This study was designed to investigate the increase in the number of circulating CD34+ cells after acute myocardial infarction (MI) and the differentiation of these cells to cardiomyocytes after transplantation into infarcted myocardium. The study involved five donor groups: MI (n=27), sham (n=26), granulocyte-colony stimulating factor (GCSF) (n=26), MI+GCSF (n=25), and control (n=25). Acute MI was induced by ligating the left anterior descending coronary arteries (LAD) of donor rats, and LAD of recipient rats were ligated on the same day. Seven days after ligation, CD34+ cells in donor rats were counted and then were directly injected into the infarcted myocardium of recipient rats. Eight weeks after the transplantation, significant differences (p<0.001) were observed in the CD34+cell counts among the 5 donor groups with the greatest increase in the MI+GCSF donor group. In rats receiving CD34+ cells, the size of the scar area smaller (p<0.001) and the thickness of the scar was greater (p=0.001) than in CD34- and saline-transplanted rats. The transplanted CD34+ cells differentiated into cardiomyocytes in the scar. This study suggests that CD34+ cells may be a potential source of stem cells and that they may be useful in strategies aimed at the regeneration of infarcted myocardium.
Animals ; Antigens, CD34/*analysis ; Body Weight ; Cell Differentiation ; Heart/*physiology ; Myocardial Infarction ; Myocardium/*cytology/pathology ; Myocytes, Cardiac/*physiology ; Rats ; *Regeneration ; *Stem Cell Transplantation ; Stem Cells/*immunology/metabolism ; Support, Non-U.S. Gov't

The P19 embryonal carcinoma cell line is a useful model cells for studies on cardiac differentiation. However, its low efficacy of differentiation hampers its usefulness. We investigated the effect of 5-azacytidine (5-aza) on P19 cells to differentiate into a high-efficacy cardiomyocytes. Embryoid-body-like structures were formed after 6 days with 1 micrometer of 5-aza in a P19 cell monolayer culture, beating cell clusters first observed on day 12, and, the production of beating cell clusters increased by 80.1% (29 of 36-wells) after 18 days. In comparison, the spontaneous beating cells was 33.3% (12 of 36-wells) for the untreated control cells. In response to 1 micrometer of 5-aza, P19 cells expressed bone morphogenetic protein-2 (BMP-2), BMP-4, Bmpr1a and Smad1 at day 6 or 9, and also cardiac markers such as GATA-4, Nkx2.5, cardiac troponin I, and desmin were up-regulated in a time-dependent manner after induction of BMP signaling molecules. Immunocytochemistry revealed the expression of smooth muscle a-actin, sarcomeric a-actinin, cardiac myosin heavy chain, cardiac troponin T and desmin, respectively. The proportion of sarcomeric a-actinin positive cells accounted for 6.48% on day 15 after 5-aza exposure as measured by flow cytometry. This study has demonstrated that 5-aza induces differentiation of P19 cells into cardiomyocytes in a confluent monolayer culture in the absence of prior embryoid formation and dimethyl sulfoxide exposure, depending in part on alteration of BMP signaling molecules. These results suggest that 5-aza treatment could be used as a new method for cardiac differentiation in P19 cells.
Animals ; Azacitidine/*pharmacology ; Bone Morphogenetic Proteins/genetics/metabolism ; Cell Differentiation/drug effects/genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; DNA-Binding Proteins/genetics/metabolism ; Embryo/cytology ; Gene Expression ; Homeodomain Proteins/genetics/metabolism ; Mice ; Muscle Proteins/analysis/genetics/metabolism ; Myocytes, Cardiac/*cytology/immunology/physiology ; Research Support, Non-U.S. Gov't ; Stem Cells/*drug effects/metabolism ; Transcription Factors/genetics/metabolism