Brief reviews
Characterization and Culture of Human Embryonic Stem Cells

https://doi.org/10.1016/S1050-1738(03)00125-7Get rights and content

Abstract

Human embryonic stem (ES) cells are cultured cell lines derived from the inner cell mass of the blastocyst that can be grown indefinitely in their undifferentiated state, yet also are capable of differentiating into all cells of the adult body as well as extraembryonic tissue. Detailed investigation of the properties of embryonal carcinoma cells of both the mouse and human as well as mouse and primate ES cells led to the initial isolation and subsequent culture of human ES cells. The methodologies that were developed to culture and characterize these cell lines have provided a template for the development of human ES cells. The existing data illustrate a number of important differences and similarities between human ES cells and the other cell lines. This review aims to provide a brief historic account of the development of the mammalian pluripotent stem cell field; describe how this led to the isolation, culture, and characterization of human ES cells; and discuss the potential implications of recent advances.

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

References (58)

  • P.W. Andrews

    From teratocarcinomas to embryonic stem cells

    Philos Trans R Soc Lond B Biol Sci

    (2002)
  • P.W. Andrews et al.

    Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells

    Hybridoma

    (1984)
  • A. Bradley et al.

    Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines

    Nature

    (1984)
  • R.L. Brinster

    The effect of cells transferred into the mouse blastocyst on subsequent development

    J Exp Med

    (1974)
  • A.A. Caricasole et al.

    Human growth-differentiation factor 3 (hGDF3)developmental regulation in human teratocarcinoma cell lines and expression in primary testicular germ cell tumours

    Oncogene

    (1998)
  • L. Cheng et al.

    Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture

    Stem Cells

    (2003)
  • S. Cooper et al.

    Biochemical properties of a keratan sulphate/chondroitin sulphate proteoglycan expressed in primate pluripotent stem cells

    J Anat

    (2002)
  • M.J. Evans et al.

    Establishment in culture of pluripotential cells from mouse embryos

    Nature

    (1981)
  • B.W. Finch et al.

    Retention of multiple developmental potentialities by cells of a mouse testicular teratocarcinoma during prolonged culture in vitro and their extinction upon hybridization with cells of permanent lines

    Proc Natl Acad Sci USA

    (1967)
  • L.A. Hanna et al.

    Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo

    Genes Dev

    (2002)
  • C. Hansis et al.

    Oct-4 expression in inner cell mass and trophectoderm of human blastocysts

    Mol Hum Reprod

    (2000)
  • J.K. Henderson et al.

    Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens

    Stem Cells

    (2002)
  • K. Illmensee et al.

    Totipotency and normal differentiation of single teratocarcinoma cells cloned by injection into blastocysts

    Proc Natl Acad Sci USA

    (1976)
  • N.B. Ivanova et al.

    A stem cell molecular signature

    Science

    (2002)
  • R. Kannagi et al.

    Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells

    EMBO J

    (1983)
  • L.J. Kleinsmith et al.

    Multipotentiality of single embryocarcinoma cells

    Cancer Res.

    (1964)
  • G.R. Martin

    Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells

    Proc Natl Acad Sci USA

    (1981)
  • M. Nishimoto et al.

    The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2

    Mol Cell Biol

    (1999)
  • M. Oka et al.

    CD9 is associated with leukemia inhibitory factor-mediated maintenance of embryonic stem cells

    Mol Biol Cell

    (2002)
  • Cited by (44)

    • Application of human induced pluripotent stem cells for modeling and treating neurodegenerative diseases

      2015, New Biotechnology
      Citation 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].

    • Embryonic Versus Adult Stem Cells

      2015, Stem Cell Biology and Tissue Engineering in Dental Sciences
    View all citing articles on Scopus
    View full text