Identification of the Factor That Leads Human Mesenchymal Stem Cell Lines into Decellularized Bone

Abstract

Hematopoiesis is maintained by the interaction of hematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (MSCs) in bone marrow microenvironments, called niches. Certain genetic mutations in MSCs, not HSCs, provoke some hematopoietic neoplasms, such as myelodysplastic syndrome. An in vivo bone marrow niche model using human MSC cell lines with specific genetic mutations and bone scaffolds is necessary to elucidate these interactions and the disease onset. We focused on decellularized bone (DCB) as a useful bone scaffold and attempted to induce human MSCs (UE7T-9 cells) into the DCB. Using the CRISPR activation library, we identified SHC4 upregulation as a candidate factor, with the SHC4 overexpression in UE7T-9 cells activating their migratory ability and upregulating genes to promote hematopoietic cell migration. This is the first study to apply the CRISPR library to engraft cells into decellularized biomaterials. SHC4 overexpression is essential for engrafting UE7T-9 cells into DCB, and it might be the first step toward creating an in vivo human-mouse hybrid bone marrow niche model.

Keywords: CRISPR SAM activation library; bone marrow niche; decellularized bone marrow; mesenchymal stem cells.

Figure 1

Finding candidate factors of intraosseous progression using CRISPR SAM library. (A) Schematic strategy for finding candidate factors. We introduced the CRISPR SAM library into the cell lines to randomly induce gene expression. We cultured these cells on DCB for 3 weeks and performed DNA sequencing analysis on the cellular components that infiltrated into DCB. (B) HE-stained specimen prepared after 3 weeks of culture. The UE7T-9 cells, expressing the CRISPR-SAM library, infiltrated along the bone trabeculae (black arrow). The bottom figure is a high-power field of the above.

Figure 2

Infiltration into DCB. (A) HE-stained specimen of DCB, after 8-weeks of culture over UE7T-9 cells. UE7T-9 cells remained on the surface of the DCB. (B) HE-stained specimen of DCB after 8-weeks of culture of UE7T-9 SHC4. There were infiltrating cells around the bone trabecular (black arrow). (C) The infiltrating distance measured in UE7T-9 cells (n = 117) and UE7T-9 SHC4 cells (n = 96). The cells were randomly selected for distance measurement. Graph showing the distance that UE7T-9 cells infiltrated from the surface of DCB (red dot line). * indicates a significant difference of p < 0.0001 between the results of the two groups.

Figure 3

Evaluation of migration and proliferation. (A,B) Photographs of HE staining of UE7T-9 cells after 18 h of culture in scratch assay (A: UE7T-9 Cont B: UE7T-9 SHC4). (C) The graph shows the cell-free area of the scratched UE7T-9 cell culture after 18 h of culture, as measured by Image J. The cell-free area of UE7T-9 SHC4 was significantly decreased compared to the control (p < 0.05), indicating enhanced migration due to SHC4 overexpression. (D) Evaluation of proliferative potential by MTS assay. No significant difference in proliferation was observed between the UE7T-9 control and the UE7T-9 SHC4.

Figure 4

Results of RNA sequence analysis of UE7T-9 cell lines with increased SHC4 gene expression. (A) Volcano plot showing the distribution of genes expressed in UE7T-9 SHC4 according to their expression levels when compared to UE7T-9 Cont. The x-axis (log2FoldChange) shows the fold change in gene expression between different samples, and the y-axis (-log10(padj)) shows the statistical significance of the difference. Red dots represent upregulated genes, and green dots represent downregulated genes. (B) Pathway enrichment analysis shows the pathways to which the differential genes are most related, with reference to the Gene Ontology database. (C) Graphical views of the enrichment plot of the GSEA results for GOBP_Positive_Regulation_of_Blood_Vessel_Endothelial_Cell_Migration.