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Enhancement of the propagation of human embryonic stem cells by modifications in the gel architecture of PMEDSAH polymer coatings.

ABSTRACT: Well-defined culture conditions are essential for realizing the full potential of human embryonic stem cells (hESCs) in regenerative medicine where large numbers of cells are required. Synthetic polymers such as poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] (PMEDSAH), offer multiple advantages over mouse embryonic fibroblasts (MEFs) and Matrigel™ for hESC culture and expansion. However, there is limited understanding of the mechanisms by which hESCs are propagated on synthetic polymers coatings. Here, the effects of PMEDSAH gel architecture on hESC self-renewal were determined. By increasing the atom transfer radical polymerization (ATRP) reaction time, the thickness of PMEDSAH was increased and its internal hydrogel architecture was modified, while maintaining its overall chemical structure. A 105 nm thick ATRP PMEDSAH coating showed a significant increase in the expansion rate of hESCs. Theoretical calculations suggested that 20,000 hESCs cultured on this substrate could be expanded up to 4.7 × 10(9) undifferentiated cells in five weeks. In addition, hESCs grown on ATRP PMEDSAH coatings retained pluripotency and displayed a normal karyotype after long-term culture. These data demonstrate the importance of polymer physical properties in hESC expansion. This modification of PMEDSAH coatings may be used to obtain large populations of hESCs required for many applications in regenerative medicine.

SUBMITTER: Qian X

PROVIDER: S-EPMC4164049 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Enhancement of the propagation of human embryonic stem cells by modifications in the gel architecture of PMEDSAH polymer coatings.

Qian Xu X Villa-Diaz Luis G LG Kumar Ramya R Lahann Joerg J Krebsbach Paul H PH

Biomaterials 20140902 36

Well-defined culture conditions are essential for realizing the full potential of human embryonic stem cells (hESCs) in regenerative medicine where large numbers of cells are required. Synthetic polymers such as poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] (PMEDSAH), offer multiple advantages over mouse embryonic fibroblasts (MEFs) and Matrigel™ for hESC culture and expansion. However, there is limited understanding of the mechanisms by which hESCs are propagated o ...[more]

PMID: 25189518

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Project description:Myocardial infarction is accompanied by a significant loss of cardiomyocytes (CMs). Functional CMs, differentiated from human embryonic stem cells (hESCs), offer a potentially unlimited cell source for cardiac disease therapies and regenerative cardiovascular medicine. However, conventional production methods on monolayer culture surfaces cannot adequately supply the large numbers of cells required for such treatments. To this end, an integrated microcarrier (MC) bioprocessing system for hESC propagation and subsequent CM differentiation was developed.Production of hESC-derived CMs was initially established in monolayer cultures. This control condition was compared against hESC expansion on laminin-coated MC with cationic surface charge, in a stirred serum-free defined culture. Following expansion, the hESC/MC aggregates were placed in a CM differentiation medium, using Wnt signalling modulators in four different culture conditions. This process eliminated the need for manual colony cutting. The final optimized protocol was tested in stirred spinner flasks, combining expansion and differentiation on the same MC, with only media changes during the culture process.In the propagation phase, a 15-fold expansion of viable pluripotent HES-3 was achieved, with homogeneous sized aggregates of 316?±?11 ?m. Of the four differentiation conditions, stirred spinner flask cultures (MC-Sp) provided the best controlled aggregate sizes and yielded 1.9?×?10? CM/ml, as compared to 0.5?×?10? CM/ml using the monolayer cultures method: a four-fold increase in CM/ml. Similar results (1.3?×?10? CM/ml) were obtained with an alternative hESC H7 line. The hESC/MC-derived CM expressed cardiac-specific transcription factors, structural, ion channel genes, and exhibited cross-striations of sarcomeric proteins, thus confirming their cardiac ontogeny. Moreover, E-4031 (0.3 ?M) prolonged the QT-interval duration by 40% and verapamil (3 ?M) reduced it by 45%, illustrating the suitability of these CM for pharmacological assays.We have demonstrated a robust and scalable microcarrier system for generating hESC-derived CM. This platform is enabled by defined microcarrier matrices and it integrates cell propagation and differentiation within a continuous process, in serum-free culture media. It can generate significant numbers of CM, which are potentially suitable for future clinical therapies.

Dataset Information

Enhancement of the propagation of human embryonic stem cells by modifications in the gel architecture of PMEDSAH polymer coatings.

Publications

Enhancement of the propagation of human embryonic stem cells by modifications in the gel architecture of PMEDSAH polymer coatings.

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