Chaoying Yin

Stoichiometry of Gata4, Mef2c, and Tbx5 Influences the Efficiency and Quality of Induced Cardiac Myocyte Reprogramming

Rationale: Generation of induced cardiac myocytes (iCMs) directly from fibroblasts offers great opportunities for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate of fibroblasts to fully reprogrammed iCMs, which could in part be attributed to unbalanced expression of reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) using the current gene delivery approach. Objective: We aimed to establish a system to express distinct ratios of G, M, T proteins in fibroblasts and determine the effect of G, M, T stoichiometry on iCM reprogramming. Methods and Results: We took advantage of the inherent feature of the polycistronic system and generated all possible combinations of G, M, T with identical 2A sequences in a single transgene. We demonstrated that each splicing order of G, M, T gave rise to distinct G, M, T protein expression levels. Combinations that resulted in higher protein level of Mef2c with lower levels of Gata4 and Tbx5 significantly enhanced reprogramming efficiency compared with separate G, M, T transduction. Importantly, after further optimization, the MGT vector resulted in more than 10-fold increase in the number of mature beating iCM loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiac myocyte markers. Conclusions: Our results demonstrate that stoichiometry of G, M, T protein expression influences the efficiency and quality of iCM reprogramming. The established optimal G, M, T expression condition will provide a valuable platform for future iCM studies.

Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming

Direct reprogramming of induced cardiomyocytes (iCMs) suffers from low efficiency and requires extensive epigenetic repatterning, although the underlying mechanisms are largely unknown. To address these issues, we screened for epigenetic regulators of iCM reprogramming and found that reducing levels of the polycomb complex gene Bmi1 significantly enhanced induction of beating iCMs from neonatal and adult mouse fibroblasts. The inhibitory role of Bmi1 in iCM reprogramming is mediated through direct interactions with regulatory regions of cardiogenic genes, rather than regulation of cell proliferation. Reduced Bmi1 expression corresponded with increased levels of the active histone mark H3K4me3 and reduced levels of repressive H2AK119ub at cardiogenic loci, and de-repression of cardiogenic gene expression during iCM conversion. Furthermore, Bmi1 deletion could substitute for Gata4 during iCM reprogramming. Thus, Bmi1 acts as a critical epigenetic barrier to iCM production. Bypassing this barrier simplifies iCM generation and increases yield, potentially streamlining iCM production for therapeutic purposes.

Re-patterning of H3K27me3, H3K4me3 and DNA methylation during fibroblast conversion into induced cardiomyocytes

Direct conversion of fibroblasts into induced cardiomyocytes (iCMs) offers an alternative strategy for cardiac disease modeling and regeneration. During iCM reprogramming, the starting fibroblasts must overcome existing epigenetic barriers to acquire the CM-like chromatin pattern. However, epigenetic dynamics along this reprogramming process have not been studied. Here, we took advantage of our recently generated polycistronic system and determined the dynamics of two critical histone marks, H3K27me3 and H3K4me3, in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. We observed reduced H3K27me3 and increased H3K4me3 at cardiac promoters as early as day 3, paralleled by a rapid significant increase in their mRNA expression. In contrast, H3K27me3 at loci encoding fibroblast marker genes did not increase until day 10 and H3K4me3 progressively decreased along the reprogramming process; these changes were accompanied by a gradual decrease in the mRNA expression of fibroblast marker genes. Further analyses of fibroblast-enriched transcription factors revealed a similarly late deposition of H3K27me3 and decreased mRNA expression of Sox9, Twist1 and Twist2, three important players in epithelial-mesenchymal transition. Our data suggest early rapid activation of the cardiac program and later progressive suppression of fibroblast fate at both epigenetic and transcriptional levels. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that not every single CpG was equally demethylated during early stages of iCM reprogramming. Rather, there are specific CpGs, whose demethylation states correlated tightly with transcription activation, that we propose are the major contributing CpGs. Our work thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci during iCM reprogramming and could provide future genome-wide epigenetic studies with important guidance such as the appropriate time window and loci to be utilized as positive and negative controls.

In vivo cardiac reprogramming using an optimal single polycistronic construct

Direct cardiac reprogramming holds great promise for cardiac regeneration and disease modelling.1–15 We recently reported that stoichiometry of Gata4 (G), Mef2c (M), and Tbx5 (T) influences the efficiency and quality of induced cardiomyocyte (iCM) reprogramming.16 We generated a full set of polycistronic vectors to manipulate the relative levels of G, M, and T protein expression. Among the six combinations, MGT that expresses a relative high expression of M and low expressions of G and T resulted in the most efficient in vitro iCM reprogramming.16 Here, we performed genetic lineage tracing in a murine myocardial infarction (MI) model to determine whether MGT could improve in vivo iCM reprogramming efficiency and result in a further improvement in ventricular contractile function compared with the traditional separate G, M, and T (G/M/T) delivery. We took advantage of the unique feature that retrovirus infects only dividing cells to deliver MGT, G/M/T, or dsRed into actively dividing cardiac fibroblasts, but not CMs, in MI hearts.3 We prepared retrovirus encoding the single-triplet MGT as well as G/M/T and dsRed control viruses. These viruses were subjected to ultracentrifugation and were concentrated at 1 × 1010 p.f.a./mL. According to the previously established protocol,3 8–12 week old male mice were treated with 10 μL of ultra-high-titre retrovirus (∼1010 p.f.a./mL) by injection into the myocardial wall immediately following coronary artery ligation. To determine whether MGT enhances reprogramming of non-myocytes into iCMs in vivo , we performed genetic lineage tracing experiments using Periostin-Cre; R26R-lacZ mice3 to quantify the number of iCMs that had a fibroblast origin. Consistent with previous reports,3 we did not detect any β-galactosidase activity in the CMs of control …

Single-cell transcriptomics reconstructs fate conversion from fibroblast to cardiomyocyte

Direct lineage conversion offers a new strategy for tissue regeneration and disease modelling. Despite recent success in directly reprogramming fibroblasts into various cell types, the precise changes that occur as fibroblasts progressively convert to the target cell fates remain unclear. The inherent heterogeneity and asynchronous nature of the reprogramming process renders it difficult to study this process using bulk genomic techniques. Here we used single-cell RNA sequencing to overcome this limitation and analysed global transcriptome changes at early stages during the reprogramming of mouse fibroblasts into induced cardiomyocytes (iCMs). Using unsupervised dimensionality reduction and clustering algorithms, we identified molecularly distinct subpopulations of cells during reprogramming. We also constructed routes of iCM formation, and delineated the relationship between cell proliferation and iCM induction. Further analysis of global gene expression changes during reprogramming revealed unexpected downregulation of factors involved in mRNA processing and splicing. Detailed functional analysis of the top candidate splicing factor, Ptbp1, revealed that it is a critical barrier for the acquisition of cardiomyocyte-specific splicing patterns in fibroblasts. Concomitantly, Ptbp1 depletion promoted cardiac transcriptome acquisition and increased iCM reprogramming efficiency. Additional quantitative analysis of our dataset revealed a strong correlation between the expression of each reprogramming factor and the progress of individual cells through the reprogramming process, and led to the discovery of new surface markers for the enrichment of iCMs. In summary, our single-cell transcriptomics approaches enabled us to reconstruct the reprogramming trajectory and to uncover intermediate cell populations, gene pathways and regulators involved in iCM induction.

Improved generation of induced cardiomyocytes using a polycistronic construct expressing optimal ratio of Gata4, Mef2c And Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

Generation of an inducible fibroblast cell line for studying direct cardiac reprogramming.

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) through forced expression of cardiac‐lineage specific transcription factors holds promise as an alternative strategy for cardiac regeneration. To facilitate research in iCM reprogramming, we generated a suite of new tools. We developed a transformed cell line derived from mouse embryonic fibroblasts (MEF). This fibroblast cell line (MEF‐T) harbors an αMHC‐eGFP reporter transgene for rapid detection of newly derived iCMs. The MEF‐T cell line is highly proliferative and easily transfected and transduced, making it an ideal tool for transgene expression and genetic manipulation. Additionally, we generated a Tet‐On inducible polycistronic iCM reprogramming construct for the temporal regulation of reprogramming factor expression. Furthermore, we introduced this construct into MEF‐T and created an inducible reprogrammable fibroblast cell line. These tools will facilitate future research in cell fate reprogramming by enabling the temporal control of reprogramming factor expression as well as high‐throughput screening using libraries of small molecules, noncoding RNAs, and siRNAs. genesis 54:398–406, 2016.

ErbB2 is required for cardiomyocyte proliferation in murine neonatal hearts

It has been long recognized that the mammalian heart loses its proliferative capacity soon after birth, yet, the molecular basis of this loss of cardiac proliferation postnatally is largely unknown. In this study, we found that cardiac ErbB2, a member of the epidermal growth factor receptor family, exhibits a rapid and dramatic decline in expression at the neonatal stage. We further demonstrate that conditional ablation of ErbB2 in the ventricular myocardium results in upregulation of negative cell cycle regulators and a significant reduction in cardiomyocyte proliferation during the narrow neonatal proliferative time window. Together, our data reveal a positive correlation between the expression levels of ErbB2 with neonatal cardiomyocyte proliferation and suggest that reduction in cardiac ErbB2 expression may contribute to the loss of postnatal cardiomyocyte proliferative capacity.

A Loss of Function Screen of Epigenetic Modifiers and Splicing Factors during Early Stage of Cardiac Reprogramming

Direct reprogramming of cardiac fibroblasts (CFs) to induced cardiomyocytes (iCMs) is a newly emerged promising approach for cardiac regeneration, disease modeling, and drug discovery. However, its potential has been drastically limited due to the low reprogramming efficiency and largely unknown underlying molecular mechanisms. We have previously screened and identified epigenetic factors related to histone modification during iCM reprogramming. Here, we used shRNAs targeting an additional battery of epigenetic factors involved in chromatin remodeling and RNA splicing factors to further identify inhibitors and facilitators of direct cardiac reprogramming. Knockdown of RNA splicing factors Sf3a1 or Sf3b1 significantly reduced the percentage and total number of cardiac marker positive iCMs accompanied with generally repressed gene expression. Removal of another RNA splicing factor Zrsr2 promoted the acquisition of CM molecular features in CFs and mouse embryonic fibroblasts (MEFs) at both protein and mRNA levels. Moreover, a consistent increase of reprogramming efficiency was observed in CFs and MEFs treated with shRNAs targeting Bcor (component of BCOR complex superfamily) or Stag2 (component of cohesin complex). Our work thus reveals several additional epigenetic and splicing factors that are either inhibitory to or required for iCM reprogramming and highlights the importance of epigenetic regulation and RNA splicing process during cell fate conversion.