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Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture.

ABSTRACT: 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.

SUBMITTER: Lam AT

PROVIDER: S-EPMC4183116 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture.

Lam Alan Tin-Lun AT Chen Allen Kuan-Liang AK Li Jian J Birch William R WR Reuveny Shaul S Oh Steve Kah-Weng SK

Stem cell research & therapy 20140915 5

<h4>Introduction</h4>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) bioprocessi ...[more]

PMID: 25223792

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Project description:Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation.Murine D3-MHC-neo(r) ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics.This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells.

Project description:Human embryonic stem (hES) cells are pluripotent, capable of differentiating into any cell type of the body, and therefore have the ability to provide insights into mechanisms of human development and disease, as well as to provide a potentially unlimited supply of cells for cell-based therapy and diagnostics. Knowledge of the adhesion receptors that hES cells employ to engage extracellular matrix (ECM) proteins is of basic biological interest and can enhance the design of cell culture and implantation systems to enable these biomedical applications. Although hES cells express a variety of cell surface receptors, little is known about which integrins are involved during subculture and passage. Matrigel is broadly used as a cell adhesive matrix for hES cell culture. Here, we sought to identify which integrins hES cells exploit for adhesion to Matrigel-coated surfaces in defined medium conditions. Using RT-PCR, flow cytometry, and fluorescence immunochemistry, we found that numerous integrins were expressed by H1 hES cells; however, antibody blocking assays indicated that only alpha(v)beta(3), alpha(6), beta(1), and alpha(2)beta(1) played a significant role in the initial adhesion of the hES cells to Matrigel in defined medium conditions. We subsequently identified a cohort of synthetic peptides that, when adsorbed to the culture surface, promoted H1 hES cell attachment and proliferation, as well as maintained a pluripotent phenotype. Peptides designed to engage with alpha(v)beta(3), alpha(6), beta(1), and alpha(2)beta(1) integrins and syndecan-1 were tested both individually and in various combinations. A combination of two integrin-engaging peptides (AG-10, C-16) and one syndecan-engaging peptide (AG-73) was sufficient to promote hES cell adhesion, maintenance, and proliferation. We propose that a specific integrin "fingerprint" is necessary for maintenance of hES cell self-renewal, and synthetic culture systems must capture this engagement profile for hES cells to remain undifferentiated.-Meng, Y., Eshghi, S., Li, Y. J., Schmidt, R., Schaffer, D. V., Healy, K. E. Characterization of integrin engagement during defined human embryonic stem cell culture.

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Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture.

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Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture.

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