Introduction

The first human embryonic stem (hES) cells were only isolated some 6 yr ago (1,2). Although we have learned much about these cells in the intervening period, the key molecules that control their self-renewal remain unidentified and present culture systems are almost certainly suboptimal. As a consequence, there are a variety of protocols available for the routine culture of hES cells that variously require feeder layers, conditioned medium, disaggregation by several enzymatic regimes, or mechanical dissociation. It seems likely that the various lines isolated under different conditions have adapted to those conditions and the best advice to those who are new to hES culture is to begin with the precise protocols provided at source. But this is not to say that any single protocol is in a general sense "best." This chapter provides the protocols presently used in our laboratory to culture the hES lines H1 and H9 that are available through the University of Wisconsin (Wi-cell). They are not the protocols provided by Wi-cell, and the H1 and H9 cell lines have required a period of adaptation.

To date, hES lines have been established by mechanical dissociation of explanted inner cell masses or blastocysts on a fibroblast feed layer. At least in

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

their early passages, hES cells are highly sensitive to the method of disaggregation and some lines are maintained routinely by mechanical dissociation. However, this is a very cumbersome method of handling hES cells and precludes many large-scale culture experiments. Other lines have traditionally been passaged using collagenase treatment, which leads to the release of cell clumps from the culture surface, but not to a single-cell suspension, and in our hands is associated with poor plating efficiency. We have therefore set out to develop passage procedures that provide for the simplified expansion of hES cell numbers within a shorter period. In doing so, we have benefited from the underpinning work of others in developing a conditioned medium system that allows culture of hES cells without direct contact with feeders (3). In our hands, hES cells passaged with collagenase frequently do not recover well and cultures frequently contain a second, fibroblast-like, population (Fig. 1A). We have found this problem greatly reduced when using a disaggregation regime comprising trypsin and ethyleneglycol tetraacetate (EGTA) (Fig. 1B).

There must be an accompanying warning that goes along with these and all other hES protocols—we have no standard assay of normality for hES cells. Protocols optimized for experimental convenience are not necessarily those that best retain the pluripotential, wild-type nature of the cells. In this respect, hES cells are at a disadvantage to their murine counterparts where germ line transmission provides a convincing indicator of normality. Nevertheless, our presently favored protocol for routine hES maintenance has been shown to sustain normal karyotype at extended passage (Fig. 1C) and to sustain the potential to differentiate widely when hES cells are injected into severe combined immunodeficiency mice (data not shown).

This is not necessarily true of all passage regimes, and we urge caution in introducing modifications to culture conditions without careful characterization of the cells over time. Figure 1C reveals the appearance of the translocation (iso7q) when cells were disaggregated for 10 passages using ethylenediamine-tetraacetic acid (EDTA) alone. This translocation had overtaken the population within 10 passages. The repeatability of this result is under investigation and it is premature to draw conclusions about the effect of EDTA. However, others have reported recurrent gain of chromosomes 17q and 12 (4), suggesting that under certain conditions, there can be strong selection pressure for chromosomal abnormalities. Whether these phenomena arise as a result of enzymatic disaggregation or as a consequence of culture to high cell density has been the subject of some debate (5). It is intriguing that different results appear recurrently in different labs such that Buzzard et al. (5) report no abnormalities over 80 passages, Thomson and Andrews (4) report different recurrent duplications in their respective laboratories while using the same cells and culture systems, and we, using the same line but disaggregating in EDTA alone see yet a third duplication. On balance, the advantages of enzymatic disaggregation make it

Fig. 1. Human embryonic stem cells 72 h after passage in (A) collagenase (B) trypsin/ethyleneglycol tetraacetate (EGTA), revealing the greater plating efficiency, growth rate, and reduced differentiation we observe with trypsin/EGTA. Cells maintain a stable karyotype for up to 54 passages with trypsin/EGTA (C, left karyotype), but abnormalities are observed when using ethylenediaminetetraacetic acid alone (C, right karyotype) (Please see the companion CD for the color versions of this figure.)

Fig. 1. Human embryonic stem cells 72 h after passage in (A) collagenase (B) trypsin/ethyleneglycol tetraacetate (EGTA), revealing the greater plating efficiency, growth rate, and reduced differentiation we observe with trypsin/EGTA. Cells maintain a stable karyotype for up to 54 passages with trypsin/EGTA (C, left karyotype), but abnormalities are observed when using ethylenediaminetetraacetic acid alone (C, right karyotype) (Please see the companion CD for the color versions of this figure.)

difficult to justify mechanical disaggregation for large-scale experiments, but the prudent researcher will avoid culturing hES cells to too high a density and will periodically assess the karyotypic stability of the cells.

Data are now accumulating regarding the patterns of gene expression that characterize hES cells (6,7). Encouragingly, hES lines of differing origins do show common expression patterns, suggesting that it may soon be possible to develop an expression "fingerprint" of normality for hES cells and this will go some way toward the further optimization of these protocols.

Experimental differentiation of hES cells often begins with the formation of embryoid bodies (EBs), structures that mimic the cellular interactions during embryogenesis and generate inductive signals. EBs are generated by partial disaggregation of hES cells and by plating onto nonadherent plastic in non-conditioned medium and in the absence of supplementary basic fibroblast growth factor (bFGF) (Fig. 2). For our own work with bone-forming cells, we have found that the prior formation of embryoid bodies is unnecessary for hES cells to respond to osteogenic-inducing agents. However, this method may be essential for other lineages and is a useful way of verifying the potentiality of cultures.

Finally, as an example of directed differentiation, we also include our protocol for osteogenic induction of hES cells (8). Human ES cells enter the osteogenic pathway within a similar time scale to human mesenchymal stem cells, suggesting that the commitment of hES cells to something similar to a mesenchymal stem cell may occur rapidly. Figure 3 shows the accumulation of alizarin red-stained areas of the culture well over time when hES cells are exposed to osteogenic factors (dexamethasone, b-glycerophosphate, and ascorbic acid).

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