Background: MEIS1 is an important transcriptional regulator in hematopoietic development. Previous studies illustrated MEIS1 was silenced by DNA methylation during differentiation of hematopoietic progenitors, suggesting a critical role of MEIS1 in the maintenance of the undifferentiated state of HSCs. Our previous studies have indicated that the expression of MEIS1 was upregulated upon osteogenic and adipogenic differentiation of human BMSCs.

Objective: The objective is to investigate the role of MEIS1 in osteogenic differentiation and ECM mineralization of hMSC-TERTs.

Methods: Two sgRNAs were designed for MEIS1 exon 6 and transfected into Cas9 expressing hMSC-TERTs (hMSC-TERTs^Cas9). Cells were single cell sorted and clonally expanded. Subsequently, cells were differentiated into osteoblast and extracellular matrix (ECM) mineralization was determined using Alizarin Red Staining at day 21. Alkaline phosphatase activity (ALP), DNA, protein and calcium were measured at 7, 14, and 21 days after osteogenic induction.

Results: Using CRISPR-Cas9 we generated a homo- (MEIS1^-/-) and heterozygous (MEIS1^+/-) disruption of MEIS1. The ALP activity in extracts from MEIS1^-/- osteoblast differentiating cells was significantly increased (1.2-1.5 fold) at each time point compared to MEIS1^+/+ and MEIS1^+/- hMSC-TERTs^Cas9. Moreover, Alizarin Red staining of the mineralized matrix and Ca²⁺ measurement in de media illustrated that the ECM mineralization in MEIS1^-/- cells was enhanced during osteoblast mineralization. Whereas heterozygous disruption of MEIS1 was similar compared to the MEIS1^+/+ hMSC-TERTs^Cas9.

Conclusion: Our findings indicate that the homeodomain containing transcription factor MEIS1 is involved in osteoblast differentiation. Further research will be necessary to elucidate the molecular function of MEIS1 in differentiating BMSCs.