Committing Bone Mesenchymal Stem Cells To Differentiate Into Thyroid Follicular Epithelial Cells

In recent years,there is a dramatical increase in the incidence of thyroid cancer, and thyroid total or subtotal surgery easily lead to permanent hypothyroidism.How to help these patients to survive without using thyroid hormone replacement therapy lifelong has become a serious problem.With the development of stem cell research,the world in the field of stem cell therapy after a large number of exploration made a series of important achievements, and some research has been well applied to clinical trials,which provides a new idea for the treatment of thyroid diseases.Bone mesenchymal stem cells (BMSC) strongly proliferate and differentiate, and their ability of autotransfusion can avoid immune rejection.These merits can explane the situation that BMSCs have a extensive application prospects in regenerative medicine seed cells. Studies in vitro have shown that, BMSCs owning multipotent differentiation potential, can differentiate into cells of the three germ layers under specific induction conditions.In this study, under different culture conditions in vitro,we induce BMSCs to differentiate into thyroid follicular cells, to explore what is their role in the progress of thyroid follicular cells’differentiation. The study of stem cell-derived thyroid follicular cells may provide a new experimental model and explore the key factors affecting BMSCs’ differentiation towards thyroid follicular cells on the basis of experiment. On the basis of induction of differentiation, based on the BMSCs derived thyroid cells grown in three-dimensional scaffolds, explore the feasibility of thyroid tissue in vitro constructed to transplant transplant by means of post- implantation rat model of hypothyroidism, thyroid detected possible effect of hypothyroidism.Methods:(1) To isolate, purify, and subculture BMSCs. Detect the expression of P3 BMSCs surface markers CD44, CD90 and CD34 with flow cytometry,and Examine its osteogenic and adipogenic differentiation differentiation.(2) The cryopreservation, anabiosis and cultivation of FRTL-5(Fisher Rat Thyroid Cell Line, rat thyroid cell line).Immunofluorescence identified these cells by detecting the expression of transcription factor TTF1 and PAX8 and thyroid -specific proteins NIS, TPO and Tg.(3) Different culture conditions were experimental groups. C+F group (co-culture+inducing factor group):The BMSCs indirect co-cultured with FRTL-5 through transwells chamber, adding medium containing inducible factors, including TSH, insulin, transferrin, somatostatin and hydrocortisone; C group (co-culture group):the BMSCs and FRTL-5 indirect co-culture through transwells chamber indirect contact,adding inducible factors-free medium; F group (inducible factors-group):the BMSCs directly exposed to a medium containing inducible factor s, including TSH, insulin, transferrin, somatostatin and hydrocortisone; Negative control group:BMSCs; Positive control group:FRTL-5.(4)BMSCs of each experimental group were observed:changes of induced cell morphology were recorded by the inverted microscope; in molecular level,the expression of thyroid-specific markers were dete ct ed by cell immunostaining; RT-PCR analysis from the genetic level to detect the thyroid cell-related genes; The secretory function of induced cells were identified by ECLIA(electrochemilu minescene immunoassay).(5) Using the BMSCs-derived thyroid follicular cells as seed cells,we exploring the suitable growth conditions in three-dimensional collagen sponge scaffold.Result:1.. The different conditions,under which the third generation BMSCs were inducted one week, provide discriminating results.(1) Immunofluorescence analysis showed that the expression of TTF1, PAX8, NIS, TPO and Tg was different among each experimental group, and the effect of co-culture+ inducible factor-group was more significant;(2) RT-PCR analysis of the experimental groups was detected at different levels of TTF1, PAX8, TSHR, NIS, Tg and TPO gene, and for Tg gene level,the co-culture+ inducible factor-group is best;(3) by the ECLIA assay the amounts of T3 and T4,co-culture+inducible factor group was found super in all experimental groups.2、Transplant the co-culture+inducible factor-group’s cells into collagen sponge scaffold.We found collagen sponge was favor to the growth of these cells,and when the seeding density is 106/ml, the incubation time in two weeks is appropriate.Conclusion:In vitro co-culture system, BMSCs indirect contacted with FRTL-5, adding inducible factors including TSH, insulin, transferrin, somatostatin and hydrocortisone, is more conducive to induce BMSCs derived thyroid follicular cells.Three-dimensional collagen sponge scaffold is suitable for the growth of these cells.