Brief reviewsCharacterization and Culture of Human Embryonic Stem Cells
Section snippets
Pluripotent Stem Cells: Embryonal Carcinoma Cells
The intense scientific and general interest in the prospective uses of human embryonic stem (ES) cells was preceded by years of research investigating cells isolated from testicular germ cell tumors called embryonal carcinoma (EC) cells. EC cell lines were derived first from teratocarcinomas of the mouse (Finch and Ephrussii 1967, for a review, see Andrews 2002). This followed from the first formal proof that a single EC cell was pluripotential—that is, capable of becoming many differentiated
Pluripotent Stem Cells: Mouse ES and Embryonic Germ Cells
Pluripotent mouse ES cell lines were isolated first in 1981 Evans and Kaufman 1981, Martin 1981 based on experience with EC cell culture. Mouse ES cell lines are obtained by immunosurgically digesting the outer layer (trophectoderm) of mouse blastocysts to isolate the inner cell mass. The inner cell mass is comprised of the cells that eventually will form the entire adult mouse. These mouse ES cell lines are maintained indefinitely in vitro and are morphologically and phenotypically similar to
Pluripotent Stem Cells: Isolation of Primate and Human ES (and EG) Cells
ES cell lines have been established from primate embryos including rhesus (Thomson et al. 1995), marmoset (Thomson et al. 1996), and cynomolgus monkeys (Suemori et al. 2001). Invaluable information gained from the studies by Thomson et al. 1995, Thomson et al. 1996, including the notion that primate ES cells do not grow well after being made into a single-cell suspension, enabled the first human ES cell lines to be isolated. Human ES cell lines were obtained from immunosurgically dissociated
Culture of Human ES Cells
The technology for successfully propagating pluripotent human ES cells evolved from the observation that murine EC and ES cells, when cultured on feeder layers, grew with higher efficiency and capacity for differentiation Martin and Evans 1974, Reubinoff et al. 2000, Thomson et al. 1998. Mitotically inactivated MEF feeders thus also were used for the establishment and growth of ES cells of human origin Pera et al. 2003, Reubinoff et al. 2000, Thomson et al. 1998. The initial protocols for the
Characterization of Human ES Cells
Human ES cells in their undifferentiated state are characterized by a distinct morphology (see Figure 1) and by the presence of molecular and antigenic markers typical of mammalian pluripotent cells. Human ES cells also are defined functionally by their ability to form cell types characteristic of the three distinct tissue types that arise in mammalian development during gastrulation: the endoderm, mesoderm, and ectoderm. These three tissue layers are named EG layers and human ES cells are
Cell-Surface Markers for Human ES Cells
Cell-surface markers for human ES cells can be used to isolate (or deplete) pure populations of live cells either via fluorescence-activated cell sorting or by using magnetic bead technology (Pera et al. 2003), thus making them an extremely useful tool for investigating the biology of pluripotent cell populations in addition to providing the ability to remove undesirable cells prior to transplantation. Unfortunately, no specific cell-surface marker (or combination of markers) has been
Molecular Characterization of Human ES Cells
Currently, there exists only a short list of genes that are rapidly downregulated upon differentiation of pluripotent cells. Oct-4 is a transcription factor that is downregulated during the stage of mammalian development called gastrulation, when the three EG layers begin to form (Nichols et al. 1998). All pluripotent stem cells from mouse or primate (EC or ES) express Oct-4 Reubinoff et al. 2000, Thomson et al. 1996, Thomson et al. 1998 and, in the mouse, the formation of pluripotent cells
Conclusion
Human ES cells show an unlimited capacity for undifferentiated proliferation while maintaining a normal karyotype. They also are pluripotent and their potential uses in research and therapy are evident from the wide range of cell types derived from them in vitro. However, in the vast majority of these studies, no functional characterization of the human ES cell–derived cells has been carried out and, as yet, there are no published reports of human ES–derived cells being used to treat models of
Acknowledgements
The authors would like to apologize to those authors whose work was not cited directly in this review due to space constraints. Work in our laboratory is supported by grants to M.F.P. from ES Cell International Pte., the National Health and Medical Research Council (Australia), the National Institute of Health (USA) GM 068417-01 and DK 63400-01, and the Juvenile Diabetes Research Foundation. The authors would like to thank Drs. Souheir Houssami and Ernst Wolvetang for their review of the
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2015, New BiotechnologyCitation Excerpt :These markers, although useful for the characterization of hiPSC lines following derivation and during culture, are also known to be immunoreactive in embryonic tissues or in more mature cell types, and are therefore required to be used in definitive context of stem cell commitment and differentiation (discussed in [26]). In recent years, there have been only a few additional markers developed that are reported to be highly specific for detecting human pluripotent cell surface antigens [34–38]. As well as the use of panels of good antibody markers to detect antigens that will definitively isolate or purge hiPSCs from end point cell populations, glycan-binding lectin proteins also offer promise as a useful tool for detecting the glycoprotein and glycolipid moieties associated with hPSCs [39,40].
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