Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces

Previous studies have shown that cultivation of undifferentiated human embryonic stem (hES) cells requires human fibroblasts (hF) or mouse embryonic fibroblast (mEF) feeders or a coating matrix such as laminin, fibronectin or Matrigel in combination with mEF or hF conditioned medium. We here demonstrate a successful feeder-free and matrix-free culture system in which undifferentiated hES cells can be cultured directly on plastic surfaces without any supportive coating, in a hF conditioned medium. The hES cells cultured directly on plastic surfaces grow as colonies with morphology very similar to cells cultured on Matrigel(TM). Two hES cell lines SA167 and AS034.1 were adapted to matrix-free growth (MFG) and have so far been cultured up to 43 passages and cryopreserved successfully. The lines maintained a normal karyotype and expressed the expected marker profile of undifferentiated hES cells for Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and SSEA-1. The hES cells formed teratomas in SCID mice and differentiated in vitro into derivates of all three germ layers. Thus, the MFG-adapted hES cells appear to retain pluripotency and to remain undifferentiated. The present culture system has a clear potential to be scaleable up to a manufacturing level and become the preferred culture system for various applications such as cell therapy and toxicity testing.     

Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers

Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. Here we showed that the bone morphogenetic protein antagonist noggin is critical in preventing differentiation of hES cells in culture. Furthermore, we found that the combination of noggin and basic fibroblast growth factor (bFGF) was sufficient to maintain the prolonged growth of hES cells while retaining all hES cell features. Since both noggin and bFGF are expressed in MEF, our findings suggest that they may be important factors secreted by MEF for maintaining undifferentiated pluripotent hES cells. Our data provide new insight into the mechanism how hES cell self-renewal is regulated. The newly developed feeder-free culture system will provide a more reliable alternative for future therapeutic applications of hES cells.     

Feeder-free growth of undifferentiated human embryonic stem cells

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.     

人胚胎干细胞优化培养的进展

人胚胎干细胞(humanembryonicstemcell,hEScell)是来源于着床前人囊胚内细胞团(innercellmass,ICM)的、具有自我更新能力和分化全能性的细胞。由于hES细胞能在一定条件下分化成三个胚层来源的各种细胞,所以它具有重要的基础研究价值和巨大的临床应用前景,可应用于人早期胚胎发育过程的研究、药物毒物筛选、细胞移植治疗、基因治疗等领域。目前,世界上已经建立了多株hES细胞系,最早建立的hES细胞系是生长在小鼠胚胎成纤维(mouseembryonicfibroblast,MEF)细胞上的,培养体系中含血清等动物源性成分,这些成分可能引起动物源性病原体或支原体的污染,从而限制了hES细胞的临床应用。近年来,科学家们在优化hES细胞的体外培养体系方面做出了很大的努力并取得了长足进展,已经开始采用无血清、无饲养层细胞、无外源性蛋白、成分明确的培养体系进行hES细胞建系及培养,从而在一定程度上解决了上述问题。本文主要从饲养层细胞、无饲养层培养体系、培养基质、细胞因子等方面综述了hES细胞建系和维持其未分化状态的优化培养所取得的最新进展和存在的问题。     

Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage

Human ES (hES) cell lines are considered to be a valuable resource for medical research and for applications in cell therapy and drug discovery. For such utilization of hES cells to be realized, however, protocols involved in the use of hES cells, such as those for establishment, propagation, and cryopreservation, have still to be improved. Here, we report on an efficient method for the establishment of hES cell lines and its detailed characterization. Additionally, we developed a new bulk-passaging technique that preserves the karyotypic integrity of hES cell lines when maintained in culture for up to 2 years. Finally, we show that a simplified vitrification cryopreservation technique is vastly superior to standard slow-cooling methods with respect to cell viability. These results provide valuable information that will assist in achieving the goal of the large-scale hES cell culture required for the application of hES cells to disease therapy.     

Feeder layer- and serum-free culture of human embryonic stem cells

In addition to their contribution to the research on early human development, human embryonic stem (hES) cells may also be used for cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast feeder layers, which allow their continuous growth in an undifferentiated state. However, the use of hES cells in human therapy requires an animal-free culture system, in which exposure to mouse retroviruses is avoided. In this study we present a novel feeder layer-free culture system for hES cells, based on medium supplemented with 15% serum replacement, a combination of growth factors including transforming growth factor beta1 (TGFbeta1), leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix. Human ES cells grown in these conditions maintain all ES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. The culture system presented here has two major advantages: 1) application of a well-defined culture system for hES cells and 2) reduced exposure of hES cells to animal pathogens. The feeder layer-free culture system reported here aims at facilitating research practices and providing a safer alternative for future clinical applications of hES cells.     

Enhancement of cell recovery for dissociated human embryonic stem cells after cryopreservation

Due to widespread applications of human embryonic stem (hES) cells, it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation, and further investigated the role of the combination of Rho‐associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow‐freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture, we found out that hES cell recovery was significantly enhanced by around 30 % (P < 0.05) by the new freezing solution. Moreover, at the first day of post‐thaw culture, the presence of 10 μM ROCK inhibitor (Y‐27632) and 1 μM pifithrin‐μ together further significantly improved cell recovery by around 20% (P < 0.05) either for feeder‐dependent or feeder‐independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore, this protocol is a scalable cryopreservation method for handling large quantities of hES cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Highly efficient mRNA-based gene transfer in feeder-free cultured H9 human embryonic stem cells

Several protocols have been described for virus-based gene transfer in human embryonic stem (hES) cells, while efficient non-viral methods are currently non-existing. In this study, we investigated the efficiency of mRNA-based gene transfer in feeder-free cultured H9 hES cells, based on electroporation of in vitro transcribed mRNA encoding the enhanced green fluorescent protein (EGFP). Optimisation of culture and electroporation conditions for feeder-free cultured H9 hES cells resulted a highly pure, transgene-expressing (90% positive cells) H9 hES cell population.     

人胚胎干细胞培养体系的研究进展

人胚胎干细胞具有广泛的研究前景,建立一个理想的人胚胎干细胞培养系统是利用它的前提.较详细地对目前关于人胚胎干细胞培养体系的研究进展、一些细胞因子对人胚胎干细胞的作用和影响以及体外长期培养对人胚胎干细胞核型的影响进行了综述.     

A defined glycosaminoglycan-binding substratum for human pluripotent stem cells

To exploit the full potential of human pluripotent stem cells for regenerative medicine, developmental biology and drug discovery, defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed, but finding chemically defined, robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides, which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells.     

Efficient derivation of new human embryonic stem cell lines

Human embryonic stem (hES) cells, unlike most cells derived from adult or fetal human tissues, represent a potentially unlimited source of various cell types for basic clinical research. To meet the increased demand for characterized hES cell lines, we established and characterized nine new lines obtained from frozen-thawed pronucleus-stage embryos. In addition, we improved the derivation efficiency from inner cell masses (to 47.4%) and optimized culture conditions for undifferentiated hES cells. After these cell lines had been maintained for over a year in vitro, they were characterized comprehensively for expression of markers of undifferentiated hES cells, karyotype, and in vitro/in vivo differentiation capacity. All of the cell lines were pluripotent, and one cell line was trisomic for chromosome 3. Improved culture techniques for hES cells should make them a good source for diverse applications in regenerative medicine, but further investigation is needed of their basic biology.     

CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells

The application of human embryonic stem (hES) cells in regenerative medicine will require rigorous quality control measures to ensure the safety of hES cell-derived grafts. During propagation in vitro, hES cells can acquire cytogenetic abnormalities as well as submicroscopic genetic lesions, such as small amplifications or deletions. Many of the genetic abnormalities that arise in hES cell cultures are also implicated in human cancer development. The causes of genetic instability of hES cells in culture are poorly understood, and commonly used cytogenetic methods for detection of abnormal cells are capable only of low-throughput analysis on small numbers of cells. The identification of biomarkers of genetic instability in hES cells would greatly facilitate the development of culture methods that preserve genomic integrity. Here we show that CD30, a member of the tumor necrosis factor receptor superfamily, is expressed on transformed but not normal hES cells, and that CD30 expression protects hES cells against apoptosis.     

Efficient derivation of Chinese human embryonic stem cell lines from frozen embryos

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research as well as a potential cell resource for therapy. However, each hES cell line demonstrates different identity. It is desirable to obtain more fully characterized hES cell lines with newly developed technologies associated with hES cell culture. Here, we report our experience of efficient derivation of three new Chinese hES cell lines (SHhES2, SHhES3, and SHhES4) from in vitro fertilization discarded embryos donated by women with polycystic ovary syndrome. These cell lines were derived under conditions minimizing exposure to animal components and maintained at an undifferentiated state for long-term culture. They retained a normal karyotype and expressed ALP, OCT4, SOX2, SSEA-4, TRA-1-60 and TRA-1-81. RT-PCR analysis also revealed high expression levels of pluripotency markers such as OCT4, LEFTY A, SOX2, TDGF-1, THY1, FGF4, NANOG, and REX1. When suspended in low-attachment culture dishes, embryoid bodies formed and were comprised of various differentiated cell types from all three embryonic germ layers. However, well-shaped teratomas were only harvested from line SHhES2, not from SHhES3 and SHhES4, indicating that the differentiation ability in vivo differs among the three cell lines. Collectively, the three new hES cell lines were established and fully characterized. The effort paves the way toward generating hES cell lines without contamination by animal components. All of these cell lines are available by contact Ying Jin at yjin@sibs.ac.cn.     

Human embryonic stem cells: caught between a ROCK inhibitor and a hard place

Since their derivation, human embryonic stem (hES) cells have been used for a variety of applications including developmental biology, pathology, chemical biology, genomics, and proteomics. However, their most important potential application is the generation of cells and tissues, which can be used for cell‐based therapies. One of the main drawbacks of hES cell culture is that they are particularly sensitive to dissociation, which is required for passaging, expansion, cryopreservation, and other applications. Recently, it has been discovered that an inhibitor of Rho kinase (ROCKi; Y‐27632) increases the survival rate of dissociated, single hES cells. This breakthrough has allowed new methods in hES cell culture to be developed, with the promise of increasing hES cell numbers into the realm of clinical relevance. In our studies demonstrating that ROCKi dramatically increases hES cell cryopreservation efficiency, we have observed that ROCKi treatment does not decrease hES cell's susceptibility to apoptosis. Rather, we hypothesize that ROCKi treatment desensitizes single hES cells to their environment reducing the odds that individual cells will undergo anoikis.     

Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells

In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2), and transforming growth factor β (TGF-β), thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.     

New culture system for human embryonic stem cells: autologous mesenchymal stem cell feeder without exogenous fibroblast growth factor 2

Human embryonic stem (hES) cells have been successfully maintained using human-cell feeder systems or feeder-free systems. However, despite advances in culture techniques, hES cells require supplementation with fibroblast growth factor 2 (FGF-2), an exogenous stemness factor, which is needed to sustain the authentic undifferentiated status. We developed a new culture system for hES cells; this system does not require supplementation with FGF-2 to obtain hES cells that are suitable for tissue engineering and regenerative medicine. This culture system employed mesenchymal stem cells derived from hES cells (hESC-MSCs) as autologous human feeder cells in the absence of FGF-2. The hES cell line SNUhES3 cultured in this new autologous feeder culture system maintained the typical morphology of hES cells and expression of pluripotency-related proteins, SSEA-4, TRA-1-60, OCT4, and alkaline phosphatase, without development of abnormal karyotypes after more than 30 passages. RNA expression of the pluripotency-related genes OCT4 and NANOG was similar to the expression in SNUhES3 cells maintained on xenofeeder STO cells. To identify the mechanism that enables the cells to be maintained without exogenous FGF-2, we checked the secretion of FGF-2 from the mitomycin-C treated autofeeder hESC-MSCs versus xenofeeder STO cells, and confirmed that hESC-MSCs secreted FGF-2 whereas STO cells did not. The level of FGF-2 in the media from the autofeeder system without exogenous FGF-2 was comparable to that from the xenofeeder system with addition of FGF-2. In conclusion, our new culture system for hES cells, which employs a feeder layer of autologous hESC-MSCs, supplies sufficient amounts of secreted FGF-2 to eliminate the requirement for exogenous FGF-2.     

人胚胎干细胞定向分化为神经前体细胞

采用单层贴壁分化的方法在无血清条件下诱导同源饲养层培养的人胚胎干细胞定向分化,得到了高比例的神经前体细胞(97.5±0.83)%(P<0.05)。这些神经前体细胞具有分化为神经元、星形胶质细胞和少突胶质细胞的能力。在长期的传代培养中发现,随着培养时间的延长,nestin阳性的神经前体细胞比例下降,同时发育能力也发生了变化。在传代培养的早期,神经前体细胞发育为神经元的比例很高,几乎没有胶质细胞分化出来。随着培养时间的延长,胶质细胞的比例逐渐上升。这与体内神经系统的发育过程非常相似。进一步研究发现具有bHLH(basic helix-loop-helix)结构域的转录因子neurogenein2(Ngn2)和Olig2可能在这一变化中起重要作用。因此,人胚胎干细胞来源的神经前体细胞能够模拟体内神经发育的模式,为在体外研究人的神经发育和再生医学奠定了基础。     

Self-Contained Induction of Neurons from Human Embryonic Stem Cells

Background

Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins.

Methodology/Principal Findings

We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES) cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM) could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation.

Conclusion/Significance

By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.     

Hemato-endothelial differentiation from lentiviral-transduced human embryonic stem cells retains durable reporter gene expression under the control of ubiquitin promoter

Human embryonic stem (hES) cells are able to give rise to a variety of cell lineages under specific culture condition. An effective strategy for stable genetic modification in hES cells may provide a powerful tool for study of human embryogenesis and cell-based therapies. However, gene silences are documented in hES cells. In current study, we investigated whether genes controlled under ubiquitin promoter are expressed during hematopoietic-endothelial differentiation in hES cells. Undifferentiated hES cells (H1) were transduced by lentivirus encoding green fluorescent protein (GFP) gene under ubiquitin promoter. GFP-expressing hES cells (GFP-H1) were established after several rounds of mechanical selection under fluorescence microscope. GFP gene was stably expressed in hES cells throughout prolonged (> 50 passages) cultivation, and in differentiated embryo body (EB) and teratoma. Hematopoietic and endothelial markers, including KDR (VEGFR2), CD34, CD31, Tie-2, GATA-1 and GATA-2, were expressed at similar levels during hES cell differentiation in parent hES cells and GFP-H1 hES cells. CD34⁺ cells isolated from GFP-H1 hES cells were capable to generate hematopoietic colony-forming cells and tubular structure-forming cells. Differentiated GFP-EB formed vasculature structures in a semi-solid sprouting EB model. These results indicated that a transgene under ubiquitin promoter in lentiviral transduced hES cells retained its expression in undifferentiated hES cells and in hES-derived hematopoietic and endothelial cells. With the view of embryonic mesodermal developing events in humans, genetic modification of hES cells by lentiviral vectors provides a powerful tool for study of hematopoiesis and vasculogenesis.     

Human feeder layers for human embryonic stem cells

Human embryonic stem (hES) cells hold great promise for future use in various research areas, such as human developmental biology and cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast (MEF) feeder layers, which permit continuous growth in an undifferentiated stage. To use these unique cells in human therapy, an animal-free culture system must be used, which will prevent exposure to mouse retroviruses. Animal-free culture systems for hES cells enjoy three major advantages in the basic culture conditions: 1). the ability to grow these cells under serum-free conditions, 2). maintenance of the cells in an undifferentiated state on Matrigel matrix with 100% MEF-conditioned medium, and 3). the use of either human embryonic fibroblasts or adult fallopian tube epithelial cells as feeder layers. In the present study, we describe an additional animal-free culture system for hES cells, based on a feeder layer derived from foreskin and a serum-free medium. In this culture condition, hES cells maintain all embryonic stem cell features (i.e., pluripotency, immortality, unlimited undifferentiated proliferation capability, and maintenance of normal karyotypes) after prolonged culture of 70 passages (>250 doublings). The major advantage of foreskin feeders is their ability to be continuously cultured for more than 42 passages, thus enabling proper analysis for foreign agents, genetic modification such as antibiotic resistance, and reduction of the enormous workload involved in the continuous preparation of new feeder lines.