Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and POT1a deleted MSPCs Transcriptomes.

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to evaluate POT1a deleted MSPCs functions by using NGS-derived MSPCs transcriptome profiling (RNA-seq) in vitro and vivo. Methods: In vitro, bone marrow cells flushed out from mouse femurs and tibiae were cultured, and adherent cells were collected. CD51+/PDGFRɑ+ cells were sorted and further cultured, and Cre Recombinase Gesicles were applied. After 48 hours culture, tdTomato fluorescently colored cells were re-sorted, cultured for about 4 weeks, and used for gene analyses. In vivo, bone marrow cells weer flushed out from mouse femurs and tibiae, and the flow-through fraction with tdTomato signals was sorted as an MSPC-enriched population. Results: Gene set enrichment analysis of the differentially expressed genes revealed that several pathways related to bone development were significantly affected. Conclusions: POT1a is essential for bone marrow MSPCs to maintain osteo- differentiation potential. In vitro, cultured POT1a deleted MSPCs mRNA profiles. In vivo, sorted bone marrow MSPCs mRNA profiles of NG2-cre / loxp-tdTomato / Pot1awt/fl and NG2-cre / loxp-tdTomato / Pot1afl/fl mice.