Isolation and phenotyping of cardiac-derived progenitor cells from neonatal mice

Dysfunctions of resident progenitor cells play a significant role in the pathogenesis of decreased myocardial contractility in heart failure, so the most promising approaches for the treatment of heart disease are cardiac-derived stem/progenitor cells (CSCs). Materials and methods. Protocols for progenitor cell cultures from different parts of the heart of newborn FVB/N mice have been developed and their proliferative potential has been characterized. Comparative analysis of the expression of CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD309 and troponin I by cells from native myocardial biopsies and in the obtained cultures was performed by flow cytometric immunophenotyping. Results. The expression of mesenchymal markers CD44 and CD90 in the absence of the hematopoietic marker CD45 was demonstrated in early passages in mouse myocardial progenitor cell cultures. Relatively high expression of CD34 and CD31 was found. The presence of a minor population of CD44+117+ cells which correspond to the phenotype of cardiac progenitor cells, was detected. Expression of troponin I as one of the key markers of cardiomyocytes as well as the vascular endothelial growth factor receptor has been confirmed in terminally differentiated cultures of cells with contractile activity. Conclusions. It was found that newborn mice in the myocardial tissue contain more cells with the expression of markers of cardiac progenitors than in adult animals. The relative content of such cells is higher in the atria than in the ventricles. Cardiac progenitor cells in neonatal mice derived from the atrial appendages have better proliferative potential than cell cultures isolated from the ventricles.

Distinct Mitochondrial Remodeling During Mesoderm Differentiation in a Human-Based Stem Cell Model

Given the considerable interest in using stem cells for modeling and treating disease, it is essential to understand what regulates self-renewal and differentiation. Remodeling of mitochondria and metabolism, with the shift from glycolysis to oxidative phosphorylation (OXPHOS), plays a fundamental role in maintaining pluripotency and stem cell fate. It has been suggested that the metabolic “switch” from glycolysis to OXPHOS is germ layer-specific as glycolysis remains active during early ectoderm commitment but is downregulated during the transition to mesoderm and endoderm lineages. How mitochondria adapt during these metabolic changes and whether mitochondria remodeling is tissue specific remain unclear. Here, we address the question of mitochondrial adaptation by examining the differentiation of human pluripotent stem cells to cardiac progenitors and further to differentiated mesodermal derivatives, including functional cardiomyocytes. In contrast to recent findings in neuronal differentiation, we found that mitochondrial content decreases continuously during mesoderm differentiation, despite increased mitochondrial activity and higher levels of ATP-linked respiration. Thus, our work highlights similarities in mitochondrial remodeling during the transition from pluripotent to multipotent state in ectodermal and mesodermal lineages, while at the same time demonstrating cell-lineage-specific adaptations upon further differentiation. Our results improve the understanding of how mitochondrial remodeling and the metabolism interact during mesoderm differentiation and show that it is erroneous to assume that increased OXPHOS activity during differentiation requires a simultaneous expansion of mitochondrial content.

Generation and characterization of cardiac valve endothelial-like cells from human pluripotent stem cells

The cardiac valvular endothelial cells (VECs) are an ideal cell source that could be used for making the valve organoids. However, few studies have been focused on the derivation of this important cell type. Here we describe a two-step chemically defined xeno-free method for generating VEC-like cells from human pluripotent stem cells (hPSCs). HPSCs were specified to KDR+/ISL1+ multipotent cardiac progenitors (CPCs), followed by differentiation into valve endothelial-like cells (VELs) via an intermediate endocardial cushion cell (ECC) type. Mechanistically, administration of TGFb1 and BMP4 may specify VEC fate by activating the NOTCH/WNT signaling pathways and previously unidentified targets such as ATF3 and KLF family of transcription factors. When seeded onto the surface of the de-cellularized porcine aortic valve (DCV) matrix scaffolds, hPSC-derived VELs exhibit superior proliferative and clonogenic potential than the primary VECs and human aortic endothelial cells (HAEC). Our results show that hPSC-derived valvular cells could be efficiently generated from hPSCs, which might be used as seed cells for construction of valve organoids or next generation tissue engineered heart valves.

Abstract P515: Dynamics Of Cardiomyocyte Differentiation In Isogenic Induced Pluripotent Stem Cells Derived From Trisomy21 Patients Hints Molecular Pathology Of Congenital Heart Defects In Down Syndrome

Down syndrome (DS) is the most frequently occurring human chromosomal disorder and is responsible for a range of both congenital defects and progressive, degenerative conditions. For instance, an estimated 50% DS neonates are born with congenital heart defects (CHD) and more than 50% of DS adults develop early onset Alzheimer’s. Using induced pluripotent stem cells (iPSCs) derived from DS patients and isogenic controls we previously demonstrated the presence of a hyper-metabolic, hyper-fused mitochondrial network in trisomic iPSCs (3S-iPSCs) compared to disomic (2S-iPSCs) controls. Furthermore, mitochondrial function was normalized by siRNA depletion of RCAN1, an inhibitor of the protein phosphatase calcineurin (CN). Both CN signaling and mitochondrial metabolism have been implicated in a variety of steps during the progression from embryonic stem cells to cardiac progenitors, including self-renewal, exit from pluripotency, and commitment to cardiac verses hematopoietic lineages. Based on this, we hypothesized that the dynamics of many of these processes will be altered over the course of differentiation of 3S-iPSCs to cardiomyocytes when compared to 2S-iPSCs. Here, we investigate the temporal expression of pluripotency associated genes and lineage associated genes as well as cardiac mesoderm and mature cardiomyocyte specific genes. We also define and compare changes in CN activity, expression of specific CN isoforms, mitochondrial expansion, ROS generation, and activation of stress responses. Our study identifies early developmental and metabolic sequelae capable of contributing to CHD in DS that may result from a disruption in the normal balance in crosstalk between CN and RCAN1.

Abstract P331: Upregulated TBX1 by genetic variants are associated with human congenital heart disease

Congenital heart disease (CHD) is the most common human birth defect worldwide. The cause of CHD is so far not well understood. Uncovering genetic factors leading to CHD is still a pressing task to be solved. TBX1 is one of the transcription factors early expressed in embryonic cardiac progenitors. In animal models, imbalanced TBX1 activity leads to cardiac defects. Given the dosage effect of TBX1, it is possible that genetic variant altering TBX1 function or expression level would affect heart development and contribute to CHD. In order to study the association of genetic variants of TBX1 and CHD susceptibility, we performed genetic screening in 409 CHD patients and 213 healthy controls. Bioinformatic and in vitro functional studies were performed to evaluate the impact of genetic variants. One single nucleotide polymorphism (SNP), rs41260844, in TBX1 promoter region was identified to be associated with CHD. The minor allele of rs41260844 is associated with higher CHD risk and shows increased TBX1 promoter activity (Fig A). Further study showed the minor allele attenuates TBX1 promoter binding affinity with nuclear protein(s) (Fig B). In addition, a novel case-specific missense rare mutation, p.P164L, in T-box domain was identified and predicted as a deleterious mutation. Functional analysis showed a trend of increased TBX1 function with the rare mutation. In summary, we concluded that a higher TBX1 expression level or activity is associated with CHD susceptibility, which could affect TBX1 downstream targets and thus disrupt the balance of the complex regulation network during cardiogenesis.

Distinct mitochondrial remodeling during early cardiomyocyte development in a human-based stem cell model

Post-mitotic tissues with high-energy demand rely on ATP generated by the mitochondrial respiratory chain through the process of oxidative phosphorylation (OXPHOS). There is common agreement that mitochondrial content and OXPHOS activity increase as cells exit from pluripotency state to meet the higher energy requirement of differentiated tissues such as heart. In this study, we examined the hypothesis that mitochondrial expansion during differentiation is necessary to compensate for higher energy demand in differentiated cells. We assessed mitochondrial and cellular metabolism during differentiation of human pluripotent stem cells to cardiac progenitors and further to functional cardiomyocytes. Contrary to expectations, we found that mitochondrial content decreased progressively during mesoderm differentiation. Nevertheless, we found that there was increased mitochondrial activity and higher levels of ATP-linked respiration, which we suggest more than compensate for the lower mitochondrial number. Our findings support a model whereby mitochondrial maturation during cardiomyocyte differentiation depends on increased efficiency of ATP generation through OXPHOS not increased mitochondrial biogenesis. Thus, the timing of the mitochondria expansion during cardiomyocyte differentiation will have to be revisited in light of these findings.