Localized decrease of beta-catenin contributes to the differentiation of human embryonic stem cells

Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, β-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of β-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down β-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of β-catenin contributes to the spatial pattern of differentiation in hESC colonies.     

人诱导性多潜能干细胞(iPS细胞)系的建立

掌握建立人iPS细胞系(induced pluripotent stem cells,iPSCs)的技术,以便为人肿瘤细胞重编程为iPS细胞建立技术平台.在人胚胎干细胞的培养条件下,通过携带Oct4、Sox2、c-Myc、Klf44个混合因子的慢病毒感染人皮肤成纤维细胞(CCD-1079SK细胞),从而诱导成干细胞样的克隆.根据人胚胎干细胞的特性进行如下鉴定:克隆形态、碱性磷酸酶活性、核型和CCD-1079SK细胞来源的克隆拟胚体(embryoid bodies,EBs)形成及分化等.结果显示,在人胚胎干细胞的培养环境中,导入Oct4、Sox2、c-Myc、Klf44个因子的CCD-1079SK细胞产生了一株iPSC克隆,这株iPSC克隆在细胞形态、增殖能力、胚胎细胞特异性表面抗原以及基因表达与人胚胎干细胞相似,此外,iPSC克隆在体外悬浮培养中形成拟胚体并分化成3个胚层.人iPS细胞系的成功建立为利用iPS细胞技术开展肿瘤细胞重编程研究奠定了坚实基础.     

Kinetics of cell death of frozen-thawed human embryonic stem cell colonies is reversibly slowed down by exposure to low temperature

A major challenge in the widespread application of hES (human embryonic stem) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of less than 5% as reported by previous studies. This study characterized cell death in frozen-thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (approximately 98%), as assessed by the trypan blue exclusion test. However, when the freshly thawed hES colonies were placed in a 37 degrees C incubator, there was a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed down by keeping the freshly thawed hES colonies at 4 degrees C, with more than 90% of cells remaining viable after 90 min of incubation at 4 degrees C. This effect was reversible upon re-exposing the cells to physiological temperatures. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 degrees C. Hence, our observations would strongly suggest involvement of a self-induced apoptotic mechanism, as opposed to cellular necrosis arising from cryoinjury.     

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.     

Gi-Coupled GPCR Signaling Controls the Formation and Organization of Human Pluripotent Colonies

Background

Reprogramming adult human somatic cells to create human induced pluripotent stem (hiPS) cell colonies involves a dramatic morphological and organizational transition. These colonies are morphologically indistinguishable from those of pluripotent human embryonic stem (hES) cells. G protein-coupled receptors (GPCRs) are required in diverse developmental processes, but their role in pluripotent colony morphology and organization is unknown. We tested the hypothesis that G_i-coupled GPCR signaling contributes to the characteristic morphology and organization of human pluripotent colonies.

Methodology/Principal Findings

Specific and irreversible inhibition of G_i-coupled GPCR signaling by pertussis toxin markedly altered pluripotent colony morphology. Wild-type hES and hiPS cells formed monolayer colonies, but colonies treated with pertussis toxin retracted inward, adopting a dense, multi-layered conformation. The treated colonies were unable to reform after a scratch wound insult, whereas control colonies healed completely within 48 h. In contrast, activation of an alternative GPCR pathway, G_s-coupled signaling, with cholera toxin did not affect colony morphology or the healing response. Pertussis toxin did not alter the proliferation, apoptosis or pluripotency of pluripotent stem cells.

Conclusions/Significance

Experiments with pertussis toxin suggest that G_i signaling plays a critical role in the morphology and organization of pluripotent colonies. These results may be explained by a G_i-mediated density-sensing mechanism that propels the cells radially outward. GPCRs are a promising target for modulating the formation and organization of hiPS and hES cell colonies and may be important for understanding somatic cell reprogramming and for engineering pluripotent stem cells for therapeutic applications.     

Neurotrophins mediate human embryonic stem cell survival

Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family, which mediate antiapoptotic signals. We show that three TRK ligands, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins, TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation.     

Human T lymphocyte colonies in agar: a comparison with other T cell assays in healthy subjects and cancer patients.

Colonies of human lymphocytes with T cell characteristics will grow in agar from repeated mitotic divisions with phytohaemagglutinin (PHA) stimulation. The colonies comprise spheres of tightly-packed cells with up to 500-1,000 blast-like cells in each colony. 65% of cells from pooled colonies bound AET-treated sheep red cells. 1,100-2,500 colonies/10(6) peripheral blood lymphocytes developed when cell donors were healthy but lower numbers (350-1,000 colonies/10(6) lymphocytes) were detected in blood from cancer patients. Comparison with other non-specific assays of cell-mediated immunity showed that while 66% of cancer patients were anergic (to five recall antigens) and 78% exhibited depressed mitotic activity in standard cultures with low dose PHA, 100% of these patients revealed T cell colony formation below normal. It is suggested that further studies of T lymphocyte colony-forming cells in healthy people and in a number of disease states may significantly advance our understanding of mechanisms of cell-mediated immunity.     

A proposed design for the cryopreservation of intact and adherent human embryonic stem cell colonies

Summary Recently, it was demonstrated that the application of slow-cooling cryopreservation protocols to adherent human embryonic stem (hES) cell colonies, cultured on matrigel or murine embryonic fibroblast feeder layers, resulted in marked improvement in postthaw viability and reduction in cell differentiation. However, the use of commercially available culture plates for this purpose presents several limitations. Most obviously, these plates are not designed for cryopreservation or to withstand the low temperatures encountered during liquid nitrogen cryopreservation, or both. The physical storage of cryopreserved plates is another consideration, in addition to difficulty in maintaining sterile conditions in liquid nitrogen storage and during the thaw phase in a water bath. Hence, a redesign of the cell culture plate for the cryopreservation of adherent hES cell colonies is proposed. In this model, a culture plate made of synthetic materials resistant to storage at −196° C of liquid nitrogen is designed, with readily attachable screw-cap culture wells that function as a replacement for cryovial storage. The detachable wells facilitate storage and after thawing can easily be reattached to a specially designed holding plate. Currently, there are no commercially available cell culture plates using this design concept. The proposed design is envisioned to facilitate the cryopreservation of intact adherent hES cell colonies that could assist the development of automated systems for handling bulk quantities of cells.     

Comparative characteristics of three human embryonic stem cell lines

Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.     

Semilethal spotted mutants of Chlorella]

Mutant colonies of Chorella vulgaris Beijer, having the region of colourless cells either in the center or in the periphery of a colony, are found. Spontaneous mutation rate varied within 1-40-10(5). The size of a mutant colony comprised about 1/2-1/3 of the diameter of a middle initial strain colony. Colonies with a mutant sector were also observed besides completely mutant colonies. Ways of the formation of mutant phenotypes are studied. It is found that from the initial spotted green colony those of the type "death in the periphery" and, through a number of intermediate stages, "death in the center" are formed. The colony development can stop at some successive stage or to result in the death of all the cells. The period of forming the mutant phenotype is found to depend on the colony growth rate.     

Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro

Colonies of chick retinal pigmented epithelial (RPE) cells offer an excellent model system for studying the organization of cytoskeleton in sheets of differentiating epithelial cells. The cells occupying the center of the colony resemble RPE cells in vivo and are cuboidal, pigmented, and relatively nonadherent while those toward the periphery gradually become flatter, nonpigmented, motile, and strongly adherent to the substratum. Immunofluorescence microscopy with antiserum against chicken erythrocyte alpha-spectrin reveals that this protein is present in the cortex of RPE cells in all parts of the colony. It is neither concentrated in, nor excluded from the regions occupied by the major microfilament bundles, and its distribution is not related to the adhesion patterns visualized by surface reflection interference microscopy. In contrast, the distribution of vinculin is closely correlated with the adhesiveness of RPE cells in different parts of the colony. Immunofluorescence microscopy reveals that in the RPE cells vinculin may be diffusely distributed in the cytoplasm; present in a cortical band outlining the cell borders; and present in focal contacts and adhesions. The distribution of vinculin is affected by the length of time the colonies grow in culture, by the degree of cell packing and by the adhesiveness of cells to the substratum. In RPE cells grown in vitro for short periods (less than or equal to 3 days) vinculin is found in focal contacts and adhesions in both the undifferentiated, well spread peripheral cells as well as in the differentiated, polygonally packed central cells of the colony. In RPE cells cultured for longer periods (greater than or equal to 14 days) vinculin is present in focal contacts and adhesions only in strongly adherent, undifferentiated cells at the edge of the colony. In packed central cells of both short- and long-term cultures vinculin is found in the cortical band which circumscribes the apical ends of cells at the level of the adherens type intercellular junctions. Its appearance in the cortical bands does not depend on the length of time the colonies are grown in vitro but on the presence of cell-cell contacts resulting from an increased degree of cell packing within the central part of the colony. These results are discussed in relation to the development and the role of extracellular matrix in determining the adhesiveness of RPE cells in vitro.     

ROCK inhibition enhances the recovery and growth of cryopreserved human embryonic stem cells and human induced pluripotent stem cells

Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study, we demonstrate that Y‐27632, a specific inhibitor of Rho kinase (ROCK) activity, significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore, treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably, hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y‐27632. Additionally, we determined that Y‐27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus, Y‐27632 provides a means to “kick‐start” slow‐growing human pluripotent stem cells, especially after being thawed from frozen stocks. Together, these results argue that Y‐27632 is a useful tool in overcoming obstacles to studies involving the cultivation of both hES cells and human iPS cells. Mol. Reprod. Dev. 76: 722–732, 2009. © 2009 Wiley‐Liss, Inc.     

Persistence of cell cycle times over many generations as determined by heritability of colony sizes of ras oncogene-transformed and non-transformed cells

Abstract. The persistence of cell lifetimes during about 10 successive cell generations was investigated by comparing the number of cells in primary colonies and in secondary colonies derived from primary colonies. Primary colonies were grown from single cells for 3 or 4 days (a time equivalent to an average of five cell generations) and the number of cells in each primary colony determined. Cells in each primary colony were dispersed to initiate secondary colonies, grown for the same time, and the number of cells in secondary colonies determined. Several criteria were used to compare primary and related secondary colonies, the most informative was found to be regression and correlation coefficients between number of cells in primary colonies and mean numbers of cells in related secondary colonies. For two non-transformed mouse fibroblast cell lines, NIH 3T3 and BALB 3T3, the regression and correlation coefficients of cell number in primary and secondary colonies were positive. This suggests inheritance of cell lifetimes over many cell generations. After the addition of an activated ras oncogene (human cellular Harvey ras , or viral Kirsten ras ) some regression and correlation coefficients changed in magnitude but all remained positive. Comparison of experimental data and the results of computer simulations suggest that several models of inheritance of cell lifetimes are not adequate to explain the results, including a model of independence between lifetimes of mother and daughter cells and the common model that describes daughter cells as inheriting the lifetime of their mother with deviation. Simulations do suggest that cell lifetimes are inherited within clones as deviation from the lifetime of the initial cell, and that the ras oncogene does not destroy persistence within clones but does increase heterogeneity of cell lifetimes.     

The Transforming Parasite Theileria Co-opts Host Cell Mitotic and Central Spindles to Persist in Continuously Dividing Cells

The protozoan parasite Theileria inhabits the host cell cytoplasm and possesses the unique capacity to transform the cells it infects, inducing continuous proliferation and protection against apoptosis. The transforming schizont is a multinucleated syncytium that resides free in the host cell cytoplasm and is strictly intracellular. To maintain transformation, it is crucial that this syncytium is divided over the two daughter cells at each host cell cytokinesis. This process was dissected using different cell cycle synchronization methods in combination with the targeted application of specific inhibitors. We found that Theileria schizonts associate with newly formed host cell microtubules that emanate from the spindle poles, positioning the parasite at the equatorial region of the mitotic cell where host cell chromosomes assemble during metaphase. During anaphase, the schizont interacts closely with host cell central spindle. As part of this process, the schizont recruits a host cell mitotic kinase, Polo-like kinase 1, and we established that parasite association with host cell central spindles requires Polo-like kinase 1 catalytic activity. Blocking the interaction between the schizont and astral as well as central spindle microtubules prevented parasite segregation between the daughter cells during cytokinesis. Our findings provide a striking example of how an intracellular eukaryotic pathogen that evolved ways to induce the uncontrolled proliferation of the cells it infects usurps the host cell mitotic machinery, including Polo-like kinase 1, one of the pivotal mitotic kinases, to ensure its own persistence and survival.     

Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.     

Human embryonic stem cells are pre‐mitotically committed to self‐renewal and acquire a lengthened G1 phase upon lineage programming

Self‐renewal of human embryonic stem (hES) cells proceeds by a unique abbreviated cell cycle with a shortened G1 phase and distinctions in molecular cell cycle regulatory parameters. In this study, we show that early lineage‐commitment of pluripotent hES cells modifies cell cycle kinetics. Human ES cells acquire a lengthened G1 within 72 h after lineage‐programming is initiated, as reflected by loss of the pluripotency factor Oct4 and alterations in nuclear morphology. In hES cells that maintain the pristine pluripotent state, we find that autocrine mechanisms contribute to sustaining the abbreviated cell cycle. Our data show that naïve and mitotically synchronized pluripotent hES cells are competent to initiate two consecutive S phases in the absence of external growth factors. We conclude that short‐term self‐renewal of pluripotent hES cells occurs autonomously, in part due to secreted factors, and that pluripotency is functionally linked to the abbreviated hES cell cycle. J. Cell. Physiol. 222:103–110, 2010. © 2009 Wiley‐Liss, Inc.