Limits...
Cardiomyocyte formation by skeletal muscle-derived multi-myogenic stem cells after transplantation into infarcted myocardium.

Tamaki T, Akatsuka A, Okada Y, Uchiyama Y, Tono K, Wada M, Hoshi A, Iwaguro H, Iwasaki H, Oyamada A, Asahara T - PLoS ONE (2008)

Bottom Line: Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells.Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution.Skeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

View Article: PubMed Central - PubMed

Affiliation: Muscle Physiology & Cell Biology Unit, Tokai University School of Medicine, Isehara, Kanagawa, Japan. tamaki@is.icc.u-tokai.ac.jp

ABSTRACT

Background: Cellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported.

Methods and results: Skeletal muscle interstitium-derived CD34(+)/CD45(-) (Sk-34) cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI) model (left ventricle). Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed.

Conclusions and significance: Skeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

Show MeSH

Related in: MedlinePlus

Expression of skeletal muscle (red), ion channel (green), smooth muscle (purple), vascular-related (light-blue), cardiac muscle (black) and cell proliferation (orange) markers before and after cell division of single Sk-34 cells.Cells were cultured clonally in collagen gels. Bar chart shows percentage of samples expressing target mRNA and total numbers are given in parentheses. Percentage of skeletal and smooth muscle, and two ion channel marker-expressing cells gradually increased following cellular divisions. However, cardiac and vascular-related makers did not change during clonal cell culture. M-cad, M-cadherin; Scn1b, sodium channel voltage gated type1-b; Cacnb1, calcium channel voltage-dependent beta-1 subunit; α-SMA, α-smooth muscle actin; TEK, tyrosine kinase-endothelial.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2262151&req=5

pone-0001789-g003: Expression of skeletal muscle (red), ion channel (green), smooth muscle (purple), vascular-related (light-blue), cardiac muscle (black) and cell proliferation (orange) markers before and after cell division of single Sk-34 cells.Cells were cultured clonally in collagen gels. Bar chart shows percentage of samples expressing target mRNA and total numbers are given in parentheses. Percentage of skeletal and smooth muscle, and two ion channel marker-expressing cells gradually increased following cellular divisions. However, cardiac and vascular-related makers did not change during clonal cell culture. M-cad, M-cadherin; Scn1b, sodium channel voltage gated type1-b; Cacnb1, calcium channel voltage-dependent beta-1 subunit; α-SMA, α-smooth muscle actin; TEK, tyrosine kinase-endothelial.

Mentions: In order to further analyze the cardiomyogenic commitment of Sk-34 cells, we also performed single and 2–8 clonal cells RT-PCR after 3 days of culture (Fig. 3). In this analysis, single cell refers to the stage before cell division, 2 cells refers to the stage after the first cell division, 3–4 cells refers to the second cell division, and 5–8 cells refers to the stage after the third division. Single Sk-34 cells typically showed no expression of Myf5, Pax3, Pax7, VE-cadherin or Nkx2.5, lower % expression of MyoD, M-cadherin, Cacn-1b, TEK, cardiac actin, GATA-4, Mef2c, Hand2 and isl-1, and higher (>50%) expression of c-met, Scn-1b, α-SMA, smoothelin and Nucleostemin mRNAs. This single cell analysis (before cell division) was similar to that for freshly isolated bulk Sk-34 cells, in which elevated markers were positive, reduced markers were sometimes positive or negative, and unexpressed marker were negative (Fig. 2). After the third cellular division, expression of c-met, Scn-1b, α-SMA, smoothelin and Nucleostemin was relatively constant, and MyoD, Myf5, M-cadherin and Cacn-1b gradually appeared after cell division. However, cardiac differentiation-related mRNAs did not dramatically change. These results suggest that cardiac differentiation of Sk-34 cells is not sufficiently induced by clonal cell culture, while skeletal and smooth myogenic differentiation progresses.


Cardiomyocyte formation by skeletal muscle-derived multi-myogenic stem cells after transplantation into infarcted myocardium.

Tamaki T, Akatsuka A, Okada Y, Uchiyama Y, Tono K, Wada M, Hoshi A, Iwaguro H, Iwasaki H, Oyamada A, Asahara T - PLoS ONE (2008)

Expression of skeletal muscle (red), ion channel (green), smooth muscle (purple), vascular-related (light-blue), cardiac muscle (black) and cell proliferation (orange) markers before and after cell division of single Sk-34 cells.Cells were cultured clonally in collagen gels. Bar chart shows percentage of samples expressing target mRNA and total numbers are given in parentheses. Percentage of skeletal and smooth muscle, and two ion channel marker-expressing cells gradually increased following cellular divisions. However, cardiac and vascular-related makers did not change during clonal cell culture. M-cad, M-cadherin; Scn1b, sodium channel voltage gated type1-b; Cacnb1, calcium channel voltage-dependent beta-1 subunit; α-SMA, α-smooth muscle actin; TEK, tyrosine kinase-endothelial.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2262151&req=5

pone-0001789-g003: Expression of skeletal muscle (red), ion channel (green), smooth muscle (purple), vascular-related (light-blue), cardiac muscle (black) and cell proliferation (orange) markers before and after cell division of single Sk-34 cells.Cells were cultured clonally in collagen gels. Bar chart shows percentage of samples expressing target mRNA and total numbers are given in parentheses. Percentage of skeletal and smooth muscle, and two ion channel marker-expressing cells gradually increased following cellular divisions. However, cardiac and vascular-related makers did not change during clonal cell culture. M-cad, M-cadherin; Scn1b, sodium channel voltage gated type1-b; Cacnb1, calcium channel voltage-dependent beta-1 subunit; α-SMA, α-smooth muscle actin; TEK, tyrosine kinase-endothelial.
Mentions: In order to further analyze the cardiomyogenic commitment of Sk-34 cells, we also performed single and 2–8 clonal cells RT-PCR after 3 days of culture (Fig. 3). In this analysis, single cell refers to the stage before cell division, 2 cells refers to the stage after the first cell division, 3–4 cells refers to the second cell division, and 5–8 cells refers to the stage after the third division. Single Sk-34 cells typically showed no expression of Myf5, Pax3, Pax7, VE-cadherin or Nkx2.5, lower % expression of MyoD, M-cadherin, Cacn-1b, TEK, cardiac actin, GATA-4, Mef2c, Hand2 and isl-1, and higher (>50%) expression of c-met, Scn-1b, α-SMA, smoothelin and Nucleostemin mRNAs. This single cell analysis (before cell division) was similar to that for freshly isolated bulk Sk-34 cells, in which elevated markers were positive, reduced markers were sometimes positive or negative, and unexpressed marker were negative (Fig. 2). After the third cellular division, expression of c-met, Scn-1b, α-SMA, smoothelin and Nucleostemin was relatively constant, and MyoD, Myf5, M-cadherin and Cacn-1b gradually appeared after cell division. However, cardiac differentiation-related mRNAs did not dramatically change. These results suggest that cardiac differentiation of Sk-34 cells is not sufficiently induced by clonal cell culture, while skeletal and smooth myogenic differentiation progresses.

Bottom Line: Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells.Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution.Skeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

View Article: PubMed Central - PubMed

Affiliation: Muscle Physiology & Cell Biology Unit, Tokai University School of Medicine, Isehara, Kanagawa, Japan. tamaki@is.icc.u-tokai.ac.jp

ABSTRACT

Background: Cellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported.

Methods and results: Skeletal muscle interstitium-derived CD34(+)/CD45(-) (Sk-34) cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI) model (left ventricle). Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed.

Conclusions and significance: Skeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

Show MeSH
Related in: MedlinePlus