Review articleCellular reprogramming of fibroblasts in heart regeneration
Graphical abstract
Introduction
>5 million Americans live with heart failure (HF), a devastating condition responsible for the high death rate and hospitalization [1]. One of the leading causes of HF is ischemic heart disease (IHD) caused by reduced blood flow to the heart [2]. Insufficient supply of oxygen and other nutrients in IHD causes the death of cardiomyocytes (CMs), weakening heart contraction. Myocardial infarction (MI) causes the death of CMs and the activation of cardiac fibroblasts (CFs) [3,4]. Activated CFs continuously proliferate and express extracellular matrix (ECM) proteins. Deposition of excessive ECM proteins results in cardiac fibrosis. CM loss and cardiac fibrosis synergistically impair cardiac function and structure. Around 40% of patients who suffer from MI eventually develop HF. CM loss is permanent because adult humans lack the sufficient regenerative capacity to remuscularization of infarct myocardium [5]. Advances in surgical and imaging techniques, pharmacotherapies, and implantable medical devices significantly improve outcomes and patient's quality of life [6]. However, current interventions can only slow the disease progression but cannot regenerate damaged myocardium completely [7]. Various strategies for heart regeneration (e.g., stimulation of endogenous CM proliferation, transplantation of stem cell-derived CMs, and direct reprogramming of CFs into CMs) have been intensively investigated during the past decade [8].
Humans and mice have low rates of CM proliferation in adulthood [[9], [10], [11], [12]]. Mice can regenerate their damaged hearts during a short time window after birth when CMs have high dividing rates [13]. Regeneration of the damaged heart by promoting CM proliferation attracts attention. After screening a human whole-genome microRNA (miRNA) library, Eulalio et al. identified two miRNAs (has-miR-590 and has-miR-199a) that facilitated the proliferation of both neonatal and adult CMs in vitro and in vivo [14]. After ligation of the left anterior descending coronary artery (LAD), adeno-associated virus (AAV) serotype 9 (AAV9) vectors expressing one miRNA (AAV9-miR-590 or AAV9-miR-199a) were injected into the peri-infarct area of adult mice. Overexpression of miR-590 or miR-199a in MI-hearts promoted CM proliferation, improved cardiac function, and decreased infarct size [14]. Gabisonia et al. examined the regenerative capacity of has-miR-199a in pigs [15]. Pigs were subjected to 90-min occlusion of the left anterior coronary artery followed by reperfusion. AAV6-miR-199a was injected into the left ventricle wall. Overexpression of miR-199a promoted CM proliferation, increased cardiac muscle mass, reduced scar size, and improved contractility of the heart one month after MI. However, continuous expression of miR-199a in the heart leads to sudden arrhythmic death of most pigs after 40 days [15], suggesting the detrimental effect of the over-proliferation of CMs. Inhibition of the Hippo pathway (e.g., by knockout or knockdown of Mst, sav, or Lats) leads to nuclear translocation of transcription factors Yap and Taz to stimulate CM proliferation in the mouse heart [[16], [17], [18], [19]]. AAV9-Sav-shRNA viral particles were transendocardially injected into the border zone of the infarct pig heart using a catheter-based delivery method two weeks after ischemia/reperfusion (I/R) injury [20]. Knockdown of Sav leads to increased CM proliferation and reduced scar size in MI pigs. MI-pigs treated with AAV9-Sav-shRNA displayed a ∼ 14% improvement in left ventricular ejection fraction three months after injection. The investigators did not observe adverse events, including uncontrolled CM proliferation, organ overgrowth, cardiac arrhythmias, and sudden cardiac death, possibly due to catheter-based gene delivery [20,21]. These studies in swine models [15,20,21] indicate that cardiac regeneration can be achieved by enhancement of CM proliferation if the proliferation event is tightly controlled.
It has been hypothesized that implanted stem cells can differentiate into CMs to regenerate the myocardium in the injured heart. However, clinical trials of stem cell therapy, including transplantation of mesenchymal stem cells, for heart regeneration have produced neutral or marginally positive outcomes [22,23]. The development of alternative approaches for heart regeneration is clinically urgent. Expression of lineage-specific factor cocktails or treatment with small molecules and/or growth factors can reprogram fibroblasts into many cell types, e.g., induced pluripotent stem cells or iPSCs [24,25], induced CMs (iCMs) [[26], [27], [28]], induced cardiac progenitor cells (iCPCs) [29], induced cardiac tissue-like structures (rCVT) [30], skeletal muscles [[31], [32], [33]], induced neuronal cells [34], induced hepatocytes [35], and induced dendritic cells [36]. Fibroblasts exist in all organs, becoming an ideal cell resource to generate cardiac cells. Importantly, autologous cardiac cells generated by fibroblast reprogramming have the potential to overcome immune rejection and physiologically couple with surrounding cardiovascular tissue after they repopulate in the damaged heart. This review summarizes recent findings in the application of iPSC-derived CMs (iPSC-CMs), iCPCs, iCMs, and rCVT to repair the damaged heart and discusses the limitations and challenges of individual approaches (Fig. 1).
Section snippets
Transplantation of CMs derived from pluripotent stem cells into the injured heart
Takahashi et al. reprogrammed human and mouse fibroblasts into iPSCs by overexpressing pluripotent factors, Oct3/4, Sox2, Klf4, and c-Myc (also known as Yamanaka factors) (24, 25). Treatment with various combinations of small molecules robustly induces the differentiation of human pluripotent stem cells (PSC) such as iPSC into CMs (PSC-CMs) [37,38]. The advantage is that self-renewal PSCs can produce an unlimited amount of CMs. Transplantation of human PSC-CMs to small animals' injured hearts
Reprogramming fibroblasts into induced cardiomyocytes
The adult mammalian heart is composed of ∼30% CMs, ∼40% endothelial cells, ∼7% hematopoietic-derived cells, and ∼ 10% CFs [10,52]. Resident CFs provide scaffold support and express ECM proteins to maintain the structure and mechanoproperties of the heart. In addition to CM necrosis, MI also causes the activation of CFs, which leads to CF proliferation and increases the expression of ECM proteins [53,54]. Activated CFs play an essential role in wound healing after acute MI to prevent heart
Reprogramming fibroblasts into induced cardiac progenitor cells
Compared to terminally differentiated iCMs, cardiac progenitor cells (CPCs) are more scalable due to their self-renewal capacity. Furthermore, CPCs can be purified based on CPC markers, such as insulin gene enhancer protein 1 (Isl1), fetal liver kinase 1 (Flk-1), platelet-derived growth factor receptor (PdgfR) α, stage-specific embryonic antigen 1 (SSEA-1), and NK2 homeobox 5 (Nkx2.5) [[122], [123], [124], [125]]. In addition, CPCs can differentiate into three types of cardiac-lineage cells (
Conclusion and perspectives
Advances in cellular reprogramming make it possible to apply precision medicine approaches for heart regeneration. Patient-specific iPSC-CMs, iCPCs, iCMs, and rCVT derived from a patient's fibroblasts can avoid immune rejection after they repopulate in the damaged heart. There are several ongoing clinical trials using human iPSC-CMs to treat heart failure (NCT04945018 and NCT04396899) or ischemic cardiomyopathy/heart failure (NCT04696328 and NCT05566600). The other reprogramming-based
Declaration of Competing Interest
None.
Acknowledgments
This work was supported by National Institute of Health HL133230 and HL159086.
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