Enhanced Osteogenic Potential Of Mouse Embryonic Stem Cells By Targeted Disruption of Sclerostin with Crispr-Cas9

Pluripotent stem cells are widely used in preclinical studies of bone regeneration due to their unique biological characteristics, diverse functions, and potential therapeutic value. With the discovery of genes important for repairing damaged tissues, pluripotent stem cells modified by these genes have increased therapeutic potential. In this study, we generated a homozygous sclerostin knockout mouse embryonic stem cell line, SOST-KO, using the CRISPR-Cas9 genome editing method. We observed that in SOST-KO cells, there were increased mineral nodule formation and elevated mRNA levels of the early osteogenic marker RUNX2 and the late osteogenic markers osteocalcin (OCN) and integrin binding sialoprotein (BSP). The osteogenic cells derived from SOST-KO were then combined with HA/TCP scaffolds and transplanted into subcutaneous tissue of nude mice. Six weeks post-surgery, radiological and histological analysis showed sclerostin knockout effectively promoted the deposition of mature bone tissue. Moreover, we used Sleeping Beauty transposon to obtain a mouse embryonic stem cell line SOST-KOGFP that stably expresses green fluorescent protein. Six weeks after the tracked cell-scaffold constructs were implanted into the subcutaneous tissue of nude mice, GFP labeled seed cells were detected on the implanted scaffold, indicating that the implants survived and successfully implanted into the mouse tissue. Our results demonstrate that sclerostin knockout facilitates the differentiation of embryonic stem cells towards osteoblast-lineage cells and promotes ectopic bone formation, providing a strategy for the generation of higher quality transplants for bone regeneration.