2019 Fiscal Year Research-status Report
Mechanistic basis of new generation laminin (NGL) for developing paraxial mesoderm cell lineages from hiPSCs
Project/Area Number |
19K20671
|
Research Institution | Kyoto University |
Principal Investigator |
Zhao Mingming 京都大学, iPS細胞研究所, 特定研究員 (50754206)
|
Project Period (FY) |
2019-04-01 – 2021-03-31
|
Keywords | 再生医学 / 次世代ラミニン / iPS細胞 / 骨格筋幹細胞 |
Outline of Annual Research Achievements |
Not only in 201B7 cell line, NGL increased myogenic progenitors and muscle stem cells population in two Myf5-tdtomato reproter lines. NGL increased myogenic differentiation in both of duchenne muscular dystrophy and Miyoshi myopathy patients derived hiPSCs. The bFGF in hiPSCs culture medium, bound to NGL, then stimulated hiPSCs differentiation.The experiments of dCas9 Knocking down and chemical blocked integrin pathway indicated that bFGF did not transduce signaling through bFGF-integrin pathways. The down stream of FGFR pathway was remarkably stimulated by NGL.
|
Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Using different cell lines, we confirmed NGL are widely used in hiPSCs differentiation, that is useful for disease modeling and cell therapy. After blocking the effect of integrin by dCas9 and integrin inhibitor, we confirm that the integrin did not transduce signaling in NGL, that indicating the effect of bFGF-FGFR in NGL regulating hiPSCs differentiation. Furthermore, the bFGF-FGFR inhibitor and the downstream of FGFR inhibitor remarkably decreased the NGL's effects, also indicating the dominative effect of bFGF-FGFR in NGL regulating differentiation.
|
Strategy for Future Research Activity |
Depend on the results we already received, we will continue to elucidate the mechanism of HS in NGL regulating FGFR signaling pathway. To confirm the function of bFGF-FGFR in our system, FGFR binding mutation bFGF and heparan sulfate binding mutation bFGF will be used in our culture system. RNA-seq experiment will be performed to elucidate the pathway of NGL regulation myogenic differentiation. Following our initial strategy, we will continue to establish the strategy to purify iPSCs derived myocytes and muscle stem cells, then confirm the characterization of myocytes and muscle stem cells in vitro and in vivo.
|
Causes of Carryover |
Incurring amount will be used for the reagent for cell culture, RNA-seq, antibody and other experiment consumables which will be acquired, and for domestic and international conference expenses. The reagents and consumables which will be acquired for this project will be used in accordance with our stipulated research plan in a timely and efficient manner.
|
Research Products
(3 results)