Exposure to Tissue Culture Conditions Can Adversely Affect Myoblast Behavior in Vivo in Whole Muscle Grafts: Implications for Myoblast Transfer Therapy

Abstract

The effects of tissue culture conditions on the viability of myoblasts in whole muscles transplanted in vivo were investigated. Whole male (SJL/J) donor muscles were exposed to various tissue culture reagents and proteolytic enzymes, and allografted into female (SJL/J) host mice. Desmin immunohistochemistry was used to assess the numbers of myogenic cells (as an index of myoblast viability and the extent of regeneration) in tissue sections of whole-muscle grafts sampled on days 7 and 14. DNA quantitation with a Y-chromosome-specific probe was used to determine the total Y-l sequence DNA (as an index of myoblast survival and proliferation) in whole-muscle grafts sampled on days 1, 3, and 7. In grafts exposed to serum-free medium, there was a delay in myoblast fusion at 7 days that was recovered by 14 days, but exposure to serum (10% or 20%) had a prolonged adverse effect on myotube formation at 14 days. DNA quantitation demonstrated that either serum-free culture medium or 10% serum enhanced the number of male cells within whole-muscle grafts at 7 days. Proteolytic digestion (even for 5 min) of whole muscles prior to grafting was extremely detrimental to myoblast survival and viability at 7 and 14 days. The unexpected finding of adverse effects of tissue culture conditions on the regeneration of whole-muscle grafts in vivo appears to parallel the major problem of the rapid death of isolated cultured donor myoblasts after injection in myoblast transfer therapy. The use of whole-muscle grafts provides an alternative and sensitive model to analyze the crucial effects of various tissue culture components on the subsequent survival and proliferation of myogenic cells in vivo.

Overexpression of SOX9 in mouse embryonic stem cells directs the immediate chondrogenic commitment

“…mES cells undergo chondrogenic differentiation by cytokine stimulation or different culture conditions (Kramer et al, 2000;Hegert et al, 2002;Tanaka et al, 2004) under a long-term culture system. However, the prolonged differentiation systems would delay the treatment to patients or could alter the immunogenicity of cultured autologous cells, ultimately leading to immunorejection upon transplantation (Hodgetts et al, 2000;Smythe et al, 2000;Heng et al, 2004). To enhance the chondrogenic differentiation through genetic modification, we successfully established stable cell lines constitutively expressing exogenous SOX9 in mES cells.…”

Therapeutic Potentials of Human Embryonic Stem Cells in Parkinson’s Disease

“…70 Ex vivo cultures with long duration of autologous stem cells may result in altered immunogenicity, which could lead to rejection of the cells upon transplantation. 71,72 This is likely because the self no longer recognizes its own cell once it has been immunogenically changed.…”

Noncultured, Autologous, Skeletal Muscle Cells Can Successfully Engraft Into Ovine Myocardium

“…It is even possible that avoiding the tissue-culture phase improves the efficacy of engraftment into the myocardium, as was previously reported in skeletal muscle. 30,31 Are noncultured cell muscle preparations more potent than cultured muscle cells? Which method yields the greatest number of implanted cells in vivo?…”