Study On C-Myc And CyclinA2 Gene-induced Cochlea Progenitor Cells Proliferation In Vitro

In adult mammalian cochlea, hair cells and spiral ganglion cells can not regenerate spontaneously after damaged. This is the major cause of permanent hearing loss. Cochlear progenitor cells have been isolated from the early postnatal rats and mice in previous studies and can differentiate into neurons, astrocytes, hair cells and supporting cells in vitro. We isolated the progenitor cells from the cochleae of 1-, 7-, and 14-day-old rats, and compared with the proliferative capacity and ultrastructure of the cells from each age group using flow cytometry and transmission electron microscopy, respectively. Our study suggests that the dormant state of the cochlear progenitor cells after birth. Although these progenitor cells displayed some features of stem cells, they lost their''stemness''and the capacity to robustly generate spheres. The cochlear progenitor cells are remnants of the stem cells that originally gave rise to the sensory epithelium. The disappearance of the cochlear progenitor cells in adult mammalian cochleae might result from their differentiation and/or apoptosis.Our study suggests that it may be possible to keep these cells in a progenitor cell state or to activate the progenitor cells by triggering the cell cycle with the expression of cell cycle-related molecules. Hence, several cell cycle regulators, including c-myc and cyclin A2, may be involved in this progress. For better understanding of cochlea development mechanism, the level of c-myc was assessed in the embryonic and postnatal cochleae, the cochlear progenitor cells and the differentiated cells. Then, we established the adenoviral vector of c-myc and cyclinA2 gene and successfully delivered the target genes into the cochlear progenitor cells in vitro. The changes of the proliferative capacity were observed and the corresponding mechanism was further studied. We found c-myc and cyclinA2 gene may induce the progenitor cells proliferation and reenter into cell cycle. The mechanism is the classic way to CKI-cyclin-CDK.1. Isolation and culture of cochlear progenitor cells from newborn ratsObjective: To isolate and culture the CPCs. Methods: The CPCs were isolated from the P7 rat cochlea tissues and cultured. We identified progenitor cells in postnatal rat cochlea and their potential to differentiate into multiple lineages using immunocytochemistry. Results: After 1 day of culture, the acutely isolated cells formed floating otospheres. After a 3-day culture, the floating otospheres expanded gradually in both volume and cell number. Five days later, adherent and differential cells were found in some parts of the spheres. Acutely dissociated cells from the postnatal rat cochleae expressed nestin and musashi1 and incorporated BrdU after 7 days of culture. This demonstrates of these spheres not only expressed the specific markers of stem cells but also were actively undergoing mitosis. The progenitor cell-derived differentiated cells expressed myosin VIIA, phalloidin, NeuN, Tuj1, GFAP, galactocerebroside and GluR-1. The cells generated from the cochlea-derived spheres contained hair cells, neurons, neuroglial cells, and glutamic neurons. Conclusions: The progenitor cells were present in the cochlea of newborn SD rats. They had the capacity of self-renew and possess potential to differentiate into cell types of the inner ear in vitro. This experiment provided a good model in vitro for studying the mechanisms controlling the domant state of the CPCs in adult. 2. A comparison of the proliferative capacity and ultrastructure of progenitor cells from the cochleae of newborn rats of different agesObjective: The progenitor cells that are capable of proliferation and regeneration are present in mammalian cochleae. However, none progenitor cells has been isolated from the adult cochlea. We examined the proliferative potential of cells derived from neonatal rats of various ages. The determination of the differences between the progenitor cells from rats of different ages may provide clues to the mechanisms controlling the destiny of these cells. Methods: The progenitor cells were isolated from the cochleae of 1-, 7-, and 14-day-old rats, and total cells and spheres were counted after 7 days of culture. The proliferative capacity and ultrastructure of the cells from each age group were assessed using flow cytometry and transmission electron microscopy, respectively. Results: During the first two postnatal weeks, the number of progenitor cells gradually fell to zero. This decrease occurred in parallel with the impairment of the proliferative capacity of the cells and the accumulation of progenitor cells in G0/G1. In addition, some of the cells exited the cell cycle by means of gradual maturity and apoptosis. Conclusions: Our study suggests that CPCs are remnants of the stem cells that originally gave rise to the sensory epithelium. The disappearance of the CPCs in adult mammalian cochleae may result from their differentiation and/or apoptosis.3. Decreased level of c-myc in rat cochlea development and cochlear progenitor cell differentiationObjective: The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea development is still unclear. To explore the role of the gene in cochleae, the c-myc expression was assessed in rat cochlea tissues of different ages, the CPCs and the progenitor cell-derived differentiated cells. Methods: We used RT-PCR to detect c-myc mRNA expression in the cochlea tissues, the CPCs and the differentiated cells. The cochlea tissues were excised from E10, E15, P1, P7 and P14 rats respectively. The differentiated cells were cultured for 7 days and the cell spheres were collected for further assay. Western blotting was used to detect c-myc protein expression in the CPCs, the differentiated cells cultured for 7 days, and the cochlea tissues (E10, E15, P1, P7 and P14). We also used immunocytochemistry to detect c-myc protein expression in the differentiated cells and the cell spheres. Results: The results indicated that c-myc level in cochlea tissues decreased gradually during embryo to newborn. Furthermore, c-myc level fell down after differentiation of CPCs. Conclusions: It was suggested that c-myc might be involved in the modulation of rat cochlea development and CPCs differentiation.4. Construction and identification of recombinant adenovirus and determinating the suitable MOI in the transfection of the CPCsObjective: We respectively established the adenoviral vector of c-myc and cyclinA2 gene. The adenovirus was used to transfect the CPCs in vitro. By observing the transfection efficiency and the cell viability, the suitable MOI was determined. Methods: Recombinant adenovirus were constructed. The complementary DNA sequence of c-myc and cyclinA2 gene was respectively obtained from GenBank. The sequence was subcloned into pDC316-CMV-EGFP and recombined with backbone pAdEasy-1 in BJ5183 bacteria. The adenovirus generation, amplifcation, and titer process was completed sequentially. The vector containing EGFP alone was also constructed. The CPCs were infected by the recombinant adenovirus with Enhance Green fluorescence protein (Ad-EGFP) in different MOI (50,100,150,200,250). After the CPCs re-cultured, we observed the expression of EGFP and the cell morphology through fluorescent microscopy. To determine the proliferation point, we used MTT to assess the cell viability. The MOI was ultimately determined and the infection rate was observed through Flow cytometry. Results: The recombinant adenovirus construction was testified through the detection of the purpose fragment in PCR. A positive dose-response relationship was observed between the expression of EGFP gene ranging from 50 ~250 MOI. The virus infecting rat CPCs at a MOI of 50, 100 and 150 can not impair the cell vitality and morphology. However, the cell growth inhibition occurs at a MOI of 200 and the cells necrosis occurs at a MOI of 250. According to the morphological evidence, we ultimately determined the MOI of 150. Conclusions: Adenovirus can efficiently transfect rat CPCs with EGFP gene in vitro. It will be a powerful tool for gene therapy and cell therapy based on CPCs.5. The influence of c-myc and cyclinA2 on the gene-induced CPCs proliferation and cell cycle in VitroObjective: We established the adenoviral vector of c-myc and cyclinA2 gene and successfully delivered the target genes into the CPCs in vitro. Then, the changes of the proliferative capacity were observed. Methods: Plated on 6-well plates (Corning) at 2×106 cells/well, the CPCs were incubated with medium (1 ml) containing different adenoviruses (Ad-c-myc-EGFP, Ad-cyclinA2-EGFP, Ad-c-myc-EGFP and Ad-cyclinA2-EGFP, Ad-EGFP), each at the MOI of 150 using standard techniques. Flow cytometry was used for assessing the influence of the cell cycle and the capacity of proliferation. To assessing the capacity of mitosis, the CPCs cells after transfection were incorporated BrdU. Using immunocytochemistry we observed the expression of BrdU. We also observed the change of propagation. Results: Our data from flow cytometry analysis showed that the cell cycle distribution of the CPCs was significantly affected by the co-overexpression of cyclinA2 and c-myc protein. By contrast, those cells were transfected with Ad-EGFP, Ad-cyclinA2-EGFP and Ad-c-myc-EGFP respectively, and no significant differences were observed. The percentage of incorporated BrdU increased in the progenitor cells co-transfected with Ad-c-myc-EGFP and Ad-cyclinA2-EGFP. The capacity of propagation had no obviously change. Conclusions: These results suggested that cyclinA2 and c-myc induced the proliferation of CPCs and reentered into cell cycle together. However, these two genes had no alone significant effects on cell cycle and the capacity of proliferation.6. The mechanism of c-myc and cyclinA2 inducing the CPCs proliferationObjective: In order to examine the target gene expression and explore the underlying molecular mechanism of cyclinA2 and c-myc inducing the CPCs to reenter into the cell cycle, we detected the expression of target gene, cell cycle-related molecules and PCNA. Methods: We used real-time PCR and westen bloting to detect c-myc, cyclinA2, PCNA, CDK2 and P27kip1 mRNA and protein expression between the Ad-EGFP transfection group and the co-transfect c-myc and cyclinA2 group. Results: The expression of c-myc and cylinA2 protein and the mRNA levels in the Ad-c-myc-EGFP and the Ad-cyclinA2-EGFP infected the CPCs also showed obviously increasing trend than that of the in Ad-EGFP infected cells. At the same time, the up-regulation of cyclinA2 and c-myc protein was associated with the exaltation of CDK2 and PCNA but with the reduction of p27Kip1 in mRNA level and protein expression. Conclusions: These results suggested a successful transfection to the progenitor cells and a significant expression of target gene in these cells, including mRNA level and protein levels. The mechanism of cyclinA2 and c-myc gene inducing the CPCs proliferation and reentering into the cell cycle is the classic way through CKI-cyclin-CDK.