The Production and Directed Differentiation of Human Embryonic Stem Cells
Alan Trounson*
Director, Monash Immunology and Stem Cell Laboratories, STRIP Building Monash University, and Australian Stem Cell Centre, Wellington Road, Clayton, Victoria, 3800, Australia
Human embryonic stem cells (hESCs) are being rapidly producedfrom chromosomally euploid, aneuploid and mutant human embryos,that are available from in vitro fertilization (IVF) clinicstreating patients for infertility or preimplantation geneticdiagnosis (PGD). These hESC lines are an important resourcefor functional genomics, drug screening and, perhaps eventually,cell and gene therapy. The methods for deriving hESCs are wellestablished and repeatable, and are relatively successful witha ratio of 1:10 to 1:2 new hESC lines produced from 4-8 dayold morula and blastocysts and from isolated inner cell masscell (ICM) clusters of human blastocysts.
The hESCs can be formedand maintained on human somatic cells in humanized serum-freeculture conditions, and for several passages in cell-free culturesystems. The hESCs can be transfected with DNA constructs. Theirgene expression profiles are being described and immunologicalcharacteristics determined. They may be grown indefinitely invitro while maintaining their original karyotype and epigeneticstatus but this needs to be confirmed from time to time in long-termcultures.
hESCs spontaneously differentiate in the absence ofthe appropriate cell feeder layer, when overgrown in cultureand when isolated from the ESC colony. All three major embryoniclineages are produced in differentiating flat attachment culturesand in unattached embryoid bodies. Cell progenitors of interestcan be identified by markers, expression of reporter genes andcharacteristic morphology, and the cells thereafter enrichedfor progenitor types and further culture to more mature celltypes. Directed differentiation systems are well developed forectodermal pathways that result in neural and glial cells andthe mesendodermal pathway for cardiac muscle cells, and manyother cell types including hematopoietic progenitors and endothelialcells. Directed differentiation into endoderm has been moredifficult to achieve perhaps because of the lack of markersof early progenitors in this lineage. There are reports of enrichedcultures of keratinocytes, pigmented retinal epithelium, neuralcrest cells and motor neurones, hepatic progenitors and cellsthat have some markers of gut tissue and pancreatic islet-likecells. The prospects for use of hESC derivatives in regenerativemedicine are significant and there is much optimism for theirpotential contributions to human regenerative medicine.
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