Spontaneous Cardiac Differentiation Research Articles

Heme oxygenase-1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells.

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1-/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2-/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018.

184 Establishment of Porcine Induced Pluripotent Stem Cell Lines by Adding LIN 28 Transcription Factor

The establishment of porcine induced pluripotent stem cells (piPSC) is important in the field of human biomedical regenerative medicine. The pig model is a more representative model than current rodent models because it better mimics human physiology in different organ systems. The piPSC can be traditionally generated by reprogramming somatic cells using 4 transcription factors (4TF: OCT4, SOX2, KLF4, and c-MYC), similarly in human. However, it is difficult to maintain the pluripotent state of reprogrammed cells and they exhibit poor differentiation capacity. Hence, the 4TF may be not enough to reprogram porcine somatic cells. This study aimed to establish piPSC by adding LIN28 (referred to as 5TF) to the traditional 4TF, via retroviral vector. Here, we report the successful establishment of 3 piPSC lines by using the 5TF. All 5TF-piPSC lines exhibited a normal karyotype (38, XY) and a typical mouse embryonic stem cell (ESC) morphology, including tightly packed and dome-like shape, even after they were propagated over 40 passages. All 5TF-piPSC lines were positive for alkaline phosphatase staining and expressed high levels of ESC-like markers (OCT4, SOX2, NANOG, and SSEA-1). Importantly, the 5TF-piPSC lines showed pluripotent capacity, as evidenced by differentiation into 3 germ layers in vitro following cystic embryoid body formation, as well as by efficiently forming teratomas containing all 3 embryonic germ layers in vivo. Moreover, the 5TF-piPSC lines showed spontaneously contractile cardiomyocytes and expressed cardiomyocyte markers (cardiac troponin T) during spontaneous cardiac differentiation using cell aggregation into spherical-like structures referred to as embryoid bodies. Thus, the addition of LIN28 TF promoted long-term pluripotency of piPSC and enhanced the ability to differentiate towards 3 embryonic germ layers and cardiac lineage. This research project is supported by grants from the Mahidol University, Thailand.

MEK/ERK signaling is involved in the role of VEGF and IGF1 in cardiomyocyte differentiation of mouse adipose tissue-derived stromal cells

BackgroundWe found that mouse brown adipose tissue-derived stromal cells (BATDCs), but not white adipose tissue-derived stromal cells (WATDCs), could spontaneously differentiate into cardiomyocyte-like cells in a simple culture medium. This study would find out some critical trophic factors that were responsible for such difference in differentiation, and further determine the involved signaling pathway. Methods and resultsThe cardiomyocyte differentiation capacity of cells was identified by morphological observations, immunofluorescence staining, and evaluation of expression of cardiomyocyte specific markers. The amount of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF1) secreted by cells was determined by ELISA analysis. Results indicated that BATDCs secreted higher levels of VEGF and IGF1 than WATDCs. Supplementation of BATDCs with antibodies against VEGF receptor Flt-1 or IGF1 receptor Igf-1rα significantly suppressed the cardiac differentiation capacity of the cells. Additionally, anti-Flt-1 and anti-Igf-1rα antibodies decreased phosphorylation of ERK1/2 in BATDCs. Inhibition of MEK/ERK activity by the inhibitor PD0325901 or by RNA interference blunted the cardiac differentiation of BATDCs. Loading recombinant VEGF and IGF1, or transfecting their expression vectors into WATDCs, promoted cardiac differentiation of the cells. Preincubation with PD0325901, before VEGF and IGF1 supplementation or vector transfections, blocked the stimulation of cardiac differentiation in WATDCs. ConclusionsThese findings indicated that VEGF and IGF1 were critical factors for the spontaneous cardiac differentiation of BATDCs, and MEK/ERK signaling was involved in the role of VEGF and IGF1. VEGF and IGF1 could be used to promote the development of cardiomyocyte phenotype in WATDCs.

Graphene induces spontaneous cardiac differentiation in embryoid bodies.

Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.

Fullerene mediates proliferation and cardiomyogenic differentiation of adipose-derived stem cells via modulation of MAPK pathway and cardiac protein expression.

Zero-dimensional fullerenes can modulate the biological behavior of a variety of cell lines. However, the effects and molecular mechanisms of proliferation and cardiomyogenic differentiation in brown adipose-derived stem cells (BADSCs) are still unclear. In this study, we report the initial biological effects of fullerene-C60 on BADSCs at different concentrations. Results suggest that fullerene-C60 has no cytotoxic effects on BADSCs even at a concentration of 100 μg/mL. Fullerene-C60 improves the MAPK expression level and stem cell survival, proliferation, and cardiomyogenesis. Further, we found that the fullerene-C60 modulates cardiomyogenic differentiation. Fullerene-C60 improves the expression of cardiomyocyte-specific proteins (cTnT and α-sarcomeric actinin). At elevated concentration, fullerene-C60 reduces the incidence of diminished spontaneous cardiac differentiation of BADSCs with time. At the genetic level, fullerene-C60 (5 μg/mL) also improves the expression of cTnT. In addition, fullerene-C60 promotes the formation of gap junction among cells. These findings have important implications for clinical application of fullerenes in the treatment of myocardial infarction.

62 EFFECT OF PROGESTERONE ON CALCIUM REGULATION DURING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES

During spontaneous cardiac differentiation of mouse embryonic stem cells (mESCs, cell line E14), the effect of progesterone on calcium regulation was investigated. Calcium (Ca2+) release from sarcoplasmic reticulum (SR) regulates various cellular functions including the smooth or skeletal muscle contraction. The cardiac L-type Ca2+ channel plays a key role in excitation-contraction coupling of cardiomyocytes and contraction-related gene expression. The mESCs formed mouse embryonic bodies (mEBs) by hanging-drop for 4 days, and mEBs were suspended for 2 days in differentiation medium; DMEM/F:12, 15% charcoal-dextran-treated FBS, β-mercaptoethanol, minimal essential medium NEAA, and penicillin/streptomycin. Then, mEBs were attached onto 6-well culture plates and differentiated into cardiomyocytes. We analysed mRNA expressions for the cardiac lineage markers and calcium-regulating genes. Percentage of beating mEBs was time-dependently increased during differentiation. Differentiated mEBs showed the highest beating ratio (92.11 ± 2.98%) after attachment for 12 days. Beating ratio was decreased to 64.86 ± 4.25% in progesterone-treated mEBs. The mRNA levels of cardiac markers such as Tbx20, Isl1, Foxh1, cTn1, and RyR2 were increased, and troponin protein was observed in beating mEBs via immunocytochemistry. Expression of calcium/contraction regulating genes including Trpv2, Ryr2, CaM2, and Mlck3 was down-regulated by progesterone treatment. These results indicate that progesterone has influences on cardiac differentiation and contraction of cardiomyocytes through regulating intercellular calcium ion. This research was supported by a grant (15182MFDS460) from the Ministry of Food and Drug Safety in 2015.

Induced expression of Fndc5 significantly increased cardiomyocyte differentiation rate of mouse embryonic stem cells

Fibronectin type III domain-containing 5 protein (Fndc5) is an exercise hormone and its transcript profile in mouse showed high degree of expression in heart, skeletal muscle and brain. Our previous studies indicated a significant increase (approximately 10 fold) in mRNA level of Fndc5 when embryonic stem cells were differentiated into beating bodies. As a step closer to identify the involvement of Fndc5 in the process of cardiomyocyte differentiation, we generated a stably inducible transduced mouse embryonic stem cell (mESC) line that overexpressed Fndc5 following Doxycycline induction. Our results indicated that the overexpression of Fndc5 during spontaneous cardiac differentiation significantly increased not only at RNA levels for mesodermal markers but also at the transcriptional levels for cardiac progenitor and cardiac genes. These data suggest that Fndc5 may be involved in cardiomyocyte differentiation. Therefore, a new hope will be arisen for potential application of this myokine for regeneration of damaged cardiac tissues especially in cardiac failure.

Cardiac progenitor cells from the left atrial appendage may originate from a resident non-hematopoietic myeloid progenitor population

Purpose: We recently identified three distinct cardiac progenitor cell (CPC) populations from the left atrial appendages (LAAs) of adult murine hearts. These populations have distinct phenotypes and characteristics. Type A CPCs are c-kit+/Sca-1+, Type B CPCs are c-kit+/CD45+, and more mature Type C CPCs can be derived from Type B CPCs in vitro. The purpose of this study was to analyze the embryologic origin and the connection between the different CPC populations. Methods: We performed a whole-genome expression array to the cell sorted Type A, Type B and Type C CPC populations. The LAAs were analyzed using immunohistochemistry. Tissue culture derived cells were analyzed using immunostaining and FACS. Neural crest lineage tracing was performed with Pax3-cre eYFP reporter mice. We investigated the spontaneous cardiac differentiation process from Type B to Type C using live microscopy and FACS. Results: Transcriptome comparison demonstrated a significant up-regulation of the neural crest differentiation –pathway genes in Type A and C CPCs. Type B CPCs expressed a gene profile, which matches that of non-hematopoietic, resident macrophage –like cells found in other tissues. Pax3 lineage tracing demonstrated traced cells in the subepicardial zone of the LAA, co-stained with F4/80 macrophage marker and c-kit. Closely attached were epicardially lined F4/80+ cells with no eYFP signal. In tissue culture, eYFP signal was strongest in the Type B CPCs and lower in the Type A CPCs. Fibroblasts in the culture were eYFP negative. The CD45 expression in the Type B CPCs was stable (∼90% of the cells) up to five passages folllowed by a rapid decline of CD45, c-kit and Flk-1 expression. In live microscopy, formation of giant Sca-1+ and Gata-4+ cells by cell fusion was followed by asymmetric divisions creating Type C CPCs, which expressed cardiac myosin heavy chain and atrial natriuretic factor. The transcriptome was significantly skewed toward a cardiomyocyte phenotype with the spontaneous differentiation, although this differentiation was not complete, as seen by electron microscopy. The partially differentiated Type C CPC population could be grown for >60 passages. Conclusion: Our results suggest that rather than having distinct embryonic origins, the different types of CPCs in the LAA originate from a resident, non-hematopoietic myeloid progenitor cell population. The neural crest differentiation pathway can be activated in these cells during adulthood, possibly in cardiac stress situations, enabling differentiation to cardiac lineage with a novel mechanism consisting of cell fusion and asymmetric division.

Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body development

Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for the differentiation of early mesoderm and cardiomyocytes. The addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5-25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on day 6 of expansion and then plateaued until day 20. In real-time PCR analysis, the cardiac development-related genes MESP1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. The activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smad1/5/8 and by the nuclear localization of phospho-Smad1/5/8 and Smad4. The addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. The cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and a course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promotes cardiomyocyte induction from human ES cells.

Cardiac mesoangioblasts are committed, self-renewable progenitors, associated with small vessels of juvenile mouse ventricle

Different cardiac stem/progenitor cells have been recently identified in the post-natal heart. We describe here the identification, clonal expansion and characterization of self-renewing progenitors that differ from those previously described for high spontaneous cardiac differentiation. Unique coexpression of endothelial and pericyte markers identify these cells as cardiac mesoangioblasts and allow prospective isolation and clonal expansion from the juvenile mouse ventricle. Cardiac mesoangioblasts express many cardiac transcription factors and spontaneously differentiate into beating cardiomyocytes that assemble mature sarcomeres and express typical cardiac ion channels. Cells similarly isolated from the atrium do not spontaneously differentiate. When injected into the ventricle after coronary artery ligation, cardiac mesoangioblasts efficiently generate new myocardium in the peripheral area of the necrotic zone, as they do when grafted in the embryonic chick heart. These data identify cardiac mesoangioblasts as committed progenitors, downstream of earlier stem/progenitor cells and suitable for the cell therapy of a subset of juvenile cardiac diseases.

INTERFERENTIAL ELECTRIC FIELD TREATMENT REVEALED A LOW INCREASE OF SPONTANEOUS CARDIAC DIFFERENTIATION BUT NO CYCLIC AMP CHANGES NOR INDUCTION OF CARDIAC-SPECIFIC GENE EXPRESSION IN PLURIPOTENT EMBRYONAL CARCINOMA P19 CELLS

Earlier studies showed that interferential currents (IFC) can modulate intracellular cAMP levels in fibroblasts. Based on these findings, an open prospective study to treat palmar psoriasis showed that IFC leads to an improvement of psoriatic skin. Here, we investigated whether 4 kHz interferential electric fields modulated at 10, 50, and 100 Hz are able to induce cardiac differentiation in pluripotent embryonal carcinoma (EC) P19 cells. P19 cells differentiated as embryoid bodies (EBs) in suspension culture were exposed to interferential fields during cultivation days 4 to 6 between capacitor plates (field strength 100 V/m). Signals were applied for 5 min at 3 hr intervals for 24, 48, or 72 hr (protocol 1) or 24 hr during day 4, 5, or 6 (protocol 2). Spontaneous cardiac differentiation in sham-exposed control embryoid bodies occurred in 4.3% compared to 100% in DMSO-treated variants (positive control). However, 4 kHz modulated at 100 Hz applied for 72 hr induced differentiation in 8% of the embryoid bodies. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed no elevated α- and β-cardiac MHC-mRNA levels. 2D-electrophoresis experiments did not indicate changes in protein expression patterns. We conclude that interferential electric fields are able to induce cardiac differentiation, but only in a small subpopulation of EC P19 cells. RT-PCR analysis and 2D-electrophoresis experiments could not resolve significant differences.