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A, Heart samples were stained for cardiomyocyte-specific marker cardiac troponin-I (green) one, two, and four weeks after cell transplantation. Transplanted nanocrystal-positive cells (red) accumulated into infarction area and could not detect in remote area at any time point. Bar = 200 µm. B, Representative immunostaining of cTn-I (green) after transplantation of nanocrystal-labeled cells (red) to detect direct regeneration of cardiomyocytes from transplanted cells at one, two, and four weeks after cell transplantation. Bar = 50 µm. C, Representative immunostaining results for cTn-I (green) using confocal microscope four weeks after transplantation of nanocrystal-labeled cells (red). Arrows indicate double positive cells for cTn-I and nanocrystal. Bar = 20 µm. D, Representative immunostaining of α-sarcomeric actinin (green) and DAPI (blue) in co-culture system. White arrows in left panels indicate cardiomyocyte (magnified from the white box) formed from multipotent cardiac cell (double positive for nanocrystal (red) and α-sarcomeric actinin; green) in co-culture system with living cardiomyocytes. Bar = 50 µm. White arrows in right panels indicate double positive cell in co-culture system with PFA-fixed cardiomyocytes. Bar = 50 µm.
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Methods and Results
The cardiac stem/progenitor cells from adult mice were seeded at low density in serum-free medium. The colonies thus obtained were expanded separately and assessed for expression of stem cell antigen-1 (Sca-1). Two colonies each with high Sca-1 (CSH1; 95.9%; CSH2; 90.6%) and low Sca-1 (CSL1; 37.1%; CSL2; 17.4%) expressing cells...
Citations
... All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati. Sca1 + CSCs were isolated from mouse hearts according to the protocols developed and modified by us [21,51]. Briefly, 12-week-old male C57BL6 mice (Harlan) were anesthetized by intraperitoneal injection of ketamine/xylazine (87-100 mg and 13-15 mg/kg, respectively). ...
... We examined the impact of HAX1 on the capabilities of stem cells through gain-of-function studies by the use of lentiviruses (Supplemental Fig. 1A). As previously reported [51], mouse Sca1 + cardiac stromal cells (CSCs) were successfully isolated and purified as evidenced by over 99% percent Sca1 + cell population (Fig. 1A). Fluorescent microscopy (Supplemental Fig. 1B) and flow cytometry analysis (Fig. 1A) showed a pronounced green fluorescence signal indicative of HAX1-overexpressing CSCs (CSCs HAX1 ). ...
Although stem/progenitor cell therapy shows potential for myocardial infarction repair, enhancing the therapeutic efficacy could be achieved through additional genetic modifications. HCLS1-associated protein X-1 (HAX1) has been identified as a versatile modulator responsible for cardio-protective signaling, while its role in regulating stem cell survival and functionality remains unknown. In this study, we investigated whether HAX1 can augment the protective potential of Sca1⁺ cardiac stromal cells (CSCs) for myocardial injury. The overexpression of HAX1 significantly increased cell proliferation and conferred enhanced resistance to hypoxia-induced cell death in CSCs. Mechanistically, HAX1 can interact with Mst1 (a prominent conductor of Hippo signal transduction) and inhibit its kinase activity for protein phosphorylation. This inhibition led to enhanced nuclear translocation of Yes-associated protein (YAP) and activation of downstream therapeutic-related genes. Notably, HAX1 overexpression significantly increased the pro-angiogenic potential of CSCs, as demonstrated by elevated expression of vascular endothelial growth factors. Importantly, implantation of HAX1-overexpressing CSCs promoted neovascularization, protected against functional deterioration, and ameliorated cardiac fibrosis in ischemic mouse hearts. In conclusion, HAX1 emerges as a valuable and efficient inducer for enhancing the effectiveness of cardiac stem or progenitor cell therapeutics.
Graphical Abstract
... CSCs are distinguished by the presence of surface markers such as c-Kit, Sca1, or PDGFR, as well as the expression of the transcription factor Isl1 and the ability to form cardiospheres in culture. CSCs can differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells (Tzahor and Poss 2017;Takamiya et al. 2011). The potential of CSCs to regenerate is attributed to the expression of transcription factors Nkx2.5 and GATA-4. ...
... Mehr als zwei Jahrzehnte experimenteller Studien an Tiermodellen (Haider et al. 2004a(Haider et al. , b, 2008Takamiya et al. 2011;Ozdemir et al. 2012;Wu et al. 2013;Ionescu et al. 2012;Fang et al. 2012) und klinischer Studien an Patienten (Veronesi et al. 2013;Herreros et al. 2012;Yau et al. 2019) sowie mechanistische Studien in vitro (Liu et al. 2011;Sassoli et al. 2012;Vishnubalaji et al. 2012) haben gezeigt, dass stammzellbasierte Interventionen eine sichere und wirksame Alternative zu herkömmlichen Behandlungsmethoden darstellen. Skelettmuskelmyoblasten (SkMs), BMSCs und CSCs gehören zu den am umfassendsten untersuchten Zelltypen mit nachgewiesener therapeutischer Wirksamkeit sowohl in experimentellen Tiermodellen als auch in klinischen Studien. ...
... Mehr als zwei Jahrzehnte experimenteller Studien an Tiermodellen (Haider et al. 2004a(Haider et al. , b, 2008Takamiya et al. 2011;Ozdemir et al. 2012;Wu et al. 2013;Ionescu et al. 2012;Fang et al. 2012) und klinischer Studien an Patienten (Veronesi et al. 2013;Herreros et al. 2012;Yau et al. 2019) sowie mechanistische Studien in vitro (Liu et al. 2011;Sassoli et al. 2012;Vishnubalaji et al. 2012) haben gezeigt, dass stammzellbasierte Interventionen eine sichere und wirksame Alternative zu herkömmlichen Behandlungsmethoden darstellen. Skelettmuskelmyoblasten (SkMs), BMSCs und CSCs gehören zu den am umfassendsten untersuchten Zelltypen mit nachgewiesener therapeutischer Wirksamkeit sowohl in experimentellen Tiermodellen als auch in klinischen Studien. ...
... Mehr als zwei Jahrzehnte experimenteller Studien an Tiermodellen (Haider et al. 2004a(Haider et al. , b, 2008Takamiya et al. 2011;Ozdemir et al. 2012;Wu et al. 2013;Ionescu et al. 2012;Fang et al. 2012) und klinischer Studien an Patienten (Veronesi et al. 2013;Herreros et al. 2012;Yau et al. 2019) sowie mechanistische Studien in vitro (Liu et al. 2011;Sassoli et al. 2012;Vishnubalaji et al. 2012) haben gezeigt, dass stammzellbasierte Interventionen eine sichere und wirksame Alternative zu herkömmlichen Behandlungsmethoden darstellen. Skelettmuskelmyoblasten (SkMs), BMSCs und CSCs gehören zu den am umfassendsten untersuchten Zelltypen mit nachgewiesener therapeutischer Wirksamkeit sowohl in experimentellen Tiermodellen als auch in klinischen Studien. ...
... Mehr als zwei Jahrzehnte experimenteller Studien an Tiermodellen (Haider et al. 2004a(Haider et al. , b, 2008Takamiya et al. 2011;Ozdemir et al. 2012;Wu et al. 2013;Ionescu et al. 2012;Fang et al. 2012) und klinischer Studien an Patienten (Veronesi et al. 2013;Herreros et al. 2012;Yau et al. 2019) sowie mechanistische Studien in vitro (Liu et al. 2011;Sassoli et al. 2012;Vishnubalaji et al. 2012) haben gezeigt, dass stammzellbasierte Interventionen eine sichere und wirksame Alternative zu herkömmlichen Behandlungsmethoden darstellen. Skelettmuskelmyoblasten (SkMs), BMSCs und CSCs gehören zu den am umfassendsten untersuchten Zelltypen mit nachgewiesener therapeutischer Wirksamkeit sowohl in experimentellen Tiermodellen als auch in klinischen Studien. ...
... In stem cell-based therapy for cardiac tissue regeneration, different types of stem cells can be used, such as embryonic stem cells (ESCs), adult stem cells including mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), cardiac stem cells (CSCs), endothelial progenitor cells, and skeletal myoblasts (Takamiya et al. 2011;Lu et al. 2009;Ibrahim et al. 2016;Rufaihah et al. 2010;Haider et al. 2017;Afjeh-Dana et al. 2022;Sung et al. 2020). Each type of these cells has its advantages and disadvantages. ...
... Unlike pluripotent stem cells, resident CSCs have a low risk of tumor formation (Rikhtegar et al. 2019), and their use in clinical trials has proven their safety for human use (Bolli et al. 2011). In addition, the time-required for CSCs cultures and expansion is shorter before successful differentiation into cardiomyocytes (Smits et al. 2009;Takamiya et al. 2011). However, the accessibility of these cells is limited, and the cells can be obtained via invasive manners from myocardial biopsies (Velichko et al. 2022). ...
Cardiovascular disorder is one of the most important causes of suffering and mortality in today’s society. Common heart disease treatment methods are complicated, plus they are still not compelling enough. These limitations attracted the interest of scientists in the field to develop novel treatment strategies. One of the most promising methods in cardiac tissue revitalization is stem cell therapy. This chapter will discuss several approaches to differentiating stem cells into cardiomyocytes and various methods of stem cell therapy. The research results suggest that stem cell therapy is a promising and effective method for cardiac tissue regeneration and cardiovascular disease treatments.Graphical AbstractKeywordsCardiacHydrogelHeartMyocardial infarctionRegenerationStem cells
... After 12 weeks, Sca1 + SP cells improved EF significantly compared to mice with MI with no treatment, however, like our results, EF remained significantly impaired compared to shams [17]. Furthermore, a study that transplanted 2 × 10 5 Sca-1 + cardiac progenitor cells in MI mice significantly improved EF compared to MI mice without treatment [27], also corroborating our findings. The importance of using Sca-1 + stem/progenitors was elegantly demonstrated by Wang et al. (2006), where post-MI, mice transplanted with cardiac-derived Sca-1 + /CD31cells showed significant cardiac functional improvement compared to mice treated with cardiac-derived Sca-1 -/CD31cells, emphasising the importance of Sca-1 + cells for cardiac reparative therapies [15]. ...
We have previously shown that skeletal muscle-derived Sca-1+/PW1+/Pax7− interstitial cells (PICs) are multi-potent and enhance endogenous repair and regeneration. Here, we investigated the regenerative potential of PICs following intramyocardial transplantation in mice subjected to an acute myocardial infarction (MI). MI was induced through the ligation of the left anterior descending coronary artery in 8-week old male C57BL/6 mice. 5 × 105 eGFP-labelled PICs (MI + PICs; n = 7) or PBS (MI-PBS; n = 7) were injected intramyocardially into the border zone. Sham mice (n = 8) were not subjected to MI, or the transplantation of PICs or PBS. BrdU was administered via osmotic mini-pump for 14 days. Echocardiography was performed prior to surgery (baseline), and 1-, 3- and 6-weeks post-MI and PICs transplantation. Mice were sacrificed at 6 weeks post-MI + PICs transplantation, and heart sections were analysed for fibrosis, hypertrophy, engraftment, proliferation, and differentiation of PICs. A significant (p < 0.05) improvement in ejection fraction (EF) and fractional shortening was observed in the MI-PICs group, compared to MI + PBS group at 6-weeks post MI + PICs transplantation. Infarct size/fibrosis of the left ventricle significantly (p < 0.05) decreased in the MI-PICs group (14.0 ± 2.5%), compared to the MI-PBS group (32.8 ± 2.2%). Cardiomyocyte hypertrophy in the border zone significantly (p < 0.05) decreased in the MI-PICs group compared to the MI-PBS group (330.0 ± 28.5 µM2 vs. 543.5 ± 26.6 µm2), as did cardiomyocyte apoptosis (0.6 ± 0.9% MI-PICs vs. 2.8 ± 0.8% MI-PBS). The number of BrdU+ cardiomyocytes was significantly (p < 0.05) increased in the infarct/border zone of the MI-PICs group (7.0 ± 3.3%), compared to the MI-PBS group (1.7 ± 0.5%). The proliferation index (total BrdU+ cells) was significantly increased in the MI-PICs group compared to the MI-PBS group (27.0 ± 3.4% vs. 7.6 ± 1.0%). PICs expressed and secreted pro-survival and reparative growth factors, supporting a paracrine effect of PICs during recovery/remodeling. Skeletal muscle-derived PICs show significant reparative potential, attenuating cardiac remodelling following transplantation into the infarcted myocardium. PICs can be easily sourced from skeletal muscle and therefore show promise as a potential cell candidate for supporting the reparative and regenerative effects of cell therapies.
... Extra-embryonic tissue-derived stem cells, i.e., umbilical cord or placental tissue-derived stem cells, are endowed with restricted differentiation potential; however, their paracrine activity and immune-modulatory potential make them suitable for clinical use (Kim et al. 2013;Ullah et al. 2015;Rohban and Pieber 2017). Adult stem cells, i.e., bone marrow-derived mesenchymal stem cells (BM-MSCs), resident cardiac stem cells (CSCs), skeletal myoblasts (SkMs), etc., have been extensively studied and characterized in vitro as well as in preclinical studies either naïve or after genetically modulation (Haider et al. 2004a, b;Lei et al. 2005;Jiang et al. 2002;Haider et al. 2008;Lei et al. 2011;Elmadbouh et al. 2011;Takamiya et al. 2011) and have advanced to clinical trials (Haider et al. 2004a, b;Han et al. 2019;Sid-Otmane et al. 2020). Innovative cellular manipulating technologies using CRISPR/Cas 9 were employed to engineer stem cells for editing their characteristic profile supporting cardiac repair. ...
Cardiomyocytes (CMs) are mitotically inactive but metabolically highly active
cells that constitute a major population in the heart. The cumbersome isolation
and purification protocols combined with the difficulty in their in vitro propagation as a primary culture remain the major impediments in their biological characterization. Therefore, understanding the molecular events that drive the successful differentiation of stem cells to become morphofunctionally competent CMs via advanced cellular technology is essential to better manipulate them for human applications. Cardiomyogenic differentiation of stem cells is relatively inefficient with a limited success rate due to the complexity of the molecular circuit involved therein. Akin to the embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) have vast prospective to give rise to any type of cell from the three germ layers without ethical concerns. Mesenchymal stem cells have limited differentiation potential toward cardiomyocyte lineage. Given their
developmental aspects, the epigenomic signature often directs the cell’s function
which in turn governs their expression profile. Such epigenetic mechanisms
include DNA methylation, histone core modifications, and miRNA regulations,
which are associated with gene expression or silencing in the cell’s progression
toward a CM’s fate. This chapter elucidates in-depth the regulatory process
underlying CMs’ differentiation at the genetic as well as epigenetic levels via
the modulation of chromatin architecture. Specifically in this chapter, we will
elaborate on the gene circuit and their expression profile in the various stem cells,
heading toward cardiomyocyte lineage under different culture technologies from
2D, 3D, and even up to the single-cell level.
... Typical features of Sca1 + /CD31 -CPCs are high clonogenic efficiency [25], a primitive undifferentiated phenotype, long term proliferation, and the ability to differentiate into different cardiac lineages in vitro, such as smooth muscle and endothelial cells [26]. The Sca1 + /CD31population expresses Nanog and the telomerase reverse transcriptase (TERT), two genes associated with pluripotent phenotypes and not expressed by differentiated fibroblasts ( Figure 2) [27]. In addition, CPCs express the embryonic heart markers Islet-1 (ISL-1) and TBX5 [28], as well as cardiac-specific transcription factors GATA-4, Nkx-2.5, and MEF2C (Table 1). ...
Cardiac stromal cells have been long underestimated in their functions in homeostasis and repair. Recent evidence has changed this perspective in that many more players and facets than just "cardiac fibroblasts" have entered the field. Single cell transcriptomic studies on cardiac interstitial cells have shed light on the phenotypic plasticity of the stroma, whose transcriptional profile is dynamically regulated in homeostatic conditions and in response to external stimuli. Different populations and/or functional states that appear in homeostasis and pathology have been described, particularly increasing the complexity of studying the cardiac response to injury. In this review, we outline current phenotypical and molecular markers, and the approaches developed for identifying and classifying cardiac stromal cells. Significant advances in our understanding of cardiac stromal populations will provide a deeper knowledge on myocardial functional cellular components, as well as a platform for future developments of novel therapeutic strategies to counteract cardiac fibrosis and adverse cardiac remodeling.
