Establishment Of A Cell Line Simultaneously Expressing Three Active Growth Factors And The Experimental Research Of It’s Efect On The Inner Ear Differentiation

Background:Sensorineural hearing loss is the most common sensory defect worldwide. Approximately one per thousand children is born with severe-to-profound sensorineural hearing loss. Hearing impairment severely affects the daily activities of the patients. The treatments available for severe-to-profound sensorineural hearing loss are very limited, mostly depending on hearing aids and electronic cochlear implants. Hearing aids can help patients with moderate deafness. However, only one-fifth patients can benefit from it. For patients with severe-to-profound sensorineural hearing loss, the only effective treatment is surgical electronic cochlear implantation, which directly stimulates the auditory nerve by inserting an electrode in tympanic scala of cochlear to restore the hearing function. So far there have been more than300000patients around the world benefit from the surgery. However, electronic cochlear implantation is an invasive surgery, and the speech recognition of the patients with the implant under noisy environment is still not very satisfactory. Electronic cochlear implant can neither cure the damaged auditory nerve nor help it recover its physiologic functions, instead the electronic cochlear implant is only able to stimulate residual neuron cells of spiral ganglion. Nevertheless, recent investigations on biotherapy for inner ear disease seem to be promising, such as stem cell transplantations, gene therapies, and growth factor administrations.Bone marrow mesenchymal stem cells (MSCs) are a kind of adult stem cells with potential of multiple differentiation and strong renewability. Under the suitable microenvironment, MSCs can be induced to differentiate into various tissue and cell types, such as osteoblast, cartilage cells, fat cells, nerve cells, myocardial cells, vascular endothelial cells and liver cells, etc. MSCs have been widely used in stem cell transplantations and gene therapies because they are easy to obtain and cultivate, and characterized by its weak immunogen during transplantations.Growth factors are humoral cytokins, which can stimulate the growth of specific cells. By binding to its specific receptors on the surface of the cell membrane, growth factors trigger some intracellular signaling pathways and promote growth, proliferation and differentiation of variety of cells. The expression of three growth factors and their receptors can be detected during cell maturation of the inner ear. They are IGF1(Insulin-like Growth Factor1), EGF (Epidermal Growth Factor) and FGF2(Fibroblast Growth Factor2). These factors can protect the hair cells of inner ear from being damaged and promote them proliferation and differentiation.In the research of biotherapy approaches for sensorineural hearing loss, co-culture of stem cells with other cells from specific tissues or adding exogenous growth factors is often used to induce the stem cell to differentiate into the hair cells of inner ear. In an attempt to combine these two strategies for further investigation, we designed a lentiviral vector which could simultaneously express active forms of those three growth factors, and the later was used to establish stable transfected cells in this work, hoping to provide the basis for the biotherapy of sensorineural hearing loss using stem cell transplantation combined with growth factors. 1、Establishment of a cell line expressing three active growth factorsThe coding sequence for active epidermal growth factor (EGF) was cloned by RT-PCR with RNA from human colorectal cancer SW620cells, and the coding sequences for active insulin-like growth factor1(IGF1) and fibroblast growth factor2(FGF2) were artificially synthesized. The coding sequence for the signal peptide of human immunoglobulin (IgK) responsible for protein secretion and sequence for internal ribosome entry site (IRES) responsible for translation initiation were cloned from plasmids pSecTag2A and pIRES2-EGFP, respectively. The coding sequences for Igκ and three growth factors were fused respectively with IRES spacers in between, cloned into a lentiviral expression plasmid with green fluorescent protein (GFP) reporter, pLVX-IRES-ZsGreenl, and packaged into recombinant lentiviruses. HEK293T cells were infected by the lentiviruses and screened for single cell clones with GFP expression by limited dilution and named as HEK293T/3GF. The expression of IGF1, EGF and FGF2were examined by Western blot, and proliferation assay of human lung cancer A549cells was used to evaluate the activity of the secreted growth factors.2. Induction of bone marrow mesenchymal stem cells to differentiation into the inner ear hair cells using co-culture systemTo investate the potential on inducing MSCs to differentiate into the inner ear hair cells by HEK239T/3GF cells, which could simultaneously express three active growth factors, a co-culture system was established. Rat MSCs were separated from whole bone marrow and cultured for four passages for purification and propagation. MSCs were co-cultured with HEK293T/3GF and HEK293T cells, with or without retinoic acid. After21days, the morphologic changes of MSCs were examined by microscope, which showed the nerve cell-like phenotype. RT-PCR results showed the expression of hair cells markers by MSCs after co-culture. These results suggested that MSCs could be induced to differentiate into inner ear hair cells when co-culture with HEK293T/3GF. Conclusion:A lentiviral vector with three active forms of growth factors IGF1, EGF and FGF2was constructed. A HEK293T/3GF cell line was successfully established which could simultaneously express three functionally active growth factors, and showed the potential in inducing differentiation of MSCs when co-cultured with the cells. This work provides a new approach for further investigation on biotherapy for inner ear diseases.