Generation of PTEN knockout bone marrow mesenchymal stem cell lines by CRISPR/Cas9-mediated genome editing

Abstract

The tumor suppressor PTEN is involved in the regulation of cell proliferation, lineage determination, motility, adhesion and apoptosis. Loss of PTEN in the bone mesenchymal stem cells (BMSCs) was shown to change their function in the repair tissue. So far, the CRISPR/Cas9 system has been proven extremely simple and flexible. Using this system to manipulate PTEN gene editing could produce the PTEN-Knocking-out (PTEN-KO) strain. We knocked out PTEN in MSCs and validated the expression by PCR and Western blot. To clarify the changes in proliferation, CCK-8 assay was applied. In support, living cell proportion was assessed by Trypan blue staining. For osteogenic and adipogenic induction, cells were cultured in different media for 2 weeks. Oil red staining and alizarin red staining were performed for assessment of osteogenic or adipogenic differentiation. The expression of Id4, Runx2, ALP and PPARγ was examined by qPCR and immunocytochemistry staining. The PTEN-KO strain was identified by sequencing. The PTEN-KO cells had an increased cell viability and higher survival compared with the wild type. However, decreased expression of Runx2 and PPARγ was found in the PTEN loss strain after induction, and consistently decreased osteogenic or adipogenic differentiation was observed by alizarin and oil red staining. Together, PTEN-KO strain showed an increased proliferation capability but decreased multi-directional differentiation potential. When BMSCs serve as seed cells for tissue engineering, the PTEN gene may be used as an indicator.

Keywords: Adipogenic differentiation; Bone mesenchymal stem cells; CRISPR/Cas9; Osteogenic differentiation; PTEN.

Fig. 1

PTEN-KO validation of the MSCs. a The designed target site exon region. b The map of empty vector (from Addgene, Massachusetts, USA). c T7E1 digestion products were analyzed by agarose gel electrophoresis. The pSpCas9(BB)-2A-GFP-T2 (px458-T2) plasmid showed the highest Knock-out performance. d DNA was extracted from plasmid-transfected BMSCs and amplified through PCR. The PTEN bands did not exist after transfection e Western blot confirmed PTEN was knocked out

Fig. 2

PTEN-KO BMSCs have an increased proliferation and decreased multipotent capability. a CCK-8 assay indicated an increased viable cell number compared to the WT (day 0 = 100% for each group). b Colony formation assay. The identical seeding number of PTEN-KO cells formed much more clonies than WT. c Alizarin red staining. d Oil red staining. e PTEN-KO BMSCs showed a decreased Runx2 mRNA level (normalized by the WT level). f PTEN-KO BMSCs showed a decreased PPARγ mRNA level (normalized by the WT level). g PTEN-KO BMSCs showed a decreased Runx2 protein level assessed by Western blot. h PTEN-KO BMSCs showed a decreased PPARγ protein level assessed by Western blot. i PTEN-KO BMSCs showed a decreased Runx2 protein level assessed by immunochemistry. j PTEN-KO BMSCs showed a decreased PPARγ protein level assessed by immunochemistry. *P < 0.05 versus WT, **P < 0.01 versus WT