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Nesprin-1 has key roles in the process of mesenchymal stem cell differentiation into cardiomyocyte-like cells in vivo and in vitro.

Yang W, Zheng H, Wang Y, Lian F, Hu Z, Xue S - Mol Med Rep (2014)

Bottom Line: Surface-associated antigens of MSCs were detected by flow cytometry.The results of the present study indicated that MSCs may be differentiated in vitro and in vivo into cells with characteristics commonly attributed to cardiomyocytes.The results also suggested that, consistent with the results of previous studies, the expression of nesprin-1 protein was higher during the differentiation process of MSCs and may have an important role in mediating MSC differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P.R. China.

ABSTRACT
The aim of the present study was to investigate the expression of nesprin-1 protein in MSCs and its effects on the differentiation of rat bone-marrow mesenchymal stem cells (MSCs). Surface-associated antigens of MSCs were detected by flow cytometry. MSC differentiation was induced by treatment with 10 µmol/l 5-azacytidine. Sprague-Dawley rats were anesthetized prior to thoracotomy and subsequent ligation of the left anterior descending coronary artery to establish a model of myocardial infarction. Two weeks following myocardial infarction, DAPI-marked MSCs were injected into the infarcted region in the experimental group, while DMEM was injected into the infarcted region of the control group. Characteristics of the putative cardiac-myogenic cells were evaluated using immunohistochemical and immunofluorescent analysis. The messenger RNA expression levels of cardiac-myogenic specific genes; desmin, α-actinin and cardiac troponin I (cTnI) were detected by reverse transcription quantitative polymerase chain reaction. The expression of nesprin-1 protein in MSCs was identified by immunofluorescence and western blot analysis, prior to and following MSC differentiation. Following differentiation, the MSCs appeared spindle-shaped with irregular processes and were positive for CD90 and CD29, but negative for CD45. Cardiomyocyte-like cells were positive for desmin, α-sarcomeric actin and cTnI. Nesprin protein was detected in the nuclear membrane via immunofluorescence, and following MSC differentiation into cardiomyocyte-like cells, the expression of nesprin protein was significantly higher (*P=0.03<0.05). The results of the present study indicated that MSCs may be differentiated in vitro and in vivo into cells with characteristics commonly attributed to cardiomyocytes. Cardiomyocyte-like cells cultured from bone marrow sources may be potentially useful for repairing the injured myocardium. The results also suggested that, consistent with the results of previous studies, the expression of nesprin-1 protein was higher during the differentiation process of MSCs and may have an important role in mediating MSC differentiation. Elucidation of the role of nesprin-1 in MSC differentiation may aid in the development of novel therapies for the treatment of myocardial ischemia and nesprin-1 genetic deficiencies.

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Characterization of MSCs and differentiated cardiomyocyte-like cells in vitro. (A) Morphological observation of rat MSCs following 3 d of culture. The cells were spindle shaped with one nucleus (magnification, ×100). (B) Morphological observation of rat MSCs following 7 d of culture (magnification, ×100). (C) Morphological observation of rat MSCs following culture for 10 d (magnification, ×100). The cells displayed as long spindle-shaped fibroblastic cells and began to form colonies (D) Morphological observation of rat MSCs following treatment with 5-azacytidine for 7 d (magnification, ×200). The cells were enlarged and assumed ball- or stick-like morphologies. (E) Morphological observation of rat MSCs following treatment with 5-azacytidine for 14 days (magnification, ×200). The cells were connected with adjoining cells. MSCs, mesenchymal stem cells.
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f1-mmr-11-01-0133: Characterization of MSCs and differentiated cardiomyocyte-like cells in vitro. (A) Morphological observation of rat MSCs following 3 d of culture. The cells were spindle shaped with one nucleus (magnification, ×100). (B) Morphological observation of rat MSCs following 7 d of culture (magnification, ×100). (C) Morphological observation of rat MSCs following culture for 10 d (magnification, ×100). The cells displayed as long spindle-shaped fibroblastic cells and began to form colonies (D) Morphological observation of rat MSCs following treatment with 5-azacytidine for 7 d (magnification, ×200). The cells were enlarged and assumed ball- or stick-like morphologies. (E) Morphological observation of rat MSCs following treatment with 5-azacytidine for 14 days (magnification, ×200). The cells were connected with adjoining cells. MSCs, mesenchymal stem cells.

Mentions: Following discarding the non-adherent cells by the first medium change and by washing with PBS three times at 24 h of primary culture, ~80% MSCs had attached to culture dishes. The medium was subsequently changed to remove the suspended hematopoietic stem cells. Following three days of primary culture, MSCs adhered to the plastic surface and presented a small population of single cells. The cells were spindle shaped with one nucleus (Fig. 1A). Seven to ten days following initial plating, the cells developed into long spindle-shaped fibroblastic cells and began to form colonies (Fig. 1B and C). Following replating, 100% of the cells had attached to the culture dishes and were polygonal or spindle-shaped, with long processes.


Nesprin-1 has key roles in the process of mesenchymal stem cell differentiation into cardiomyocyte-like cells in vivo and in vitro.

Yang W, Zheng H, Wang Y, Lian F, Hu Z, Xue S - Mol Med Rep (2014)

Characterization of MSCs and differentiated cardiomyocyte-like cells in vitro. (A) Morphological observation of rat MSCs following 3 d of culture. The cells were spindle shaped with one nucleus (magnification, ×100). (B) Morphological observation of rat MSCs following 7 d of culture (magnification, ×100). (C) Morphological observation of rat MSCs following culture for 10 d (magnification, ×100). The cells displayed as long spindle-shaped fibroblastic cells and began to form colonies (D) Morphological observation of rat MSCs following treatment with 5-azacytidine for 7 d (magnification, ×200). The cells were enlarged and assumed ball- or stick-like morphologies. (E) Morphological observation of rat MSCs following treatment with 5-azacytidine for 14 days (magnification, ×200). The cells were connected with adjoining cells. MSCs, mesenchymal stem cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237090&req=5

f1-mmr-11-01-0133: Characterization of MSCs and differentiated cardiomyocyte-like cells in vitro. (A) Morphological observation of rat MSCs following 3 d of culture. The cells were spindle shaped with one nucleus (magnification, ×100). (B) Morphological observation of rat MSCs following 7 d of culture (magnification, ×100). (C) Morphological observation of rat MSCs following culture for 10 d (magnification, ×100). The cells displayed as long spindle-shaped fibroblastic cells and began to form colonies (D) Morphological observation of rat MSCs following treatment with 5-azacytidine for 7 d (magnification, ×200). The cells were enlarged and assumed ball- or stick-like morphologies. (E) Morphological observation of rat MSCs following treatment with 5-azacytidine for 14 days (magnification, ×200). The cells were connected with adjoining cells. MSCs, mesenchymal stem cells.
Mentions: Following discarding the non-adherent cells by the first medium change and by washing with PBS three times at 24 h of primary culture, ~80% MSCs had attached to culture dishes. The medium was subsequently changed to remove the suspended hematopoietic stem cells. Following three days of primary culture, MSCs adhered to the plastic surface and presented a small population of single cells. The cells were spindle shaped with one nucleus (Fig. 1A). Seven to ten days following initial plating, the cells developed into long spindle-shaped fibroblastic cells and began to form colonies (Fig. 1B and C). Following replating, 100% of the cells had attached to the culture dishes and were polygonal or spindle-shaped, with long processes.

Bottom Line: Surface-associated antigens of MSCs were detected by flow cytometry.The results of the present study indicated that MSCs may be differentiated in vitro and in vivo into cells with characteristics commonly attributed to cardiomyocytes.The results also suggested that, consistent with the results of previous studies, the expression of nesprin-1 protein was higher during the differentiation process of MSCs and may have an important role in mediating MSC differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P.R. China.

ABSTRACT
The aim of the present study was to investigate the expression of nesprin-1 protein in MSCs and its effects on the differentiation of rat bone-marrow mesenchymal stem cells (MSCs). Surface-associated antigens of MSCs were detected by flow cytometry. MSC differentiation was induced by treatment with 10 µmol/l 5-azacytidine. Sprague-Dawley rats were anesthetized prior to thoracotomy and subsequent ligation of the left anterior descending coronary artery to establish a model of myocardial infarction. Two weeks following myocardial infarction, DAPI-marked MSCs were injected into the infarcted region in the experimental group, while DMEM was injected into the infarcted region of the control group. Characteristics of the putative cardiac-myogenic cells were evaluated using immunohistochemical and immunofluorescent analysis. The messenger RNA expression levels of cardiac-myogenic specific genes; desmin, α-actinin and cardiac troponin I (cTnI) were detected by reverse transcription quantitative polymerase chain reaction. The expression of nesprin-1 protein in MSCs was identified by immunofluorescence and western blot analysis, prior to and following MSC differentiation. Following differentiation, the MSCs appeared spindle-shaped with irregular processes and were positive for CD90 and CD29, but negative for CD45. Cardiomyocyte-like cells were positive for desmin, α-sarcomeric actin and cTnI. Nesprin protein was detected in the nuclear membrane via immunofluorescence, and following MSC differentiation into cardiomyocyte-like cells, the expression of nesprin protein was significantly higher (*P=0.03<0.05). The results of the present study indicated that MSCs may be differentiated in vitro and in vivo into cells with characteristics commonly attributed to cardiomyocytes. Cardiomyocyte-like cells cultured from bone marrow sources may be potentially useful for repairing the injured myocardium. The results also suggested that, consistent with the results of previous studies, the expression of nesprin-1 protein was higher during the differentiation process of MSCs and may have an important role in mediating MSC differentiation. Elucidation of the role of nesprin-1 in MSC differentiation may aid in the development of novel therapies for the treatment of myocardial ischemia and nesprin-1 genetic deficiencies.

Show MeSH
Related in: MedlinePlus