A major drawback in the derivation and propagation of human embryonic stem (hES) cells on mouse embryonic fibroblasts (MEFs) is the risk of transmitting pathogens; in particular, small viruses from the animal feeder cells to the hES cells because of direct contact. Reliance on a xeno-support system introduces considerable disadvantages with respect to exploiting the therapeutic potential of hES cells. Human feeders have been successfully used to grow human inner cell masses and isolate hES cells as far back as 1994 (1,2). It was recently shown that both commercially available and in-house-derived human feeders support the derivation and propagation of hES cell lines (3,4). Additionally, the animal-based xenoproteins in the culture media nourishing the hES cells and feeders can be

From: Methods in Molecular Biology, vol. 331: Human Embryonic Stem Cell Protocols Edited by: K. Turksen © Humana Press Inc., Totowa, NJ

substituted with human-based ingredients, making it a completely xeno-free humanized in vitro system. Unfortunately, the commercial feeders have the disadvantage that they have been grown in the presence of calf sera and other animal proteins and hence exposed to xenoproteins. Feeder-free in vitro systems using noncellular matrices (e.g., Matrigel, collagen) with conditioned media or hES media substituted with specific growth factors support hES cell growth for only six to seven passages and are xenosupport systems because many of the matrices are of animal origin (3,5). Additionally it was recently suggested that feeder-free conditions could induce chromosome anomalies (recurrent gain of chromosomes 12 and 17q) similar to embryonal carcinoma karyotypes at late passages (6). Thus until such time as a feeder-free system that is xeno-free, and has no risk of inducing embryonal carcinoma-like genotypes is available, in-house-derived human feeders appear to be the best choice. Of the in-house-derived human feeders, human fetal muscle, human fetal skin, and human adult skin appear to be the best support cells for hES cells (3,4). Although ranked third, human adult skin offers the advantage of obtaining biopsies by a noninvasive, simple procedure for feeder derivation and propagation. Additionally, a skin biopsy from the same patient donating in vitro fertilization embryos for hES cell derivation will be the perfect autologous disease-free in vitro system because the patient would have already been screened for HIV, hepatitis B, and other communicable diseases before enrolling for in vitro fertilization.

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