Molecular remodelling of gene expression profile during differentiation of equine bone marrow-derived MSCs towards adipocytes and chondrocytes

Background - The mesenchymal stem cells (MSCs) have a unique self-renewal and developmental potential resulting from their multipotency – the ability to differentiate into particular cell types. It has been confirmed that MSCs have a huge potential and outstanding therapeutic effects in variety of clinical treatments due to the their abilities to differentiate towards ectodermal, endodermal, and mesodermal cell lines. Objectives - the project aims to identify changes in MSCs transcriptome profile during the process of losing multipotency and acquiring the ability to differentiate cells into adipocytes (ADM) and chondrocytes (CDM) equine cell lines. Methods - Bone marrow-derived MSCs were differentiated towards adipocytes and chondrocytes using different procedures. The specificity of obtained cell lines was checked using qPCR, cytometry, and staining approaches. The transcriptome analysis was performed using NGS transcript sequencing on the Illumina HiSeq 3000 platform. Results - The differential expression analysis allowed us to identify the 2325 DEGs between MSC and ADM groups, while 803 DEGs were detected between MCS and CDM cell lines. According to the comparison of both DEG sets the expression of 320 genes was significantly modified despite the direction of differentiation. The direct related to the transformation of MSCs into adipocytes seems to be fat cell differentiation represented by 47 DEGs and included additional GOs as regulation of fat cell differentiation, regulation of cell differentiation, brown fat cell differentiation, and adipocyte fat development. The results indicated the top DEGs involved in differentiation into adipocytes and previously established as related to adipogenesis: down-regulated - MEOX1, CYYR1, and up-regulated – CYP24A1; LEP; TBX5; FABP4 and GPER1. The obtained data indicated molecular mechanism altered during equine cell remodelling towards chondrocyte differentiation. One of identified GOs was ossification process represented by 35 DEGs with the highest up regulation of two genes was observed - IHH (6.46) and PHOSPHO1 (5.46) involved in chondrocyte proliferation and matrix mineralisation; respectively. The highest down regulation was detected for BMPR1B gene, critical for bone and cartilage formation (-5.98) and together with BMP2 and BMP4 (2.26 and 2.48; respectively) triggered BMP signaling. Conclusions - The obtained picture of transcriptome differences between primary donor cells and reprogrammed cell lines revealed a range of transcripts important for molecular alterations resulting from substantial phenotypic change of reprogramed cells under special ex-vivo procedures. The study showed the effects of the radical change of cell fate decisions on the molecular level and reinforced further work on efficient procedures to produce MSC-derived reprogrammed cells that have the potential for regenerative medicine of hard connective tissue in horses. Bone marrow-derived MSCs were differentiated towards adipocytes and chondrocytes using different procedures. The specificity of obtained cell lines was checked using qPCR, cytometry, and staining approaches. RNA was isolated from differentiated lines (n=3 per each line ADM and CDM) and control MSCs line (n=3) the using PureLink™ RNA Mini Kit (Ambion) with DNAse digestion between washing. Its quality and quantity were measured on a NanoDrop2000 and a TapeStation 2200 system (RNA tapes, Agilent). Acceptable samples had an RIN over 9.5. According to the manufacturer's protocol, cDNA libraries cells were prepared using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina from 300 ng aliquots of purified RNA samples. The final cDNA library concentration was 10 nM. The quality and quantity of cDNA libraries was assessment using D1000 tapes on TapeStation2200 and on Quibit. The cDNA were sequenced over 150PE cycles on a HiSeq 3000 System (Illumina, San Diego, CA, USA) in outdoor facilities (Admera Health Biopharma Services). The qPCR method and Pearson correlation were applied to validate the RNA-seq results.