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The Production and Directed Differentiation of Human Embryonic Stem Cells

Monash Immunology and Stem Cell Laboratories, Monash University, and Australian Stem Cell Centre, Clayton, Victoria 3800, Australia

Correspondence: Address all correspondence and requests for reprints to: Alan Trounson, Ph.D., Director, Monash Immunology and Stem Cell Laboratories, STRIP Building, Monash University, and Australian Stem Cell Centre, Wellington Road, Clayton, Victoria 3800, Australia. E-mail: alan.trounson{at}med.monash.edu.au

Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps 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 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. 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 vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. 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 flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of early progenitors in this lineage. There are reports of enriched cultures of keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurons, hepatic progenitors, and cells that have some markers of gut tissue and pancreatic islet-like cells. The prospects for use of hESC derivatives in regenerative medicine are significant, and there is much optimism for their potential contributions to human regenerative medicine.

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