Biological characteristics and phenotype of rabbit muscle-derived stem cells

Jinming ZHANG; Tao HE; Chenyang JI; Weiqiang LIANG; Hongjun HUANG

ABSTRACT

BACKGROUND:

Bone marrow mesenchymal stem cells (BMSCs) have a remarkable differentiation potential and superiority as a type of seed cells,but their application is limited in the presence of certain diseases,such as aplastic anemia and myelogenous neoplasm.The present studies have found that seed cells called muscle-derived stem cells (MDSCs) have brought more and more attention,because of their capability of stir-renewal and multi-diffcrentiation like B MSCs.

OBJECTIVE:

To explore the biological characterization of the muscle-derived stem cells (MDSCs) from rabbits,and analyze the phenotype.DESIGN,TIME AND

SETTING:

Cell in vitro observation experiment was performed at the Medical Research Center of Second Affiliated Hospital of Sun Yat-sen University from August 2005 to March 2006.MATERIALS A New Zealand rabbit (1.5 months old,clean grade) was enrolled for the preparation of Muscle-derived stem cells.Growth medium was DMED-LG added with 10% fetal bovine serum and 10% horse serum and fusion medium was DMEM-LG added with 2% fetal bovine serum.

METHODS:

The muscle mass was removed from the anesthetized rabbit to isolate MDSCs.These cells were dissociated using three enzymes (collagenase XI,dispase and trypsin) respectively.Sediment was resuspended.Then preplate technique was used.The muscle cell extract was plated on a collagen-coated culture flask with growth medium.The flask was called PP1.PPI was kept overnight in a 37 ℃ incubator containing 5% CO2,After that,the suspension was transferred to another collagen-coated culture flask,which was called PP2.PP3,PP4,PP5 and PP6 were constructed later following the same procedures.The cells adhered in PP6 were collected,plated in 6-well plates,and divided into 2 groups.Growth medium was used in one group,in which the cells were kept growing at a degree of confluence beyond 50%,and fusion medium was used in the other one,in which the cells were passaged with a degree up to 30%.MAIN OUTCOME

MEASURES:

The cells from PP1 to PP6 were collected,and the characterization was identified preliminary by Flow cytomctry,Immunocytochemistry and Western Blotting.The fusion of cells in PP6 was detected at different confluence degrees and concentration of medium.

RESULTS:

The cells in PP6 showed ＞ 80% desmin+,＞ 70% Bcl-2+,＞ 95% CD45,which indicated that MDSCs were in a high concentration.The expressions of α-SMA in the cells were decreasing with the Preplate technique used and the cells in PP6 almost had no α -SMA expression.When passaged at a high confluence (＞ 50%) or cultured with low concentrations of serum (2% serum),the cells in PP6 had a strong tendency of fusing into myotubes or cell chains and were skeletal myosin+.

CONCLUSION:

MDSCs,which are capable of multi-differentiation under a high fusion or low serum conditions,express dcsmin and Bcl-2 highly,but extreruelv little CD45 and no α -SMA.