Melissa K. Carpenter

Characterization and culture of human embryonic stem cells.

Human embryonic stem cells have been defined as self-renewing cells that can give rise to many types of cells of the body. How and whether these cells can be manipulated to replace cells in diseased tissues, used to screen drugs and toxins, or studied to better understand normal development, however, depends on knowing more about their fundamental properties. Many different human embryonic stem cell lines—which are pluripotent, proliferate indefinitely in vitro and maintain a normal, euploid karyotype over extended culture—have now been derived, but whether these cell lines are in fact equivalent remains unclear. It will therefore be important to define robust criteria for the assessment of both existing and newly derived cell lines and for the validation of new culture conditions.

X‐Inactivation Status Varies in Human Embryonic Stem Cell Lines

Human embryonic stem cells (hESCs) derived from human blastocysts have an apparently unlimited proliferative capacity and can differentiate into ectoderm, mesoderm, and endoderm. As such, hESC lines have enormous potential for use in cell replacement therapies. It must first be demonstrated, however, that hESCs maintain a stable karyotype and phenotype and that gene expression is appropriately regulated. To date, different hESC lines exhibit similar patterns of expression of markers associated with pluripotent cells. However, the evaluation of epigenetic status of hESC lines has only recently been initiated. One example of epigenetic gene regulation is dosage compensation of the X chromosome in mammalian females. This is achieved through an epigenetic event referred to as X‐chromosome inactivation (XCI), an event initiated upon cellular differentiation. We provide the first evidence that undifferentiated hESC lines exhibit different patterns of XCI.

Normal Human Pluripotent Stem Cell Lines Exhibit Pervasive Mosaic Aneuploidy

Human pluripotent stem cell (hPSC) lines have been considered to be homogeneously euploid. Here we report that normal hPSC – including induced pluripotent - lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation.

Human embryonic stem cell stability.

Human embryonic stem cells (hESCs) are derived from human preimplantation embryos, and exhibit the defining characteristics of immortality and pluripotency. Indeed, these cell populations can be maintained for several years in continuous culture, and undergo hundreds of population doublings (see refs. 1,2). hESCs are thus likely candidates for source of cells for cell replacement therapies. Although hESC lines appear stable in their expression of cytokine markers, expression of telomerase, ability to differentiate, and maintenance of a stable karyotype, several other aspects of stability have not yet been addressed, including mitochondrial sequencing, methylation patterns, and fine resolution cytogenetic analysis. Because of the potential utility of hESCs, it will be of utmost importance to evaluate the stability of these aspects of ESC biology.

Feeder-Free Culture

Because of their remarkable proliferative capacity and differentiation potential, human embryonic stem (hES) cells may provide a source of cells for cell therapies, drug screening, and functional genomics applications. Derivation of hES cell lines has been accomplished in several laboratories by culturing cells from the inner cell mass of preimplantation embryos on mouse or human embryonic feeder cells ( Thomson et al., 1998 ; Reubinoff et al., 2000 ; Amit and Itskovitz-Eldor, 2002 ; Richards et al., 2002 ). In these conditions, the cells are maintained in the undifferentiated state and show stability in long-term culture ( Amit et al., 2000 ; Carpenter et al., 2003 ). hES cells have been maintained on feeders in media containing serum or serum replacement supplemented with basic fibroblast growth factor (bFGF). In addition, we have found that hES cells maintained on or off feeders express integrins a6 and b1, which may form a laminin-specific receptor, suggesting that the cells may interact with matrix components (Xu et al., 2001). These findings indicate that hES cells require both soluble factors and matrix proteins.

Feeder-Free and Defined Culture Conditions for hESC Growth

Because of their remarkable proliferative capacity and differentiation potential, human pluripotent stem cells (hPSCs) may provide a cell source for cell replacement therapies, drug screening, and disease modeling. The use of hPSCs in these applications requires a scalable, reproducible and controlled process. An adherent cell culture using cell feeder layers has been traditionally employed for the growth of human embryonic stem cells (hESCs). The utilization of defined culture components allows for reagent standardization, consistent cultures, and reduces risks from undefined components or undetectable animal pathogens. We firstly demonstrated that hESCs could be maintained on Matrigel or laminin-coated plates in serum-free medium conditioned by mouse feeders. We later developed culture techniques using defined media. Cells maintained in these culture systems show stable karyotype and proliferation rates, express pluripotency markers and differentiate into all three germ layers. In this chapter, we present culture methods for hPSC cultures and an approach for their characterization.