The Isolation, Cultivation And Identification Of Laryngeal Mucosa Mesenchymal Stem Cells

Lesions of vocal fold usually can be treated by laser under the microscope in current clinical treatment. However, the operation also makes the lamina propria of vocal fold missing and scars formation which affects patients with their pronunciation in the end. At the moment, the main MSC for preventative treatment of vocal scar include: bone marrow mesenchymal stem cells (BMSCs); muscle-derived stem cells (MDSCs); adipose-derived stem cells (ASCs). But so far, the most suitable seed cells still cannot be found. The main difficulty exists which is the long-term results are not ideal in vivo because the planted cells are not able to form the original structure.In recent years, with the development of the stem cell research, people found that different organizations existed extensively mesenchymal stem cells, and they had participated in the formation and development of different tissues.We tried to look for a new kind of MSC in the laryngeal mesenchymal organization, hoping to use its' characteristics derived from laryngeal tissue to reconstruct laryngeal original structure.1 ObjectiveTo explore laryngeal mucosa and vocal fold lamina propria whether there are mesenchymal stem cells and to carry on the separation, cultivation and identification for laryngeal organization's repair to provide new seed cells.2 Methods2.1 Isolation,cultivation and identification of laryngeal mucosa mesenchymal stem cellsVocal fold mesenchymal stem cells: Vocal fold mesenchymal stem cells (VF-MSC) were isolated from patients undergone laryngocarcinoma surgery by tissue culture method. Flow cytometry was performed for the expression of the cell surface marker profiles. Multiple differentiation potentials were confirmed by adipogenic and osteogenic lineages induction.Epiglottis mucosa mesenchymal stem cells: Epiglottis mucosa mesenchymal stem cells (EP-MSC) were isolated from patients undergone laryngocarcinoma surgery by digestive method. Flow cytometry was performed for the expression of the cell surface marker profiles. Multiple differentiation potentials were confirmed by adipogenic, osteogenic and neural lineages induction.2.2 Compare hVF-MSC with hEP-MSCThe cell growth curves of hVF-MSC and hEP-MSC were evaluated by MTT assay. Colony Forming Cell Assays (CFC) were used to evaluate clone formation ability. hVF-MSC and hEP-MSC were cultured by medium containing with VitC. The cell sheets were ripped by microscopic tweezers after 2 weeks to continue to culture for round states in petri dishes. The round states were transplanted into the nude mouse subcutaneous for 2 weeks and then were taken out to observe survival and growth of cells by H&E and immunohistochemical staining.2.3 Characteristics of hEP-MSC in the condition of inflammationIL-1βand TNF-αwere added in DMEM/F12 medium as inflammation medium to simulate inflammation microenvironment in vitro. There were two groups in the Experiment. One group adopted inflammatory medium as test group and another group used normal medium as control. The main contents including:①The influence of acute inflammation on hEP-MSC proliferation by using MTT assay was studied.②The apoptosis and cell-cycle were determined by flow cytometric analysis in the 7th inflammatory microenvironment.③The change of collagen gene in inflammatory conditions in different time points was studied by using qPCR test.3 Results3.1 Isolation,cultivation and identification of laryngeal mucosa mesenchymal stem cellsVocal fold mesenchymal stem cells: By tissue culture method we had successfully obtained hVF-MSC from patients undergone laryngocarcinoma surgery. Flow cytometry analysis revealed that the hVF-MSC were positive for CD29(57.3%), CD44(99.1%), CD90(90-97%), CD105(26%), CD146(4.8%),but negative for CD34(0.9%) and CD45(0.4%). hVF-MSC undergone adipogenic, osteogenic lineages induction were positive for oil red staining and alizarin red staining respectively.Epiglottis mucosa mesenchymal stem cells: By digestion culture method we had successfully obtained hEP-MSC from patients undergone laryngocarcinoma surgery. Flow cytometry analysis revealed that the hEP-MSC were positive for CD29(16.9%), CD44(97.4%), CD90(89.5%), CD105(85.9%), CD146(2.5%) and Stro-1(20.4%) but negative for CD34 and CD45. hEP-MSC undergone adipogenic, osteogenic and neural lineages induction were positive for oil red staining, alizarin red staining and S100 staining respectively. hEP-MSC showed high clone formation and proliferation capacity.3.2 Compare hVF-MSC with hEP-MSCMTT assay and colony forming cell assay showed that hEP-MSC and hVF-MSC had a relatively rapid proliferation capacity and a relatively high clone formation capacity (clone formation rate 7.1% and 8.2%). The cell sheets of hEP-MSC and hVF-MSC were cultured by medium containing with VitC. Animals were sacrificed for H&E staining and immunohistochemical analysis at 2 weeks (4 animals) after implantation. There were no evident inflammatory infiltrates in the H&E staining. Vimentin presented a positive staining indicated that the hEP-MSC could be survival in a long time.3.3 Characteristics of hEP-MSC in the condition of inflammationThe influence of acute inflammation on hEP-MSC proliferation by using MTT assay was studied. Comparison of the proliferation rates of hEP-MSC in the inflammation condition or not in 5 days, different times of optical values was drawn into the growth curve. The proliferation of normal group was slower than inflammation group. The differences in proliferation rates of both were statistically signi?cant (p<0.05). Histograms of flow cytometry at day 7 revealed a typical distribution of three phases. A lower percentage of cells in S(29.0%) and G2M(1.45%) phases and a higher percentage in G0G1 phase (69.55%) were detected in induced cells compared with untreated cells (S = 29.4%, G2M = 7.52%, G0G1 = 63.08%), suggesting that part of the induced cells shifted from G0G1 phase to G2M or S phases. In the apoptosis inflammation factors induced significant cell apoptosis. The apoptosis rate of induced group was higher than normal group.The hEP-MSC was induced for 4 time points (6h, 12h, 24h, 48h) as test group and normal group as control. Gene expression of COL-1 was declined in all time points. Gene expression of COL-3 was declined in 3 time points (6h, 12h, 24h) and the 48h was risen obviously(P < 0.05).4 Conclusions4.1 This study successfully obtained MSC from epiglottis mucosa and vocal fold. They were isolated from patients undergone laryngocarcinoma surgery by digestion culture method and tissue culture method. Multiple differentiation potentials were confirmed by adipogenic and osteogenic lineages induction.4.2 MTT assay and colony forming cell assay showed that hVF-MSC had a relatively rapid proliferation capacity and a relatively high clone formation capacity than hEP-MSC. The results of naked mouse trial showed that hEP-MSC and hVF-MSC could be used in trail in vivo, and the cells could be normal growth and differentiation for later large animal tests providing evidence.4.3 It was found that the normal mesenchymal stem cells showed a lower proliferation rate than the inflammatory mesenchymal stem cells in 5 days. The change of collagen gene in inflammatory conditions in different time points was studied by using RT-PCR test. Through the analysis of the results, we found the expression of collagen can be used to explained the reasons of scar formation.