Cardiac reprogramming via chromatin remodeling by CRISPR activation
Authors: James W.S. Jahng, Joseph C. Wu
Journal: Molecular Therapy (2021)

Cardiovascular progenitor cells (CPCs) are the resident cells in the heart that can generate different cardiovascular cells, such as cardiomyocytes (CMs), smooth muscle cells (SMCs), and endothelial cells (ECs). The exogenous production of CPCs in vitro has been of immense interest in the field of heart regenerative medicine with numerous studies highlighting the therapeutic potential of CPCs to treat myocardial infarction.1Amini H. Rezaie J. Vosoughi A. Rahbarghazi R. Nouri M. Cardiac progenitor cells application in cardiovascular disease.J. Cardiovasc. Thorac. Res. 2017; 9: 127-132https://doi.org/10.15171/jcvtr.2017.22Crossref PubMed Google Scholar In this issue of Molecular Therapy, Jiang et al.2Jiang L. Liang J. Huang W. Ma J. Park K.H. Wu Z. Chen P. Zhu H. Ma J.J. Cai W. et al.CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy.Mol. Ther. 2021; https://doi.org/10.1016/j.ymthe.2021.10.015Abstract Full Text Full Text PDF Scopus (3) Google Scholar detail how CPCs can be efficiently generated from non-cardiac fibroblasts by clustered regularly interspaced short palindromic repeat (CRISPR) activation of three cardiogenic transcription factors. Unlike conventional reprograming strategies that overexpress the exogenous reprogramming factors, CRISPR activation uses a recombinant nuclease-deficient Cas9 (dCas9) to enhance endogenous gene expression by recruiting transcriptional activators (e.g., VP64) at specific loci directed by single-guide RNAs.3Nishiga M. Qi L.S. Wu J.C. Therapeutic genome editing in cardiovascular diseases.Adv. Drug Deliv. Rev. 2021; 168: 147-157https://doi.org/10.1016/j.addr.2020.02.003Crossref PubMed Scopus (13) Google Scholar Jiang et al.2Jiang L. Liang J. Huang W. Ma J. Park K.H. Wu Z. Chen P. Zhu H. Ma J.J. Cai W. et al.CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy.Mol. Ther. 2021; https://doi.org/10.1016/j.ymthe.2021.10.015Abstract Full Text Full Text PDF Scopus (3) Google Scholar systematically tested 11 cardiogenic transcription factors to better define the minimal cardiogenic reprogramming factors (Gata4, Nkx2.5, and Tbx5-GNT) with an Nkx2.5 cardiac enhancer-driven eGFP reporter system (Nkx2.5eGFP). Simultaneous activation of GNT robustly increased Nkx2.5eGFP expression at more than 80% efficiency, with the resultant Nkx2.5eGFP+ cells being able to give rise to different cardiovascular cells (CMs, ECs, and SMCs) (Figure 1). Jiang et al. noticed the challenge in maintaining CPCs because the mechanobiological environment could significantly affect the cardiogenic potential of CPCs. Nkx2.5eGFP expression was maintained when cells were cultured in suspension, whereas it rapidly dissipated when cells were re-plated in the monolayer. To massively expand Nkx2.5eGFP+ CPCs, a chemical cocktail (comprising leukemia inhibitory factor, basic FGF, and SB431542 [TGF-β inhibitor]) was formulated to improve the self-renewal and proliferative capacity of CPCs through serial passaging. Interestingly, the addition of insulin-like growth factor 1, a well-known cell-cycle inducer, did not enhance the proliferative capacity of CPCs, whereas adding CHIR99021 (a GSK3β inhibitor) did. This is consistent with a recent report finding that mechanobiology modulation with CHIR99021 treatment robustly induced CM proliferation.4Buikema J.W. Lee S. Goodyer W.R. Maas R.G. Chirikian O. Li G. Miao Y. Paige S.L. Lee D. Wu H. et al.Wnt activation and reduced cell-cell contact synergistically induce massive expansion of functional human iPSC-derived cardiomyocytes.Cell Stem Cell. 2020; 27: 50-63.e55https://doi.org/10.1016/j.stem.2020.06.001Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar Although the precise mechanisms underlying CPC expansion (e.g., the AKT signaling pathway or alternative pathways, such as via oxidative stress) have not been delineated, the chemical cocktail developed by Jiang et al.2Jiang L. Liang J. Huang W. Ma J. Park K.H. Wu Z. Chen P. Zhu H. Ma J.J. Cai W. et al.CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy.Mol. Ther. 2021; https://doi.org/10.1016/j.ymthe.2021.10.015Abstract Full Text Full Text PDF Scopus (3) Google Scholar was able to successfully expand Nkx2.5eGFP+ CPCs over 20 passages (∼1014-fold expansion) (Figure 1). Jiang et al.2Jiang L. Liang J. Huang W. Ma J. Park K.H. Wu Z. Chen P. Zhu H. Ma J.J. Cai W. et al.CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy.Mol. Ther. 2021; https://doi.org/10.1016/j.ymthe.2021.10.015Abstract Full Text Full Text PDF Scopus (3) Google Scholar next investigated the cardiogenic potential of CPCs after serial passaging by terminally differentiating CPCs into different cardiovascular lineages. CPCs at early passage (<10) expressed cardiac markers (cTnT, αMHC), but only sparsely expressed vascular markers (αSMA, CD31, and Cdh5). In contrast, CPCs at late passage expressed both cardiac and vascular markers. CPCs at late passage readily differentiated into vascular lineages (SMCs and ECs) upon angiogenic factor (VEGFA and/or PDGF-BB) treatment, yet they were inefficient at generating CMs even in the absence of angiogenic factors (∼20% CMs versus ∼50% SMCs or ECs). A transcriptomic analysis of CPCs at late passage showed a high abundance of transcripts related to CMs (e.g., sarcomere organization and cardiac muscle contraction) yet the current CPC expansion method could condition CPCs to favorably commit to vascular lineage over the cardiac lineage (Figure 1). Nonetheless, CPCs after serial passaging could be engrafted to re-muscularize the hearts of infarcted mice so as to restore contractile function. Aligning with the in vitro experiment, the engrafted CPCs predominantly gave rise to vascular (∼24% SMCs and ∼39% ECs) over cardiac lineages (∼36% CMs). However, to further restore contractile function, the derivation of greater numbers of CMs relative to vascular cells from CPCs may be required. Understanding the epigenetic basis of lineage commitment is critical to modulating intrinsic cell fate determination and cellular interaction with the extrinsic microenvironment. Jiang et al.2Jiang L. Liang J. Huang W. Ma J. Park K.H. Wu Z. Chen P. Zhu H. Ma J.J. Cai W. et al.CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy.Mol. Ther. 2021; https://doi.org/10.1016/j.ymthe.2021.10.015Abstract Full Text Full Text PDF Scopus (3) Google Scholar used a doxycycline-inducible system to examine the epigenetic remodeling that occurs during and after CPC induction. After 21 days of GNT activation by doxycycline treatment, chromatin accessibility and expression of GNT were enhanced. Withdrawing doxycycline over 14 days following GNT activation was found to maintain chromatin accessibility and expression of GNT and led to a significant increase in active chromatin marking (H3K4me3) at the GNT loci. Such epigenetic remodeling establishes an autoregulatory loop of transcription factor-to-promoter interaction to maintain CPCs after reprogramming. It remains unknown whether the epigenetic remodeling remains stable after serial passaging or whether it is linked to the propensity of the CPCs to differentiate to the cardiac/vascular lineages. Future studies should focus on the application of global epigenetic profiling and/or single cell profiling (e.g., scRNA-seq, scATAC-seq) to explore the complex gene regulatory networks within CPCs/CPC-derivatives and their interaction with the microenvironment. One potential key contributor is metabolism. Emerging evidence suggests that energy substrate availability in the culture medium and intrinsic cell metabolism(s) may significantly affect the cell fate determination via epigenetic modification in cardiac reprogramming.5Jahng J.W.S. Zhang M. Wu J.C. The role of metabolism in directed differentiation versus trans-differentiation of cardiomyocytes.Semin. Cell Dev. Biol. 2021; https://doi.org/10.1016/j.semcdb.2021.05.018Crossref PubMed Scopus (1) Google Scholar Jiang et al.2Jiang L. Liang J. Huang W. Ma J. Park K.H. Wu Z. Chen P. Zhu H. Ma J.J. Cai W. et al.CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy.Mol. Ther. 2021; https://doi.org/10.1016/j.ymthe.2021.10.015Abstract Full Text Full Text PDF Scopus (3) Google Scholar predominantly used culture medium supplemented with high glucose, a major energy source for non-CMs, which may have conditioned CPCs to the vascular lineage throughout serial passaging. In summary, the work by Jiang et al.2Jiang L. Liang J. Huang W. Ma J. Park K.H. Wu Z. Chen P. Zhu H. Ma J.J. Cai W. et al.CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy.Mol. Ther. 2021; https://doi.org/10.1016/j.ymthe.2021.10.015Abstract Full Text Full Text PDF Scopus (3) Google Scholar is a novel application of CRISPR-Cas9 genome editing and a beautiful example of direct reprogramming without using exogenous gene delivery. A better understanding of the transcriptional and epigenetic regulation of CPC reprogramming, maintenance, and cardiovascular lineage differentiation will further refine the production of CPCs at a therapeutic scale. The current work is supported by National Institute of Health (NIH) grants R01 HL126527, R01 HL130020, and R01 HL146990 (J.C.W.), and the Translational Research Institute for Space Health (TRISH) through Cooperative Agreement NNX16AO69A (J.W.S.J). We thank Blake Wu for proof-reading of our manuscript. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapyJiang et al.Molecular TherapyOctober 19, 2021In BriefThis study from Jiang et al. identifies essential loci controlling CPC formation and helps understanding of the mechanisms underlying cardiac gene autoregulation during cell lineage reprogramming. It also provides a new epigenetic reprogramming system of CPC generation using CRISPR activation approaches for myocardial infarction therapy. Full-Text PDF Open Access