Realization of the multitude of potential hPSC applications is dependent upon our ability to design novel culture conditions which are chemically defined, robust, cost-effective, and devoid of animal-derived components. Much of the research carried out on hPSCs thus far has focused on soluble signals and their effects on hPSC self renewal [3
], thus providing a well-defined alternative combination of soluble factors to replace conditioned medium for hPSC expansion [1
]. Since signaling molecule responses are affected by interactions between the cell and its surrounding matrix, we [29
] and others [37
] have investigated the effect of the extracellular matrix components on influencing hPSC fate. Subsequently, various ECMP combinations have been developed for the expansion of hPSCs [33
]. However, these approaches utilize purified or recombinant proteins, which are expensive, thus making the use of these systems for large-scale expansion of hPSCs cost-prohibitive. Compared to ECMPs and synthetic peptides, polymer biomaterials are inexpensive and provide a robust platform to produce large numbers of hPSCs.
In this study, we employed an unbiased high-throughput screening approach to systematically screen in a concentration-varying manner, a library of synthetic polymers to develop a chemically defined, cost-effective, robust culture system for in vitro expansion of hPSCs. From our initial screens, we identified several polymers that were able to support short-term hPSC proliferation and maintenance of pluripotency. Although most of these polymers could not support long-term self-renewal of hPSCs, we identified one polymer, PMVE-alt-MA, that was able to sustain the long-term culture of two hESC lines (HUES1 and HUES9) and one hiPSC line, while maintaining their morphology, expression of stem cell markers, genetic integrity, and in vitro pluripotency. Furthermore, we verified that this polymer substrate was compatible with the defined medium StemPro™ (Life Technologies). Our results also show a molecular weight-dependent effect of the polymer on hPSC growth supporting the notion that the physicochemical properties of polymers play an important role in modulating cell-matrix interactions. There could also be topographical changes associated with the molecular weight-dependent entanglement of polymer chains and/or changes in interaction between the polymer-coated matrix and its surrounding medium.
The specific mechanism by which PMVE-alt-MA supports self-renewal of hPSCs is not entirely clear. Cell surface integrins of the hPSCs play an important role in their interactions with the surrounding matrix and thus playing a role in their self-renewal [35
]. The hPSCs cultured on PMVE-alt-MA exhibited higher expression levels of integrin α5 and αv
. These integrins have been shown to mediate adhesion of hPSCs to various ECMPs and Matrigel [35
]. It has also been shown that synthetic peptides designed to engage these integrins and cell surface heparan sulfates support self-renewal of hESCs in vitro
We also found that hPSCs cultured free of exogenous ECMPs on PMVE-alt-MA show increased expression of several endogenous ECMPs. This suggests that although PMVE-alt-MA fosters initial hPSC adhesion independent of non-specific adsorption of proteins from the surrounding medium, there are additional contributions of proteins secreted by the growing hPSCs. Even though a defined medium free of exogenous ECMPs was used in these experiments, growth factors in the medium, such as bFGF can bind to the polymer. In an aqueous environment, PMVE-alt-MA undergoes hydrolysis and forms poly[(methyl vinyl ether)-alt-(maleic acid)] [46
]. Such anionic polymers bearing carboxyl and sulfonyl groups have been shown to mimic functional features of heparin [47
]. Heparin-mimicking polymers have been shown to bind to various growth factors, such as bFGF, mainly through favorable energetic interactions [49
]. In fact, maleic acid-based heparin-mimetic polymers have been used in vivo
for regulating various heparin binding growth factors [51
]. Furthermore, heparin is an important component of the microenvironment, which has recently been recognized for its role in regulating self-renewal of hESCs [52
]. Taken together, these findings suggest a possible mechanism by which PMVE-alt-MA promotes proliferation of hPSCs while maintaining their undifferentiated state in a manner similar to heparin in MEF-coculture or MEF-conditioned medium.
A recent report by Villa-Diaz et al.
demonstrated the use of synthetic polymer coatings for the growth of hESCs [20
]. The authors observed five polymer coatings that were able to support short term self-renewal of hESCs while only one polymer, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), was able to sustain long-term growth of hESCs without inducing differentiation. Although we did not test PMEDSAH in our screen, we did test several chemically similar polymers containing sulfonyl groups, and found that two of these polymers, poly(acrylamido-methyl-propane sulfonate) (PAMPS) and poly(sodium 4-styrenesulfonate) (PSSS), were able to support short-term self-renewal of hPSCs. However, unlike PMVE-alt-MA, PSSS and PAMPS were unable to support long-term culture of hPSCs. Nonetheless, like PMVE-alt-MA, PAMPS, PSSS, and PMEDSAH are anionic polymers which may assist in hPSC self renewal by mimicking heparin activities, such as bFGF binding. Our study distinguishes itself from that by Villa-Diaz et al. in several ways: 1) PMVE-alt-MA was identified from an unbiased screening approach in which we analyzed a large number of polymers at varying concentrations. 2) PMVE-alt-MA is available off the shelf with controlled molecular weights. As seen from our findings, polymer molecular weight plays a significant role in hPSC attachment and growth. 3) PMVE-alt-MA supports the long-term maintenance of hiPSCs in addition to hESCs.