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OPEN
Received: 2 February 2017 Accepted: 12 July 2017 Published: xx xx xxxx
Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells Cláudia Correia1,2, Alexey Koshkin1,2, Patrícia Duarte1,2, Dongjian Hu3,4, Ana Teixeira1,2,5, Ibrahim Domian3,4, Margarida Serra1,2 & Paula M. Alves1,2 The immature phenotype of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) constrains their potential in cell therapy and drug testing. In this study, we report that shifting hPSCCMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. Integrated “-Omics” analyses showed that addition of galactose to culture medium improves total oxidative capacity of the cells and ameliorates fatty acid oxidation avoiding the lipotoxicity that results from cell exposure to high fatty acid levels. This study provides an important link between substrate utilization and functional maturation of hPSC-CMs facilitating the application of this promising cell type in clinical and preclinical applications. In the last decade remarkable progress has been made towards the establishment of protocols for directed differentiation of human pluripotent stem cells (hPSCs, including hiPSCs and hESCs) into cardiomyocytes (hPSC-CMs)1. However, hPSC-CMs are often immature, showing metabolic, structural and functional characteristics that more closely resemble fetal CMs rather than adult CMs2. hPSC-CMs commonly display disarrayed sarcomeres, irregular shapes, underdeveloped mitochondria and use glucose (Glc) as major energy source, contrasting with adult CMs which present organized sarcomere structures, rod-shaped morphologies, well-developed mitochondria with mature lamellar cristae and rely on fatty acid (FA) β-oxidation for energy production2, 3. Although some efforts have been made recently towards the development of methods for enhancing hPSC-CM maturity (by increasing time in culture4, applying mechanical and electrical stimulation5–7, adding chemicals or small molecules8, adjusting substrate stiffness9, using genetic approaches10, 11 or growth as 3-dimensional (3D) tissues12–15) the outcomes have been variable. The use of distinct sets of analyses for CM maturation profile characterization has also limited the direct comparison between different studies. CM maturation in vivo has been associated with a transition from an embryonic-like glycolytic to an adult-like oxidative metabolism16. In a normal heart, 70% of ATP generation comes from FA oxidation, whereas Glc, lactate (Lac) and pyruvate (Pyr) provide only 30% of the energy produced17. It has been shown that hPSC-CMs that are metabolically dependent on FA β-oxidation, in vitro, recapitulate cellular activities of more mature CMs, 1
iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal. 2Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB-NOVA), Oeiras, 2780-157, Portugal. 3Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA, 02114, USA. 4Harvard Medical School, Boston, MA 02115, USA, Harvard Stem Cell Institute, Cambridge, MA, 02138, USA. 5Present address: ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058, Basel, Switzerland. Correspondence and requests for materials should be addressed to M.S. (email: [email protected]) or P.M.A. (email: [email protected]) ScIentIfIc ReporTS | 7: 8590 | DOI:10.1038/s41598-017-08713-4
1
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Figure 1. Effect of culture medium composition on central carbon metabolism of hiPSC-CMs. (A) Representative scheme illustrating the experimental set-up. The metabolome of hiPSC-CMs was evaluated before (d0) and after 10 (d10) and 20 days (d20) of culture in different media: GLCM (black), FAM (Orange), GFAM (Red) and LACM&GFAM (Grey). (B) Heatmap image of metabolome data illustrating specific consumption (blue) and production (red) rates of metabolites (qMET; nmol/(106cell.h)). (C) Ratio of Lac production to Glc consumption (YLac/Glc) in GLCM through culture time. (D) Percentage of labelled M2 isotopomers from [1,2-13C]Glc in Lac and in TCA intermediates (Cit, Fum, Mal). (E) Percentage of labelled M4 isotopomers from [U-13C]Gln in TCA intermediates. M2 and M4, reflect the first round of TCA cycling of [1,213 C]Glc and [U-13C]Gln, respectively. (F) Basal oxygen consumption rate (OCR). (G) Extracellular acidification rate (ECAR). (H) OCR/ECAR ratio reflecting the relative contribution of OXPHOS over glycolysis for energy generation. (I–K) Pie charts indicating the contribution of the main substrates of each media, for TCA cycle pools. The percentages were determined based on the incorporation of each labelled substrate in Cit, Fum and Mal. OA: Oleic Acid and PA: Palmitic acid. Data are represented as mean ± SD of 12–24 wells, n ≥ 3 separate experiments. *p
OPEN
Received: 2 February 2017 Accepted: 12 July 2017 Published: xx xx xxxx
Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells Cláudia Correia1,2, Alexey Koshkin1,2, Patrícia Duarte1,2, Dongjian Hu3,4, Ana Teixeira1,2,5, Ibrahim Domian3,4, Margarida Serra1,2 & Paula M. Alves1,2 The immature phenotype of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) constrains their potential in cell therapy and drug testing. In this study, we report that shifting hPSCCMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. Integrated “-Omics” analyses showed that addition of galactose to culture medium improves total oxidative capacity of the cells and ameliorates fatty acid oxidation avoiding the lipotoxicity that results from cell exposure to high fatty acid levels. This study provides an important link between substrate utilization and functional maturation of hPSC-CMs facilitating the application of this promising cell type in clinical and preclinical applications. In the last decade remarkable progress has been made towards the establishment of protocols for directed differentiation of human pluripotent stem cells (hPSCs, including hiPSCs and hESCs) into cardiomyocytes (hPSC-CMs)1. However, hPSC-CMs are often immature, showing metabolic, structural and functional characteristics that more closely resemble fetal CMs rather than adult CMs2. hPSC-CMs commonly display disarrayed sarcomeres, irregular shapes, underdeveloped mitochondria and use glucose (Glc) as major energy source, contrasting with adult CMs which present organized sarcomere structures, rod-shaped morphologies, well-developed mitochondria with mature lamellar cristae and rely on fatty acid (FA) β-oxidation for energy production2, 3. Although some efforts have been made recently towards the development of methods for enhancing hPSC-CM maturity (by increasing time in culture4, applying mechanical and electrical stimulation5–7, adding chemicals or small molecules8, adjusting substrate stiffness9, using genetic approaches10, 11 or growth as 3-dimensional (3D) tissues12–15) the outcomes have been variable. The use of distinct sets of analyses for CM maturation profile characterization has also limited the direct comparison between different studies. CM maturation in vivo has been associated with a transition from an embryonic-like glycolytic to an adult-like oxidative metabolism16. In a normal heart, 70% of ATP generation comes from FA oxidation, whereas Glc, lactate (Lac) and pyruvate (Pyr) provide only 30% of the energy produced17. It has been shown that hPSC-CMs that are metabolically dependent on FA β-oxidation, in vitro, recapitulate cellular activities of more mature CMs, 1
iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal. 2Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB-NOVA), Oeiras, 2780-157, Portugal. 3Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA, 02114, USA. 4Harvard Medical School, Boston, MA 02115, USA, Harvard Stem Cell Institute, Cambridge, MA, 02138, USA. 5Present address: ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058, Basel, Switzerland. Correspondence and requests for materials should be addressed to M.S. (email: [email protected]) or P.M.A. (email: [email protected]) ScIentIfIc ReporTS | 7: 8590 | DOI:10.1038/s41598-017-08713-4
1
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Figure 1. Effect of culture medium composition on central carbon metabolism of hiPSC-CMs. (A) Representative scheme illustrating the experimental set-up. The metabolome of hiPSC-CMs was evaluated before (d0) and after 10 (d10) and 20 days (d20) of culture in different media: GLCM (black), FAM (Orange), GFAM (Red) and LACM&GFAM (Grey). (B) Heatmap image of metabolome data illustrating specific consumption (blue) and production (red) rates of metabolites (qMET; nmol/(106cell.h)). (C) Ratio of Lac production to Glc consumption (YLac/Glc) in GLCM through culture time. (D) Percentage of labelled M2 isotopomers from [1,2-13C]Glc in Lac and in TCA intermediates (Cit, Fum, Mal). (E) Percentage of labelled M4 isotopomers from [U-13C]Gln in TCA intermediates. M2 and M4, reflect the first round of TCA cycling of [1,213 C]Glc and [U-13C]Gln, respectively. (F) Basal oxygen consumption rate (OCR). (G) Extracellular acidification rate (ECAR). (H) OCR/ECAR ratio reflecting the relative contribution of OXPHOS over glycolysis for energy generation. (I–K) Pie charts indicating the contribution of the main substrates of each media, for TCA cycle pools. The percentages were determined based on the incorporation of each labelled substrate in Cit, Fum and Mal. OA: Oleic Acid and PA: Palmitic acid. Data are represented as mean ± SD of 12–24 wells, n ≥ 3 separate experiments. *p
