Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Sharma, Arun; Li, Guang; Rajarajan, Kuppusamy; Hamaguchi, Ryoko; Burridge, Paul W.; Wu, Sean M.

doi:10.3791/52628

Cited by 80 publications

(70 citation statements)

References 13 publications

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“…The in vitro cardiac differentiation from PSCs is similar to heart development involving the primitive streak formation, the specification of the lateral mesoderm, and the generation of cardiac progenitor cells and cardiac maturation. Cardiac progenitor cell formation mainly occurred during days 3 to 5 in the chemically defined methods [52]. In this study, through the administration of histamine in different time periods, we revealed that histamine enhanced cardiac differentiation mainly through promoting cardiac progenitor cell generation.…”

Section: Discussionmentioning

confidence: 53%

Disruption of histamine/H1R signaling pathway represses cardiac differentiation and maturation of human induced pluripotent stem cells

Zhu

Ding

et al. 2020

Stem Cell Res Ther

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Background:The efficiency and quality of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are crucial for regenerative medicine, disease modeling, drug screening, and the study of the development events during cardiac specification. However, their applications have been hampered by the differentiation efficiency, poor maturation, and high interline variability. Recent studies have reported that histamine plays important roles in hematopoietic stem cell proliferation and neutrophil maturation. However, its roles in cardiovascular tissue regeneration have not been thoroughly investigated. In the current study, we identified a novel physiological function of the histamine/histamine 1 receptor (H 1 R) signal in regulating the differentiation of hiPSC-CMs and heart development.Methods: Transgenic zebrafish model (cmlc2: mCherry) was treated with histamine and histamine receptor (HR) antagonists. Histological morphology and ultrastructure of zebrafish heart were measured. Histamine-deficient pregnant mice (HDC −/− ) were treated with H 1 R antagonist (pyrilamine) by intragastric administration from E8.5 to E18.5. Cardiac histological morphology and ultrastructure were analyzed in neonatal mice, and cardiac function in adult mice was measured. In vitro, histamine and HR antagonists were administrated in the culture medium during hiPSC-CM differentiation at different stages. The efficiency and maturation of cardiac differentiation were evaluated. Finally, histamine-treated hiPSC-CMs were transplanted into ischemic myocardium to detect the possible therapeutic effect.Results: Administration of H 1 R antagonist during heart development induced cardiac dysplasia in zebrafish. Furthermore, using histidine decarboxylase (HDC) knockout mice, we examined abnormal swelling of myocardial mitochondria and autophagy formation under the condition of endogenous histamine deficiency. Histamine significantly promoted myocardial differentiation from human induced pluripotent stem cells (hiPSCs) with better structure and function via a H 1 R-dependent signal. The activation of histamine/H 1 R signaling pathway augmented hiPSC-derived cardiomyocyte (hiPSC-CM) differentiation through the ERK1/2-STAT3 signaling pathway. In addition, histamine-pre-treated hiPSC-CMs were transplanted into the ischemic hearts of myocardial injured mice and exhibited better survival and myocardial protection. Conclusions: Thus, these findings indicated that histamine/H 1 R and its downstream signals were not only involved in cardiac differentiation but also provided a better survival environment for stem cell transplanted into ischemic myocardium.

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Section: Discussionmentioning

confidence: 53%

Disruption of histamine/H1R signaling pathway represses cardiac differentiation and maturation of human induced pluripotent stem cells

Zhu

Ding

et al. 2020

Stem Cell Res Ther

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“…To enrich the population of cardiomyocytes, metabolic selection was used (98). On days 12-17, the cells were cultured in glucose-free media (glucose-free RPMI-1640 supplemented with B-27 supplement with insulin).…”

Section: Methodsmentioning

confidence: 99%

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Clippinger¹,

Cloonan²,

Greenberg³

et al. 2019

Preprint

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27Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a 28 major indicator for heart transplant. The disease is frequently caused by mutations of 29 sarcomeric proteins; however, it is not well understood how these molecular mutations 30 lead to alterations in cellular organization and contractility. To address this critical gap in 31 our knowledge, we studied the molecular and cellular consequences of a DCM mutation 32 in troponin-T, DK210. We determined the molecular mechanism of DK210 and used 33 computational modeling to predict that the mutation should reduce the force per 34 sarcomere. In mutant cardiomyocytes, we found that DK210 not only reduces contractility, 35 but also causes cellular hypertrophy and impairs cardiomyocytes' ability to adapt to 36 changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and 37 disease). These results link the molecular and cellular phenotypes and implicate 38 alterations in mechanosensing as an important factor in the development of DCM. 39Results 100 DK210 decreases calcium sensitivity in an in vitro motility assay 101We set out to decipher the molecular mechanism of the DK210 mutation in vitro. 102The molecular effects of cardiomyopathy mutations depend on the myosin isoform (7-9, 103 35-37) and therefore, we used porcine cardiac ventricular myosin (38). Porcine ventricular 104 cardiac myosin (MYH7) is 97% identical to human, while murine cardiac myosin (MYH6) 105 is only 92% identical. Porcine cardiac myosin has very similar biophysical properties to 106 human cardiac myosin, including the kinetics of the ATPase cycle, step size, and 107 sensitivity to load (38-41), making it an ideal myosin for biophysical studies. 108Given the role of troponin-T in thin filament regulation, we first determined whether 109 the DK210 mutation affects calcium-based regulation of myosin binding to thin filaments 110 using an in vitro motility assay (42). Reconstituted thin filaments, consisting of porcine 111 cardiac actin and recombinantly expressed human troponin and tropomyosin, were added 112 to a flow cell coated with porcine cardiac myosin in the presence of ATP. The speed of 113 filament translocation was measured as a function of added calcium. As has been 114 reported previously, the speed of regulated thin filament translocation increased 115 sigmoidally with increasing Ca 2+ concentration (43), ( Figure 1B). Data were fit with the Hill 116 equation to obtain the pCa50 (i.e., the concentration of calcium necessary for half-117 maximal activation). Consistent with previous studies using mouse cardiac, rabbit cardiac, 118 and rabbit skeletal muscle fibers (31, 33, 44), DK210 shows a right-shifted curve (pCa50 119 = 5.7 ± 0.1) compared to the WT (pCa50 = 6.1 ± 0.1; p < 0.0001), meaning more calcium 120 is needed for the same level of activation. This suggests that the mutant could show 121 impaired force production during a calcium transient. 122 7 123 Molecular mechanism of DK210-induced changes in thin filament regulat...

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“…To further increase cardiomyocyte purity, the differentiated cells were subjected to subsequent glucose starvation using non-glucose-supplemented RPMI/B27 medium for three times (two days per time) to decrease non-cardiomyocyte cells, since cardiomyocytes are more tolerant to glucose starvation. 10 …”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Temporal Profiling of Transcriptome and Open Chromatin of Early Cardiomyocyte Differentiation Derived From hiPSCs and hESCs

Liu

Jiang

et al. 2017

Circulation Research

111

103

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Rationale Recent advances have improved our ability to generate cardiomyocytes from human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). However, our understanding of the transcriptional regulatory networks underlying early stages (i.e. from mesoderm to cardiac mesoderm) of cardiomyocyte differentiation remains limited. Objective To characterize transcriptome and chromatin accessibility during early cardiomyocyte differentiation from hiPSCs and hESCs. Methods and Results We profiled the temporal changes in transcriptome and chromatin accessibility at genome-wide levels during cardiomyocyte differentiation derived from two hiPSC lines and two hESC lines at four stages: pluripotent stem cells, mesoderm, cardiac mesoderm, and differentiated cardiomyocytes. Overall, RNA-seq analysis revealed that transcriptomes during early cardiomyocyte differentiation were highly concordant between hiPSCs and hESCs, and clustering of four cell lines within each time-point demonstrated that changes in genome-wide chromatin accessibility were similar across hiPSC and hESC cell lines. Weighted gene co-expression network analysis (WGCNA) identified several modules that were strongly correlated with different stages of cardiomyocyte differentiation. Several novel genes were identified with high weighted-connectivity within modules and exhibited co-expression patterns with other genes, including non-coding RNA LINC01124 and uncharacterized RNA AK127400 in the module related to the mesoderm stage; and ZEB1 in the module correlated with post-cardiac mesoderm. We further demonstrated that ZEB1 is required for early cardiomyocyte differentiation. In addition, based on integrative analysis of both WCGNA and TF-motif enrichment analysis, we determined numerous TFs likely to play important roles at different stages during cardiomyocyte differentiation, such as T and EOMES (mesoderm); LEF1 and MESP1 (from mesoderm to cardiac mesoderm); MEIS1 and GATA4 (post-cardiac mesoderm); JUN and FOS families, and MEIS2 (cardiomyocyte). Conclusions Both hiPSCs and hESCs share similar transcriptional regulatory mechanisms underlying early cardiac differentiation, and our results have revealed transcriptional regulatory networks and new factors (e.g. ZEB1) controlling early stages of cardiomyocyte differentiation.

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Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Cited by 80 publications

References 13 publications

Disruption of histamine/H1R signaling pathway represses cardiac differentiation and maturation of human induced pluripotent stem cells

Disruption of histamine/H1R signaling pathway represses cardiac differentiation and maturation of human induced pluripotent stem cells

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Genome-Wide Temporal Profiling of Transcriptome and Open Chromatin of Early Cardiomyocyte Differentiation Derived From hiPSCs and hESCs

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