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Improvement of cardiac function in mouse myocardial infarction after transplantation of epigenetically-modified bone marrow progenitor cells.

Rajasingh J, Thangavel J, Siddiqui MR, Gomes I, Gao XP, Kishore R, Malik AB - PLoS ONE (2011)

Bottom Line: Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes.We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes.Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Center of Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois, United States of America. rjohnson9@kumc.edu

ABSTRACT

Objective: To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice.

Methods and results: We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells.

Conclusion: Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells.

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Characterization of Day-7 BPCs and their conversion into multipotent progenitor cells using Aza and TSA.Immunofluoresent analysis of BPCs stained with DiI-acLDL (red), Isolectin B4 (green), nuclei (blue,) and co-localized cells (yellow) (A); FACS analysis of BPCs for specific progenitor markers, (B); Dose-response relationship of Aza- or TSA-treated BPCs. RT-PCR analysis of Oct4 and Nanog transcripts after treatment of BPCs with Aza (0, 10, 25, 50, 100 nM) or TSA (0, 5, 10, 25, 50 nM) for 48 hours, (C); RT-PCR analysis of Oct4, Nanog and Sox2 transcripts after treatment of BPCs with combination of Aza (0, 10, 25, 50 nM) and TSA (0, 5, 10, 25 nM) for 48 hours, (D); Day-10 Oct4 protein expression by immunofluoresence, (E); Day-10 Oct4 protein expression by Western blotting, (F); RT-PCR analysis of endothelial markers eNOS and VE-cadherin transcripts after treatment of BPCs with Aza (50 nM) or TSA (25 nM) or combination of both drugs for 48 hours, (G); RT-PCR analysis; each bar represents mean ± S.E of 3 replicate experiments. Fold expression was calculated as ratio of experimental cell expression-to-expression in control cells. *p<0.01 vs. control, †p<0.001 vs. control.
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pone-0022550-g001: Characterization of Day-7 BPCs and their conversion into multipotent progenitor cells using Aza and TSA.Immunofluoresent analysis of BPCs stained with DiI-acLDL (red), Isolectin B4 (green), nuclei (blue,) and co-localized cells (yellow) (A); FACS analysis of BPCs for specific progenitor markers, (B); Dose-response relationship of Aza- or TSA-treated BPCs. RT-PCR analysis of Oct4 and Nanog transcripts after treatment of BPCs with Aza (0, 10, 25, 50, 100 nM) or TSA (0, 5, 10, 25, 50 nM) for 48 hours, (C); RT-PCR analysis of Oct4, Nanog and Sox2 transcripts after treatment of BPCs with combination of Aza (0, 10, 25, 50 nM) and TSA (0, 5, 10, 25 nM) for 48 hours, (D); Day-10 Oct4 protein expression by immunofluoresence, (E); Day-10 Oct4 protein expression by Western blotting, (F); RT-PCR analysis of endothelial markers eNOS and VE-cadherin transcripts after treatment of BPCs with Aza (50 nM) or TSA (25 nM) or combination of both drugs for 48 hours, (G); RT-PCR analysis; each bar represents mean ± S.E of 3 replicate experiments. Fold expression was calculated as ratio of experimental cell expression-to-expression in control cells. *p<0.01 vs. control, †p<0.001 vs. control.

Mentions: On day 7, BPCs obtained from C57BL/6J mouse bone marrow as described [12] were analyzed by immunofluorescence and flowcytometry (Figure 1A, B). Most cells were positive for Dil-acLDL and showed binding to fluorescein isothicyanate-conjugated Banderia simplicifolia lectin 1 (FITC-BS1). FACS analysis also showed BPCs were a heterogeneous population. They were positive for progenitor cell markers such as CD34 (17%), CD133 (10%), Sca1 (100%), and c-kit (12%) and some were also positive for endothelial cell markers CD31 (10%) and VE-cadherin (16%). BPCs were treated for 4 days with Aza alone, TSA alone, or both Aza and TSA at various concentrations. BPCs treated with Aza or TSA alone (Figure 1C) or with both agents in combination induced expression of Oct4, Nanog and Sox2 (Figure 1D, p<0.001 control vs. Aza+TSA treatment group). Maximum expression of these factors occurred at combined concentrations of 50 nM Aza and 25 nM TSA. Immunofluorescence analysis of Oct4 expression showed that BPCs treated with Aza+TSA showed significantly up-regulated expression of pluripotent genes at passage 0 (Figure 1E) but expression decreased markedly by passage 6 (data not shown). The expression of Oct4 gene transcript was supported by Oct4 Western blot protein expression data (Figure 1F). The results shown in Figure 1A-E demonstrate that pluripotent genes in BPCs were activated by co-treatment with Aza and TSA in a concentration-dependent manner. We also observed by qRT-PCR that BPCs treated with 50 nM Aza and 25 nM TSA resulted in silencing of eNOS and VE-cadherin genes compared to control cells (Figure 1G), suggesting that these drugs promoted de-differentiation of endothelial cells present in the BPC population.


Improvement of cardiac function in mouse myocardial infarction after transplantation of epigenetically-modified bone marrow progenitor cells.

Rajasingh J, Thangavel J, Siddiqui MR, Gomes I, Gao XP, Kishore R, Malik AB - PLoS ONE (2011)

Characterization of Day-7 BPCs and their conversion into multipotent progenitor cells using Aza and TSA.Immunofluoresent analysis of BPCs stained with DiI-acLDL (red), Isolectin B4 (green), nuclei (blue,) and co-localized cells (yellow) (A); FACS analysis of BPCs for specific progenitor markers, (B); Dose-response relationship of Aza- or TSA-treated BPCs. RT-PCR analysis of Oct4 and Nanog transcripts after treatment of BPCs with Aza (0, 10, 25, 50, 100 nM) or TSA (0, 5, 10, 25, 50 nM) for 48 hours, (C); RT-PCR analysis of Oct4, Nanog and Sox2 transcripts after treatment of BPCs with combination of Aza (0, 10, 25, 50 nM) and TSA (0, 5, 10, 25 nM) for 48 hours, (D); Day-10 Oct4 protein expression by immunofluoresence, (E); Day-10 Oct4 protein expression by Western blotting, (F); RT-PCR analysis of endothelial markers eNOS and VE-cadherin transcripts after treatment of BPCs with Aza (50 nM) or TSA (25 nM) or combination of both drugs for 48 hours, (G); RT-PCR analysis; each bar represents mean ± S.E of 3 replicate experiments. Fold expression was calculated as ratio of experimental cell expression-to-expression in control cells. *p<0.01 vs. control, †p<0.001 vs. control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3142193&req=5

pone-0022550-g001: Characterization of Day-7 BPCs and their conversion into multipotent progenitor cells using Aza and TSA.Immunofluoresent analysis of BPCs stained with DiI-acLDL (red), Isolectin B4 (green), nuclei (blue,) and co-localized cells (yellow) (A); FACS analysis of BPCs for specific progenitor markers, (B); Dose-response relationship of Aza- or TSA-treated BPCs. RT-PCR analysis of Oct4 and Nanog transcripts after treatment of BPCs with Aza (0, 10, 25, 50, 100 nM) or TSA (0, 5, 10, 25, 50 nM) for 48 hours, (C); RT-PCR analysis of Oct4, Nanog and Sox2 transcripts after treatment of BPCs with combination of Aza (0, 10, 25, 50 nM) and TSA (0, 5, 10, 25 nM) for 48 hours, (D); Day-10 Oct4 protein expression by immunofluoresence, (E); Day-10 Oct4 protein expression by Western blotting, (F); RT-PCR analysis of endothelial markers eNOS and VE-cadherin transcripts after treatment of BPCs with Aza (50 nM) or TSA (25 nM) or combination of both drugs for 48 hours, (G); RT-PCR analysis; each bar represents mean ± S.E of 3 replicate experiments. Fold expression was calculated as ratio of experimental cell expression-to-expression in control cells. *p<0.01 vs. control, †p<0.001 vs. control.
Mentions: On day 7, BPCs obtained from C57BL/6J mouse bone marrow as described [12] were analyzed by immunofluorescence and flowcytometry (Figure 1A, B). Most cells were positive for Dil-acLDL and showed binding to fluorescein isothicyanate-conjugated Banderia simplicifolia lectin 1 (FITC-BS1). FACS analysis also showed BPCs were a heterogeneous population. They were positive for progenitor cell markers such as CD34 (17%), CD133 (10%), Sca1 (100%), and c-kit (12%) and some were also positive for endothelial cell markers CD31 (10%) and VE-cadherin (16%). BPCs were treated for 4 days with Aza alone, TSA alone, or both Aza and TSA at various concentrations. BPCs treated with Aza or TSA alone (Figure 1C) or with both agents in combination induced expression of Oct4, Nanog and Sox2 (Figure 1D, p<0.001 control vs. Aza+TSA treatment group). Maximum expression of these factors occurred at combined concentrations of 50 nM Aza and 25 nM TSA. Immunofluorescence analysis of Oct4 expression showed that BPCs treated with Aza+TSA showed significantly up-regulated expression of pluripotent genes at passage 0 (Figure 1E) but expression decreased markedly by passage 6 (data not shown). The expression of Oct4 gene transcript was supported by Oct4 Western blot protein expression data (Figure 1F). The results shown in Figure 1A-E demonstrate that pluripotent genes in BPCs were activated by co-treatment with Aza and TSA in a concentration-dependent manner. We also observed by qRT-PCR that BPCs treated with 50 nM Aza and 25 nM TSA resulted in silencing of eNOS and VE-cadherin genes compared to control cells (Figure 1G), suggesting that these drugs promoted de-differentiation of endothelial cells present in the BPC population.

Bottom Line: Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes.We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes.Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Center of Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois, United States of America. rjohnson9@kumc.edu

ABSTRACT

Objective: To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice.

Methods and results: We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells.

Conclusion: Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells.

Show MeSH
Related in: MedlinePlus