US 12,324,817 B2
Method of inducing dedifferentiation of somatic cells with small molecules to prepare rejuvenated mesenchymal stem cells and uses thereof
Min Hu, Yunnan (CN); Yanjiao Li, Yunnan (CN); and Junyuan Hu, Guangdong (CN)
Assigned to SHENZHEN ALPHA BIOPHARMACEUTICAL CO. LTD., Shenzhen (CN)
Filed by SHENZHEN ALPHA BIOPHARMACEUTICAL CO. LTD., Guangdong (CN)
Filed on Oct. 31, 2020, as Appl. No. 17/086,397.
Application 17/086,397 is a continuation of application No. PCT/CN2019/085401, filed on May 1, 2019.
Claims priority of application No. 201810407290.X (CN), filed on May 1, 2018.
Prior Publication US 2021/0213069 A1, Jul. 15, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. A61K 35/33 (2015.01); C12N 5/077 (2010.01)
CPC A61K 35/33 (2013.01) [C12N 5/0656 (2013.01); C12N 2501/01 (2013.01); C12N 2501/03 (2013.01); C12N 2501/155 (2013.01); C12N 2501/165 (2013.01); C12N 2501/21 (2013.01); C12N 2501/2302 (2013.01); C12N 2501/2306 (2013.01); C12N 2501/24 (2013.01); C12N 2501/25 (2013.01); C12N 2501/999 (2013.01)] 12 Claims
 
1. A method of preparing rejuvenated and regenerative fibroblasts, comprising:
treating fibroblasts sequentially with a first composition and a second composition;
wherein the first composition comprises high glucose DMEM (HG-DMEM) supplemented with 10% fetal bovine serum (FBS) and a first small molecule mixture consisting of WNT/β-catenin agonist CHIR99021, histone deacetylase (HDAC) inhibitor valproic acid (VPA), cyclic adenosine monophosphate (cAMP) agonist Forskolin, and transforming growth factor (TGF)-β inhibitor Repsox; and
the second composition comprises HG-DMEM supplemented with 10% FBS and a second small molecule mixture consisting of HDAC inhibitor VPA, TGF-β inhibitor Repsox, WNT/β-catenin agonist CHIR99021, cAMP agonist Forskolin, retinoic acid receptor (RAR) agonists AM580 and TTNPB, histone methyltransferase (HMT) inhibitor EPZ004777, ascorbate, protein kinase C (PKC) inhibitor Go 6983, Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632, and c-Jun N-terminal kinase (JNK) inhibitor SP600125;
wherein the fibroblasts are treated in the first composition for 5 days, and the fibroblasts are treated in the second composition for 7 days.