Skeletal muscle-derived CD34+/45- and CD34-/45- stem cells are situated hierarchically upstream of Pax7+ cells.
The hierarchical relationship of skeletal muscle-derived multipotent stem cells sorted as CD34(+)/CD45(-) (Sk-34) and CD34(-)/CD45(-) (Sk-DN) cells, which have synchronized reconstitution capacities for blood vessels, peripheral nerves, and muscle fibers, was examined. Expression of Sca-1 and CD34 (typical state of freshly isolated Sk-34 cells) in Sk-DN cells was examined using in vitro culture and in vivo cell implantation. Sk-DN cells sequentially expressed Sca-1 and CD34 during cell culture showing self-maintenance and/or self-renewal-like behavior, and are thus considered hierarchically upstream of Sk-34 cells in the same lineage. Sk-34 and Sk-DN cells were further divided into small and large cell fractions by cell sorting. Immunocytochemistry using anti-Pax7 was performed at the time of isolation (before culture) and revealed that only 1% of cells in the large Sk-DN cell fraction were positive for Pax7, while Sk-34 cells and 99% of Sk-DN cells were negative for Pax7. Therefore, putative satellite cells were possibly present in the large Sk-DN cell fraction. However, serial analysis of Pax7 expression by RT-PCR and immunocytochemistry for single and 2 to >40 clonally proliferated Sk-34 and Sk-DN cells revealed that both cell types expressed Pax7 after several asymmetric cellular divisions during clonal-cell culture. In addition, production of satellite cells was seen after muscle fiber formation following Sk-34 or Sk-DN cell transplantation into damaged muscle, and even in the nonmuscle tissue environment (beneath the renal capsule). Thus, Sk-DN cells are situated upstream of Sk-34 cells and both cells can produce Pax7+ cells (putative satellite cells) after cellular division.
Tamaki
Tetsuro
T
Muscle Physiology and Cell Biology Unit, Department of Regenerative Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, Japan. tamaki@is.icc.u-tokai.ac.jp
Okada
Yoshinori
Y
Uchiyama
Yoshiyasu
Y
Tono
Kayoko
K
Masuda
Maki
M
Nitta
Masahiro
M
Hoshi
Akio
A
Akatsuka
Akira
A
D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D000950:Antigens, Ly / Q000096:biosynthesis; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D007150:Immunohistochemistry; D017493:Leukocyte Common Antigens; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D008822:Mice, Transgenic; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D051763:PAX7 Transcription Factor / Q000096:biosynthesis; D013234:Stem Cells / Q000166:cytology*
D018952:Antigens, CD34; D000950:Antigens, Ly; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D051763:PAX7 Transcription Factor; C497322:Pax7 protein, mouse; D017493:Leukocyte Common Antigens; C516770:Ptprc protein, mouse