Preliminary Study On Molecular Mechanism And Microenvironment Of Cochlear Stem Cell Differentiation

In adult mammalian cochlea, the hair cells cannot regenerate spontaneously after damaged. This is the major cause of permanent hearing loss. Cochlear stem cells have been isolated from the early postnatal rat and mouse in previous studies. These stem cells cultured in vitro can differentiate into neurons, astrocytes, hair cells and supporting cells. Differentiation of cochlear stem cells is critical to the development of cochlea tissue. Study of cochlear stem cells may help to learn more about cochlear development.Microenvironment is critical to differentiation of stem cells. But little is known about microenvironment of cochlear stem cell differentiation. Microenvironment of differentiation consists of all molecules in molecular network. We need to know more molecules involved in this network. Cell cycle exit participates in stem cell differentiation. Several cell cycle regulators, including cyclin A2 and CRP2, may be involved in this network. Hence, we isolated cochlear stem cells from newborn rats and induced differentiation of these cells. The level of cyclin A2 and CRP2 were assessed in stem cells and differentiated cells for better understanding of cochlea development mechanism. Then we cultured neural stem cells in the microenvironment of cochlear stem cell differentiation to see the effect of microenvironment on neural stem cell differentiation.1. Isolation and culture of cochlear stem cells from newborn ratStem cells were isolated from the P2 rat cochlea tissues. Stem cells were incubated at 37°C in 5% CO2 atmosphere. The culture medium consisting of DMEM/F12 supplemented with B27, N2, EGF and FGF-2. For cell expansion and testing for self-renewal, we plated the cells at very low density to ensure that cell spheres were formed from single cells. For analysis of cell differentiation, we transferred cell spheres into 6-well dishes with poly-l-lysine treated coverslips and filled with 10% FCS. After 16 h, we replaced the medium with serum-free DMEM/F12 supplemented with N2 and B27. Half of the medium was replaced every second day. The differentiated cells were identified after 14 days in culture by immunocytochemistry. The cell spheres and differentiated cells were fixed with 4% paraformaldehyde, and incubated at 4°C overnight with primary antibodies: mouse anti-nestin monoclonal antibody, mouse anti-BrdU monoclonal antibody, rabbit anti-myosin VIIA (hair cell marker) polyclonal antibody, rabbit anti-GFAP (astrocyte marker) polyclonal antibody, mouse anti-NeuN (neuron marker) monoclonal antibody. Cy3 conjugated second antibodies were used to detect primary antibodies. Hoechst 33342 was used to visualize nuclei. For BrdU detection, stem cells were incubated with 10μM BrdU in the culture medium for 24 h, fixed for 30 min, treated with 2 N HCl for 30 min before antibody incubation. The differentiated cells cultured for 21 d were analyzed by SEMWe found sphere-forming capacity of isolated cells after 7 days in culture. These cells were further identified with nestin and BrdU antibodies. The stem cell-derived differentiated cells expressed myosin VIIA, GFAP and NeuN. SEM was performed in order to determine the shape and surface structure of hair cells. Hair cells in culture were identified with stereocilia of cell surface. We found that hair cells with stereocilia were flat and irregularly shaped. We also found bipolar neuron-like cells.2. Decreased level of cyclin A2 in rat cochlea development and cochlear stem cell differentiationIn order to detect the distribution of cyclin A2 protein in the rat cochlea, the immunohistochemical staining method was used in our study. P2 (postnatal day 2), P10 and adult (P42) rats were deeply anesthetized and exsanguinated, then decapitated. The cochlea tissues were excised carefully and then fixed in 4% paraformaldehyde in PBS at 4°C overnight. The P2 samples were decalcified for 1 day. The P10 and P42 samples were decalcified for 10 days. All samples were then immersed in 30% sucrose in PBS for 1 day and embedded carefully in optimal cutting temperature (OCT) compound. Sections (10μm thick) were cut on a cryostat at -27°C and mounted on poly-l-lysine treated glass slides. The immunohistochemistry of cyclin A2 in the rat cochlea was performed by streptavidin–biotin complex (SABC) staining. The primary antibody was mouse anti-cyclin A monoclonal antibody, and the second antibody was biotinylated horse anti-mouse IgG. For negative control, an equivalent dilution of normal mouse IgG was used in place of the primary antibody. We used immunocytochemistry to detect cyclin A2 protein expression in differentiated cells and cell spheres.We used RT-PCR to detect cyclin A2 mRNA expression in cochlea tissues, cochlear stem cells and differentiated cells. The cochlea tissues were excised from P0, P2, P14 and P42 rats respectively. The differentiated cells cultured for 6 or 14 days and cell spheres were collected for further assay. Total RNA was extracted. Then cDNA was amplified by PCR. The cyclin A2 PCR product was sequenced.Western blotting was used to detect cyclin A2 protein expression in cochlear stem cells, differentiated cells cultured for 14 days, and cochlea tissues (P0 and P14). Each protein sample was electrophoresed onto 12% SDS-polyacrylamide gel and transferred onto the nitrocellulose membranes. The membranes were incubated with mouse anti-cyclin A monoclonal antibody. The membranes were then incubated with the HRP-conjugated goat anti-mouse IgG. The membranes were also probed with anti-β-actin as internal control. The reaction product was visualized with enhanced chemiluminescence.With immunohistochemical staining, cyclin A2 was observed localizing in the spiral limbus of the P2 rat cochlea, but not in the cochlea of P10, P42 and the negative control. Cyclin A2 mRNA and protein levels in cultured cells fell down after differentiation of cochlear stem cells. Cyclin A2 level in cochlea tissues decreased from newborn to adult. The sequence of PCR product was determined to be identical to the originally published sequence of rat cyclin A2.3. Expression of CRP2 and CRIP2 in rat cochlea development and cochlear stem cell differentiationIn order to detect the distribution of CRP2 and CRIP2 proteins in the rat cochlea of P2, P6 and P10, the immunohistochemical staining method was used. We used immunocytochemistry to detect CRP2 and CRIP2 proteins in differentiated cells and cell spheres.CRP2 protein level fell down during the development of cochlea tissues and differentiation of cochlear stem cells. While we found no difference of CRIP2 protein level in cochlea development and cochlear stem cell differentiation.4. Microenvironment of cochlear stem cell differentiation induces neural stem cells into myosin VIIA-positive cells and bipolar neuron-like cellsCocultures were performed on poly-L-lysine-coated coverslips in 6-well plates mixed with 1×10~5 CPCs and 1×10~4 NPCs per well. Cell spheres of NSCs and CPCs were dissociated by 0.025% trypsin and mechanical procedures. Then dissociated cells were mixed and allowed to attach for 16 h in wells filled with DMEM/F12 containing 10% FCS. After the cells were attached, we replaced the medium with serum-free DMEM/ F12 supplemented with N2 and B27 solutions. Half of the medium was replaced every second day. Cocultured cells were analyzed after 21 d by immunocytochemistry. Primary antibodies were as follows: rabbit anti-myosin VIIA polyclonal antibody, rabbit anti-peripherin polyclonal antibody. Cy3 conjugated second antibodies were used to detect primary antibodies. Hoechst 33342 was used to visualize nuclei. For negative controls, 1×10~5 NPCs were dissociated and cultured in 6-well plates with the same medium and methods as above.After 21 d in coculture, about 8% differentiated GFP-labeled NSCs expressed myosin VIIA. Myosin VIIA-positive cells were flat and irregularly shaped. About 1% differentiated NSCs expressed peripherin, a marker of peripheral neuron. Some of them were bipolar neuron-like cells. We also observed that spontaneous neural cell apoptosis which occurred in adherent cultures was delayed in the presence of differentiated CPCs. After 21 d in culture, no differentiated NPC was alive in negative controls, while the number of living differentiated NSCs among cocultured cells was about 8×103 per well.5. Methods for transplantation of stem cells into the cochlea of chickens and mature guinea pigsNSCs labeled with GFP were transplanted into chicken's cochlea via cochlear window after exposure of middle ear cavity. For cell transplantation into cochlear perilymphatic space of guinea pigs, NSCs were inoculated via a small hole drilled on the bony wall of scala tympani in the basal turn of the cochlea. For cell transplantation into scala media, a small hole was made to penetrate through the stria vascularis in the third turn of the cochlea. NSCs were then injected into the scala media through the fenestra. One day after transplantation, the transplant cells were examined with fluorescent microscope.After transplantation, NSCs labeled with GFP were observed in chicken's cochlea. In the cochlea tissues of guinea pigs, NSCs were detected in scala tympani and scala media, respectively. The methods for transplantation of stem cells into the cochlea of chickens and mature guinea pigs were established, which provides a basis for application of stem cells in studies of sensorineural hearing loss.