Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces

Abstract

Realization of the full potential of human pluripotent stem cells (hPSCs) in regenerative medicine requires the development of well-defined culture conditions for their long-term growth and directed differentiation. Current practices for maintaining hPSCs generally utilize empirically determined combinations of feeder cells and other animal-based products, which are expensive, difficult to isolate, subject to batch-to-batch variations, and unsuitable for cell-based therapies. Using a high-throughput screening approach, we identified several polymers that can support self-renewal of hPSCs. While most of these polymers provide support for only a short period of time, we identified a synthetic polymer poly(methyl vinyl ether-alt-maleic anhydride) (PMVE-alt-MA) that supported the long-term attachment, proliferation and self-renewal of HUES1, HUES9, and iPSCs. The hPSCs cultured on PMVE-alt-MA maintained their characteristic morphology, expressed high levels of markers of pluripotency, and retained a normal karyotype. Such cost-effective, polymer-based matrices that support long-term self-renewal and proliferation of hPSCs will not only help to accelerate the translational perspectives of hPSCs, but also provide a platform to elucidate the underlying molecular mechanisms that regulate stem cell proliferation and differentiation.

Figure 1

Schematic representation of polymer array assay. (a) Polymers were printed using a contact microarray spotter onto glass slides that have been coated with polyacrylamide gels. Cells were globally seeded onto polymer arrays consisting of spotted polymers arranged in 16 subarrays consisting of a 20 × 20 matrix of spots. (b) Each polymer was printed in five replicates (scale bar = 450 μm). (c) Each polymer spot had a diameter of 150 μm (scale bar = 75 μm). Prior to seeding onto the array, hPSCs were cultured in feeder-free conditions and assessed for their (d) characteristic morphology and (e, f) maintenance of markers of pluripotency. hESCs were grown on the polymer arrays for 5 days and imaged in real time by automated microscopy. hESCs were assessed for their (g) characteristic morphology and proliferation (Hoechst) and (h) maintenance of pluripotency (OCT4).

Figure 2

Screening paradigm for identification of polymers that support hPSC attachment, growth, and maintenance of pluripotency.

Figure 3

Hit polymers and their chemical structures.

Figure 4

Polymer molecular weight influences hESC proliferation. Hues9 cells were cultured on arrays of (a) PMVE-alt-MA and (b) PAA with varying molecular weights and concentrations for 5 days. Quantitative analysis (relative Hoechst staining) reveals that hESC proliferation on (c) PMVE-alt-MA and (d) PAA is influenced by the polymer’s molecular weight. Data are representative of 3 independent array experiments. All values are presented as mean ± standard error of the mean of 5 replicate spots.

Figure 5

PMVE-alt-MA supports long-term culture of hESCs in defined medium. (a) Hues1 and (b) Hues9 were cultured on PMVE-alt-MA for 5 passages in StemPro^™. Immunofluorescence of (c) Hues1 and (d) Hues9 cultured on PMVE-alt-MA showing expression of markers of pluripotency, NANOG and OCT4. Quantitative RT-PCR of (e) Hues1 and (f) Hues9 cultured on PMVE-alt-MA demonstrates similar expression levels of markers of pluripotency compared to hESCs cultured on Matrigel. Delta C_t values were calculated as C_t^target − C_t^18s. Data presented as mean ± standard error of the mean of three independent experiments. Karyotype analysis reveals a normal euploid karyotype of (g) Hues1 and (h) Hues9 cultured on PMVE-alt-MA. Immunofluorescence of in vitro differentiation of (i) Hues1 and (j) Hues9 cultured on PMVE-alt-MA shows expression of ectoderm (beta III tubulin; B3T), mesoderm (smooth muscle actin; SMA), and endoderm (Sox17) cell derivatives.

Figure 6

Culture of hESCs on PMVE-alt-MA leads to increased expression of endogenous ECMPs and integrins. Hues9 cells were cultured on Matrigel and PMVE-alt-MA for 24, 48, and 72 hr. Quantitative RT-PCR of expression levels of ECMPs (collagen I:COL I, collagen III: COL III, collagen IV: COL IV, collagen V:COL V, fibronectin: FN, laminin: LN, and vitronectin: VTN) and integrins (integrin alpha 5: ITGA5, integrin alpha V: ITGAV) was performed. Data are displayed in a heat map where each block represents (Delta C_t^PMVE-alt-MA − Delta C_t^Matrigel) at the same time point.