SRRM4 Knockout Helps the Human Mesenchymal Stem Cell Line to Penetrate Decellularized Cancellous Bone.

BACKGROUND: Analyzing the human bone marrow microenvironment requires an in vivo model that reflects the human bone marrow microenvironment. Introducing a human bone marrow mesenchymal stem cell (MSC) line into decellularized cancellous bone (DCB) is a first step in forming such a bone marrow model. Our goal with this research is identifying factors that promote the penetration of MSCs into DCBs in an ex vivo setting. METHODS: We introduced the CRISPR Knock Out (GeCKO v2) library to identify candidate genes in UE7T-9 cell line (MSC line) for DCB penetration. We established a candidate gene-knockout UE7T-9 cell for validation and evaluated its penetration into DCB (measured distance of randomly selected 100 cells), proliferation (MTS assay), migration (scratch assay), and ancorage-independent growth (soft agar assay). RNA sequencing was performed to analyze changes in gene expression comprehensively. RESULTS: We identified Serine/Arginine Repetitive Matrix 4 (SRRM4) knockout (KO) in the UE7T-9 cell as a candidate factor for bone penetration. SRRM4 KO promoted DCB penetration (3.1-7.1 times deeper, each p ≤ 1.91 × 10-24), cell migration (p = 0.039), and ancorage-independent growth (2.5 times in colony count, 7.1 times in colony size, each p = 0.001) but retained stem cell characteristics. CONCLUSIONS: SRRM4 KO is a newly defined factor of UE7T-9 cell penetrating into DCB. SRRM4 KO UE7T-9 cells may be used to analyze hematological diseases such as myelodysplastic neoplasms.