chir-99021 and 4-(2-(5-6-7-8-tetrahydro-5-5-8-8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic-acid

A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5-9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and β-catenin association with DNA showed that retinoic acid and Wnt/β-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation.

Topics: Animals; Benzoates; beta Catenin; Cartilage; Cell Differentiation; Chondrocytes; Chondrogenesis; Chromatin; Collagen Type X; Gene Expression; Humans; Mice; Mice, Inbred NOD; Pluripotent Stem Cells; Pyridines; Pyrimidines; Receptors, Retinoic Acid; Retinoids; Wnt Signaling Pathway

The direct conversion, or transdifferentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provides promising approaches for cardiac regeneration. However, genetic manipulations raise safety concerns and are thus not desirable in most clinical applications. The discovery of full chemically induced pluripotent stem cells suggest the possibility of replacing transcription factors with chemical cocktails. Here, we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts using only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage. Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.

Topics: Actinin; Animals; Benzoates; Calcium; Cell Transdifferentiation; Cells, Cultured; Cellular Reprogramming; Colforsin; Electrophysiological Phenomena; Fibroblasts; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Patch-Clamp Techniques; Pyrazoles; Pyridines; Pyrimidines; Retinoids; Transcriptome; Tranylcypromine; Troponin I; Troponin T