Primary human bone marrow stromal cells (BMSCs) have been considered as an ideal source for the use in regenerative medicine. However,  studying these cells in vitro is challenging due to inter donor variations, their limited expansion capacity and the induction of cellular senescence. Although gene knockdown studies using siRNA/shRNA have been applied successfully, it may result in residual gene activity with heterogeneous effects between different knockdown constructs and individual cells. The aim of the current study is to generate spCas9 expressing BMSCs that can be used to efficiently generate genomic gene disruptions and applied in functional studies of osteoblast differentiation.

Immortalized BMSCs (hMSC-TERTs) were transduced with a lentiviral construct expressing spCas9 in the absence of gRNAs.  hMSC-TERTs with high spCas9 expression were selected by puromycin. For the generation of gene disruptions, hMSC-TERTsCas9 were transiently transfected with 1 or more sgRNA constructs that target the genomic region of interest. Cells are single cell sorted, clonally expanded and gene disruptions were analysed by Sanger sequencing.

We have applied hMSC-TERTsCas9 to knockout several transcription factors and a cell surface receptor by generating indels within the codon region resulting in a premature stopcodon. Even larger genomic deletion (200bp) can be established by co-transfection of 2 different sgRNA constructs. Complete removal of the vitamin D receptor illustrated that these cells do not respond anymore to vitamin D and were unable to differentiate into mineralizing osteoblasts. Additionally, homo- and heterozygous gene disruptions have been established for FGFR2 and indicated that the presence of 2 functionally intact alleles are essential for normal osteoblast differentiation in hMSC-TERTsCas9.