Terapia celular

Circulation. 2022 Dec 20;146(25):1950-1967. doi: 10.1161/CIRCULATIONAHA.122.061960. Epub 2022 Nov 24.

ABSTRACT

BACKGROUND: Cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes (CMs). However, certain animals readily regenerate lost myocardium through a process involving dedifferentiation, which unlocks their proliferative capacities.

METHODS: We bred mice with inducible, CM-specific expression of the Yamanaka factors, enabling adult CM reprogramming and dedifferentiation in vivo.

RESULTS: Two days after induction, adult CMs presented a dedifferentiated phenotype and increased proliferation in vivo. Microarray analysis revealed that upregulation of ketogenesis was central to this process. Adeno-associated virus-driven HMGCS2 overexpression induced ketogenesis in adult CMs and recapitulated CM dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue, and HMGCS2 knockout mice showed impaired cardiac function and response to injury. Finally, we showed that exogenous HMGCS2 rescues cardiac function after ischemic injury.

CONCLUSIONS: Our data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to CM injury, thus allowing cell dedifferentiation and proliferation as a regenerative response.

PMID:36420731 | DOI:10.1161/CIRCULATIONAHA.122.061960

Mol Pharm. 2022 Nov 22. doi: 10.1021/acs.molpharmaceut.2c00650. Online ahead of print.

ABSTRACT

With permanent heart muscle injury or death, myocardial infarction (MI) is complicated by inflammatory, proliferation and remodeling phases from both the early ischemic period and subsequent infarct expansion. Though in situ re-establishment of blood flow to the infarct zone and delays of the ventricular remodeling process are current treatment options of MI, they fail to address massive loss of viable cardiomyocytes while transplanting stem cells to regenerate heart is hindered by their poor retention in the infarct bed. Equipped with heart-specific mimicry and extracellular matrix (ECM)-like functionality on the network structure, hydrogels leveraging tissue-matching biomechanics and biocompatibility can mechanically constrain the infarct and act as localized transport of bioactive ingredients to refresh the dysfunctional heart under the constant cyclic stress. Given diverse characteristics of hydrogel including conductivity, anisotropy, adhesiveness, biodegradability, self-healing and mechanical properties driving local cardiac repair, we aim to investigate and conclude the dynamic balance between ordered architectures of hydrogels and the post-MI pathological milieu. Additionally, our review summarizes advantages of heart-tailored architectures of hydrogels in cardiac repair following MI. Finally, we propose challenges and prospects in clinical translation of hydrogels to draw theoretical guidance on cardiac repair and regeneration after MI.

PMID:36413809 | DOI:10.1021/acs.molpharmaceut.2c00650

J Mol Cell Cardiol. 2022 Nov 18;174:77-87. doi: 10.1016/j.yjmcc.2022.11.004. Online ahead of print.

ABSTRACT

Advances in stem cell biology have facilitated cardiac regeneration, and many animal studies and several initial clinical trials have been conducted using human pluripotent stem cell-derived cardiomyocytes (PSC-CMs). Most preclinical and clinical studies have typically transplanted PSC-CMs via the following two distinct approaches: direct intramyocardial injection or epicardial delivery of engineered heart tissue. Both approaches present common disadvantages, including a mandatory thoracotomy and poor engraftment. Furthermore, a standard transplantation approach has yet to be established. In this study, we tested the feasibility of performing intracoronary administration of PSC-CMs based on a commonly used method of transplanting somatic stem cells. Six male cynomolgus monkeys underwent intracoronary administration of dispersed human PSC-CMs or PSC-CM aggregates, which are called cardiac spheroids, with multiple cell dosages. The recipient animals were sacrificed at 4 weeks post-transplantation for histological analysis. Intracoronary administration of dispersed human PSC-CMs in the cynomolgus monkeys did not lead to coronary embolism or graft survival. Although the transplanted cardiac spheroids became partially engrafted, they also induced scar formation due to cardiac ischemic injury. Cardiac engraftment and scar formation were reasonably consistent with the spheroid size or cell dosage. These findings indicate that intracoronary transplantation of PSC-CMs is an inefficient therapeutic approach.

PMID:36403760 | DOI:10.1016/j.yjmcc.2022.11.004

Elife. 2022 Nov 18;11:e77014. doi: 10.7554/eLife.77014.

ABSTRACT

Cardiovascular disease is the leading cause of death worldwide due to the inability of adult heart to regenerate after injury. N6-methyladenosine (m6A) methylation catalyzed by the enzyme methyltransferase-like 3 (Mettl3) plays an important role in various physiological and pathological bioprocesses. However, the role of m6A in heart regeneration remains largely unclear. To study m6A function in heart regeneration, we modulated Mettl3 expression in vitro and in vivo. Knockdown of Mettl3 significantly increased the proliferation of cardiomyocytes and accelerated heart regeneration following heart injury in neonatal and adult mice. However, Mettl3 overexpression decreased cardiomyocyte proliferation and suppressed heart regeneration in postnatal mice. Conjoint analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq identified Fgf16 as a downstream target of Mettl3-mediated m6A modification during postnatal heart regeneration. RIP-qPCR and luciferase reporter assays revealed that Mettl3 negatively regulates Fgf16 mRNA expression in an m6A-Ythdf2-dependent manner. The silencing of Fgf16 suppressed the proliferation of cardiomyocytes. However, the overexpression of ΔFgf16, in which the m6A consensus sequence was mutated, significantly increased cardiomyocyte proliferation and accelerated heart regeneration in postnatal mice compared with wild-type Fgf16. Our data demonstrate that Mettl3 post-transcriptionally reduces Fgf16 mRNA levels through an m6A-Ythdf2-dependen pathway, thereby controlling cardiomyocyte proliferation and heart regeneration.

PMID:36399125 | PMC:PMC9674341 | DOI:10.7554/eLife.77014

iScience. 2022 Oct 20;25(11):105402. doi: 10.1016/j.isci.2022.105402. eCollection 2022 Nov 18.

ABSTRACT

Currently, there are no treatments that ameliorate cardiac cell death, the underlying basis of cardiovascular disease. An unexplored cell type in cardiac regeneration is cardiac Purkinje cells; specialized cells from the cardiac conduction system (CCS) responsible for propagating electrical signals. Purkinje cells have tremendous potential as a regenerative treatment because they may intrinsically integrate with the CCS of a recipient myocardium, resulting in more efficient electrical conduction in diseased hearts. This study is the first to demonstrate an effective protocol for the direct reprogramming of human cardiomyocytes into cardiac Purkinje-like cells using small molecules. The cells generated were genetically and functionally similar to native cardiac Purkinje cells, where expression of key cardiac Purkinje genes such as CNTN2, ETV1, PCP4, IRX3, SCN5a, HCN2 and the conduction of electrical signals with increased velocity was observed. This study may help to advance the quest to finding an optimized cell therapy for heart regeneration.

PMID:36388958 | PMC:PMC9646947 | DOI:10.1016/j.isci.2022.105402

Nanomedicine. 2023 Jan;47:102616. doi: 10.1016/j.nano.2022.102616. Epub 2022 Oct 29.

ABSTRACT

In recent decades, myocardial regeneration through stem cell transplantation and tissue engineering has been viewed as a promising technique for treating myocardial infarction. As a result, the researcher attempts to see whether co-culturing modified mesenchymal stem cells with Au@Ch-SF macro-hydrogel and H9C2 may help with tissue regeneration and cardiac function recovery. The gold nanoparticles (Au) incorporated into the chitosan-silk fibroin hydrogel (Au@Ch-SF) were validated using spectral and microscopic examinations. The most essential elements of hydrogel groups were investigated in detail, including weight loss, mechanical strength, and drug release rate. Initially, the cardioblast cells (H9C2 cells) was incubated with Au@Ch-SF macro-hydrogel, followed by mesenchymal stem cells (2 × 105) were transplanted into the Au@Ch-SF macro-hydrogel+H9C2 culture at the ratio of 2:1. Further, cardiac phenotype development, cytokines expression and tissue regenerative performance of modified mesenchymal stem cells treatment were studied through various in vitro and in vivo analyses. The Au@Ch-SF macro-hydrogel gelation time was much faster than that of Ch and Ch-SF hydrogels, showing that Ch and SF exhibited greater intermolecular interactions. The obtained Au@Ch-SF macro-hydrogel has no toxicity on mesenchymal stem cells (MS) or cardiac myoblast (H9C2) cells, according to the biocompatibility investigation. MS cells co-cultured with Au@Ch-SF macro-hydrogel and H9C2 cells also stimulated cardiomyocyte fiber restoration, which has been confirmed in myocardial infarction rats using -MHC and Cx43 myocardial indicators. We developed a novel method of co-cultured therapy using MS cells, Au@Ch-SF macro-hydrogel, and H9C2 cells which could promote the regenerative activities in myocardial ischemia cells. These study findings show that co-cultured MS therapy might be effective for the treatment of myocardial injury.

PMID:36374915 | DOI:10.1016/j.nano.2022.102616

Polymers (Basel). 2022 Oct 28;14(21):4571. doi: 10.3390/polym14214571.

ABSTRACT

Myocardial infarction is a leading cause of death worldwide and has severe consequences including irreversible damage to the myocardium, which can lead to heart failure. Cardiac tissue engineering aims to re-engineer the infarcted myocardium using tissues made from human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to regenerate heart muscle and restore contractile function via an implantable epicardial patch. The current limitations of this technology include both biomanufacturing challenges in maintaining tissue integrity during implantation and biological challenges in inducing cell alignment, maturation, and coordinated electromechanical function, which, when overcome, may be able to prevent adverse cardiac remodeling through mechanical support in the injured heart to facilitate regeneration. Polymer scaffolds serve to mechanically reinforce both engineered and host tissues. Here, we introduce a novel biodegradable, customizable scaffold composed of wet-spun polycaprolactone (PCL) microfibers to strengthen engineered tissues and provide an anisotropic mechanical environment to promote engineered tissue formation. We developed a wet-spinning process to produce consistent fibers which are then collected on an automated mandrel that precisely controls the angle of intersection of fibers and their spacing to generate mechanically anisotropic scaffolds. Through optimization of the wet-spinning process, we tuned the fiber diameter to 339 ± 31 µm and 105 ± 9 µm and achieved a high degree of fidelity in the fiber structure within the scaffold (fiber angle within 1.8° of prediction). Through degradation and mechanical testing, we demonstrate the ability to maintain scaffold mechanical integrity as well as tune the mechanical environment of the scaffold through structure (Young's modulus of 120.8 ± 1.90 MPa for 0° scaffolds, 60.34 ± 11.41 MPa for 30° scaffolds, 73.59 ± 3.167 MPa for 60° scaffolds, and 49.31 ± 6.90 MPa for 90° scaffolds), while observing decreased hysteresis in angled vs. parallel scaffolds. Further, we embedded the fibrous PCL scaffolds in a collagen hydrogel mixed with hiPSC-CMs to form engineered cardiac tissue with high cell survival, tissue compaction, and active contractility of the hiPSC-CMs. Through this work, we develop and optimize a versatile biomanufacturing process to generate customizable PCL fibrous scaffolds which can be readily utilized to guide engineered tissue formation and function.

PMID:36365565 | PMC:PMC9657938 | DOI:10.3390/polym14214571

Front Cardiovasc Med. 2022 Oct 10;9:953211. doi: 10.3389/fcvm.2022.953211. eCollection 2022.

ABSTRACT

BACKGROUND: Mechanisms contributing to tissue remodeling of the infarcted heart following cell-based therapy remain elusive. While cell-based interventions have the potential to influence the cardiac healing process, there is little direct evidence of preservation of functional myocardium.

AIM: The aim of the study was to investigate tissue remodeling in the infarcted heart following human embryonic stem cell-derived endothelial cell product (hESC-ECP) therapy.

MATERIALS AND METHODS: Following coronary artery ligation (CAL) to induce cardiac ischemia, we investigated infarct size at 1 day post-injection in media-injected controls (CALM, n = 11), hESC-ECP-injected mice (CALC, n = 10), and dead hESC-ECP-injected mice (CALD, n = 6); echocardiography-based functional outcomes 14 days post-injection in experimental (CALM, n = 13; CALC, n = 17) and SHAM surgical mice (n = 4); and mature infarct size (CALM and CALC, both n = 6). We investigated ligand-receptor interactions (LRIs) in hESC-ECP cell populations, incorporating a publicly available C57BL/6J mouse cardiomyocyte-free scRNAseq dataset with naive, 1 day, and 3 days post-CAL hearts.

RESULTS: Human embryonic stem cell-derived endothelial cell product injection reduces the infarct area (CALM: 54.5 ± 5.0%, CALC: 21.3 ± 4.9%), and end-diastolic (CALM: 87.8 ± 8.9 uL, CALC: 63.3 ± 2.7 uL) and end-systolic ventricular volume (CALM: 56.4 ± 9.3 uL, CALC: 33.7 ± 2.6 uL). LRI analyses indicate an alternative immunomodulatory effect mediated via viable hESC-ECP-resident signaling.

CONCLUSION: Delivery of the live hESC-ECP following CAL modulates the wound healing response during acute pathological remodeling, reducing infarct area, and preserving functional myocardium in this relatively acute model. Potential intrinsic myocardial cellular/hESC-ECP interactions indicate that discreet immunomodulation could provide novel therapeutic avenues to improve cardiac outcomes following myocardial infarction.

PMID:36299872 | PMC:PMC9588936 | DOI:10.3389/fcvm.2022.953211

Theranostics. 2022 Oct 3;12(16):7009-7031. doi: 10.7150/thno.74563. eCollection 2022.

ABSTRACT

Rationale: Chronic pressure overload is a major trigger of cardiac pathological hypertrophy that eventually leads to heart disease and heart failure. Understanding the mechanisms governing hypertrophy is the key to develop therapeutic strategies for heart diseases. Methods: We built chronic pressure overload mice model by abdominal aortic constriction (AAC) to explore the features of Yes-associated protein 1 (YAP1). Then AAV-cTNT-Cre was applied to Yap1F/F mice to induce mosaic depletion of YAP1. Myh6CreERT2; H11CAG-LSL-YAP1 mice were involved to establish YAP1 overexpression model by Tomaxifen injection. ATAC-seq and bioChIP-seq were used to explore the potential targets of YAP1, which were verified by a series of luciferase reporter assays. Dnm1l and Mfn1 were re-expressed in AAC mice by AAV-cTNT-Dnm1l and AAV-cTNT-Mfn1. Finally, Verteprofin was used to inhibit YAP1 to rescue cardiac hypertrophy. Results: We found that pathological hypertrophy was accompanied with the activation of YAP1. Cardiomyocyte-specific deletion of Yap1 attenuated AAC-induced hypertrophy. Overexpression of YAP1 was sufficient to phenocopy AAC-induced hypertrophy. YAP1 activation resulted in the perturbation of mitochondria ultrastructure and function, which was associated with the repression of mitochondria dynamics regulators Dnm1l and Mfn1. Mitochondrial-related genes Dnm1l and Mfn1, are significantly targeted by TEAD1/YAP complex. Overexpression of Dnm1l and Mfn1 synergistically rescued YAP1-induced mitochondrial damages and cardiac hypertrophy. Pharmacological repression of YAP1 by verteporfin attenuated mitochondrial damages and pathological hypertrophy in AAC-treated mice. Interestingly, YAP1-induced mitochondria damages also led to increased reactive oxidative species, DNA damages, and the suppression of cardiomyocyte proliferation. Conclusion: Together, these data uncovered YAP signaling as a therapeutic target for pressure overload-induced heart diseases and cautioned the efforts to induce cardiomyocyte regeneration by activating YAP.

PMID:36276651 | PMC:PMC9576622 | DOI:10.7150/thno.74563

Stem Cell Rev Rep. 2022 Oct 22. doi: 10.1007/s12015-022-10468-z. Online ahead of print.

ABSTRACT

BACKGROUND: Ischemic heart disease, often caused by an acute myocardial infarction (AMI) is one of the leading causes of morbidity and mortality worldwide. Despite significant advances in medical and procedural therapies, millions of AMI patients progress to develop heart failure every year.

METHODS: Here, we examine the combination therapy of human mesenchymal stromal cells (MSCs) and endothelial colony-forming cells (ECFCs) to reduce the early ischemic damage (MSCs) and enhance angiogenesis (ECFCs) in a pre-clinical model of acute myocardial infarction. NOD/SCID mice were subjected to AMI followed by transplantation of MSCs and ECFCs either alone or in combination. Cardiomyocyte apoptosis and cardiac functional recovery were assessed in short- and long-term follow-up studies.

RESULTS: At 1 day after AMI, MSC- and ECFC-treated animals demonstrated significantly lower cardiomyocyte apoptosis compared to vehicle-treated animals. This phenomenon was associated with a significant reduction in infarct size, cardiac fibrosis, and improvement in functional cardiac recovery 4 weeks after AMI.

CONCLUSIONS: The use of ECFCs, MSCs, and the combination of both cell types reduce cardiomyocyte apoptosis, scar size, and adverse cardiac remodeling, compared to vehicle, in a pre-clinical model of AMI. These results support the use of this combined cell therapy approach in future human studies during the acute phase of ischemic cardiac injury.

PMID:36271311 | DOI:10.1007/s12015-022-10468-z

ACS Appl Bio Mater. 2022 Nov 21;5(11):5218-5230. doi: 10.1021/acsabm.2c00659. Epub 2022 Oct 20.

ABSTRACT

Cardiac patches are widely investigated to repair or regenerate diseased and aging cardiac tissues. While numerous studies looked into engineering the biochemical/biomechanical/cellular microenvironment and components in the heart tissue, the changes induced by cardiac patches and how they should be controlled to promote cardiac tissue repair/regeneration remains an important yet untapped direction, especially immunological responses. In this study, we designed and fabricated a bilaminated cardiac patch based on extracellular matrix (ECM) materials loaded with the extracellular vesicles (EVs) derived from mesenchymal stromal cells. The function of the biological material to modulate the injury-related microenvironment in a cardiac infarction model in mice was investigated. The study showed that the treatment of EV-ECM patches to the infarcted area increased the level of immunomodulatory major histocompatibility complex class IIlo macrophages in the early stage of myocardial injury to mitigate excessive inflammatory responses due to injury. The intensity of the acquired proinflammatory immune response in systemic immune organs was reduced. Further analyses indicated that the EV-ECM patches exhibited proangiogenic functions and decreased the infarct size with improved cardiac recovery in mice. The study provided insights into shaping the injury-related microenvironment through the incorporation of extracellular vesicles into cardiac patches, and the EV-ECM material is a promising design paradigm to improve the function of cardiac patches to treat myocardial injuries and diseases.

PMID:36265007 | DOI:10.1021/acsabm.2c00659

Sci Rep. 2022 Oct 18;12(1):17409. doi: 10.1038/s41598-022-22225-w.

ABSTRACT

Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs-namely human organotypic cardiac microtissues (hOCMTs)-that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.

PMID:36257968 | PMC:PMC9579206 | DOI:10.1038/s41598-022-22225-w

Int J Biol Macromol. 2022 Dec 1;222(Pt B):3045-3056. doi: 10.1016/j.ijbiomac.2022.10.079. Epub 2022 Oct 12.

ABSTRACT

Cell therapy is one of the promising approaches for cardiac repair, subsequently after infarction or injury. However, contemporary mesenchymal stromal/stem cell (MSCs) delivery strategies result in low retention and poor engraftment of donor cells, thus limiting the therapeutic efficacy. Here, we developed an engineered biomimetic cardiogel patch (EBCP) comprising of the native decellularized cardiac extracellular matrix (ECM) "cardiogel" and chitosan, leading to the efficient regeneration of injured myocardium. We also developed novel bio-adhesive that is capable of suture-free epicardial placement of EBCP to injured myocardium. We have illustrated the potential of the mussels-inspired bioadhesive system, which comprises gelatin catechol and partially oxidized chitosan, which relies on self-crosslinking capability, to promote wet adhesion. In vitro studies with isolated cardiogel promoted cell proliferation, adhesion, and migration while aiding cardiomyogenic differentiation. The EBCP's ability to protect cells from abrasion due to surrounding tissues in the myocardial infarction (MI) rat model makes it more desirable. Furthermore, the epicardial implantation of the EBCP loaded with MSCs improves the initial retention of cells and subsequent functional cardiac recovery with enhanced myocardial tissue restoration. Histological examination showed the presence of EBCP and infiltration of cells to the infarcted heart tissue. The fast and facile synthesis of bioadhesive and major therapeutic benefits of EBCP make it a potential candidate for recuperating the ailing heart.

PMID:36243159 | DOI:10.1016/j.ijbiomac.2022.10.079

Int J Mol Sci. 2022 Sep 29;23(19):11490. doi: 10.3390/ijms231911490.

ABSTRACT

This review discusses a novel experimental approach for the regeneration of original tissue structure by recruitment of endogenous stem-cells to injured sites following administration of α-gal nanoparticles, which harness the natural anti-Gal antibody. Anti-Gal is produced in large amounts in all humans, and it binds the multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) presented on α-gal nanoparticles. In situ binding of anti-Gal to α-gal nanoparticles activates the complement system and generates complement cleavage chemotactic-peptides that rapidly recruit macrophages. Macrophages reaching anti-Gal coated α-gal nanoparticles bind them via Fc/Fc receptor interaction and polarize into M2 pro-reparative macrophages. These macrophages secrete various cytokines that orchestrate regeneration of the injured tissue, including VEGF inducing neo-vascularization and cytokines directing homing of stem-cells to injury sites. Homing of stem-cells is also directed by interaction of complement cleavage peptides with their corresponding receptors on the stem-cells. Application of α-gal nanoparticles to skin wounds of anti-Gal producing mice results in decrease in healing time by half. Furthermore, α-gal nanoparticles treated wounds restore the normal structure of the injured skin without fibrosis or scar formation. Similarly, in a mouse model of occlusion/reperfusion myocardial-infarction, near complete regeneration after intramyocardial injection of α-gal nanoparticles was demonstrated, whereas hearts injected with saline display ~20% fibrosis and scar formation of the left ventricular wall. It is suggested that recruitment of stem-cells following anti-Gal/α-gal nanoparticles interaction in injured tissues may result in induction of localized regeneration facilitated by conducive microenvironments generated by pro-reparative macrophage secretions and "cues" provided by the extracellular matrix in the injury site.

PMID:36232789 | PMC:PMC9569695 | DOI:10.3390/ijms231911490

Front Cardiovasc Med. 2022 Sep 26;9:901396. doi: 10.3389/fcvm.2022.901396. eCollection 2022.

ABSTRACT

Promoting cardiomyocyte proliferation is a promising strategy to regenerate the heart. Yet, so far, it is poorly understood how cardiomyocyte proliferation is regulated, and no factor identified to promote mammalian cardiomyocyte proliferation has been translated into medical practice. Therefore, finding a novel factor will be vital. Here, we established a live cell screening based on mouse embryonic stem cell-derived cardiomyocytes expressing a non-functional human geminin deletion mutant fused to Azami Green (CM7/1-hgem-derived cardiomyocytes). We screened for a subset of compounds of the small molecule library Spectrum Collection and identified 19 potential inducers of stem cell-derived cardiomyocyte proliferation. Furthermore, the pro-proliferative potential of identified candidate compounds was validated in neonatal and adult rat cardiomyocytes as well as human induced pluripotent stem cell-derived cardiomyocytes. 18 of these compounds promoted mitosis and cytokinesis in neonatal rat cardiomyocytes. Among the top four candidates were two cardiac glycosides, peruvoside and convallatoxin, the flavonoid osajin, and the selective α-adrenoceptor antagonist and imidazoline I1 receptor ligand efaroxan hydrochloride. Inhibition of PTEN and GSK-3β enhanced cell cycle re-entry and progression upon stimulation with cardiac glycosides and osajin, while inhibition of IP3 receptors inhibited the cell cycle-promoting effect of cardiac glycosides. Collectively, we established a screening system and identified potential compounds to promote cardiomyocyte proliferation. Our data suggest that modulation of calcium handling and metabolism promotes cardiomyocyte proliferation, and cardiac glycosides might, besides increasing myocardial contraction force, contribute to cardiac repair by inducing cardiomyocyte proliferation.

PMID:36225954 | PMC:PMC9549374 | DOI:10.3389/fcvm.2022.901396

Curr Stem Cell Res Ther. 2022 Oct 11. doi: 10.2174/1574888X17666221011085745. Online ahead of print.

ABSTRACT

Introduction of an animal experimental model for myocardial infarction (MI) has particular importance. Research done on large animals provides valuable information for the researchers because of the similar characteristics of their hearts compared with human, but the cost of purchasing and maintenance of them is high. In comparison, using small animals has advantages such as they are easy to work with and have low purchase and maintenance cost. However, in some of these animals, due to less similarity of the heart to human, cannot simulate the natural pathogenesis of human MI. Moreover, there are different methods for induction of MI in animals, each has its own advantages and disadvantages. However, the method must be chosen that can simulate the natural pathogenesis of MI with minimal complication. Today, attempts are being made for myocardial regeneration after MI using the direct transplantation of stem cells or with an engineered scaffold. The scaffold creates a 3D ambience for the cultured cells. The task of tissue engineering is to optimize the scaffold with appropriate systems for separation, proliferation, and differentiation of the desired cells until they are capable to promote the three-dimensional and appropriate growth of the tissue.The purpose of tissue engineering in cardiac is use of scaffold and cell in the damaged area, followed by the improvement of the heart function through the automatic pulsation, communication with the host vessels, and electrical coupling with the myocardium, eventually creating a force to increase the heart function.

PMID:36221884 | DOI:10.2174/1574888X17666221011085745

Elife. 2022 Oct 11;11:e76781. doi: 10.7554/eLife.76781.

ABSTRACT

Each heartbeat is triggered by the sinoatrial node (SAN), the primary pacemaker of the heart. Studies in animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into 'transitional', 'tail', and 'head' subtypes. However, the underlying molecular mechanisms, especially of human pacemaker cell development, are poorly understood. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCMs) to construct a roadmap of transcriptional changes and lineage decisions. In differentiated SANCM, we identified distinct clusters that closely resemble different subpopulations of the in vivo SAN. Moreover, the presence of a side population of proepicardial cells suggested their shared ontogeny with SANCM, as also reported in vivo. Our results demonstrate that the divergence of SANCM and proepicardial lineages is determined by WNT signaling. Furthermore, we uncovered roles for TGFβ and WNT signaling in the branching of transitional and head SANCM subtypes, respectively. These findings provide new insights into the molecular processes involved in human pacemaker cell differentiation, opening new avenues for complex disease modeling in vitro and inform approaches for cell therapy-based regeneration of the SAN.

PMID:36217819 | PMC:PMC9553210 | DOI:10.7554/eLife.76781

FASEB J. 2022 Nov;36(11):e22587. doi: 10.1096/fj.202200289RR.

ABSTRACT

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by the cell cycle inhibitors Cdkn2a, Cdkn1a, and Trp53. Senescent cells are implicated in chronic diseases and tissue repair through their increased secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP). Here, we use spatial transcriptomics and single-cell RNA sequencing (scRNAseq) to demonstrate that cells displaying senescent characteristics are "transiently" present within regenerating skeletal muscle and within the muscles of D2-mdx mice, a model of Muscular Dystrophy. Following injury, multiple cell types including macrophages and fibrog-adipogenic progenitors (FAPs) upregulate senescent features such as senescence pathway genes, SASP factors, and senescence-associated beta-gal (SA-β-gal) activity. Importantly, when these cells were removed with ABT-263, a senolytic compound, satellite cells are reduced, and muscle fibers were impaired in growth and myonuclear accretion. These results highlight that an "acute" senescent phenotype facilitates regeneration similar to skin and neonatal myocardium.

PMID:36190443 | DOI:10.1096/fj.202200289RR

World J Stem Cells. 2022 Sep 26;14(9):700-713. doi: 10.4252/wjsc.v14.i9.700.

ABSTRACT

BACKGROUND: Heart diseases are the primary cause of death all over the world. Following myocardial infarction, billions of cells die, resulting in a huge loss of cardiac function. Stem cell-based therapies have appeared as a new area to support heart regeneration. The transcription factors GATA binding protein 4 (GATA-4) and myocyte enhancer factor 2C (MEF2C) are considered prominent factors in the development of the cardiovascular system.

AIM: To explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs).

METHODS: hUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry, and by their potential to differentiate into osteocytes and adipocytes. hUC-MSCs were transfected with GATA-4, MEF2C, and their combination to direct the differentiation. Cardiac differentiation was confirmed by semiquantitative real-time polymerase chain reaction and immunocytochemistry.

RESULTS: hUC-MSCs expressed specific cell surface markers CD105, CD90, CD44, and vimentin but lack the expression of CD45. The transcription factors GATA-4 and MEF2C, and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e., GATA-4, MEF2C, NK2 homeobox 5 (NKX2.5), MHC, and connexin-43, and cardiac proteins GATA-4, NKX2.5, cardiac troponin T, and connexin-43.

CONCLUSION: Transfection with GATA-4, MEF2C, and their combination effectively induces cardiac differentiation in hUC-MSCs. These genetically modified MSCs could be a promising treatment option for heart diseases in the future.

PMID:36188117 | PMC:PMC9516467 | DOI:10.4252/wjsc.v14.i9.700

EBioMedicine. 2022 Nov;85:104274. doi: 10.1016/j.ebiom.2022.104274. Epub 2022 Sep 28.

ABSTRACT

BACKGROUND: Homeodomain-Interacting Protein Kinase 2 (HIPK2) has been reported to maintain basal cardiac function, however, its role in pathological cardiac remodeling remains unclear.

METHODS: HIPK2 inhibitors (tBID and PKI1H) treated mice and two lines of HIPK2-/- mice were subjected to transverse aortic constriction (TAC). HIPK2 knockdown were performed in neonatal rat cardiomyocytes (NRCMs), neonatal rat cardiac fibroblasts (NRCFs), and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Microarray analysis was used to screen HIPK2 targets. Overexpression of early growth response 3 (EGR3) and C-type lectin receptor 4D (CLEC4D) were performed in NRCMs, while an activator of Smad3 was used in NRCFs, to rescue the effects of HIPK2 knockdown. Finally, the effects of EGR3 and CLEC4D knockdown by AAV9 in TAC were determined.

FINDINGS: HIPK2 was elevated in TAC mice model, as well as cardiomyocyte hypertrophy and NRCFs fibrosis model. Pharmacological and genetic inhibition of HIPK2 improved cardiac function and suppressed cardiac hypertrophy and fibrosis induced by TAC. In vitro, HIPK2 inhibition prevented cardiomyocyte hypertrophic growth and NRCFs proliferation and differentiation. At the mechanistic level, we identified EGR3 and CLEC4D as new targets of HIPK2, which were regulated by ERK1/2-CREB and mediated the protective function of HIPK2 inhibition in cardiomyocytes. Meanwhile, inhibition of phosphorylation of Smad3 was responsible for the suppression of cardiac fibroblasts proliferation and differentiation by HIPK2 inhibition. Finally, we found that inhibition of EGR3 or CLEC4D protected against TAC.

INTERPRETATION: HIPK2 inhibition protects against pathological cardiac remodeling by reducing EGR3 and CLEC4D with ERK1/2-CREB inhibition in cardiomyocytes, and by suppressing the phosphorylation of Smad3 in cardiac fibroblasts.

FUNDING: This work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to J.X.), National Natural Science Foundation of China (82020108002 and 81911540486 to J.X., 81400647 to MJ Xu), the grant from Science and Technology Commission of Shanghai Municipality (21XD1421300 and 20DZ2255400 to J.X.), the "Dawn" Program of Shanghai Education Commission (19SG34 to J.X.), and Shanghai Sailing Program (21YF1413200 to Q.Z.).

PMID:36182775 | PMC:PMC9526139 | DOI:10.1016/j.ebiom.2022.104274