Human embryonic stem cell derivation and directed differentiation.

Abstract

Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much optimism for their contribution to human medicine.