When human embryonic stem cells (hESCs) were isolated for the first time by Thomson and colleagues, the culture methods used were similar to the procedures developed earlier for embryonal carcinoma cell lines or mouse ESCs (1), i.e., coculture with mitotically inactivated mouse embryonic fibroblasts (MEFs) using medium supplemented with 20% fetal bovine serum (FBS). However, the potential use of hESCs for clinical purposes such as cell-based therapy requires their isolation and culture in an entirely animal-free environment. This will prevent their exposure to retroviruses or other pathogens potentially present in the mouse feeder layer or FBS. Additionally, for developing therapies based on hESCs and for using the cells as research models, a well-defined and reproducible

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

system is required. The use of FBS or mouse feeder layer prevents establishing such a culture system for hESCs.

To promote the development of animal-free and well-defined culture systems for hESCs, one can culture the cells either with or without human feeder layers. Both options require animal serum substitute. The simplest alternative to FBS is human serum (2), though it is not well defined and batch variation cannot be avoided. Another alternative is serum replacement when supplemented with basic fibroblast growth factor (bFGF) (3). The drawback here is that it contains "Albumax," which is a lipid-enriched bovine serum albumin mixed with additional animal products and, therefore, although relatively defined, it is not animal-free.

The first animal-free culture system for hESCs was published by Richards and colleagues based on coculture with human fetal-derived feeder layers or human adult Fallopian tube-epithelial feeder layers and medium supplement with 20% human serum (2). Using this culture system, hESCs can be cultured for prolonged periods while maintaining hESC features, and new hESC lines can be isolated. Foreskin fibroblasts were also found to support both the culture and derivation of hESCs using medium supplemented with either human serum or serum replacement (4,5).

Foreskin fibroblast has several advantages. Unlike fetal fibroblast, which can be grown to reach a certain limited passage, foreskin fibroblast can be grown continuously for at least 6 mo while maintaining the ability to support hESCs as undifferentiated cells. Their long lifespan enables their comprehensive characterization, which is essential for hESCs' future clinical use. These feeders may therefore have an advantage when large-scale growth of hESCs is concerned. Because of their importance, great effort is being invested in the optimization of hESC culture methods and in developing alternative systems based on coculture with human feeder layers (6).

In view of the large-scale and well-defined culture system required for hESC growth for clinical uses, the ideal method for growing these cells would be on matrix using serum and animal-free medium. Xu et al. were the first to demonstrate a culture system in which hESCs were cultured without direct contact with MEFs, using Matrigel matrix and 100% MEF-conditioned medium supplemented with serum replacement and bFGF (7). Using this culture system, hESCs cells may still be exposed to animal pathogens through the MEF-conditioned medium. Later, Amit et al. suggested a feeder-free culture system in which no conditioned medium is used (8), which is based on human fibronectin matrix and medium supplemented with serum replacement, bFGF, and transforming growth factor-pi (TGFpi). The hESCs cultured in these conditions maintained all ESCs characteristics for more than 1 yr. Colonies of hESCs cultured under animal or feeder layer free conditions are illustrated in

Fig. 1. Human embryonic stem cells colonies of cell line I3 cultured (A) with mouse embryonic fibroblasts, (B) with human fibronectin and medium supplemented with growth factors, (C,D) with foreskin fibroblasts. Bar: A-C = 50 pM; D = 38 pM.

Fig. 1. The existing feeder-free culture systems for hESCs are based on medium supplemented with serum replacement, a component that is not regarded as animal free. Whether these systems are able to support the derivation of hESC lines is yet to be seen.

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