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

In stem cell-regulated organs, a subset of niches is characterized by low oxygen tension. This metabolic adaptation offers a selective advantage to stem cells favoring the preservation of their quiescent undifferentiated phenotype. The objective of this work was to determine whether in the mouse heart cardiac niches constitute a heterogeneous compartment composed of hypoxic and normoxic niches, and whether differences in O 2 concentration affect the function of c-kit-positive cardiac stem cells (CSCs).To test this possibility, we studied first the in vivo uptake of the hypoxic marker pimonidazole (PIMO), which identifies intracellular O 2 concentration <10 mmHg. Mice were sacrificed 2 hours after intraperitoneal administration of PIMO, and PIMO-labeling was analyzed. By immunolabeling, 15% of cardiac niches were characterized by a hypoxic microenvironment and more than 20% of isolated CSCs were PIMO-positive, as measured by flow-cytometry. The cell cycle protein Ki67 was restricted to the PIMO-negative CSC class, which contained early committed cells expressing c-kit together with the myocyte specific transcription factors GATA4 and Nkx2.5. Mice were then administered tirapazamine, a compound that kills selectively hypoxic cells. One day later, the fraction of PIMO-positive CSCs was markedly decreased but, at 5 days, this compartment was partly reconstituted. This compensatory response was coupled with increased proliferation of PIMO-negative CSCs, suggesting that normoxic CSCs have the ability to replenish hypoxic niches following injury. Subsequently, the effects of hypoxia were studied in human CSCs (hCSCs) exposed in vitro to 1% O 2 . With respect to cells cultured in normoxia, 1% O 2 led to upregulation of HIF1α in hCSCs which also showed lower levels of BrdU incorporation. These cellular responses were accompanied by an increase in transcripts for the stemness genes c-kit, Oct4, Nanog and Sox2, and a decrease in mRNA for myocyte and vascular genes. Apoptosis, measured by TdT labeling, did not differ in normoxic and hypoxic hCSCs. In conclusion, our data indicate that hypoxic and normoxic niches coexist in the myocardium, and that intracellular hypoxia regulates the quiescent primitive CSC phenotype.

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Abstract 245: Cardiac Stem Cells and Myocyte Regeneration in Diabetic Cardiomyopathy

Circulation Research ◽

10.1161/res.113.suppl_1.a245 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Barbara Ogorek ◽

Alex Matsuda ◽

Ewa Wybieralska ◽

James Kostyla ◽

Giulia Borghetti ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Systolic Pressure ◽

Left Ventricular ◽

Dependent Diabetes Mellitus ◽

Mouse Heart ◽

Type I ◽

Cell Renewal ◽

Cardiac Stem Cells ◽

Multiple Variables

The mammalian heart contains a pool of resident c-kit-positive cardiac stem cells (CSCs), raising questions concerning their role in the etiology of the diabetic myopathy. The objective of the current study was to determine whether the negative effects of diabetes on the adult heart are dictated by defects in CSC growth and lineage commitment. Type I insulin-dependent diabetes mellitus was induced in mice by streptozotocin administration. The kinetics of CSCs and cardiomyocytes was measured 3, 7, 10, 20, and 30 days after the onset of diabetes, by implementing a hierarchically structured cell system, which allows the quantitative analysis of the rate of cell turnover. The multiple variables required to apply this mathematical model were measured by confocal microscopy. They included first the number of CSCs, LCCs (lineage committed cells: myocyte progenitors-precursors), transit amplifying myocytes, and post-mitotic myocytes in the left ventricular (LV) myocardium. Additionally, the fraction of cycling CSCs and the length of their cell cycle were measured, together with the number of cardiomyocytes dying by apoptosis and necrosis. The specificity of labeling was documented by spectral analysis. The diabetic heart was characterized by a severe time-dependent loss in LV post-mitotic myocytes, dictated primarily by a defect in cell renewal. Under control conditions, 20% of myocytes were replaced per month in the adult mouse heart through activation, cell cycle reentry, and differentiation of CSCs. This value decreased sharply to 6%, 1.4%, 0.5%, 0.07%, and 0.05% at 3, 7, 10, 20 and 30 days after the administration of streptozotocin. The myocardium showed a progressive increase in old cardiomyocytes expressing the senescence-associated protein p16INK4a and p53. Importantly, the severe impairment in myocyte regeneration was coupled with an increase in LV end-diastolic pressure, and a decrease in LV systolic pressure, LV developed pressure, positive and negative dP/dt. Thus, our data indicate that the diabetic myopathy has to be viewed as a stem cell disease in which the decrease in the number of myocytes is a secondary event, resulting from defects in replication and lineage specification of cardiac stem cells.

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Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart

Nature Protocols ◽

10.1038/nprot.2014.113 ◽

2014 ◽

Vol 9 (7) ◽

pp. 1662-1681 ◽

Cited By ~ 69

Author(s):

Andrew J Smith ◽

Fiona C Lewis ◽

Iolanda Aquila ◽

Cheryl D Waring ◽

Aurora Nocera ◽

...

Keyword(s):

Stem Cells ◽

Rat Heart ◽

Adult Mouse ◽

Cardiac Stem Cells ◽

Isolation And Characterization

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Abstract 3822: Different microRNA In Mouse Heart, Cardiac Stem Cells And Cardiac Stem Cells With GATA4, Indicate Different Sets Of microRNA Involved In Stem Cell Differentiation By Gene Repression

Circulation ◽

10.1161/circ.118.suppl_18.s_491-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Edilamar M de Oliveira ◽

Yao Liang Tang ◽

Keping Qian ◽

leping Shen ◽

Luvena Ong ◽

...

Keyword(s):

Stem Cells ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Activin A ◽

Specific Gene ◽

Mouse Heart ◽

Heart Cells ◽

Cardiac Stem Cells

Several studies suggest that miRNA have important roles in the development of the heart and cardiac function. We hypothesized that specific miRNA are involved in the differentiation steps of cardiac stem cells to heart cells. We studied 569 unique miRNAs probes in mouse heart cells (MHtC), cardiac stem cells (CSC) and CSC with GATA4 (CSCG) . Based on Sanger miRMouse -10.1, we compared miRNAs in the following groups 1) MHtC vs. CSC, 2) CSC vs.CSCG , 3) MHtCs v CSCG. We also compared CSCs under hypoxia and normoxia. miRNAs reported in the results were confirmed by PCR As previously reported miRNA 1, 133a and 133b were highly expressed in MHt. However, we also found previously unreported, relatively high expression of miRNAs 126 –3p, 145, , 451and 499 in MHtC. MicroRNAs have not previously been reported in CSCs or CSCG. We found unique expression miRNAs 10a,10b, 31, 214 and 762 in CSCs and increased expression of miRNAs 705,709,762, and 1224 in CSCG. In MHt, miR 21 was lower compared to miR21 in CSCs . Expression of miRNA-762 was expressed significantly more in CSC-G than in CSC. Therefore, miR-762 could be the direct transcriptional target of GATA-4 in CSC and a part of the differentiation control pathway from CSC to MHtC. In addition we studied miRNA from CSCs exposed to hypoxia. Hypoxia suppressed all miRs found except miR-574 –5p, which was increased in hypoxia. The results led us to identify which genes are targeted by these miRNAs. miRNA 762 is located into the BCl-7c gene and by miRNA target predictions it was found to target activin A receptor (type IB and II-like-1) gene, bone morphogenic gene and transforming growth factor, beta receptor III (TGFβIII). Embryonic stem cells differentiate into cardiomyocytes in the presence of activin A and BMP4, therefore we suggest that the miRNA 700 family ( miR762 in particluar) in cardiac stem cells inhibits activin A and BMP4 genes, possibly through TGFβIII signaling, to maintain stem cells in the undifferentiated state. The results reveal new miRNAs in cardiac stem cells differentiation to adult heart cells and suggest their role of specific gene targeting.

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?-Ca2+/calmodulin-dependent protein kinase II expression pattern in adult mouse heart and cardiogenic differentiation of embryonic stem cells

Journal of Cellular Biochemistry ◽

10.1002/1097-4644(20001101)79:2<293::aid-jcb120>3.0.co;2-q ◽

2000 ◽

Vol 79 (2) ◽

pp. 293-300 ◽

Cited By ~ 10

Author(s):

Brigitte Hoch ◽

Anna M. Wobus ◽

Ernst-Georg Krause ◽

Peter Karczewski

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Protein Kinase ◽

Expression Pattern ◽

Adult Mouse ◽

Embryonic Stem ◽

Mouse Heart ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Cardiogenic Differentiation

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Abstract 264: TNFR2-Bmx Signaling in Cardiac Stem Cells and Cardiac Repair

Circulation Research ◽

10.1161/res.115.suppl_1.264 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Huanjiao J Zhou ◽

Qunhua Huang ◽

Wang Min

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Wild Type Mouse ◽

Cardiac Repair ◽

Mouse Heart ◽

Coronary Artery Ligation ◽

Cardiac Stem Cells ◽

Organ Cultures ◽

Cell Cycle Entry ◽

And Migration

While cytokine TNF via TNFR1 induces inflammation and apoptosis, it through its second receptor TNFR2 induces cell survival and migration by activating bone marrow non-receptor tyrosine kinase Bmx. Since Bmx has been implicated in self-renewal of stem cells, we hypothesize that TNF via TNFR2 activates Bmx in cardiac stem cells (CSCs) to mediate cardiac repair. We show that in human cardiac tissue affected by ischemia heart disease (IHD), TNFR2 is expressed on intrinsic CSCs, identified as c-kit(+) /CD45(-) /VEGFR2(-) interstitial round cells, which are activated as determined by entry to cell cycle and expression of Lin-28. Wild-type mouse heart organ cultures subjected to hypoxic conditions both increase cardiac TNF expression and show induced TNFR2 and Lin-28 expression in c-kit(+) CSCs that have entered cell cycle. These CSC responses are enhanced by exogenous TNF. TNFR2(-/-) mouse heart organ cultures subjected to hypoxia increase cardiac TNF but fail to induce CSC activation. Similarly, c-kit(+) CSCs isolated from mouse hearts exposed to hypoxia or TNF show induction of Lin-28, TNFR2, cell cycle entry, and cardiogenic marker, α-sarcomeric actin (α-SA), responses more pronounced by hypoxia in combination with TNF. Knockdown of Lin-28 by siRNA results in reduced levels of TNFR2 expression, cell cycle entry, and diminished expression of α-SA (references: Stem Cells 2013;31:1881-1892). In the present study, we detect the c-kit(+)Lin28(+) CSCs populations in a mouse coronary artery ligation ischemic model. Furthermore, the c-kit(+) CSCs are reduced in TNFR2-KO and Bmx-KO mice. Mechanistically, we show a crosstalk between the TNFR2-Bmx and the c-Kit signaling pathways to mediate CSC proliferation, survival and migration. These observations suggest that TNFR2-Bmx signaling in c-kit(+) CSCs induces cardiac repair, providing a potential strategy to stimulate cardiac regeneration by TNFR2-specific agonists.

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