Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Inner Ear Hair Cell Progenitors In Vitro

Background:Sensorineural hearing loss (SHL) is quiet common in the clinic. The etiology of SHL is aging, noise, ototoxic drugs, traumas, infection, inherit diseases and immune reaction, which results in permanent hearing impairment because of the defects of cochlear hair cells, and will be hardly recovered. By going on research for the stem cells, a potential treatment is to replace the damaged hair cells and induce to regenerate it by transplantation of the stem cells. So far, it is one of the best active researches in otology.The bone marrow mesenchymal stem cells (MSCs) are the other population of stem cells in bone marrows besides the hematopoietic stem cells, which have great potentiality for self-renewal and high differentiation. Under some appropriate conditions, MSCs can be induced and transdifferentiation into multiple cell lineages, such as osteoblasts, adipocytes, chondrocytes, myoblasts, cardiomyocytes, hepatocytes and neuronal-like cells. MSCs have extensitve source, and is convenient to get and not restricted by the ethic and law. Also, MSCs have poor immunogenicity.Here, we explored the possibility of MSCs to differentiate into the hair cells of the inner ear. We tried to provide exprimental foundation for treatment of the sensorineural hearing loss by transplantation of the stem cells.Methods:We collected MSCs from rat bone marrows by plastic adherent selection method. After purification and division to the forth generation in vitro, we applied six growth factors ( including epidermal growth factor (EGF),basic fibroblast growth factor(bFGF), insulin-like growth factor-1(IGF-1),brain-derived neurotrophic factor (BDNF),neurotrophin-3 (NT-3),all-trans retinoic acid (ATRA)) and combined into eight groups as the induce factors. Then MSC were induced and differentiated in MSCs basic culture mediums containing 2% fetal bovine serum, 1% N2 supplement and Dulbecco's modified Eagle's medium-low glucose. During this period, the shape of the induced cells was observed daily, and the differentiated cells were characterized by immunocytochemistry assays using monocloned antibodies (nestin, pax-2,myosin â…¥,p27^kip1), which are some markers of the hair cell progenitors or hair cells-specific antigen.Results:1. In the group with EGF+IGF-1 or EGF+IGF-1+BDNF+ATRA, the induced cells showed short spindle-like or rectangle-like structure, but no processes formed. The former group, the expression of the cells demonstrated weak positive by myosin â…¥, and moderate positive by pax-2. The latter, the cells showed moderate expression both by myosinâ…¥ and pax-2. Moreover, the cells showed positive by nestin and p27^kip1 staining in these groups.2. In the group with NT-3,bFGF or BDNF, the induced cells showed round or slim, gradually protruding processes, and some parts of them forming net- or ring- like structure. Cells with processes showed moderate expression of nestin by ICC staining, but low or negative expressions in others.3. In all groups, the induced cells in culture medium with high density, first became round, then gradually protruding several short processes, or keeping round if therewas no space. However, the induced cells with low density, became slim, then becoming long processes in two sides of them.4. Un-induced MSCs showed low expression of nestin, short spindle-like or rectangle-like structure with the blurry boundary and more cell debris.Conclusion1. MSCs show the potential to differentiate into hair cell progenitors and neural progenitor cells.2. EGF+IGF-1+ BDNF+ATRA is the better factor induced for MSCs to differentiate into the hair cells of the inner ear.3. Culture density in vitro is one of important microenvironment for the cells, which might affect transdifferentiation of MSCs.4. MSCs has the ability to spontaneously differentiate into neural progenitor cells, but it is necessary for us to apply appropriate factors in order to induce MSCs to further mature.