Pinacidil enhances survival of cryopreserved human embryonic stem cells

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.     

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.     

以人皮肤成纤维细胞作为饲养层细胞培养人胚胎干细胞

目的：比较人皮肤成纤维细胞（humandermalfibroblasts,HDFs）与小鼠胚胎成纤维细胞（Mouseembryonicfibroblasts,MEFs）的增殖能力及研究人皮肤成纤维细胞作为饲养层支持人胚胎干细胞（humanembryonicstemcells,hESCs）未分化生长的能力。方法：利用组织贴壁法从人皮肤中分离出HDFs,通过细胞形态的观察和生长曲线的绘制比较HDFs与MEFs的体外增殖能力。将HDFs作为饲养层细胞与hESCs共培养,传代12代后,检测hESCs碱性磷酸酶（AKP）、表面特异性标志及胚胎干细胞特异性转录因子。结果：HDFs可连续传代培养15代以上,10代以下的HDFs增殖迅速,而MEFs自第4代起,增殖能力就明显下降;hESCs在HDFs饲养层上可传代培养12代以上,克隆边界清晰,细胞排列紧密,碱性磷酸酶、表面标志物检测均呈阳性,表达了hESCs特异性转录因子。结论：HDFs比MEFs具有更强的增殖能力;HDFs可作为培养hEscs的饲养层细胞。     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.     

人脐带间充质干细胞作为维持人胚胎干细胞生长饲养层细胞的研究

目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells(hESCs) can self-renew indefinitely and differentiate into all cell types in the human body.Therefore,they are valuable in regenerative medicine,human developmental biology and drug discovery.A number of hESC lines have been derived from the Chinese population, but limited of them are available for research purposes.Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin.These hESCs express alkaline phosphatase and hE...     

Proliferative feeder cells support prolonged expansion of human embryonic stem cells

Long-term proliferation of human embryonic stem (hES) cells is currently achieved by co-culturing with mitotically inactive primary mouse embryonic fibroblasts (mEFs), which serve as feeder cells. This study explores the possibility that proliferative mEFs can be used as feeder cells to maintain the prolonged expansion of hES cells. All undifferentiated hES cell clumps were re-plated on six different densities of proliferative mEFs. hES colonies cultured on 1 x 10(5) - 5 x 10(5) proliferative mEFs amplified over 130 days of continuous culture and remained undifferentiated, as did those cultured on mitotically inactive mEFs. This suggests that certain densities of proliferative mEFs can maintain the propagation of hES cells, which may be helpful for identifying the cytokines and adhesion molecules that are required for their self-renewal.     

Establishment and therapeutic use of human embryonic stem cell lines

Embryonic stem (ES) cell lines, which are derived from the inner cell mass of blastocysts, proliferate indefinitely in vitro, retaining their potency to differentiate into various cell types derived from all of the three embryonic germ layers: the ectoderm, mesoderm and endoderm. Establishment of human ES cell lines in 1998 has indicated the great potential of ES cells for applications in medical research and other purposes such as cell transplantation therapy. Careful assessment of safety and effectiveness using proper animal models is required before such therapies can be attempted on human patients. Monkey ES cell lines provide valuable models for such research.     

Membrane proteomic signatures of karyotypically normal and abnormal human embryonic stem cell lines and derivatives

Cultured human embryonic stem cells (hESCs) and derived derivatives contain heterogeneous cell populations with varying degrees of differentiation and karyotypic stability. The inability to isolate homogenous population presents a challenge toward cell-based applications and therapies. A proteomics approach was utilized to discover novel membrane proteins able to distinguish between the hESC lines BG01, WA09, and abBG02 (trisomy 12, 14, 17 and an extra copy of the X chromosome), along with WA09-derived human neural progenitor (hNP) cells. Membrane protein signatures were developed using sucrose-gradient isolation, 1-D gel electrophoresis followed by in-gel digestion and analysis by reverse phase chromatography coupled to ion trap-FT-ICR. At a ≤1.0% false discovery rate, 1918 proteins were identified; 775 were annotated as membrane proteins and 720 predicted to contain transmembrane spanning regions. Flow cytometry was used to validate cell surface expression of selected proteins. Junctional adhesion molecule 1 expression was shared by BG01, BG02 and abBG02 hESC lines. Dysferlin expression was specific to the WA09 hESC line and not the derived neural or mesenchymal progenitors. Ciliary neurotrophic factor receptor distinguished WA09-derived human neural progenitor cells from the parent hESC population, and WA09-derived mesenchymal progenitor cells. This study expands the current membrane protein data set for hESCs.     

Gene expression profiles of human inner cell mass cells and embryonic stem cells

Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of preimplantation human blastocysts obtained on days 5–6 following fertilization. Based on their derivation, they were once thought to be the equivalent of the ICM. Recently, however, studies in mice reported the derivation of mouse embryonic stem cell lines from the epiblast; these epiblast lines bear significant resemblance to human embryonic stem cell lines in terms of culture, differentiation potential and gene expression. In this study, we compared gene expression in human ICM cells isolated from the blastocyst and embryonic stem cells. We demonstrate that expression profiles of ICM clusters from single embryos and hESC populations were highly reproducible. Moreover, comparison of global gene expression between individual ICM clusters and human embryonic stem cells indicated that these two cell types are significantly different in regards to gene expression, with fewer than one half of all genes expressed in both cell types. Genes of the isolated human inner cell mass that are upregulated and downregulated are involved in numerous cellular pathways and processes; a subset of these genes may impart unique characteristics to hESCs such as proliferative and self-renewal properties.     

Diverse hematopoietic potentials of five human embryonic stem cell lines

Despite a growing body of literature concerning the hematopoietic differentiation of human embryonic stem cells (hESCs), the full hematopoietic potential of the majority of existing hESC lines remains unknown. In this study, the hematopoietic response of five NIH-approved hESC lines (H1, hSF6, BG01, BG02, and BG03) was compared. Our data show that despite expressing similar hESC markers under self-renewing conditions and initiating mesodermal differentiation under spontaneous differentiation conditions, marked differences in subsequent hematopoietic differentiation potential among these lines existed. A high degree of hematopoietic differentiation was attained only by H1 and BG02, whereas this process appeared to be abortive in nature for hSF6, BG01, and BG03. This difference in hematopoietic differentiation predisposition was readily apparent during spontaneous differentiation, and further augmented under hematopoietic-inducing conditions. This predisposition appeared to be intrinsic to the specific hESC line and independent of passage number or gender karyotype. Interestingly, H1 and BG02 displayed remarkable similarities in their kinetics of hematopoietic marker expression, hematopoietic colony formation, erythroid differentiation, and globin expression, suggesting that a similar, predetermined differentiation sequence is followed. The identification of intrinsic and extrinsic factors governing the hematopoietic differentiation potential of hESCs will be of great importance for the putative clinical utility of hESC lines.     

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     

Derivation and characterization of human embryonic stem cell lines from poor quality embryos

Poor quality embryos discarded from in vitro fertilization （IVF） laboratories are good sources for deriving human embryonic stem cell （hESC） lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses （ICMs） could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods （P〉0.05）. As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.     

Quantitative identification of teratoma tissues formed by human embryonic stem cells with TeratomEye

An automated vision system, TeratomEye, was developed for the identification of three representative tissue types: muscle, gut and neural epithelia which are commonly found in teratomas formed from human embryonic stem cells. Muscle tissue, a common structure was identified with an accuracy of 90.3% with high specificity and sensitivity greater than 90%. Gut epithelia were identified with an accuracy of 87.5% with specificity and sensitivity greater than 80%. Neural epithelia which were the most difficult structures to distinguish gave an accuracy of 47.6%. TeratomEye is therefore useful for the automated identification of differentiated tissues in teratoma sections.     

Effective surface-based cryopreservation of human embryonic stem cells by vitrification

Human embryonic stem cells (hESCs) are candidates for many applications in the areas of regenerative medicine, tissue engineering, basic scientific research as well as pharmacology and toxicology. However, use of hESCs is limited by their sensitivity to freezing and thawing procedures. Hence, this emerging science needs new, reliable preservation methods for the long-term storage of large quantities of functional hESCs remaining pluripotent after post-thawing and culturing.Here, we present a highly efficient, surface based vitrification method for the cryopreservation of large numbers of adherent hESC colonies, using modified cell culture substrates. This technique results in much better post-thaw survival rate compared to cryopreservation in suspension and allows a quick and precise handling and storage of the cells, indicating low differentiation rates.     

Cloned mice and embryonic stem cell establishment from adult somatic cell

Cloning methods are now well described and becoming routine. Yet the frequency at which cloned offspring are produced remains below 2% irrespective of nucleus donor species or cell type. Especially in the mouse, few laboratories can make clones from adult somatic cells, and most mouse strains never succeed to produce cloned mice. On the other hand, nuclear transfer can be used to generate embryonic stem (ntES) cell lines from a patient's own somatic cells. We have shown that ntES cells can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. Several reports have already demonstrated that ntES cells can be used in regenerative medicine in order to rescue immune deficient or infertile phenotypes. However, it is unclear whether ntES cells are identical to fertilized embryonic stem (ES) cells. In general, ntES cell techniques are expected to be applicable to regenerative medicine, however, these techniques can also be used for the preservation of the genetic resources of mouse strains instead of preserving such resources in embryos, oocytes or spermatozoa. This review seeks to describe the phenotype, application, and possible abnormalities of cloned mice and ntES cell lines.     

Automated maintenance of embryonic stem cell cultures

Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.     

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

We previously showed that Wnt3a could stimulate human embryonic stem （hES） cell proliferation and affect cell fate determination. In the absence of feeder cell--derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wntl and Wnt5a. While Wntl displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wntl enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wntl gene in immortalized human adult fibroblast （HAFi） cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt tmnsgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells （W3R） that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.     

Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.