Reversible Mitochondrial Fragmentation in iPSC-Derived Cardiomyocytes from Children with DCMA, a Mitochondrial Cardiomyopathy

Abstract

Background:The dilated cardiomyopathy with ataxia syndrome (DCMA) is an understudied autosomal recessive disease caused by loss-of-function mutations in the poorly characterized gene DNAJC19. Clinically, DCMA is commonly associated with heart failure and early death in affected children through an unknown mechanism.DCMA has been linked to Barth syndrome, a rare but well-studied disorder caused by deficient maturation of cardiolipin (CL), a key mitochondrial membrane phospholipid.Methods: Peripheral blood mononuclear cells from two children with DCMA and severe cardiac dysfunction were reprogrammed into induced pluripotent stem cells (iPSCs).Patient and control iPSCs were differentiated into beating cardiomyocytes (iPSC-CMs) using a metabolic selection strategy and mitochondrial structure and CL content before and after incubation with the mitochondrially-targeted peptide SS-31 were quantified.Results: Patient iPSCs carry the causative DNAJC19 mutation (rs137854888) found in the Hutterite population and the iPSC-CMs demonstrated highly fragmented and abnormally-shaped mitochondria associated with an imbalanced isoform ratio of the mitochondrial protein OPA1, an important regulator of mitochondrial fusion. These abnormalities were reversible by incubation with SS-31 for 24 hours. Differentiation of iPSCs into iPSC-CMs increased the number of CL species observed but consistent, significant differences in CL content were not seen between patients and control. Conclusions:We describe a unique and novel cellular model that provides insight into the mitochondrial abnormalities present in DCMA and identifies SS-31 as a potential therapeutic for this devastating disease.

Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells

“…Naïve hPSC aggregates were differentiated into cardiomyocytes, hepatocytes and neural rosettes using the previously published protocols 26 , 52 , 53 . The aggregates were collected on day 4 post-inoculation and were either dissociated into single cells using StemPro® Accutase® Cell Dissociation Reagent (Thermo Fisher Scientific, A1110501), or were directly used for in vitro differentiation.…”

“…Bioreactor-cultured, naïve hPSCs from H1 hESC line could also be directly differentiated into cells representing all three germ layers: cardiomyocytes (mesoderm), hepatocytes (endoderm), and neural rosettes (ectoderm) in vitro. This was done using previously published protocols for each specific differentiation 26,52,53 . Beating clusters of cardiomyocytes (Supplementary Movie 7) were purified using a lactate-based metabolic selection and, at day 20, mature cardiomyocytes showed robust expression of TNNT2 with clear appearance of linear Z-disk organocations (Fig.…”

“…The aggregates were collected on day 4 post-inoculation and were either dissociated into single cells using StemPro® Accutase® Cell Dissociation Reagent (Thermo Fisher Scientific, A1110501), or were directly used for in vitro differentiation. For both cardiomyocyte and hepatocyte differentiation, naïve hPSCs as single cells (at the density of 2E5 cells per plate)/or aggregates were cultured on Matrigel® hESC qualified Matrix-coated FluoroDish Cell Culture Dish plates (World Precision Instrument, FD35-100), containing mTeSR™1 or RSeT™ medium until they were 80-90% confluent, and then differentiation procedure was performed using the previously published protocol 52,53 . Mature cardiomyocytes and hepatocytes at day 20 of differentiation, were used for the analysis.…”

Overcoming Bioprocess Bottlenecks in the Large-Scale Expansion of High Quality hiPSC Aggregates in Vertical-Wheel Stirred Suspension Bioreactors

“…Karyogramswere analyzed according to the ISCN standards at ~450 band resolution using the Ikaros karyotyping system (Metasystems).Iodide Nucleic Acid Stain (Cat#T3605, Thermo Fisher). Cells were then rinsed twice with PBS and imaged using a Carl Zeiss Laser Scanning Microscope 700 with lasers at 488 nm and 639 nm and corresponding filter sets.Bioreactor cultured hiPSC aggregates were differentiated into cardiomyocytes, hepatocytes and neural rosettes using the previously published protocols[42][43][44]. The aggregates were collected on day 12 post-inoculation and were either dissociated into single cells as described in the above methods sections or directly plated as aggregates for in vitro differentiation.…”

“…The aggregates were collected on day 12 post-inoculation and were either dissociated into single cells as described in the above methods sections or directly plated as aggregates for in vitro differentiation. For both cardiomyocyte and hepatocyte differentiation, hiPSCs were seeded at a density of 2E5 cells/plate on Matrigel coated FluoroDish Cell Culture Dish plates (Cat#FD35-100, World Precision Instrument) containing mTeSR1 supplemented with Y-27632 and cultured to 80-90% confluency before undergoing differentiation[42,43]. Mature cardiomyocytes and hepatocytes at day 20 of differentiation, were used for the analysis.…”