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.     

The derivation of clinical-grade human embryonic stem cell lines

The pluripotent nature of human embryonic stem cells (hESC) has attracted great interest in using them as a source of cells or tissue in cell therapy. However, in order to be used in regenerative medicine, the pluripotent hESC lines should be established and propagated according to good manufacturing practice quality requirements. The cultures should be animal substance free in order to exclude the risk of infections and immunogenity. They should also be genetically and epigenetically normal. The detailed molecular mechanisms of their pluripotency are still not defined. Using human feeder cells, a medium containing only human proteins, the mechanical isolation of the inner cell mass and mechanical passaging of hESC, is a safe option until a functional defined medium containing physiological concentrations of regulatory factors is available.     

Clonal derivation and characterization of human embryonic stem cell lines

Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus should formally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning. Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the previously clonally derived cell line AS034.1, respectively. The hESC line SA002 was recently reported to have an abnormal karyotype (trisomy 13), but within this population of cells we observed rare individual cells with an apparent normal karyotype. At a cloning efficiency of 5%, we established 33 subclones from SA002, out of which one had a diploid karyotype and this subline was designated SA002.5. From AS034.1 we established one reclone designated AS034.1.1 at a cloning efficiency of 0.1%. These two novel sublines express cell surface markers indicative of undifferentiated hESC (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), Oct-4, alkaline phosphatase, and they display high telomerase activity. In addition, the cells are pluripotent and form derivatives of all three embryonic germ layers in vitro as well as in vivo. These results, together with the clonal character of SA002.5 and AS034.1.1 make these homogenous cell populations very useful for hESC based applications in drug development and toxicity testing. In addition, the combination of the parental trisomic hESC line SA002 and the diploid subclone SA002.5 provides a unique experimental system to study the molecular mechanisms underlying the pathologies associated with trisomy 13.     

Chemically defined conditions for human iPSC derivation and culture

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.     

High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis

Pre-implantation genetic diagnosis allows the characterisation of embryos that carry a gene responsible for a severe monogenic disease and to transfer to the mother’s uterus only the unaffected one(s). The genetically affected embryos can be used to establish human embryonic stem cell (hESC) lines. We are currently establishing a cell bank of ESC lines carrying specific disease-causing mutant genes. These cell lines are available to the scientific community. For this purpose, we have designed a technique that requires only minimal manipulation of the embryos. At the blastocyst stage, we just removed the zona pellucida before seeding the embryo as a whole on a layer of feeder cells. This approach gave a good success rate (>20%), whatever the quality of the embryos, and allowed us to derive 11 new hESC lines, representing seven different pathologies. Full phenotypic validation of the cell lines according to ISCI guidelines confirmed their pluripotent nature, as they were positive for hESC markers and able to differentiate in vitro in all three germ layers derivatives. Nine out of 11 stem cell lines had normal karyotypes. Our results indicate that inner cell mass isolation is not mandatory for hESC derivation and that minimal manipulation of embryos can lead to high success rate.     

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.     

Derivation of three new human embryonic stem cell lines

Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines.     

Derivation of human embryonic stem cells in defined conditions

We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.     

Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells

Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6-10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.     

Derivation of human embryonic stem cell lines

Human embryonic stem cells (hESC) are undifferentiated cells derived from an early embryo that can grow in vitro indefinitely, while retaining their capability to differentiate into specialized somatic cell types. Over the last decade there has been great interest in derivation and culture of these cells, as they can potentially provide a supply of readily available differentiated cells and tissues of all types to be used for therapeutic purposes in cell transplantation in humans, as well as for other medical uses such as drug discovery. The source of hESC lines is usually excess human embryos from in vitro fertilization treatments, although novel ways of producing hESCs have been suggested recently. The actual methods of hESC derivation have not changed greatly since the first report by Thomson et al. in 1998 . However, the main emphasis over the last several years has been in finding defined conditions for derivation and culture of hESCs, because to enable the clinical use of hESC for cell transplantation, the use of animal derived biological components is no longer acceptable. For basic research, the aim is to replace even human derived materials with completely defined systems. In this paper we describe methods utilized in our laboratory for hESC derivation and describe two studies conducted in an attempt to improve derivation efficiency and to enable research outcomes to be achieved using fewer embryos.     

Soft substrate maintains stemness and pluripotent stem cell-like phenotype of human embryonic stem cells under defined culture conditions

Cytotechnology - Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the pre-implantation blastocyst. Prior to embryo implantation, the ICM cells are surrounded by...     

Derivation and characterization of three new human embryonic stem cell lines in Finland

Human embryonic stem cell (hESC) lines can be established from the preimplantation embryos. Due to their ability to differentiate into all three embryonic layers, hESC are of significant interest as a renewable source of cell material for different applications, especially for cell replacement therapy. Since the establishment of the first hESC lines in 1998, several studies have described the derivation and culture of new hESC lines using various derivation methods and culture conditions. Our group has currently established eight new hESC lines of which three of the latest ones are described in a more detailed way in this report. The described lines have been established using mechanical derivation methods for surplus bad quality embryos and culture conditions containing human foreskin fibroblast feeder cells and serum-free culture medium. All the new lines have a normal karyotype and typical hESC characteristics analyzed in vitro. The described hESC lines are available for research purposes upon request (www.regea.fi).     

The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking

This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.     

人胚胎干细胞建系和鉴定

人胚胎干细胞是一种取自人囊胚内细胞团且具有形成所有三个胚层细胞能力的全能细胞。建立一个理想的人胚胎干细胞培养系统是研究和利用这种具有巨大潜力细胞的首要条件。本文讨论了目前建立的人胚胎干细胞培养系统,阐述了其有利的和不利的一面,并着重讨论其体外培养方法和鉴定策略。     

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.     

一种新的培养人胚胎干细胞的包被基质

目的开发一种新的培养人胚胎干细胞(hESCs)的包被基质,使hESCs的培养更加简便。方法用甲醇固定的小鼠胚胎成纤维细胞(MEF)作为包被基质,人胚胎干细胞系X-01在该基质上生长,每隔5~6 d传代一次,培养10代后,对人胚胎干细胞特性进行检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达和分化能力。结果 hESCs在新的基质上生长良好,经10次传代后仍能保持典型的hESCs克隆形态。碱性磷酸酶染色阳性,免疫荧光染色Oct4、SSEA4、Tra-1-60为阳性,体外分化可形成拟胚体。结论此种固定的基质可以大量制备,长期保存,并可以长期维持hESCs的未分化状态,为人胚胎干细胞的体外扩增探索出了一个新的途径。     

More new lines of human parthenogenetic embryonic stem cells

Two papers [1, 2] published in a recent issue of Cell Research describe the derivation of pluripotent human embryonic stem （hES）-like cell lines from parthenogeneic blastocysts.