The use of leukemia inhibitory factor immobilized on virus-derived polyhedra to support the proliferation of mouse embryonic and induced pluripotent stem cells

Human leukemia inhibitory factor (LIF) was immobilized into insect virus-derived microcrystals (polyhedra) to generate LIF polyhedra (LIF-PH) that can slowly release LIF into embryonic stem (ES) cell culture media and thus maintain ES cells in an undifferentiated state. Assays of the biological activities of LIF-PH indicated that a single addition of LIF-PH to the ES cell culture medium can support the proliferation of mouse ES and induced pluripotent stem (iPS) cells continuously for 14 days, and suggest that LIF-PH can be successfully used in the place of a periodic addition of recombinant LIF to the media every 2-3 days. The release of LIF protein from LIF-PH was determined by enzyme-linked immunosorbent assay (ELISA). Maintenance of undifferentiated state of mouse ES and iPS cells cultured with LIF-PH was determined by the detection of pluripotency-related biomarkers Oct3/4 and stage-specific embryonic antigen-1 (SSEA-1) through immunostaining and measurement of alkaline phosphatase activity. In this paper, we propose a closed culture system for mass production of ES and iPS cells that utilize a slow-releasing agent of LIF.     

Self Renewal of Embryonic Stem Cells in the Absence of Feeder Cells and Exogenous Leukaemia Inhibitory Factor

Abstract

To evaluate the role of leukaemia inhibitory factor (LIF) for maintaining pluripotent embryonic stem (ES) cells in culture, we established several exogenous LIF-independent ES cell lines by continuous passaging in culture. The newly established ES cells, Kli and CBli, sustained their growth and remained undifferentiated in LIF-deficient medium. Analysis of chimaeric animals, produced with the p-galactosidase transgenic Kli ES cells, revealed that LDF-independent ES cells can contribute to all embryonic germ layers. There was no detectable LIF protein in ES cell conditioned medium, and no upregulation of LIF mRNA was found. The addition of neutralising anti-LIF antibodies was not sufficient to abrogate the self renewal of the Kli ES cells. These studies suggest that the signalling pathway involving diffusible LIF can be bypassed for maintaining the pluripotency in culture, and indicate a considerable heterogeneity in growth factor dependence and differentiation of different ES cells.     

Biomodification of non-woven polyester fabrics by insulin and RGD for use in serum-free cultivation of tissue cells

In this study, the development of a novel cell support material was purposed as due to the serum-free cultivation of tissue cells. This material was prepared by immobilizing RGD (Arg-Gly-Asp) sequence of cell-adhesion factor, fibronectin, and cell-growth factor, insulin, to the three-dimensional non-woven polyester fabric (briefly NWPF) discs that have been used successfully in our previous cell culture studies. At first these matrices were partially hydrolyzed and then the carboxyl groups were coupled with RGD or insulin in the presence of water-soluble carbodiimide. The effectiveness of immobilization process was checked with SEM, ATR-FTIR spectroscopy and swelling studies. The maximum amount of immobilized insulin was 6.96 micorgcm(-2) and it was obtained at 200 micorgml(-1) initial insulin concentration for 60 min immobilization period. The cell culture studies which were carried out with human skin fibroblasts (HS An1) showed that, percentage of adhesion on RGD modified NWPF discs is higher than that of other surfaces. i.e., unmodified discs, polystyrene Petri dishes and insulin-immobilized discs, in serum-free culture. According to the results of growth studies, highest cell yield was obtained in the case of insulin-modified discs.     

以转染白血病抑制因子基因的人胚肺成纤维细胞为饲养层培养人胚胎生殖细胞

目的 以转染白血病抑制因子(LIF)基因的人胚肺成纤维细胞为饲养层培养人胚胎生殖细胞,为建立无动物成分污染的人胚胎生殖细胞培养体系奠定基础.方法 将LIF真核表达载体pcDNA3.1( )-LIF转染到人胚肺成纤维细胞中,通过筛选和鉴定获得表达LIF的阳性细胞.将原始生殖细胞(PGCs)种植到转染后细胞制备而成的饲养层上,在不添加外源性LIF的条件下培养,并对PGCs来源的细胞集落进行鉴定.结果 经RT-PCR及Western blotting 鉴定证实,转染pcDNA3.1( )-LIF的人胚肺成纤维细胞表达LIF基因.在转染后人胚肺成纤维细胞上生长的PGCs可形成典型的鸟巢状集落,经检测集落碱性磷酸酶活性呈强阳性,表达胚胎阶段特异性抗原SSEA-1、SSEA-4、TRA-1-60、TRA-1-81,及未分化标志Oct-4.结论 用转染LIF基因后的人胚肺成纤维细胞作为饲养层能支持PGCs来源人胚胎生殖细胞的生长,维持自我更新.     

Maintenance of murine embryonic stem cells' self-renewal and pluripotency with increase in proliferation rate by a bovine granulosa cell line-conditioned medium

Murine embryonic stem cells (mESCs) are pluripotent cells that can be propagated in an undifferentiated state in continuous culture on a feeder layer or without feeders in the presence of leukemia inhibitory factor (LIF). Although there has been a great advance since their establishment, ESC culture is still complex and expensive. Therefore, finding culture conditions that maintain the self-renewal of ESCs, preventing their differentiation and promoting their proliferation, is still an area of great interest. In this work, we studied the effects of the conditioned medium from a bovine granulosa cell line (BGC-CM) on the maintenance of self-renewal and pluripotency of mESCs. We found that this medium is able to maintain mESCs' self-renewal while preserving its critical properties without LIF addition. mESCs cultured in BGC-CM expressed the stem cell markers Oct4, Sox2, Nanog, SSEA-1, Klf4, Rex1, and ECAT1. Moreover, mESCs cultured in BGC-CM gave rise to embryoid bodies and teratomas that differentiated effectively to diverse cell populations from endoderm, mesoderm, and ectoderm. Further, we found that mESCs cultured in BGC-CM have an increased proliferation rate compared with cells grown in the mESC standard culture medium supplemented with LIF. These findings may provide a powerful tool to culture mESCs for long periods of time with high proliferation rate while preserving its basic characteristics, contributing to the application of these cells to assess potential tissue engineering and cellular therapy applications.     

Murine embryonic stem cells secrete cytokines/growth modulators that enhance cell survival/anti-apoptosis and stimulate colony formation of murine hematopoietic progenitor cells

Stromal cell-derived factor (SDF)-1/CXCL12, released by murine embryonic stem (ES) cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells. Conditioned medium (CM) from murine ES cells growing in the presence of leukemia inhibitory factor (LIF) was generated while the ES cells were in an undifferentiated Oct-4 expressing state. ES cell-CM enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased apoptosis of murine bone marrow c-kit(+)lin- cells. ES CM contained interleukin (IL)-1alpha, IL-10, IL-11, macrophage-colony stimulating factor (CSF), oncostatin M, stem cell factor, vascular endothelial growth factor, as well as a number of chemokines and other proteins, some of which are known to enhance survival/anti-apoptosis of progenitors. Irradiation of ES cells enhanced release of some proteins and decreased release of others. IL-6, FGF-9, and TNF-alpha, not detected prior to irradiation was found after ES cells were irradiated. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins, results which may be of physiological and/or practical significance.     

C57BL/6J×129/J杂交小鼠ES细胞系的建株(英)

 目的：建立C57BL/6J×129/J杂交小鼠ES细胞系。 方法： 收集3.5 d.p.c.的囊胚，培养在预先铺有小鼠成纤维细胞（MEFs）的高糖DMEM培养液中。3-4 d后，挑出内细胞团（ICM），消化后重新种到新鲜的有MEFS培养液中。等到有典型的ES样集落长出，即传代以得到永久ES细胞系。通过分析碱性磷酸酶活性，SSEA-1，Oct-4的表达和形成畸胎瘤的能力来鉴定ES细胞的多向分化能力。 结果： 获得的两个C57BL/6J×129/J杂交小鼠ES细胞系绝大多数细胞具有正常的核型（40，XY），碱性磷酸酶染色阳性，SSEA-1，Oct-4表达阳性， ES细胞注入SCID鼠后可获得来自3个胚层的组织。 结论： 建立了两株具有长期自我更新能力和多向分化潜能的C57BL/6J×129/J杂交小鼠ES细胞系。     

小鼠胚胎干细胞表面糖分子被凝集素特异性识别的研究

目的　探讨小鼠胚胎干细胞表面糖蛋白能够被特异性识别的凝集素。 方法　通过磁珠筛选出小鼠胚胎干细胞系ES-D3 和 ESC57BL/6中周期特异性胚胎抗原1(stage specific embryonic antigen-1,SSEA-1)阳性的胚胎干细胞，进行凝集素的特异性结合，流式细胞仪筛选和免疫荧光染色，观察凝集素特异性结合情况。 结果　磁珠法可以筛选出99%的SSEA-1阳性细胞，它们代表着未分化的胚胎干细胞。在SSEA-1阳性细胞中，凝集素的结合实验的流式筛选提示，凝集素DSL结合率达99%，多花紫藤凝集素(Wisteria Floribunda Lectin,WFL)的结合呈双相性，部分不结合，部分高度结合；而荆豆凝集素I( Ulex europaeus Agglutinin UEAI)几乎不结合。 结论　曼陀罗植物凝集素(Datura stramonium Lectin,DSL)可以象SSEA-1一样，作为小鼠胚胎干细胞的特异性标志物，而UEAI可以作为阴性标志物。     

A zonal pattern of neuroglial cells during the development of the intermediate lobe of the rat pituitary

In this paper the development of the intermediate lobe (IL) of the pituitary of the rat is described using alkaline phosphatase (AP) (EC 3.1.3.1.) enzyme histochemistry and stage-specific embryonic antigen 1 (SSEA-1) immunocytochemistry. SSEA-1 and AP are co-localized during late development and reveal the existence of two cytochemically different cell types within the IL, i.e. SSEA-1/AP-positive and SSEA-1/AP-negative cells. The SSEA-1/AP-positive cells are initially arranged along the hypophyseal lumen, in a number of longitudinally oriented zones. alpha-Melanocyte-stimulating hormone (alpha-MSH) immunoreactivity is expressed in the SSEA-1/AP-negative cells from E20 onwards. Eventually the SSEA-1/AP-positive cells develop into a layer of cells covering the luminal surface of the IL lobules. These cells represent the glio-epithelial or neuroglial cells of the IL.     

CD133 expression by neural progenitors derived from human embryonic stem cells and its use for their prospective isolation

The ability to generate purified neural progenitors is critical to the development of embryonic stem cell-based therapies to alleviate human neurological disorders. While many cell culture protocols for directed differentiation of human embryonic stem (hES) cells into neural cells have been described, most yield mixed populations, some containing cells of different embryonic germ layer lineages, or even undifferentiated embryonic stem cells. In this study, we describe a method for single-cell dissociation, isolation by flow cytometry, and subsequent culture of neural progenitors from hES cells. As reported earlier, hES cells treated with the bone morphogenetic protein (BMP) antagonist noggin gave rise to neurospheres at a relatively high frequency. However, these noggin-treated embryonic stem cell cultures were heterogeneous, with cells expressing embryonic stem markers still detectable even following 14 days of differentiation. In order to isolate pure human neural progenitors, we fractionated noggin-treated ES cells on the basis of their expression of the putative neural stem cell marker, CD133, and the GCTM-2, and SSEA-1 antigens, which mark pluripotent stem cells and differentiated cells respectively from hES cell culture. CD133(+) cells formed larger spheres compared to CD133(-) cells. CD133(+)SSEA1(+) cells and CD133(+)SSEA-1(-) cells expressed similar levels of neural genes and formed neurospheres at similar frequencies. By contrast, CD133(+)GCTM-2(+) cells expressed high levels of OCT4 but not neural lineage genes and failed to form neurospheres. CD133(+)GCTM-2(-) cells formed neurospheres at the relative highest frequency. Thus, negative selection with GCTM-2 may be useful for the purification of specific cell types differentiated from hES cells.     

Self-renewal of embryonic stem cells through culture on nanopattern polydimethylsiloxane substrate

Embryonic stem (ES) cells can undergo continual proliferation and differentiation into cells of all somatic cell lineages in vitro; they are an unlimited cell source for regenerative medicine. However, techniques for maintaining undifferentiated ES cells are often inefficient and result in heterogeneous cell populations. Here, we determined effects of nanopattern polydimethylsiloxane (PDMS) as a culture substrate in promoting the self-renewal of mouse ES (mES) cells, compared to commercial plastic culture dishes. After many passages, mES cells efficiently maintained their undifferentiated state on nanopattern PDMS, but randomly differentiated on commercial plastic culture dishes, as indicated by partially altered morphologies and decreases in alkaline phosphatase activity and stage-specific expression of embryonic antigen-1. Under nanopattern PDMS conditions, we found increased activities of STAT3 and Akt, important proteins involved in maintaining the self-renewal of mES cells. The substrate-cell interactions also enhanced leukemia inhibitory factor (LIF)-downstream signaling and inhibited spontaneous differentiation, concomitant with reduced focal adhesion kinase (FAK) signaling. This reduction in FAK signaling was shown to be important for promoting mES cell self-renewal. Thus, our data demonstrates that nanopattern PDMS contributes to maintaining the self-renewal of mES cells and may be applicable in the large-scale production of homogeneously undifferentiated mES cells.     

Human placenta-derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: comparison with human bone marrow-derived feeders

Co-culture of human embryonic stem (ES) cells on mouse fibroblast feeders is the commonly used method for in vitro expansion of human ES cells in an undifferentiated state. However, it has potential risks of pathogen transmission from animals; thus, human cell-derived feeders have been employed to minimize this problem. In this study, we compared human placenta-derived feeders with bone marrow to demonstrate its effectiveness as feeders for in vitro long-term culture of human ES cells. We cultured a human ES cell line, SNUhES3, on human placenta-derived mesenchymal stem cell feeders and compared their culture efficiency with human bone marrow-derived feeders and control group (mouse fibroblast feeders, STO). The mean number of human ES cell colonies was 166 +/- 35 in the placenta feeders; this was significantly higher than bone marrow-derived feeders (87 +/- 16, p < 0.05). We could propagate the culture of SNUhES3 on the placenta feeders past the 50th week similar to control group. During the culture, the maintenance of undifferentiated state of SNUhES3 was demonstrated by the expression of SSEA-4, TRA-1-81, TRA-1-60, and Oct-4. However, we failed to propagate the culture of human ES cells on the human bone marrow-derived feeders past the 5th week. The efficiency of embryoid body formation was similar between placenta and control group, indicating the preservation of differentiation ability. Thus, placenta-derived feeders are more efficient for the long-term in vitro culture of human ES cells than bone marrow-derived feeders suggesting the possible role of placenta as a source for human cell-derived feeders.     

Colonization and maintenance of murine embryonic stem cells on poly(alpha-hydroxy esters)

The aim of this study was to determine the ability of various poly(alpha-hydroxy esters) to support the in vitro propagation of murine embryonic stem (ES) cells in an undifferentiated state. To this end, ES cell colonization, growth and Oct-4 immunoreactivity following a 48 h culture period upon poly((D,L)-lactide), poly((L)-lactide), poly(glycolide) and poly((D,L)-lactide-co-glycolide) (PLGA) were assessed. By the analysis of live and dead cell number indices and Oct-4 immunoreactivity, ES cell colonization rate during a 48 h culture period was found to be significantly greater on PLGA compared to all the other unmodified poly(alpha-hydroxy esters) tested. Surface treatment of all polymers with 0.1m potassium hydroxide revealed a significant increase in ES cell live numbers when compared to all unmodified polymers, thus revealing a correlation between polymer content, hydrophilicity and colonization rate. These data suggest that surface treated poly(alpha-hydroxy esters) may be employed for ES cell scale up procedures and in tissue engineering applications requiring the colonization of scaffolds by ES cells in an undifferentiated state. According to such applications, once the designated scaffold has been colonized, ES cell directed differentiation into the desired and fully differentiated, functional adult tissue may then be effected.     

Derivation and spontaneous differentiation of human embryonic stem cells

Embryonic stem (ES) cells are unique cells derived from the inner cell mass of the mammalian blastocyst. These cells are immortal and pluripotent, retain their developmental potential after prolonged culture, and can be continuously cultured in an undifferentiated state. Many in vitro differentiation systems have been developed for mouse ES cells, including reproducible methods for mouse ES cell differentiation into haematopoietic and neural precursors, cardiomyocytes, insulin‐secreting cells, endothelial cells and various other cell types. The derivation of new human ES cell lines provides the opportunity to develop unique models for developmental research and for cell therapies. In this review we consider the derivation and spontaneous differentiation of human ES cells.     

The effects of covalently immobilized hyaluronic acid substrates on the adhesion, expansion, and differentiation of embryonic stem cells for in vitro tissue engineering

We investigated the in vitro effects of the molecular weight (MW) of hyaluronic acid (HA) on the maintenance of the pluripotency and proliferation of murine embryonic stem (ES) cells. High (1000 kDa) or low (4-8 kDa) MW HA was derivatized using an ultraviolet-reactive compound, 4-azidoaniline, and the derivative was immobilized onto cell culture cover slips. Murine ES cells were cultured on these HA surfaces for 5 days. High-MW HA interacted with murine ES cells via CD44, whereas low-MW HA interacted with these cells mostly via CD168. ES cells grown on both high- and low-MW HA appeared undifferentiated after 3 days. However, more cells adhered, proliferated, and exhibited greater amounts of phospho-p42/44 mitogen-activated-protein-kinase on low- compared with high-MW HA. Expression of Oct-3/4 and phosphorylation of STAT3 were enhanced by ES cells on low-MW HA, not on high-MW HA. After release from HA, cells cultured on low-MW HA in the presence of differentiating medium showed enhanced expression of α-SMA or CD31 compared with cells cultured on high-MW HA. It was concluded that low-MW HA substrates were effective in maintaining murine ES cells in a viable and undifferentiated state, which favors their use in the propagation of ES cells for tissue engineering.