Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation¹. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity⁴. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types^5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.     

人胚胎干细胞在3种培养体系中的生长

人胚胎干细胞(human embryonic stem cells.hESCs)的培养一直是干细胞研究的重要内容.用本实验室独立建系的两株hESCs,建立3种不同的培养体系:小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)做饲养层,永生化人成纤维细胞(immortalized human adult fibroblasts,HAFi)做饲养层,无饲养层条件培养基培养体系(condition medium,CM),观察在3种培养体系中,干细胞的增殖和分化情况.发现3种培养体系中的hESCs都可以表达一致的生物学特性,但也有不同之处,相对于CM干细胞在MEFs和HAFi饲养层体系的分化率低,增殖快;但MEFs来源于鼠类是异源细胞,HAFi虽不舍鼠源性成分却繁殖很慢;无饲养层的体系便于操作,无外源细胞存在.实验所得出的结果可以引导研究人员针对于临床、科研不同的需要,选择最适合的培养体系.     

A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers

Mouse embryonic fibroblasts (MEFs) are widely used to prepare feeder layers for culturing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. Transportation lesions and exorbitant prices make the commercially obtained MEFs unsuitable for long term research. The aim of present study is to establish a method, which enables researchers to gain MEFs from mice and establish feeder layers by themselves in ordinary laboratories. MEFs were isolated from ICR mouse embryos at 12.5–17.5 day post-coitum (DPC) and cultured in vitro. At P2–P7, the cells were inactivated with mitomycin C or by X-ray irradiation. Then they were used to prepare feeder layers. The key factors of the whole protocol were analyzed to determine the optimal conditions for the method. The results revealed MEFs isolated at 12.5–13.5 DPC, and cultured to P3 were the best choice for feeder preparation, those P2 and P4–P5 MEFs were also suitable for the purpose. The P3–P5 MEFs treated with 10 μg/ml of mitomycin C for 3 h, or irradiated with X-ray at 1.5 Gy/min for 25 Gy were the most suitable feeder cells. Treating MEFs with 10 μg/ml of mitomycin C for 2.5 h, 15 μg/ml for 2.0 h, or irradiating the cells with 20 Gy of X-ray at 2.0 Gy/min could all serve as alternative methods for P3–P4 cells. Our study provides a reliable and economical way to obtain large amount of qualified MEFs for long term research of ESCs or iPSCs.     

Targeted Proteomics of the Secretory Pathway Reveals the Secretome of Mouse Embryonic Fibroblasts and Human Embryonic Stem Cells

Proteins endogenously secreted by human embryonic stem cells (hESCs) and those present in hESC culture medium are critical regulators of hESC self-renewal and differentiation. Current MS-based approaches for identifying secreted proteins rely predominantly on MS analysis of cell culture supernatants. Here we show that targeted proteomics of secretory pathway organelles is a powerful alternate approach for interrogating the cellular secretome. We have developed procedures to obtain subcellular fractions from mouse embryonic fibroblasts (MEFs) and hESCs that are enriched in secretory pathway organelles while ensuring retention of the secretory cargo. MS analysis of these fractions from hESCs cultured in MEF conditioned medium (MEF-CM) or MEFs exposed to hESC medium revealed 99 and 129 proteins putatively secreted by hESCs and MEFs, respectively. Of these, 53 and 62 proteins have been previously identified in cell culture supernatants of MEFs and hESCs, respectively, thus establishing the validity of our approach. Furthermore, 76 and 37 putatively secreted proteins identified in this study in MEFs and hESCs, respectively, have not been reported in previous MS analyses.The identification of low abundance secreted proteins via MS analysis of cell culture supernatants typically necessitates the use of altered culture conditions such as serum-free medium. However, an altered medium formulation might directly influence the cellular secretome. Indeed, we observed significant differences between the abundances of several secreted proteins in subcellular fractions isolated from hESCs cultured in MEF-CM and those exposed to unconditioned hESC medium for 24 h. In contrast, targeted proteomics of secretory pathway organelles does not require the use of customized media. We expect that our approach will be particularly valuable in two contexts highly relevant to hESC biology: obtaining a temporal snapshot of proteins secreted in response to a differentiation trigger, and identifying proteins secreted by cells that are isolated from a heterogeneous population.Human embryonic stem cells (hESCs)¹ are pluripotent cells isolated from the inner cell mass of a pre-implantation blastocyst stage embryo (1). They have potential applications in regenerative medicine, are an attractive source of human cells for drug evaluation, and are useful models for understanding human development. The self-renewal or differentiation of hESCs is controlled by endogenous proteins secreted by hESCs and by exogenous factors present in cell culture medium (2, 3). For instance, hESCs are routinely cultured on feeder layers of mouse embryonic fibroblasts (MEFs) or on Matrigel-coated plates in mouse embryonic fibroblast–conditioned medium (MEF-CM). In these cases, cytokines secreted by MEFs and present in MEF-CM, together with cytokines and extracellular matrix (ECM) proteins secreted by hESCs, form a localized microenvironment that regulates hESC fate.The comprehensive identification of proteins secreted by MEFs and hESCs—their cellular secretome—can help unravel the molecular mechanisms that regulate hESC fate. Yet the use of MS-based approaches for secretome analysis remains challenging. In general, secretome studies of various cell types have relied on MS analysis of cell culture supernatants (reviewed in Ref. 4). However, such an approach typically results in the identification of small numbers of extracellular proteins. This was indeed the case with MS analysis of conditioned medium (CM) from MEFs or other feeder cells that support the maintenance of undifferentiated hESCs (5–8). A low abundance of secreted proteins of interest and a high concentration of serum proteins in cell culture media significantly impede MS analysis. To overcome these limitations, Bendall et al. implemented an iterative-exclusion MS (IE-MS) strategy, in conjunction with the use of medium without serum or serum replacer, for the identification of proteins secreted by MEFs and hESCs (2). Using this approach, large numbers of previously unreported proteins secreted by MEFs and hESCs could be identified, showing that IE-MS is a powerful strategy for the identification of low abundance proteins. However, the use of medium without serum or serum replacer for secretomic analysis can be problematic. Specifically, the use of a “blank” or serum-free medium might alter cellular physiology and, consequently, the profile of secreted proteins. Indeed, we observe that hESCs are highly prone to apoptosis under such growth conditions. Moreover, an analysis of the cell culture supernatant is not specifically targeted toward endogenously secreted ECM proteins, which are also an important component of the cellular microenvironment. ECM proteins form a matrix that associates with the cell and might not be present in the cell culture supernatant. Moreover, many growth factors are known to be sequestered by ECM proteins and might not be released into the culture medium (9). Here we present a rigorous evaluation of an alternate strategy to interrogate the entire cellular secretome, including cytokines and ECM proteins. Notably, our approach does not require the use of customized media lacking serum and serum replacers, and it is compatible with cell culture systems utilizing media of unknown or poorly defined composition, such as CM from MEFs.To identify the secretome of MEFs and hESCs, we carried out an MS analysis of their subcellular fractions that were enriched in secretory pathway organelles. The secretory pathway comprises the endoplasmic reticulum (ER), the Golgi apparatus, and the associated transport vesicles. Detailed MS analysis of these organelles identifies the secretory cargo (i.e. proteins destined to be secreted) in addition to the secretory pathway proteome (10). Indeed, we have previously identified several secreted proteins in hESCs as a result of contamination by the ER and Golgi (11) in our subcellular fractions. In light of these reports, we hypothesized that targeted proteomic analysis of the secretory pathway is a viable approach for comprehensive characterization of the cellular secretome. Accordingly, we developed protocols to isolate subcellular fractions enriched in the ER and Golgi compartments from MEFs and hESCs, and we subsequently carried out MS analysis on these samples. Several proteins secreted by MEFs and hESCs could be identified in this manner. Strikingly, the numbers of proteins identified were comparable to those obtained with the highly efficient IE-MS approach. Furthermore, we also show that short-term changes in medium composition affect the profile and quantitative levels of several proteins that transit through the secretory pathway, including secreted and membrane proteins. Taken together, our results validate the use of targeted secretory pathway proteomics as a powerful alternate approach to interrogate the cellular secretome.     

昆明小鼠胚胎干细胞滋养层制备条件的实验研究

目的:建立小鼠胚胎成纤维细胞(MEFS)滋养层,用于昆明小鼠胚胎干细胞的培养。方法:取妊娠13.5的胎鼠,采用组织消化法分离培养出原代成纤维细胞,对MEFs的生长形态、生长曲线及分裂指数进行观察;MTT法筛选丝裂霉素C(MMC)作用的最佳浓度和时间;取妊娠3.5d的囊胚在经MMC处理的饲养层上培养,观察胚胎干细胞集落生成情况。结果:MEFS为一种贴壁生长且增殖速度较快的细胞,第三代细胞增殖旺盛,第5代以后细胞开始变形并趋于衰老;MMC能抑制胚胎成纤维细胞的增殖,最佳的作用浓度和时间是10ug/ml作用2.5～4h,20ug/ml作用1-2.5h。妊娠3.5d小鼠囊胚在饲养层上培养能形成典型的"鸟巢"状干细胞集落,并可维持胚胎干细胞的正常形态且不发生分化。结论:这种方法制备的滋养细胞层适用于胚胎干细胞的培养。     

胰蛋白酶消化法和组织块法原代培养人包皮成纤维细胞的比较

小鼠胚胎成纤维细胞(MEFs)是目前国际上公认的人胚胎干细胞(hESCs)体外培养的饲养层细胞,但MEFs存在寿命短、动物源性污染等问题,需要探索适于hESCs体外培养且寿命长的人源性饲养层.采用胰蛋白酶消化法和组织块法分别原代培养人包皮成纤维细胞(hFFs),探索hFFs原代培养的最佳方法,为hFFs作为饲养层在hESCs研究中的应用提供可靠的科学依据;通过倒置显微镜下观察其生长状态和免疫细胞化学染色鉴定,结果显示两种原代培养方法获得的hFFs的形态及生物学特性均符合成纤维细胞;通过测定细胞生长曲线及MTT法检测,结果显示两种原代培养方法获得的不同代数的hFFs均具有较高的增殖活性,传代10余代仍能保持较好的细胞形态,可以制备成饲养层用于hESCs的研究.     

Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system

The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 μl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs.     

The piggyBac Transposon-Mediated Expression of SV40 T Antigen Efficiently Immortalizes Mouse Embryonic Fibroblasts (MEFs)

Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs’ basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.     

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.     

An In Vitro Mechanism Study on the Proliferation and Pluripotency of Human Embryonic Stems Cells in Response to Magnesium Degradation

Magnesium (Mg) is a promising biodegradable metallic material for applications in cellular/tissue engineering and biomedical implants/devices. To advance clinical translation of Mg-based biomaterials, we investigated the effects and mechanisms of Mg degradation on the proliferation and pluripotency of human embryonic stem cells (hESCs). We used hESCs as the in vitro model system to study cellular responses to Mg degradation because they are sensitive to toxicants and capable of differentiating into any cell types of interest for regenerative medicine. In a previous study when hESCs were cultured in vitro with either polished metallic Mg (99.9% purity) or pre-degraded Mg, cell death was observed within the first 30 hours of culture. Excess Mg ions and hydroxide ions induced by Mg degradation may have been the causes for the observed cell death; hence, their respective effects on hESCs were investigated for the first time to reveal the potential mechanisms. For this purpose, the mTeSR®1 hESC culture media was either modified to an alkaline pH of 8.1 or supplemented with 0.4–40 mM of Mg ions. We showed that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation. At all tested Mg ion dosages, the hESCs grew to confluency and retained pluripotency as indicated by the expression of OCT4, SSEA3, and SOX2. When the supplemental Mg ion dosages increased to greater than 10 mM, however, hESC colony morphology changed and cell counts decreased. These results suggest that Mg-based implants or scaffolds are promising in combination with hESCs for regenerative medicine applications, providing their degradation rate is moderate. Additionally, the hESC culture system could serve as a standard model for cytocompatibility studies of Mg in vitro, and an identified 10 mM critical dosage of Mg ions could serve as a design guideline for safe degradation of Mg-based implants/scaffolds.     

High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling

Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However, insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs), which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore, we investigated the effects of sodium fluoride (NaF) on the proliferation, differentiation and viability of H9 hESCs. For the first time, we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology, mitochondrial membrane potential (MMP), caspase activities, cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway, coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a p-JNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs.     

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。     

小鼠胚胎干细胞的培养

目的:建立小鼠胚胎干细胞(embryonic stem cells,ES)的培养方法。方法:制备G418抗性的原代小鼠胚胎成纤维细胞,经丝裂霉素C处理后成滋养层细胞,将小鼠胚胎干细胞复苏后,应用含白血病抑制因子的ES细胞培养液,培养小鼠ES细胞,观察集落的生长情况,并在光镜下观察细胞形态。结果:小鼠胚胎成纤维细胞生长良好,ES细胞呈克隆状生长,且保持未分化状态。结论:建立了小鼠胚胎干细胞培养的有效方法,为下一步基因打靶奠定基础。     

Attachment and growth of human embryonic stem cells on microcarriers

The use of human embryonic stem cells (hESCs) for cell-based therapies will require large quantities of genetically stable pluripotent cells and their differentiated progeny. Traditional hESC propagation entails adherent culture and is sensitive to enzymatic dissociation. These constraints hamper modifying method from 2-dimensional flat-bed culture, which is expensive and impractical for bulk cell production. Large-scale culture for clinical use will require innovations such as suspension culture for bioprocessing. Here we describe the attachment and growth kinetics of both murine embryonic stem cells (mESCs) and hESCs on trimethyl ammonium-coated polystyrene microcarriers for feeder-free, 3-dimensional suspension culture. mESCs adhered and expanded according to standard growth kinetics. For hESC studies, we tested aggregate (collagenase-dissociated) and single-cell (TrypLE-dissociated) culture. Cells attached rapidly to beads followed by proliferation. Single-cell cultures expanded 3-fold over approximately 5 days, slightly exceeding that of hESC aggregates. Importantly, single-cell cultures were maintained through 6 passages with a 14-fold increase in cell number while still expressing the undifferentiated markers Oct-4 and Tra 1-81. Finally, hESCs retained their capacity to differentiate towards pancreatic, neuronal, and cardiomyocyte lineages. Our studies provide proof-of-principle of suspension-based expansion of hESCs on microcarriers, as a novel, economical and practical feeder-free means of bulk hESC production.     

Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products

Human embryonic stem cells (hESCs) can serve as an unlimited cell source for cellular transplantation and tissue engineering due to their prolonged proliferation capacity and their unique ability to differentiate into derivatives of all three-germ layers. In order to reliably and safely produce hESCs, use of reagents that are defined, qualified, and preferably derived from a non-animal source is desirable. Traditionally, mouse embryonic fibroblasts (MEFs) have been used as feeder cells to culture undifferentiated hESCs. We recently reported a scalable feeder-free culture system using medium conditioned by MEFs. The base and conditioned medium (CM) still contain unknown bovine and murine-derived components, respectively. In this study, we report the development of a hESC culture system that utilizes a commercially available serum-free medium (SFM) containing human sourced and recombinant proteins supplemented with recombinant growth factor(s) and does not require conditioning with feeder cells. In this system, which employs human laminin coated surface and high concentration of hbFGF, the hESCs maintained undifferentiated hESC morphology and had a twofold increase in expansion compared to hESCs grown in MEF-CM. The hESCs also expressed surface markers SSEA-4 and Tra-1-60 and maintained expression of hTERT, Oct4, and Cripto genes similar to cells cultured in MEF-CM. In addition, hESCs maintained in this culture system were able to differentiate in vitro and in vivo into cells of all three-germ layers. The cells maintained a normal karyotype after prolonged culture in SFM. In summary, this study demonstrates that the hESCs cultured in defined non-conditioned serum-free medium (NC-SFM) supplemented with growth factor(s) retain the characteristics and replicative potential of hESCs. The use of defined culture system with NC-SFM on human laminin simplifies scale-up and allows for reproducible generation of hESCs under defined and controlled conditions that would serve as a starting material for production of hESC derived cells for therapeutic use.     

Epigenetic Regulation of miR-302 by JMJD1C Inhibits Neural Differentiation of Human Embryonic Stem Cells

It has been recently reported that the regulatory circuitry formed by OCT4, miR-302, and NR2F2 controls both pluripotency and neural differentiation of human embryonic stem cells (hESCs). We show here that JMJD1C, a histone 3 lysine 9 (H3K9) demethylase expressed in hESCs, directly interacts with this circuitry. hESCs with stable knockdown of JMJD1C remain pluripotent while having reduced miR-302 expression, decreased BMP signaling, and enhanced TGFβ signaling. JMJD1C binds to the miR-302 promoter and reduces H3K9 methylation. Withdrawal of basic fibroblast growth factor (bFGF) from the culture induces neural differentiation of the knockdown, but not the control, cells within 3 days, accompanied by elevated NR2F2 expression. This can be attenuated with miR-302 mimics or an H3K9 methytransferase inhibitor. Together, our findings suggest that JMJD1C represses neural differentiation of hESCs at least partially by epigenetically sustaining miR-302 expression and that JMJD1C knockdown is sufficient to trigger neural differentiation upon withdrawal of exogenous bFGF.     

Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30^Hi-GCTM-2^Hi hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30^Neg-GCTM-2^Neg). In a further study, pluripotent stem cell-free samples of differentiated TG30^Neg-GCTM-2^Neg cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30^Hi-GCTM-2^Hi hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched populations of hPSC as inputs for differentiation assays and to rid potentially tumorigenic (or residual) hESC from derivative cell populations.     

Effect of Antibiotics against Mycoplasma sp. on Human Embryonic Stem Cells Undifferentiated Status,Pluripotency, Cell Viability and Growth

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of Plasmocin^TM and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days Plasmocin^TM 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with Plasmocin^TM 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that Plasmocin^TM and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.