Generation of human mesenchymal stem cells lacking diabetoporosity-associated miRNAs using CRISPR/Cas9

Introduction “Diabetoporosity” is a new term for osteoporosis in patients with diabetes mellitus, where classical tools for the assessment of bone properties like dual x-ray absorptiometry or bone turnover markers are lacking prognostic accuracy for the determination of bone quality and fracture risk. MicroRNAs (miRNAs) are getting into the focus of research not only for their importance in the development of osteogenic lineage cells, but also as biomarkers for bone related diseases. Differentially expressed miRNA were revealed by sequencing of serum samples from elderly diabetic patients with prospective fractures, compared to control serum samples. To examine the function of these microRNAs during differentiation of osteoblasts, we have generated immortalized human mesenchymal stem cells lacking microRNAs associated with diabetic fractures.

Methods MiRNA sequencing was performed in serum samples of patients with type 2 DM (T2DM) who developed fractures within two years of follow-up (n = 6) and compared to samples of T2DM patients without fractures (n = 10). Immortalized human bone marrow derived mesenchymal stem cells expressing spCas9 (hMSC-TERTCas9) were used to delete identified miRNAs. Guide RNAs were designed and cloned into the plasmid phU6 and transfected into MSC-TERTcas9 cells. Individual cells lacking miRNAs were clonally expanded and confirmation done by PCR and sanger-sequencing.

Results miRNA sequencing analysis of serum samples revealed 16 miRNAs (FDR < 0.05) that were correlated with prospective fractures in elderly diabetic patients. MiRNAs with average relative sequencing counts between 100 and 10000 were selected for further analysis. Eight of these were expressed in hMSC-TERTs, and human primary bone marrow derived mesenchymal stem cells (hMSCs) and considered for deletion and functional analyses. We have successfully generated three hMSC lines lacking miRNAs associated with diabetic fractures, namely miR140-KO, miR25/93/106b KO and miR-363/19b-KO.

Discussion The creation of hMSC lines lacking our target miRNAs is the first step in the examination and importance during osteoblast differentiation of hMSCs. Further functional experiments may reveal the role of these miRNAs in the context of osteogenic differentiation.

Keywords diabetoporosity, miRNAs, hMSCs, CRISPR/Cas9

Korrespondenzadresse Ines Foessl, Medizinische Universität Graz, Universitätsklinik für Innere Medizin, Klinische Abteilung für Endokrinologie und Diabetologie, Auenbruggerplatz 15, 8036 Graz, Österreich, Germany

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