Appendix I. 
281 
Culture Optimization for Human ES Cells. 
Improvement of culture conditions to enable large-scale 
production and reduce safety concerns has been a major research 
focus. The first tv\^o research groups that described the derivation of 
human ES cell lines examined long-term proliferation, karyotypic 
stability, developmental potential, and cell surface marker expression 
by ES cells [1, 2]. Because these first human ES cell lines remain the 
most extensively characterized, most subsequent research has 
utilized them. These human ES cell lines were derived on mouse 
fibroblast feeder layers in the presence of fetal bovine serum. The 
exposure to these and other sources of animal proteins has raised 
concern that some yet unidentified pathogen(s) may have been 
transferred to the ES cells by contact with cells or proteins from other 
species, and that these pathogens could be transferred to patients if 
these ES cells were to be used for transplantation therapies. Thus, 
several research groups have been actively working to reduce or 
eliminate non-human cells or proteins from human ES cell culture. 
Significant progress has been made in eliminating serum, and limited 
progress has been made in eliminating fibroblasts from human ES 
cell culture. Serum is a complex, poorly defined mixture of 
components, and there is significant variation between lots [41]. 
Individual lots of serum, therefore, must be carefully screened for 
their ability to sustain undifferentiated ES cell growth. If basic 
fibroblast growth factor is added to a proprietary serum substitute 
(Gibco BRL® Knockout™ Serum Replacer), it supports human ES cells 
and significantly reduces the batch variability associated with serum 
[12]. However, this medium does not eliminate all serum products 
from human ES cell culture medium, as it still contains a bovine 
serum albumin component. With this same medium, human ES cells 
can be cultured without direct contact with feeder layers if the 
medium is first conditioned by exposure to mouse embryonic 
fibroblasts [6]. However, the medium still contains bovine serum 
products and is exposed to fibroblasts, therefore cross-species 
contamination with pathogens remains a concern. 
Recent reports demonstrate that human ES cells can be 
maintained on feeder layers of human origin. Feeder layers obtained 
from human bone marrow [9], fetal muscle or skin [7], adult human 
fallopian tube epithelial cells [7], or human foreskin [8] support 
human ES cell proliferation and maintenance of normal karyotype 
and developmental potential. These results led to the growth of 
human ES cells in the complete absence of non-human products [7]. 
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