Single cell dissection of Chemical Reprogramming from Fibroblasts into functional Chondrocytes

We used single cell analysis to systematically dissect the sequential changes of cellular phenotypes during fibroblast-chondrocyte direct reprograming.1401 single-cell transcriptomes were measured in total (232 MEFs of day0, 577 chemical-induced intermediate cells (ci-ICs) of day6, 311 ci-chons of day 34, 281 mouse primary chondrocytes (mchons) as positive control). We found that ci-chons and mchons shared similar subpopulation with specifically expression of cartilage-related markers. The findings also indicated that in the early induction, fibroblasts to lose their original features, and transit into a cartilage progenitor intermediate state. Single cells (MEFs, ci-ICs, ci-chons and primary mouse chondocytes) were captured using FluidigmTM C1 high-throughput IFC. Therefore, the sorted single-cell suspension was loaded on a microfluidic RNA-seq chip. We checked the cells by a microscope for captured cell number confirmation. Then cell lysis, reverse transcription and cDNA pre-amplification were performed on the chip according to Fluidigm’s standard protocol. Illumina libraries were Illumina Nextera XT DNA Sample Preparation kit according to the protocol supplied by Fluidigm. Cell libraries were pooled and sequenced 150 bp paired-end on one lane of Illumina HiSeq xten.