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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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Differentiation of iPSC into CMs in WAVE and stirred tank bioreactors. WAVE cultures were compared with optimized stirred tank bioreactor cultures (operated under 4 % O2 and intermittent agitation without direction change) in terms of CM differentiation efficiency. a. Phase contrast and fluorescence images showing cell aggregates composed by eGFP-positive cells (green) at days 5, 7, 9 and 11. Scale bars: 200 μm. c. Percentage of eGFP-positive aggregates during culture time. c. Percentage of eGFP-positive cells during culture time determined by flow cytometry. d. CM yields (stripped bars) and number of CMs generated per liter of culture medium throughput (CMs/L, black bars). e. Semiquantitative RT-PCR analyses showing expression of pluripotency (Oct4), endoderm (AFP), mesoderm (T-Bra) and CM-specific (Nkx2.5, cTnT, HCN4, Myl2, Myl7) genes during the time course of differentiation process in WAVE bioreactors. f. Quantitative RT-PCR analysis of cells cultured in optimized stirred tank and WAVE bioreactors, at day 6, before (day 9) and after CM selection (day 16 and 11 for stirred tank and WAVE bioreactor cultures, respectively). Expression was normalized to RNA from stirred tank bioreactor cultures operating with intermittent agitation, except for Oct4 expression that was normalized to RNA of undifferentiated cells (day 0, black bars). Data are given as mean ± SD of 3 individual bioreactors experiments. Significantly different, P < 0.05 (*) and P < 0.01 (**)
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Fig4: Differentiation of iPSC into CMs in WAVE and stirred tank bioreactors. WAVE cultures were compared with optimized stirred tank bioreactor cultures (operated under 4 % O2 and intermittent agitation without direction change) in terms of CM differentiation efficiency. a. Phase contrast and fluorescence images showing cell aggregates composed by eGFP-positive cells (green) at days 5, 7, 9 and 11. Scale bars: 200 μm. c. Percentage of eGFP-positive aggregates during culture time. c. Percentage of eGFP-positive cells during culture time determined by flow cytometry. d. CM yields (stripped bars) and number of CMs generated per liter of culture medium throughput (CMs/L, black bars). e. Semiquantitative RT-PCR analyses showing expression of pluripotency (Oct4), endoderm (AFP), mesoderm (T-Bra) and CM-specific (Nkx2.5, cTnT, HCN4, Myl2, Myl7) genes during the time course of differentiation process in WAVE bioreactors. f. Quantitative RT-PCR analysis of cells cultured in optimized stirred tank and WAVE bioreactors, at day 6, before (day 9) and after CM selection (day 16 and 11 for stirred tank and WAVE bioreactor cultures, respectively). Expression was normalized to RNA from stirred tank bioreactor cultures operating with intermittent agitation, except for Oct4 expression that was normalized to RNA of undifferentiated cells (day 0, black bars). Data are given as mean ± SD of 3 individual bioreactors experiments. Significantly different, P < 0.05 (*) and P < 0.01 (**)

Mentions: The comparison of CM differentiation efficiency in WAVE and optimized stirred tank bioreactors (operated under 4 % O2 tension and intermittent agitation without direction change) revealed that WAVE bioreactor cultures favored CM lineage commitment, enabling a reduction in the differentiation time and increased CM yields (Fig. 4). In these cultures, eGFP-positive cells and contracting areas in aggregates were observed from day 5 onwards, increasing rapidly in number and size (Fig. 4a–c, Supplementary Movie I). By day 7, 54.3 ± 5.2 % of the aggregates exhibited a considerable area of eGFP-positive and spontaneously beating cells (Fig. 4a–b). In accordance with this result, flow cytometry analyses indicated that 38.6 ± 6.0 % of the cells in aggregates were eGFP-positive, compared to less than 5 % detected in stirred tank bioreactors at this time point (Fig. 4c). Importantly, on day 9 of culture, i.e. before CM selection, the percentage of eGFP-positive cells among all cells dissociated from aggregates was almost 2-fold higher in WAVE bioreactors (76.0 ± 5.8 %) compared to stirred tank cultures (43.9 ± 6.6, Fig. 4c, Table 1). Consequently, only 2 days of antibiotic treatment were sufficient to obtain a 97.6 % pure CM population in WAVE bioreactors (Fig. 4c, Table I). In contrast, at day 11 of differentiation, aggregates in stirred tank bioreactors were not yet completely pure and additional 5 days of antibiotic treatment were required to generate a 97.4 % pure CM population (Fig. 4a, c). In terms of CM productivities, in optimized stirred tank cultures approximately 0.430 L medium throughput (total volume of medium used during the process) resulted in the generation of 0.1x109 CMs, which correspond to a coefficient of 0.2x109 CMs/L. On the other hand, in WAVE bioreactor 2.3x109 CMs were produced in 1.5 L medium throughput (1 L until day 9, 0.5 L medium exchange at day 9) obtaining 1.5x109 CMs/L (Table 1). Overall, iPSC differentiation in WAVE bioreactors resulted in an improvement of about 40 % in CM yield (60.8 ± 0.7 CMs/input iPSC) and in a 5 times higher CM production per liter of culture medium throughput (CMs/L) in comparison to optimized stirred tank cultures (Fig. 4d, Table 1).Fig. 4


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)

Differentiation of iPSC into CMs in WAVE and stirred tank bioreactors. WAVE cultures were compared with optimized stirred tank bioreactor cultures (operated under 4 % O2 and intermittent agitation without direction change) in terms of CM differentiation efficiency. a. Phase contrast and fluorescence images showing cell aggregates composed by eGFP-positive cells (green) at days 5, 7, 9 and 11. Scale bars: 200 μm. c. Percentage of eGFP-positive aggregates during culture time. c. Percentage of eGFP-positive cells during culture time determined by flow cytometry. d. CM yields (stripped bars) and number of CMs generated per liter of culture medium throughput (CMs/L, black bars). e. Semiquantitative RT-PCR analyses showing expression of pluripotency (Oct4), endoderm (AFP), mesoderm (T-Bra) and CM-specific (Nkx2.5, cTnT, HCN4, Myl2, Myl7) genes during the time course of differentiation process in WAVE bioreactors. f. Quantitative RT-PCR analysis of cells cultured in optimized stirred tank and WAVE bioreactors, at day 6, before (day 9) and after CM selection (day 16 and 11 for stirred tank and WAVE bioreactor cultures, respectively). Expression was normalized to RNA from stirred tank bioreactor cultures operating with intermittent agitation, except for Oct4 expression that was normalized to RNA of undifferentiated cells (day 0, black bars). Data are given as mean ± SD of 3 individual bioreactors experiments. Significantly different, P < 0.05 (*) and P < 0.01 (**)
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Fig4: Differentiation of iPSC into CMs in WAVE and stirred tank bioreactors. WAVE cultures were compared with optimized stirred tank bioreactor cultures (operated under 4 % O2 and intermittent agitation without direction change) in terms of CM differentiation efficiency. a. Phase contrast and fluorescence images showing cell aggregates composed by eGFP-positive cells (green) at days 5, 7, 9 and 11. Scale bars: 200 μm. c. Percentage of eGFP-positive aggregates during culture time. c. Percentage of eGFP-positive cells during culture time determined by flow cytometry. d. CM yields (stripped bars) and number of CMs generated per liter of culture medium throughput (CMs/L, black bars). e. Semiquantitative RT-PCR analyses showing expression of pluripotency (Oct4), endoderm (AFP), mesoderm (T-Bra) and CM-specific (Nkx2.5, cTnT, HCN4, Myl2, Myl7) genes during the time course of differentiation process in WAVE bioreactors. f. Quantitative RT-PCR analysis of cells cultured in optimized stirred tank and WAVE bioreactors, at day 6, before (day 9) and after CM selection (day 16 and 11 for stirred tank and WAVE bioreactor cultures, respectively). Expression was normalized to RNA from stirred tank bioreactor cultures operating with intermittent agitation, except for Oct4 expression that was normalized to RNA of undifferentiated cells (day 0, black bars). Data are given as mean ± SD of 3 individual bioreactors experiments. Significantly different, P < 0.05 (*) and P < 0.01 (**)
Mentions: The comparison of CM differentiation efficiency in WAVE and optimized stirred tank bioreactors (operated under 4 % O2 tension and intermittent agitation without direction change) revealed that WAVE bioreactor cultures favored CM lineage commitment, enabling a reduction in the differentiation time and increased CM yields (Fig. 4). In these cultures, eGFP-positive cells and contracting areas in aggregates were observed from day 5 onwards, increasing rapidly in number and size (Fig. 4a–c, Supplementary Movie I). By day 7, 54.3 ± 5.2 % of the aggregates exhibited a considerable area of eGFP-positive and spontaneously beating cells (Fig. 4a–b). In accordance with this result, flow cytometry analyses indicated that 38.6 ± 6.0 % of the cells in aggregates were eGFP-positive, compared to less than 5 % detected in stirred tank bioreactors at this time point (Fig. 4c). Importantly, on day 9 of culture, i.e. before CM selection, the percentage of eGFP-positive cells among all cells dissociated from aggregates was almost 2-fold higher in WAVE bioreactors (76.0 ± 5.8 %) compared to stirred tank cultures (43.9 ± 6.6, Fig. 4c, Table 1). Consequently, only 2 days of antibiotic treatment were sufficient to obtain a 97.6 % pure CM population in WAVE bioreactors (Fig. 4c, Table I). In contrast, at day 11 of differentiation, aggregates in stirred tank bioreactors were not yet completely pure and additional 5 days of antibiotic treatment were required to generate a 97.4 % pure CM population (Fig. 4a, c). In terms of CM productivities, in optimized stirred tank cultures approximately 0.430 L medium throughput (total volume of medium used during the process) resulted in the generation of 0.1x109 CMs, which correspond to a coefficient of 0.2x109 CMs/L. On the other hand, in WAVE bioreactor 2.3x109 CMs were produced in 1.5 L medium throughput (1 L until day 9, 0.5 L medium exchange at day 9) obtaining 1.5x109 CMs/L (Table 1). Overall, iPSC differentiation in WAVE bioreactors resulted in an improvement of about 40 % in CM yield (60.8 ± 0.7 CMs/input iPSC) and in a 5 times higher CM production per liter of culture medium throughput (CMs/L) in comparison to optimized stirred tank cultures (Fig. 4d, Table 1).Fig. 4

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