isolation-characterization-differentiation-cardiac-stem-cells-from

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

University of Nebraska Medical Center

Hydrogen sulfide (HS), a cardioprotective gas, is endogenously produced from homocysteine by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE) enzymes. However, effect of HS or homocysteine on CBS and CSE expression, and cross-talk between CBS and CSE are unclear. We hypothesize that homocysteine and HS regulate CBS and CSE expressions in a dose dependent manner in cardiomyocytes, and CBS deficiency induces cardiac CSE expression. To test the hypothesis, we treated murine atrial HL1 cardiomyocytes with increasing doses of homocysteine or NaS/GYY4137, a HS donor, and measured the levels of CBS and CSE. We found that homocysteine upregulates CSE but downregulates CBS whereas NaS/GYY4137 downregulates CSE but upregulates CBS in a dose-dependent manner. Moreover, the NaS-treatment downregulates specificity protein-1 (SP1), an inducer for CSE, and upregulates miR-133a that targets SP1 and inhibits cardiomyocytes hypertrophy. Conversely, in the homocysteine-treated cardiomyocytes, CBS and miR-133a were downregulated and hypertrophy was induced. In vivo studies using CBS+/-, a model for hyperhomocysteinemia, and sibling CBS+/+ control mice revealed that deficiency of CBS upregulates cardiac CSE, plausibly by inducing SP1. In conclusion, we revealed a novel mechanism for HS-mediated regulation of homocysteine metabolism in cardiomyocytes, and a negative feedback regulation between CBS and CSE in the heart.

HS and homocysteine control a novel feedback regulation of cystathionine beta synthase and cystathionine gamma lyase in cardiomyocytes.

Prevalence of diabetes mellitus (DM), a multifactorial disease often diagnosed with high blood glucose levels, is rapidly increasing in the world. Association of DM with multi-organ dysfunction including cardiomyopathy makes it a leading cause of morbidity and mortality. There are two major types of DM: type 1 DM (T1D) and type 2 DM (T2D). T1D is diagnosed by reduced levels of insulin and high levels of glucose in the blood. It is caused due to pancreatic beta cell destruction/loss, and mostly found in juveniles (juvenile DM). T2D is diagnosed by increased levels of insulin and glucose in the blood. It is caused due to insulin receptor dysfunction, and mostly found in the adults (adult DM). Both T1D and T2D impair cardiac muscle function, which is referred to as diabetic cardiomyopathy. We and others have reported that miRNAs, a novel class of tiny non-coding regulatory RNAs, are differentially expressed in the diabetic heart and they contribute to diabetic cardiomyopathy. Here, we elaborated the biogenesis of miRNA, how miRNA regulates a gene, cardioprotective roles of different miRNAs including miRNAs present in exosomes, underlying molecular mechanisms by which miRNA ameliorates diabetic cardiomyopathy, and the role of miRNA as a potential therapeutic target for juvenile and adult diabetic cardiomyopathy.

Targeting miRNA for Therapy of Juvenile and Adult Diabetic Cardiomyopathy.

Despite several strategies developed for replenishing the dead myocardium after myocardial infarction (MI), stem cell therapy remains the leading method to regenerate new myocardium. Although induced pluripotent stem cells (iPS) and transdifferentiation of the differentiated cells have been used as novel approaches for myocardial regeneration, these approaches did not yield very successful results for myocardial regeneration in in vivo studies. Asynchronous contractility of newly formed cardiomyocytes with the existing cardiomyocytes is the most important issue with iPS approach, while very low yield of transdifferentiated cardiomyocytes and their less chances to beat in the same rhythm as existing cardiomyocytes in the MI heart are important caveats with transdifferentiation approach. CSCs are present in the heart and they have the potential to differentiate into myocardial cells. However, the number of resident CSCs is very low. Therefore, it is important to get maximum yield of CSCs during isolation process from the heart. Increasing the number of CSCs and initiating their differentiation ex vivo are crucial for CSC-based stem cell therapy. Here, we present a better method for isolation, characterization and differentiation of CSCs from the mouse heart. We also demonstrated morphological changes in the CSCs after 2 days, 3 days, and 7 days in maintenance medium and a separate group of CSCs cultured for 12 days in differentiation medium using Phase-Contrast microscopy. We have used different markers for identification of CSCs isolated from the mouse heart such as marker for mouse CSC, Sca-1, cardiac-specific markers NKX2-5, MEF2C, GATA4, and stemness markers OCT4 and SOX2. To characterize the differentiated CSCs, we used CSCs maintained in differentiation medium for 12 days. To evaluate differentiation of CSCs, we determined the expression of cardiomyocyte-specific markers actinin and troponin I. Overall; we described an elegant method for isolation, identification, differentiation and characterization of CSCs from the mouse heart.

Article	Year
HS and homocysteine control a novel feedback regulation of cystathionine beta synthase and cystathionine gamma lyase in cardiomyocytes. Scientific reports\| PubMed ID: 28623294	2017
Targeting miRNA for Therapy of Juvenile and Adult Diabetic Cardiomyopathy. Advances in experimental medicine and biology\| PubMed ID: 29754174	2018
Isolation, Characterization and Differentiation of Mouse Cardiac Progenitor Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30196409	2018

Paras K. Mishra

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