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PubMed2005

Culture systems for pluripotent stem cells.

Pluripotent stem cells have the capacity to self renew and to differentiate to cells of the three somatic germ layers that comprise an organism. Embryonic stem cells are the most studied pluripotent stem cells. Pluripotent stem cells have also been derived from adult tissues. Both embryonic and adult stem cells represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self-renewal and differentiation capacity. In generating particular cell types for specific applications, it is important to direct their differentiation to the desired lineage. Challenges in expansion of undifferentiated stem cells for clinical applications include the removal of feeder layers and non-defined components in the culture medium. Our limited basic knowledge on the requirements for maintaining pluripotency of adult pluripotent stem cells and the lack of appropriate markers associated with pluripotency hinders the progress toward their wide spread application. In vitro differentiation of stem cells usually produces a mixed population of different cell lineages with the desired cell type present only at a small proportion. Use of growth factors that promote differentiation, expansion or survival of specific cell types is key in controlling the differentiation towards specific cell lineages. A variety of bioreactors for cell cultivation exist and can be readily adapted for stem cell cultivation and differentiation. They provide a well-controlled environment for studying the process of stem cell propagation and differentiation. Their wide use will facilitate the development of processes for stem cell application.

Animals↗
PubMed2002

A molecular view on pluripotent stem cells.

Pluripotent stem cells are undifferentiated cells that are capable of differentiating to all three embryonic germ layers and their differentiated derivatives. They are transiently found during embryogenesis, in preimplantation embryos and fetal gonads, or as established cell lines. These unique cell types are distinguished by their wide developmental potential and by their ability to be propagated in culture indefinitely, without loosing their undifferentiated phenotype. This short review intends to give a general overview on the pluripotent nature of embryo-derived stem cells with a focus on human embryonic stem cells.

Animals↗
PubMed2001

The end of the beginning for pluripotent stem cells.

Pluripotent stem cells can be expanded seemingly indefinitely in culture, maintain a normal karyotype and have the potential to generate any cell type in the body. As such they represent an incredible resource for the repair of diseased or damaged tissues in our bodies. These cells also promise to open a new window into the embryonic development of our species.

Animals↗
PubMed2004

Avian pluripotent stem cells.

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.

Animals↗
PubMed2005

Identification of developmental pluripotency associated 5 expression in human pluripotent stem cells.

Pluripotent embryonic germ cells (EGCs) can be derived from the culture of primordial germ cells (PGCs). However, there are no reports of gonocytes, following the stage of PGC development, becoming stem cell lines. To analyze the gene expression differences between PGCs and gonocytes, we performed cDNA subtractive hybridization with mouse gonads containing either of the two cell populations. We confirmed that developmental pluripotency associated 5 (Dppa5), originally found in mouse embryonic stem cells (ESCs) and mouse embryonic carcinoma cells (ECCs), was strongly expressed in mouse PGCs and the expression was rapidly downregulated during germ cell development. A human sequence homologous to Dppa5 was identified by bioinformatics approaches. Interestingly, human Dppa5 was expressed only in human PGCs, human EGCs, and human ESCs and was not detected in human ECCs. Its expression was downregulated during induced differentiation of human ESCs. These findings confirmed that Dppa5 is specifically and differentially expressed in human cells that have pluripotency. The results strongly suggest that Dppa5 may have an important role in stemness in human ESCs and EGCs and also can be used as a marker of pluripotent stem cells. Human pluripotent stem cells may have their own ways to be pluripotent, as opposed to the much uniform mouse stem cells.

Animals↗
PubMed2005

Clonal multilineage differentiation of murine common pluripotent stem cells isolated from skeletal muscle and adipose stromal cells.

Pluripotent stem cells (PSCs) with transdifferentiation capacity may provide useful therapeutic modalities in the areas of cellular restoration and regenerative medicine. The utility of PSCs depends on their ability to respond to different stimuli and to adapt to tissue-specific differentiation conditions. Given that a number of cells possessing characteristics of PSCs have been identified and isolated from several adult murine tissues, we hypothesized that a common PSC may exist in multiple murine tissues and that these cells may either reside permanently in specific sites or continue to circulate and colonize tissues as needed. Previous data from our laboratory suggest that PSCs exhibiting an immunophenotype of CD45(-)Sca-1(+)c-kit(-)Thy-1(+) can be isolated from multiple murine tissues and may represent putative common PSCs (CoPSCs). To investigate whether the multiple tissue differentiation potential observed with these cells resulted from the presence of different tissue-restricted progenitors within CD45(-)Sca-1(+)c-kit(-)Thy-1(+) cells or was the product of clonal differentiation of CoPSCs, clonality studies were performed. Single skeletal muscle (SM)-derived CoPSCs were expanded for 10 days, and progeny cells were split into three culture conditions designed to stimulate myogenic, adipogenic, and neurogenic differentiation. Analysis of 600 clones indicated that 2.16%, 0.83%, and 0.33% of the total number of plated single cells were capable of unipotent, bipotent, and tripotent differentiation, respectively, into combinations of myocytes, adipocytes, and neuronal cells. Given that SM-derived CoPSCs represent 4.78% of the total cells analyzed, tripotent CoPSCs made up 0.016% of the total muscle cells. Similar results were obtained in clonal analyses using adipose stromal cell (ASC)-derived CoPSCs, suggesting that both SM- and ASC-derived CoPSCs may be phenotypically and functionally identical. Taken together, these data demonstrate that a common PSC can be identified in different murine tissues and suggest that a small fraction of these cells are capable of clonal differentiation into multiple cell types.

Adipocytes↗
PubMed2004

Self-renewal of teratocarcinoma and embryonic stem cells.

Pluripotent stem cells derived from preimplantation embryos, primordial germ cells or teratocarcinomas are currently unique in undergoing prolonged symmetrical self-renewal in culture. For mouse embryonic stem (ES) cells, self-renewal is dependent on signals from the cytokine leukaemia inhibitory factor (LIF) and from either serum or bone morphogenetic proteins (BMPs). In addition to the extrinsic regulation of gene expression, intrinsic transcriptional determinants are also required for maintenance of the undifferentiated state. These include Oct4, a member of the POU family of homeodomain proteins and a second recently identified homeodomain protein, Nanog. When overexpressed, Nanog allows ES cells to self-renew in the absence of the otherwise obligatory LIF and BMP signals. Although Nanog can act independent of the LIF signal, a contribution of both pathways provides maximal self-renewal efficiency. Nanog function also requires Oct4. Here, we review recent progress in ES cell self-renewal, relate this to the biology of teratocarcinomas and offer testable hypotheses to expose the mechanics of ES cell self-renewal.

Animals↗
PubMed2006

The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells.

The germ cell lineage is a specified cell population that passes through a series of differentiation steps before giving rise, eventually, to either eggs or sperm. We have investigated the manner in which primordial germ cells (PGCs) are reprogrammed in vitro to form pluripotent stem cells in response to exogenous fibroblast growth factor-2 (FGF-2). The response is dependent on time of exposure and concentration of FGF-2. PGCs isolated in culture show a motile phenotype and lose any expression of a characteristic germ cell marker, mouse vasa homolog. Subsequently, some but not all of the cells show further changes of phenotype, accompanied by changes in expression of endogenous FGF-2 and up-regulation of its receptor, fibroblast growth factor receptor-3, in the nucleus. We propose that it is from this reprogrammed component of the now heterogeneous PGC population that pluripotent stem cells arise.

Animals↗
PubMed2005

A molecular basis for human embryonic stem cell pluripotency.

Embryonic stem cells (ESCs) are able to generate a wide array of differentiated cell fates while maintaining self-renewal. Understanding the biology of these choices may be central to the use of human embryonic stem cells (HESCs), both as a model for early human development as well as a resource for cell based therapies. Efforts to dissect the molecular mechanisms that mediate stem cell identity are underway, and in this review we summarize recent progress in defining the markers and pathways involved in these decisions. We discuss recent efforts to assess the molecular signature of pluripotent HESCs and highlight work demonstrating a set of genes, including representatives from the FGF, TGFbeta, and Wnt signaling pathways, that consistently mark the undifferentiated state. In addition, we describe experiments in which signaling of HESCs is augmented by chemical probing with small molecule compounds. Using these compounds, we have demonstrated an important role for Wnt signaling in HESC pluripotency and shown a requirement for TGFbeta signaling in the maintenance of the undifferentiated state. These experiments have revealed some molecular aspects of the pluripotent state and demonstrated clear differences between mouse and human ESCs in the maintenance of this identity.

Animals↗
PubMed2017

Rapid DNA replication origin licensing protects stem cell pluripotency.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

Cell Cycle↗
PubMed1998

Derivation of pluripotent stem cells from cultured human primordial germ cells.

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7-21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

Animals↗
PubMed2004

Poly(D,L lactic-co-glycolic acid) microspheres as biodegradable microcarriers for pluripotent stem cells.

The pluripotent nature and proliferative capacity of embryonic stem cells makes them an attractive cell source for tissue engineering and regeneration. In our study we investigated the use of poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres as biodegradable microcarriers of pluripotent cells and as delivery systems of bioactive factors, which influence cell differentiation. The pluripotent P19 embryonal carcinoma cell line was used as a model to study cell attachment, growth and differentiation of pluripotent stem cells on PLGA microspheres. Retinoic acid (RA) was encapsulated in the PLGA microcarriers to influence cell differentiation-more specifically, to induce P19 cell differentiation into neurons. The results revealed that P19 cells attach and grow on the surface of the RA loaded PLGA microspheres. Moreover, the RA loaded PLGA microspheres were shown to be as effective as soluble RA at inducing P19 cell differentiation into neurons. Hence, the results of these ex vivo studies clearly demonstrate the capacity of PLGA microspheres to serve a dual role as both delivery systems of bioactive factors and as scaffolds for pluripotent cells. More importantly, our study demonstrates the potential use of PLGA microspheres as transplantation matrices of pluripotent stem cells for tissue engineering and regeneration.

Absorbable Implants↗
PubMedDate not supplied

Bioactive factors affect proliferation and phenotypic expression in progenitor and pluripotent stem cells.

Progenitor and pluripotent stem cells reside within connective tissue compartments. They are also present in granulation tissue. This study examined the effects of treating these two cell populations with eight bioactive factors. Cells were assayed for DNA content as a measure of proliferation and for tissue-specific phenotypic markers as measures of lineage progression and lineage commitment. Platelet-derived endothelial growth factor and insulin-like growth factor-II did not induce proliferation in either population. However, dexamethasone, insulin, insulin-like growth factor-I, muscle morphogenetic protein, platelet-derived growth factor-AA, and platelet-derived growth factor-BB stimulated proliferation in one or both cell populations. Platelet-derived growth factor-BB was the most potent stimulator of proliferation in either population. Phenotypic expression markers were induced in the progenitor cells by insulin, insulin-like growth factor-I, insulin-like growth factor-II, dexamethasone, and muscle morphogenetic protein. However, only dexamethasone and muscle morphogenetic protein induced phenotypic expression markers in the pluripotent cells. Platelet-derived endothelial cell growth factor, platelet-derived growth factor-AA, and platelet-derived growth factor-BB did not induce phenotypic expression markers in progenitor or pluripotent cells. This study suggests the potential for using progenitor and pluripotent cells as an in vitro model to ascertain the effects of various bioactive factors on stem cells potentially involved in tissue maintenance and repair.

Animals↗
PubMed2006

Breathing chromatin in pluripotent stem cells.

Only a few factors controlling stem cell pluripotency have been identified to date, and we do not yet fully understand how they act to maintain pluripotency and control differentiation. A report in this issue of Developmental Cell (Meshorer et al., 2006) describes a new trait of pluripotent cells: hyperdynamic or "breathing" chromatin. According to this report, hyperdynamic chromatin is both specific and functionally relevant for pluripotent cells.

Animals↗
PubMed2001

Germ cells and pluripotent stem cells in the mouse.

For many years, attempts to achieve long-term culture of mouse primordial germ cells (PGCs) proved unsuccessful, even when feeder layers were used and individual growth factors were added to the medium. However, when three growth factors were added simultaneously to the medium, some of the cells continued to proliferate indefinitely. Similar to embryonic stem cell lines, these embryonic germ (EG) cell lines were capable of giving rise to embryoid bodies in vitro, and colonizing all cell lineages in chimeras, including the germline. Initially, EG cells were made from PGCs before migration, 8.5 days post coitum (dpc), and after entry into the genital ridge, 11.5 and 12.5 dpc. New EG cell lines from 9.5 dpc (migrating) and 11.5 dpc PGCs, carrying either a LacZ or GFP transgene, are described here. The developmental potential of the new EG cell lines in vitro, in vivo in chimeras, and in tissue aggregates in organ culture was studied. The EG cells were compared with PGCs at the stage from which the EG cells were derived. The two cell types show several similarities, but also some differences in gene expression and cell behaviour, which require further exploration.

Animals↗
PubMed2005

Evaluating human embryonic germ cells: concord and conflict as pluripotent stem cells.

The realization of cell replacement therapy derived from human pluripotent stem cells requires full knowledge of the starting cell types as well as their differentiated progeny. Alongside embryonic stem cells, embryonic germ cells (EGCs) are an alternative source of pluripotent stem cell. Since 1998, four groups have described the derivation of human EGCs. This review analyzes the progress on derivation, culture, and differentiation, drawing comparison with other pluripotent stem cell populations.

Animals↗
PubMed2005

Karyotypic analysis of adult pluripotent stem cells.

Three categories of precursor cells have been identified in postnatal mammals: tissue-committed progenitor cells, germ layer lineage-committed stem cells and lineage-uncommitted pluripotent stem cells. Progenitor cells are the immediate precursors of differentiated tissues. Germ layer lineage stem cells can be induced to form multiple cell types belonging to their respective ectodermal, mesodermal, and endodermal embryological lineages. Pluripotent stem cells will form somatic cell types from all three primary germ layer lineages. Progenitor cells demonstrate a finite life span before replicative senescence and cell death occur. Both germ layer lineage stem cells and pluripotent stem cells are telomerase positive and display extensive capabilities for self-renewal. Stem cells which undergo such extensive replication have the potential for undergoing mutations that may subsequently alter cellular functions. Gross mutations in the genome may be visualized as chromosomal aneuploidy and/or chromosomes that appear aberrant. This study was designed to determine whether any gross genomic mutations occurred within the adult pluripotent stem cells. Karyotypic analysis was performed using pluripotent stem cells purified from adult male rats using established procedures. Giemsa Banding was used in conjunction with light microscopy to visualize metaphase chromosome spreads. To date over 800 metaphase spreads have been analyzed. We found that the metaphase spreads averaged 42 chromosomes and concluded that these pluripotent stem cells isolated from adult rats have a normal karyotype.

Animals↗