Establishment and characteristics of new human embryonic stem cell subline SC6-FF in an allogenic feeder-free culture system

The novel human embryonic stem cell (hESC) subline SC6-FF was derived from SC6 cells in an allogenic feeder-free culture system. Key components of the feeder-free culture system were extracellular matrix proteins and conditioned medium from the mesenchymal stem cell line SC5-MSC. These conditions are allogenic for SC6-FF cells. SC6-FF subline underwent more than one hundred cell population doublings and retained a normal diploid karyotype; 46, XX. The average population doubling time was 23.7 ± 0.8 h, similar to that of the parent SC6 line. The presence of undifferentiated hESC markers (alkaline phosphatase activity, Oct-4, SSEA-4, and TRA-1-60) was verified by histochemistry and immunofluorescence. Cells were distinguished from parental cells in size and morphology as a result of spontaneous differentiation. These cells exhibited the ability to differentiate into derivates of three germ layers by expressing common markers of the ectoderm (alpha-fetoprotein), mesoderm (a-actinin) and endoderm (a-fetoprotein) cells. We could conclude that characteristics of the novel feeder-free SC6-FF subline correspond to the status of human embryonic stem cells.     

Dynamic Status of REST in the Mouse ESC Pluripotency Network

Background

REST is abundantly expressed in mouse embryonic stem cells (ESCs). Many genome-wide analyses have found REST to be an integral part of the ESC pluripotency network. However, experimental systems have produced contradictory findings: (1) REST is required for the maintenance of ESC pluripotency and loss of REST causes increased expression of differentiation markers, (2) REST is not required for the maintenance of ESC pluripotency and loss of REST does not change expression of differentiation markers, and (3) REST is not required for the maintenance of ESC pluripotency but loss of REST causes decreased expression of differentiation markers. These reports highlight gaps in our knowledge of the ESC network.

Methods

Employing biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature.

Results

We show that Rest+/− and Rest−/− AB-1 mutant ESCs, which did not exhibit a role of REST in ESC pluripotency when cultured in the presence of feeder cells, did show impaired self-renewal when compared with the parental cells under feeder-free culture conditions, but only in early passage cells. In late passage cells, both Rest+/− and Rest−/− AB-1 ESCs restored pluripotency, suggesting a passage and culture condition-dependent response. Genome-wide analysis followed by biochemical validation supported this response and further indicated that the restoration of pluripotency was associated by increased expression of the ESC pluripotency factors. E14Tg2a.4 ESCs with REST-knockdown, which earlier showed a REST-dependent pluripotency when cultured under feeder-free conditions, as well as Rest−/− AB-1 ESCs, showed no REST-dependent pluripotency when cultured in the presence of either feeder cells or laminin, indicating that extracellular matrix components can rescue REST''s role in ESC pluripotency.

Conclusions

REST regulates ESC pluripotency in culture condition- and ESC line-dependent fashion and ESC pluripotency needs to be evaluated in a context dependent manner.     

Evaluation of unrestricted somatic stem cells as a feeder layer to support undifferentiated embryonic stem cells

The use of unrestricted somatic stem cells (USSCs) holds great promise for future clinical applications. Conventionally, mouse embryonic fibroblasts (MEFs) or other animal‐based feeder layers are used to support embryonic stem cell (ESC) growth; the use of such feeder cells increases the risk of retroviral and other pathogenic infection in clinical trials. Implementation of a human‐based feeder layer, such as hUSSCs that are isolated from human sources, lowers such risks. Isolated cord blood USSCs derived from various donors were used as a novel, supportive feeder layer for growth of C4mES cells (Royan C4 ESCs). Complete cellular characterization using immunocytochemical and flow cytometric methods were performed on murine ESCs (mESCs) and hUSSCs. mESCs cultured on hUSSCs showed similar cellular morphology and presented the same cell markers of undifferentiated mESC as would have been observed in mESCs grown on MEFs. Our data revealed these cells had negative expression of Stat3, Sox2, and Fgf4 genes while showing positive expression for Pou5f1, Nanog, Rex1, Brachyury, Lif, Lifr, Tert, B2m, and Bmp4 genes. Moreover, mESCs cultured on hUSSCs exhibited proven differentiation potential to germ cell layers showing normal karyotype. The major advantage of hUSSCs is their ability to be continuously cultured for at least 50 passages. We have also found that hUSSCs have the potential to provide ESC support from the early moments of isolation. Further study of hUSSC as a novel human feeder layer may lead to their incorporation into clinical methods, making them a vital part of the application of human ESCs in clinical cell therapy. Mol. Reprod. Dev. 79: 709–718, 2012. © 2012 Wiley Periodicals, Inc.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

Comparative characteristics of new mesenchymal stem cell lines derived from human embryonic stem cells, bone marrow and foreskin

New nonimmortalized fibroblast-like cell lines SC5-MSC and SC3a-MSC, FetMSC, FRSN were obtained from human embryonic stem cells (ESC), bone marrow of a 5-6-days embryo and foreskin of a 3-years-old boy, respectively. All the lines are successfully used as the feeder at human ESC cultivation. It is determined that the average cell population doublings time varies from 25.5 h for ISC5-MSC to 38.8 h for SC3a-MSC. Active proliferation of all the lines is also shown by the corresponding growth curves. Numerical and structural karyotypic analysis showed that these lines had normal karyotype: 46,XX (SC5-MSC and SC3a-MSC) and 46,XY (FetMSC and FRSN). To determine the status of the lines, their cell surface markers were analyzed by flow cytometry. This analysis revealed the presence of surface antigens CD44, CD73, CD90, CD105 and HLA-ABC, characteristic of human MSC, and the absence of CD34 and HLA-DR. Different lines were found to express CD117(c-kit) to a different level. Immunofluorescence and flow cytometry analysis did not detect TRA-1-60 and Oct-4, characteristic of human embryonic stem cells, and revealed interlinear variations in the level of SSEA, which did not depend on the cell origin. It is not clear yet whether these interlinear variations affect functional MSC status. In all the lines, immunofluorescence analysis showed the presence of the markers of early differentiation in the derivates of three germ layers which may allow MSC to be useful, in corresponding microenvironments, for reparation of tissue injures. Adipogenic and osteogenic differentiatiation of all cell lines has been shown.     

Electric pulses to prepare feeder cells for sustaining and culturing of undifferentiated embryonic stem cells

Current challenges in embryonic-stem cell (ESC) research include the inability of sustaining and culturing of undifferentiated ESCs over time. Growth-arrested feeder cells are essential to the culture and sustaining of undifferentiated ESCs, and they are currently prepared using gamma-radiation and chemical inactivation. Both techniques have severe limitations. In this study, we developed a new, simple and effective technique (pulsed electric fields, PEFs) to produce viable growth-arrested cells (RTS34st) and used them as high-quality feeder cells to culture and sustain undifferentiated zebrafish ESCs over time. The cells were exposed to 25 sequential 10-ns electric pulses (10nsEPs) of 25, 40 and 150 kV/cm with 1-s pulse interval, or 2 sequential 50-μs electric pulses (50μsEPs) of 2.83, 1.78 and 0.78 kV/cm with 5-s pulse interval, respectively. We found that the cellular effects of PEFs depended directly upon the duration, number and electric field strength of the pulses, showing the feasibility of tuning them to produce various types of growth-arrested cells for culturing undifferentiated ESCs. Both 10nsEPs of 40 kV/cm produced by a 10nsEP generator and 50μsEPs of 1.78 kV/cm provided by inexpensive and widely available conventional electroporators, generated high-quality growth-arrested feeder cells for proliferation of undifferentiated ESCs over time. PEFs can therefore be used to replace radiation and chemical inactivation methods for preparation of growth-arrested feeder cells for advancing ESC research.     

Immortalized feeders for the scale-up of human embryonic stem cells in feeder and feeder-free conditions

Human embryonic stem cells (hESC) are pluripotent cells that proliferate indefinitely in culture, whilst retaining their capacity for differentiation into different cell types. However, hESC cultures require culture in direct contact with feeder cells or conditioned medium (CM) from feeder cells. The most common source of feeders has been primary mouse embryonic fibroblast (MEF). In this study, we immortalized a primary MEF line with the E6 and E7 genes from HPV16. The immortal line, DeltaE-MEF, was able to proliferate beyond 7-9 passages and has an extended lifespan beyond 70 passages. When tested for its ability to support hESC growth, it was found that hESC continue to maintain the undifferentiated morphology for >40 passages both in co-culture with DeltaE-MEF and in feeder-free cultures supplemented with CM from DeltaE-MEF. The cultures also continue to express the pluripotent markers, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, alkaline phosphatase and maintain a normal karyotype. In addition, these hESC formed teratomas when injected into SCID mice. Lastly, we demonstrated the feasibility of scaling-up significant quantities of undifferentiated hESC (>10(8) cells) using DeltaE-MEF in cell factories. The results from this study suggest that immortalized feeders can provide a consistent and reproducible source of feeders for hESC expansion and research.     

山羊胚胎干细胞的分离与培养

对关中奶山羊配种后6～7天的桑椹胚和囊胚,分别采用全胚培养法、酶消化法和免疫外科法进行处理.将处理后的胚胎培养于小鼠胎儿成纤维细胞(MEF)饲养层上,分离培养山羊胚胎干细胞(Embryonic stem cell,ESC).对分离传代的山羊ESCs分别进行免疫组化染色,RT-PCR检测和体外诱导分化试验.结果表明.全胚培养法易于胚胎贴壁形成原代集落,采用全胚培养法获得的ESCs有一株目前已传至18代.山羊ESCs Nanong、Oct4、SSEA-3免疫组化染色呈阳性,SSEA-1免疫组化染色呈弱阳性,SSEA-4免疫组化染色呈阴性,RT-PCR检测显示其表达Nanog、Oct4、端粒酶、CD117.山羊ESCs经DMSO体外诱导可以向心肌细胞分化.这些试验均表明该细胞具有ESCs的生物学特性.     

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications.     

The Plasminogen Activation System Modulates Differently Adipogenesis and Myogenesis of Embryonic Stem Cells

Regulation of the extracellular matrix (ECM) plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiation in vitro is unknown. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system display reduced adipogenic capacities after induction of the gene. Furthermore, the adipogenic differentiation capacities of PAI-1^−/− induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms.     

A p38mapk-p53 cascade regulates mesodermal differentiation and neurogenesis of embryonic stem cells

Embryonic stem cells (ESCs) differentiate in vivo and in vitro into all cell lineages, and they have been proposed as cellular therapy for human diseases. However, the molecular mechanisms controlling ESC commitment toward specific lineages need to be specified. We previously found that the p38 mitogen-activated protein kinase (p38MAPK) pathway inhibits neurogenesis and is necessary to mesodermal formation during the critical first 5 days of mouse ESC commitment. This period corresponds to the expression of specific master genes that direct ESC into each of the three embryonic layers. By both chemical and genetic approaches, we found now that, during this phase, the p38MAPK pathway stabilizes the p53 protein level and that interfering directly with p53 mimics the effects of p38MAPK inhibition on ESC differentiation. Anti-p53 siRNA transient transfections stimulate Bcl2 and Pax6 gene expressions, leading to increased ESC neurogenesis compared with control transfections. Conversely, p53 downregulation leads to a strong inhibition of the mesodermal master genes Brachyury and Mesp1 affecting cardiomyogenesis and skeletal myogenesis of ESCs. Similar results were found with p53^−/− ESCs compared with their wild-type counterparts. In addition, knockout p53 ESCs show impaired smooth muscle cell and adipocyte formation. Use of anti-Nanog siRNAs demonstrates that certain of these regulations result partially to p53-dependent repression of Nanog gene expression. In addition to its well-known role in DNA-damage response, apoptosis, cell cycle control and tumor suppression, p53 has also been involved in vivo in embryonic development; our results show now that p53 mediates, at least for a large part, the p38MAPK control of the early commitment of ESCs toward mesodermal and neural lineages.     

Automated Identification and Location Analysis of Marked Stem Cells Colonies in Optical Microscopy Images

Embryonic stem cells (ESCs) are characterized by two remarkable peculiarities: the capacity to propagate as undifferentiated cells (self-renewal) and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency). Although the majority of ESCs divide without losing the pluripotency, it has become evident that ESC cultures consists of multiple cell populations highlighted by the expression of early germ lineage markers during spontaneous differentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity, we developed an analysis pipeline which can automatically process images of stem cell colonies in optical microscopy. The question we try to address is to find out the statistically significant preferred locations of the marked cells. We tested our algorithm on a set of images of stem cell colonies to analyze the expression pattern of the Zscan4 gene, which was an elite candidate gene to be studied because it is specifically expressed in subpopulation of ESCs. To validate the proposed method we analyzed the behavior of control genes whose pattern had been associated to biological status such as differentiation (EndoA), pluripotency (Pou5f1), and pluripotency fluctuation (Nanog). We found that Zscan4 is not uniformly expressed inside a stem cell colony, and that it tends to be expressed towards the center of the colony, moreover cells expressing Zscan4 cluster each other. This is of significant importance because it allows us to hypothesize a biological status where the cells expressing Zscan4 are preferably associated to the inner of colonies suggesting pluripotent cell status features, and the clustering between themselves suggests either a colony paracrine effect or an early phase of cell specification through proliferation. Also, the analysis on the control genes showed that they behave as expected.