Abstract
Background Human bone marrow stromal/stem cells (hMSCs, also known as the skeletal stem cells or mesenchymal stem cells) are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes. However, mechanistic studies employing hMSC have been hampered by the difficulty of deriving genetically modified cell lines due to the low and unstable transfection efficiency. Methods We infected hMSC with a CRISPR/Cas9 lentivirus system, with specific inducible dCas9-coupled transcription activator or repressor: dCas9-KRAB or dCas9-VP64, respectively, and established two hMSC lines (hMSC-CRISPRi and hMSC-CRISPRa) that can inhibit or activate gene expression, respectively. The two cell lines showed similar cell morphology, cell growth kinetics, and similar lineage differentiation potentials as the parental hMSC line. The expression of KRAB-dCas9 or VP64-dCas9 was controlled by the presence or absence of doxycycline (Dox) in the cell culturing medium. To demonstrate the functionality of the dCas9-effector hMSC system, we tested controlled expression of alkaline phosphatase (ALP) gene through transfection with the same single ALP sgRNA. Results In the presence of Dox, the expression of ALP showed 60-90% inhibition in hMSC-CRISPRi while ALP showed more than 20-fold increased expression in hMSC-CRISPRa. As expected, the ALP was functionally active and the cells showed evidence for inhibition or enhancement of in vitro osteoblast differentiation, respectively. Conclusion hMSC-CRISPRi and hMSC-CRISPRa are useful resources to study genes and genetic pathways regulating lineage-specific differentiation of hMSC.
Highlights
Human bone marrow stromal/stem cells are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes
Human bone marrow stromal/stem cells are clonal cells present within the bone marrow stroma and are capable of differentiation into various mesoderm-type lineage cells, e.g., osteoblasts, adipocytes and chondrocytes [1]. hMSC has been extensively employed to study the molecular mechanisms of lineage commitment and differentiation and to identify novel factors regulating differentiation processes [2]
By infecting hMSC with dCas9-VP64 or dCas9-KRAB lentiviral vectors, respectively, we selected and obtained two hMSC lines to be employed for an easy and quick approach for the activation or inhibition of targeted genes by transfecting targeted sgRNA, and we showed that the regulation of gene expression is inducible by addition or removal of doxycycline (Dox) in cell culture medium
Summary
Human bone marrow stromal/stem cells (hMSCs, known as the skeletal stem cells or mesenchymal stem cells) are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes. Stem Cells International and powerful technology to manipulate gene expression It is developed from the bacterial immune system for cleaving foreign DNA [7] and is composed of Cas endonuclease and a target-identifying CRISPR RNA (single guide RNA, sgRNA). The sgRNA targets a 18-25 base pair sequence of target gene and guides Cas to specific DNA sites where it creates a blunt-ended double-stranded break (DSB) within the sequence by its endonuclease activity [8, 9]. This DSB induces the generation of mutations that may cause a frameshift in gene coding sequence [10, 11]. It can supply a repair template with homology to the cut site and facilitates targeted integration of mutation or insertion [12]
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