Establishment and functional characterization of gene activation in human mesenchymal stromal cells using the CRISPR/Cas9 synergistic activation mediator
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Abstract
Mesenchymal stromal cells (MSC) possess the combined abilities for chemotactic tumor homing and immunomodulation, which makes them highly interesting for clinical applications. Beyond that, genetic engineering of MSC has the potential to expand the scope of their possible applications.
Hence, the objective of this study was to establish a procedure for stable gene activation in primary human MSC. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) synergistic activation mediator (SAM) was chosen. This technique enables permanent induction of target gene expression and was already applied for engineering of other human stem cells. First, the SAM system was to be generally established in primary MSC by its application for activation of a gene that is naturally inducible in MSC. Moreover, it was to be tested whether SAM also enables the activation of a target gene that is presumably less susceptible for activation in MSC.
For the development of a protocol specifically for transduction and genetic modification of primary MSC, various established approaches and supplements were tested. Some of these were found to be suitable for MSC, while others were not. In the eventually established process, the transduction was done in serum-free medium with application of RetroNectin for plate coating and Polybrene as a transduction enhancer. Medium containing viral particles was concentrated and used directly for transduction of target cells (instead of frozen storage). Antibiotic selection appeared to be suboptimal for primary MSC. As the components of the SAM system are encoded in three individual plasmids, the transduction process was divided into two steps to reduce cell stress and to increase transduction efficiency.
For protocol establishment, CEBPA, the gene encoding the transcription factor CCAAT/enhancer-binding protein alpha, was chosen as an initial target for SAM-mediated activation. It is naturally induced during adipogenic differentiation of MSC and was therefore suspected to be rather susceptible for activation. Indeed, CEBPA was clearly activated in control cell lines and primary MSC, demonstrating the functionality of the established protocol.
The subsequent target for activation was the class II transactivator (CIITA) gene, encoding a regulator of human leukocyte antigen (HLA) class II transcription. It was chosen primarily to analyze whether the SAM system enables the activation of a presumably less susceptible target gene that is not naturally expressed by MSC without extrinsic stimulation. Besides, as CIITA regulates the expression of the DR isotype of HLA (HLA‑DR), amongst others, an activation of CIITA appeared to be interesting with regard to potential effects on the immunomodulatory capacity of MSC.
Initial experiments showed that the expression of CIITA as well as its downstream target gene HLA‑DR was inducible in primary MSC and a control cell line by addition of interferon gamma (IFN‑γ). Besides, IFN‑γ activated expression of HLA‑DR protein on the cell surface of MSC and reduced their capacity for T‑cell suppression, which was demonstrated via co-cultivation with peripheral blood mononuclear cells. According to a chromatin immunoprecipitation analysis of the four promoters of CIITA, promoter IV appeared to be the main IFN‑γ-inducible promoter but with a conceivable additional activity of promoter III in human MSC.
CIITA promoter III and promoter IV were consequently chosen as targets for SAM-mediated gene activation. When targeting promoter III, gene expression was induced in the control cell line, proving the general functionality of the system and the guide RNAs applied. In MSC, however, CIITA expression could not be activated, neither by addition of epigenetic inhibitors for improved promoter accessibility nor by using an alternative system for cellular delivery of the SAM components.
In contrast, when using the SAM system with guide RNAs targeting CIITA promoter IV, gene activation was achieved not only in the control cell line and an immortalized MSC line but also in primary MSC. Increased expression of CIITA and HLA‑DR was verified on mRNA as well as on protein level in all cell types. Notably, maximum levels of target gene expression observed were comparable in primary and immortalized MSC.
Taken together, a protocol for transduction and SAM-mediated gene activation in primary human MSC was established. It was successfully applied not only for activation of the control gene CEBPA but also for the presumably less susceptible target gene CIITA. Functional activation of the latter was demonstrated by the induction of HLA‑DR gene and protein expression.
In the long term, the establishment of genetic engineering in primary human MSC has a high potential to further increase their applicability in pre-clinical and clinical research.
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