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Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes.

Correia C, Serra M, Espinha N, Sousa M, Brito C, Burkert K, Zheng Y, Hescheler J, Carrondo MJ, Sarić T, Alves PM - Stem Cell Rev (2014)

Bottom Line: The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated.Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures.This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal.

ABSTRACT
Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

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Scanning-electron and confocal microscopy of iPSC-derived cell aggregates and cardiospheres. a–b. SEM micrograph of differentiating cell aggregates collected from stirred tank (a) and WAVE (b) bioreactors at day 9 of differentiation. Higher magnification images evidence the differences in aggregate surface topography. c. Immunofluorescence and confocal microscopy images of the whole-mount day 9 aggregates (left panel) and cardiospheres collected at the end of selection process (right panel). The frequency and distribution of eGFP-positive (green), Ki-67 (red) and collagen type I-positive cells (red) is shown. Nuclei were labeled with DAPI (blue). Scale bars: 100 μm
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Fig5: Scanning-electron and confocal microscopy of iPSC-derived cell aggregates and cardiospheres. a–b. SEM micrograph of differentiating cell aggregates collected from stirred tank (a) and WAVE (b) bioreactors at day 9 of differentiation. Higher magnification images evidence the differences in aggregate surface topography. c. Immunofluorescence and confocal microscopy images of the whole-mount day 9 aggregates (left panel) and cardiospheres collected at the end of selection process (right panel). The frequency and distribution of eGFP-positive (green), Ki-67 (red) and collagen type I-positive cells (red) is shown. Nuclei were labeled with DAPI (blue). Scale bars: 100 μm

Mentions: Phase-contrast and scanning-electron microscopy (SEM) analysis of aggregates on day 9 of differentiation (i.e. before cell lineage selection) revealed that aggregates from WAVE and stirred tank bioreactor cultures differed in size and morphology (Fig. 5a–b, Supplementary Table I/Fig. III). The mean size of aggregates cultured in WAVE bioreactors was higher (440.04 ± 107.89 μm) and they were less spherical and more elongated than aggregates from stirred tank bioreactors (384.41 ± 124.80 μm) (Supplementary Table I, Supplementary Fig. III). This higher size and reduced “sphericity” observed in the aggregates cultured in the WAVE bioreactor might be justified by the different hydrodynamic environment promoted by this type of bioreactor. Additionally, aggregates from WAVE bioreactors showed a smooth outer surface (Fig. 5b), whereas aggregates from stirred tank cultures exhibited a looser texture and a rough surface in which cell-cell contacts could be discerned at a higher magnification (Fig. 5a). Previous studies demonstrated that during cardiac differentiation of ESCs, the extracellular matrix (ECM) mainly composed of collagen type I is deposited on the surface of differentiating aggregates providing for a smoother aggregate surface topography [27, 42]. Thus, our findings may suggest that prior to CM selection the aggregates cultured in WAVE bioreactors present a higher deposition of extracellular matrix (ECM) than stirred tank aggregates. To further confirm these observations, we stained the whole-mount day 9 aggregates with collagen type I antibody to determine the amount and distribution of this ECM component by confocal microscopy. This analysis clearly revealed that aggregates from WAVE bioreactors contain considerably higher levels of collagen type I than aggregates from stirred tank bioreactors (Fig. 5c, left panel) and that, based on the fraction of eGFP-positive cells in whole aggregates, the WAVE aggregates presented higher CM purity than the aggregates derived in stirred tank bioreactors, before induction of CM selection. Moreover, at this timepoint a lower percentage of proliferative cells (Ki-67 positive cells) was observed in aggregates derived from WAVE bioreactors when comparing with aggregates from stirred tank bioreactor cultures (approximately 20 % vs. 58 % of the cells, respectively, Fig. 5c). Taken together, our data show that iPSCs differentiate into CMs in WAVE bioreactors with faster kinetics and higher efficiency than in stirred tank bioreactors. At the end of the differentiation process and the puromycin selection procedure (day 16 for stirred tank and day 11 for WAVE bioreactor), collagen type I staining was similar in intensity and distribution in aggregates from both types of cultures (Fig. 5c, right panel).Fig. 5


Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes.

Correia C, Serra M, Espinha N, Sousa M, Brito C, Burkert K, Zheng Y, Hescheler J, Carrondo MJ, Sarić T, Alves PM - Stem Cell Rev (2014)

Scanning-electron and confocal microscopy of iPSC-derived cell aggregates and cardiospheres. a–b. SEM micrograph of differentiating cell aggregates collected from stirred tank (a) and WAVE (b) bioreactors at day 9 of differentiation. Higher magnification images evidence the differences in aggregate surface topography. c. Immunofluorescence and confocal microscopy images of the whole-mount day 9 aggregates (left panel) and cardiospheres collected at the end of selection process (right panel). The frequency and distribution of eGFP-positive (green), Ki-67 (red) and collagen type I-positive cells (red) is shown. Nuclei were labeled with DAPI (blue). Scale bars: 100 μm
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Related In: Results  -  Collection

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Fig5: Scanning-electron and confocal microscopy of iPSC-derived cell aggregates and cardiospheres. a–b. SEM micrograph of differentiating cell aggregates collected from stirred tank (a) and WAVE (b) bioreactors at day 9 of differentiation. Higher magnification images evidence the differences in aggregate surface topography. c. Immunofluorescence and confocal microscopy images of the whole-mount day 9 aggregates (left panel) and cardiospheres collected at the end of selection process (right panel). The frequency and distribution of eGFP-positive (green), Ki-67 (red) and collagen type I-positive cells (red) is shown. Nuclei were labeled with DAPI (blue). Scale bars: 100 μm
Mentions: Phase-contrast and scanning-electron microscopy (SEM) analysis of aggregates on day 9 of differentiation (i.e. before cell lineage selection) revealed that aggregates from WAVE and stirred tank bioreactor cultures differed in size and morphology (Fig. 5a–b, Supplementary Table I/Fig. III). The mean size of aggregates cultured in WAVE bioreactors was higher (440.04 ± 107.89 μm) and they were less spherical and more elongated than aggregates from stirred tank bioreactors (384.41 ± 124.80 μm) (Supplementary Table I, Supplementary Fig. III). This higher size and reduced “sphericity” observed in the aggregates cultured in the WAVE bioreactor might be justified by the different hydrodynamic environment promoted by this type of bioreactor. Additionally, aggregates from WAVE bioreactors showed a smooth outer surface (Fig. 5b), whereas aggregates from stirred tank cultures exhibited a looser texture and a rough surface in which cell-cell contacts could be discerned at a higher magnification (Fig. 5a). Previous studies demonstrated that during cardiac differentiation of ESCs, the extracellular matrix (ECM) mainly composed of collagen type I is deposited on the surface of differentiating aggregates providing for a smoother aggregate surface topography [27, 42]. Thus, our findings may suggest that prior to CM selection the aggregates cultured in WAVE bioreactors present a higher deposition of extracellular matrix (ECM) than stirred tank aggregates. To further confirm these observations, we stained the whole-mount day 9 aggregates with collagen type I antibody to determine the amount and distribution of this ECM component by confocal microscopy. This analysis clearly revealed that aggregates from WAVE bioreactors contain considerably higher levels of collagen type I than aggregates from stirred tank bioreactors (Fig. 5c, left panel) and that, based on the fraction of eGFP-positive cells in whole aggregates, the WAVE aggregates presented higher CM purity than the aggregates derived in stirred tank bioreactors, before induction of CM selection. Moreover, at this timepoint a lower percentage of proliferative cells (Ki-67 positive cells) was observed in aggregates derived from WAVE bioreactors when comparing with aggregates from stirred tank bioreactor cultures (approximately 20 % vs. 58 % of the cells, respectively, Fig. 5c). Taken together, our data show that iPSCs differentiate into CMs in WAVE bioreactors with faster kinetics and higher efficiency than in stirred tank bioreactors. At the end of the differentiation process and the puromycin selection procedure (day 16 for stirred tank and day 11 for WAVE bioreactor), collagen type I staining was similar in intensity and distribution in aggregates from both types of cultures (Fig. 5c, right panel).Fig. 5

Bottom Line: The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated.Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures.This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal.

ABSTRACT
Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

Show MeSH
Related in: MedlinePlus