 kDa on protein blots as compared with the apparent 200-kDa molecular weight form of podocalyxin expressed in untreated cells. Furthermore, the modified form of podocalyxin no longer reacts with the TRA-1-60/TRA-1-81 antibodies. Thus, embryonal carcinoma expresses two distinct forms of podocalyxin, and the larger version is a molecular carrier of the human stem cell-defining antigens TRA-1-60 and TRA-1-81.     

Transwell共培养条件下诱导脂肪干细胞成骨能力的改变

BACKGROUND:Under co-culture conditions, mesenchymal stem cells could regulate osteogenic differentiation and osteogenesis of osteoblasts. OBJECTIVE:To observe the osteogenic efficiency of osteoblastic precursor cells co-cultured with undifferentiated bone marrow-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, or placenta-derived mesenchymal stem cells in mineralization medium. METHODS:Adipose-derived stem cells were induced in osteogenic differentiation medium for 7 days before being indirectly co-cultured with undifferentiated mesenchymal stem cells isolated from different tissues (bone marrow group, umbilical cord group and placenta group) in Transwell plates. Induced adipose-derived stem cells cultured alone served as control group. At different experimental intervals, quantitative analysis of alkaline phosphatase activity and calcified matrix was preformed to observe the effects of mesenchymal stem cells from different sources on the osteogenic efficiency of induced adipose-derived stem cells. RESULTS AND CONCLUSION:Expression of alkaline phosphatase was significantly higher in different experimental groups than the control group (P < 0.05), and it was also higher in the bone marrow group than the umbilical cord and placenta groups (P < 0.05). Quantitative analysis of calcified matrix revealed that the experimental groups were significantly higher than the control group (P < 0.05); and in experimental groups, the umbilical cord group was higher than bone marrow group and placenta group(P < 0.05). These findings indicate that the osteogenic efficiency of induced adipose-derived stem cells is improved dramatically under co-culture conditions.     

Isolation of multipotent cells from human term placenta

Current sources of stem cells include embryonic stem cells (ESCs) and adult stem cells (ASCs). However, concerns exist with either source: ESCs, with their significant ethical considerations, tumorigenicity, and paucity of cell lines; and ASCs, which are possibly more limited in potential. Thus, the search continues for an ethically conducive, easily accessible, and high-yielding source of stem cells. We have isolated a population of multipotent cells from the human term placenta, a temporary organ with fetal contributions that is discarded postpartum. These placenta-derived multipotent cells (PDMCs) exhibit many markers common to mesenchymal stem cells--including CD105/endoglin/SH-2, SH-3, and SH-4--and they lack hematopoietic-, endothelial-, and trophoblastic-specific cell markers. In addition, PDMCs exhibit ESC surface markers of SSEA-4, TRA-1-61, and TRA-1-80. Adipogenic, osteogenic, and neurogenic differentiation were achieved after culturing under the appropriate conditions. PDMCs could provide an ethically uncontroversial and easily accessible source of multipotent cells for future experimental and clinical applications.     

人胚胎干细胞H1培养条件的优化

目的:采用不同培养基和饲养层培养人胚胎干细胞H1,建立适合H1细胞增殖的最佳条件并分析其基本生物学特性。方法:鼠源性饲养层采用ICR品系小鼠胚胎成纤维细胞(MEF),人源性饲养层采用人胚胎成纤维细胞系(HFF-1)。H1基本培养基配制分别采用传统DMEM/F12和改良培养基Knock-outTM DMEM。实验共分为MEF DMEM/F12、MEF K-DMEM、HFF DMEM/F12、HFF K-DMEM组。H1基本生物学特性检测采用免疫荧光、RT-PCR、碱性磷酸酶和核型分析。结果:MEF DMEM/F12组中H1克隆形态规则,不发生分化,增殖速度快;而MEF K-DMEM组细胞克隆传代后第4日发生分化;HFF DMEM/F12组和HFF K-DMEM组细胞传代后第3日就显示出分化趋势,克隆变扁。MEF DMEM/F12组中H1细胞保持正常核型和基本生物学特性。结论:不同的人胚胎干细胞系最佳培养条件是不同的,建立的MEF DMEM/F12组培养条件最适合H1细胞增殖。     

Derivation of melanocytes from embryonic stem cells in culture.

We report that embryonic stem (ES) cells were efficiently induced to differentiate to melanocytes in vitro. When undifferentiated ES cells were cocultured with a bone marrow-derived stromal cell line, a very small but significant number of melanocytes were reproducibly generated. This process was greatly enhanced by addition of dexamethasone to the culture and strictly depended on steel factor, the ligand for the c-Kit receptor tyrosine kinase. Expression of c-Kit on the precursor cells was confirmed by using SCL/tal-1-/- ES cells, which are defective for producing hematopoietic cells, which were thus ruled out as possible sources of nonmelanogenic c-Kit-expressing cells. The morphology, reactivity to growth factors, and expression of melanogenic markers of the cells generated all indicated unequivocally that these cells were melanocytes. This culture system may provide a potent tool for the study of development and function of melanocytes.     

Culture and characterization of human embryonic stem cells

Human embryonic stem (ES) cells offer substantial opportunities for providing well-defined differentiated cells for drug discovery, toxicology, and regenerative medicine, but the development of efficient techniques for their large-scale culture under defined conditions, and for controlling and directing their differentiation, presents a substantial challenge. Markers for defining the undifferentiated cells are well established, based upon previous studies of embryonal carcinoma (EC) cells, their malignant counterparts from teratocarcinomas. These provide valuable tools for monitoring human ES cultures and their state of differentiation. However, current culture techniques are suboptimal and involve the use of poorly defined culture media and the use of feeder cells. Over time, the cells may also acquire karyotypic changes, reflecting genetic selection and adaptation to in vitro culture conditions. Nevertheless, progress is being made. Originally, human ES cells were derived and maintained in medium containing fetal calf serum. They are now widely cultured in a proprietary serum-free formulation (serum replacement from Invitrogen Corp., Carlsbad, CA), and recently we have derived a new human ES line in this medium without fetal calf serum. Human fibroblasts can also be used to replace mouse embryo fibroblasts as feeder cells. We have now found it possible to culture a subline of human ES cells on Matrigel, or purified collagen type IV, laminin, and fibronectin, without feeders or feeder-conditioned medium. These cells nevertheless retain the features of undifferentiated human ES cells, including a capacity for differentiation. Although these cells also carried karyotypic changes, further research focused upon understanding the mechanisms that control self-renewal, apoptosis, and commitment to differentiation will facilitate the development of defined culture conditions that minimize genetic change and optimize the maintenance of the undifferentiated stem cells.     

心肌细胞培养上清诱导骨髓间充质干细胞的分化

背景：前期研究发现,心肌细胞培养上清具有诱导骨髓间充质干细胞分化为心肌样细胞的能力,这可能与培养上清内的某种细胞因子或几种细胞因子有关。 目的：分析大鼠心肌细胞培养上清诱导骨髓间充质干细胞分化为心肌样细胞是否与心肌细胞培养上清内细胞因子含量不同有关。 方法：采用全骨髓贴壁培养法分离骨髓间充质干细胞并在体外培养纯化,酶消化法分离心肌细胞,分别以 1×10⁸ L^-1的细胞浓度培养72 h后收集上清。用酶联免疫吸附试验技术检测心肌细胞和骨髓间充质干细胞培养上清内的肝细胞生长因子、胰岛素样生长因子1、血小板源生长因子、干细胞因子、成纤维细胞生长因子和血管内皮生长因子的水平。 结果与结论：心肌细胞培养上清组内的胰岛素样生长因子1、血小板源生长因子和成纤维细胞生长因子水平明显高于骨髓间充质干细胞培养上清组(P < 0.01),提示心肌细胞培养上清内的胰岛素样生长因子1、血小板源生长因子和成纤维细胞生长因子可能与诱导骨髓间充质干细胞分化为心肌样细胞有关,其中主要的细胞因子是胰岛素样生长因子1。     

Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors

In the culture system using human feeder cells, the mechanism through which these cells support undifferentiated growth of embryonic stem cells (ESCs) has not been well investigated. Here, we explored the mechanisms of 3 kinds of human feeder cells, including human placental cells from the chorionic plate, human bone marrow stromal cells, and human foreskin fibroblasts. First, we determined that undifferentiated growth of 2 kinds each of human (H1 and HSF6) and mouse (D3 and CE3) ESCs was possible in all human feeder cell types tested (human placental cells, human bone marrow stromal cells, and human foreskin fibroblasts), without the need for exogenous cytokine supplementation including basic fibroblast growth factor (bFGF) and leukemia inhibitory factor. We then prepared their corresponding endogenous bFGF-knockout feeders using siRNA and tried to maintain human and mouse ESCs in their undifferentiated state; however, neither human nor mouse ESCs could be maintained in bFGF-knockout human feeder cells. The expressions of stemness markers such as Oct-4 and Nanog were significantly decreased in the bFGF-knockout group compared with those in the controls, and differentiation had already occurred, despite the undifferentiated morphologic appearance of the ESCs. In conclusion, human feeder cells are able to support the undifferentiated growth of human and mouse ESCs via bFGF synthesis. Further, a bFGF-dependent pathway might be crucial for maintaining the undifferentiated characteristics of mouse and human ESCs.     

Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation

Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.     

Homologous feeder cells support undifferentiated growth and pluripotency in monkey embryonic stem cells

In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor beta1, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.     

人胚胎干细胞系HUES4的培养和染色体核型分析

目的建立人胚胎干细胞无动物源性饲养层培养方法,同时对长时间体外培养的人胚胎干细胞核型变化进行分析。方法人胚胎干细胞系HUES4细胞分别培养于小鼠胚胎成纤维细胞和人包皮成纤维细胞饲养层,并对其干细胞特性进行鉴定;在培养传代过程中,收获P27、P34、P41和P44细胞进行染色体核型分析,P27细胞还进行DNA短串联重复序列多态性分析。结果生长于人包皮成纤维细胞饲养层的HUES4细胞碱性磷酸酶染色以及SSEA-4、TRA-1-60和TRA-1-81抗原阳性,SSEA-1抗原阴性。所检测的4代细胞中均见46,XY/46,XY,t（9;15）（q22;q26）核型嵌合现象,且异常核型百分比随传代次数增加有上升的趋势。结论培养人胚胎干细胞的饲养层细胞可由无动物源性的饲养层细胞替代;长期体外培养有增加细胞染色体核型异常的风险。     

Stem cells: you can't tell a cell by its cover

Embryonic stem (ES) cells are capable of unlimited self-renewal and have the ability to give rise to all tissue types in the body. The use of human ES cells for tissue and cell therapeutics has been suggested, but is limited by ethical concerns as these cells are derived from the inner cell mass of human embryos. In addition, the need for HLA matching of ES cell-derived tissues for allogeneic transplantation would require a bank of several thousand ES cell lines to make tissue therapeutics practical. Recently, adult stem cells-of which those in bone marrow are the best studied-have been shown to be capable of multilineage differentiation into cells of various non-blood tissues. Umbilical cord blood (UCB) haematopoietic stem cells have been shown to be equivalent to bone marrow stem cells for reconstitution of the haematopoietic system. Preliminary studies have also demonstrated that UCB haematopoietic stem cells are multipotent and capable of differentiating into non-blood cell types. This observation raises the exciting possibility of replacing human ES cells for tissue and cell therapeutics with UCB blood haematopoietic stem cells that are normally discarded with the placenta after delivery.