In this study, we report the development of a cost-effective, feeder-, and nearly xeno-free culture system for hESC propagation. Specifically, we started with KSR medium containing serum replacement, which includes a small amount of bovine albumin. Since, as discussed below, we used substrates formed from human proteins and added only recombinant products to the medium, the serum supplement was the only source of non-human proteins in our culture system.
With regard to the culture substrate, maintaining the cells on human serum eliminated the need for feeder cells, thus providing a cost-effective alternative to complex mouse matrices (e.g.
, matrigel) or purified human ECM components (e.g.
, fibronectin). Serum, the fluid by-product of clotted blood, contains ~60–80 mg protein/ml and various other constituents such as salts, lipids, amino acids, and sugars. Mass-spectrometry-based analyses of albumin- and immunoglobulin-depleted serum detected about 650 different proteins in a very broad range of concentrations (Schenk et al., 2008
). It is likely that coating tissue culture dishes with serum immobilizes, among other components, ECM constituents in this body fluid such as vitronectin, fibrinogen, perlecan, fibronectin, and collagens IV and XI (Adkins et al., 2002
; Adkins et al., 2005
). Serum is also rich in various ECM- and growth factor-binding proteoglycans such as heparan sulfate, which plays an important role in hESC self-renewal (Furue et al., 2008
). Additionally, using substrates formed from human serum increases the likelihood that the native conformation, glycosylation, and hence, activity of the bound glycoprotein substrates are preserved.
With regard to the culture medium, many previous studies have focused on the role of protein constituents – namely, growth factors and cytokines – with evidence suggesting that other molecules such as lipids also play important roles (Pebay et al., 2005
). Several groups independently reported that hbFGF is necessary and sufficient for feeder-free hESC growth in the absence of MEF-conditioned medium (Amit et al., 2004
; Wang et al., 2005
; Xu et al., 2005
). Under these conditions, increasing the concentration of hbFGF ~25-fold to 100 ng/ml sustains hESC growth in a pluripotent state (Levenstein et al., 2006
). The requirement for high concentrations of hbFGF was attributed to the low stability of hbFGF in culture medium and/or to the recruitment of additional signaling pathways. TGFβ/activin/nodal signaling also plays a significant role in regulating hESC pluripotency when the cells are cultured in the absence of feeders (James et al., 2006
; Saha et al., 2008
). Thus, our basal medium was KSR supplemented with bFGF and TGF-β.
In devising a new formulation for propagating hESCs under feeder-free conditions, we hypothesized that lactate, which was at significantly higher concentrations in CMs that supported hESC self-renewal, may have a beneficial effect. The evidence presented here supports this theory (see Supplementary Data). However, initial experiments showed that basal medium containing lactate failed to maintain hESC growth in an undifferentiated state for more than 2–3 passages. To identify other novel additives, we compared the secretomes of hPFs, which we use as hESC feeders (Genbacev et al., 2005
), and IMR90 cells, which do not sustain hESC replication in an undifferentiated state. The most obvious difference was higher production of GROα by hPFs. Accordingly, in addition to lactate, we added to the basal medium a recombinant version of this protein. In other experiments, we demonstrated the ability of H7 and H9 cells, cultured under these novel conditions for 10 and 20 passages, to differentiate into derivatives of the three primary germ layers. However, we noticed an increased propensity of the H7 line to form contractile cardiomyocytes suggesting a potential bias that could be exploited in the directed differentiation of these cells. At P10 and P20 the hESCs expressed a normal karyotype and at P15 they formed teratomas. Taken together, the results show that this novel substrate and medium combination sustains the long-term growth and proliferation of hESCs in an undifferentiated state while preserving their pluripotency.
Adding GROα, a member of the CXC chemokine family, was key to formulating this new medium. Initially, this molecule was isolated based on its ability to stimulate proliferation of malignant melanoma cells in vitro
(Anisowicz et al., 1987
; Richmond and Thomas, 1988
). However, GROα promotes the growth of many cell types (Haghnegahdar et al., 2000
; Aust et al., 2001
; Li et al., 2004
). Its receptor, CXCR2, which belongs to the G-coupled seven-transmembrane- domain family, transmits signals via the mitogen-activated protein kinase and protein kinase C pathways (Stillie et al., 2009
). Here we show that hESCs express this receptor, providing further rationale for addition of GROα to the hESC culture medium.
Because hESCs grow as polarized epithelia (Krtolica et al., 2007
), we were interested in whether single cells, cultured under the novel conditions described here, could reestablish this highly specialized form of cell–cell adhesion. Interestingly, despite the high levels of hbFGF in our defined medium, single cells that were re-plated failed to form colonies. The same concentration of hbFGF (100 ng/ml) that we used sustained self-renewal of hESCs grown as single cells on matrigel for up to 164 population doublings, i.e.
, performed very similarly to MEF CM (Levenstein et al., 2006
). This dichotomy is likely created by the presence of GROα (and perhaps other growth factors) in our novel culture medium and differences in the microenvironment provided by the serum vs. matrigel substrate (Ilic, 2006
). One possible interpretation of this result is that dissociation of colonies into single cell suspensions destroys the tight and adherent junction complexes by removing the basement membrane components that hESCs synthesize (Evseenko et al., 2009
). Consequently, when single cells are plated on substrates that are not enriched in basement membrane components, they may not be able to reestablish polarity, which is one of the essential characteristics of undifferentiated hESCs (Krtolica et al., 2007
). Interestingly, it was recently demonstrated that two chemicals that upregulate E-cadherin expression significantly increase the reprogramming efficiency of induced pluripotent stem cells (iPSCs) (Chen et al., 2010
). This finding provides additional evidence that pluripotency is associated with acquisition of a polarized phenotype. Taken together, these findings and our results suggest that minimizing the loss of cell–cell and cell–extracellular matrix contacts during passaging of hESC colonies reduces the risk of chromosomal abnormalities and renders the cells less dependent on extracellular matrix composition. This also explains why mechanical dissection of colonies into uniform sized clumps seems to be the best current practice for establishment and maintenance of hESCs (Hasegawa et al., 2010
In our system single cells continued to proliferate, but changed their morphology and downregulated hESC-associated pluripotency markers. Coincidentally, they upregulated the expression of markers of neuronal progenitors. This finding may reflect the fact that hESCs appear to be closely related to components of the epiblast (Henderson et al., 2002
) which, among other cell types, gives rise to progenitors that ultimately form central and peripheral nervous systems. The results of this study provide further evidence that various combinations of factors can support self-renewal of hESC. It is possible that specific culturing conditions may predispose hESC to more efficiently undergo directed differentiation towards specific lineages and that the choice of the optimal culturing conditions will depend on hESC application. Addressing these issues will involve an iterative process in which we use our increasing knowledge of the molecular determinants that enable self-replication of the true pluripotent hESC-like cell population in vivo
as well as the signals that are involved in early lineage decisions to improve the culture conditions that are used to propagate pluripotent hESCs in vitro