G.G. van den Akker

A4.02 Linking inhibition of chondrogenesis and inflammation through the smad linker domain

Background and objectives Cartilage repair using mesenchymal stem cells is a promising new strategy for the treatment of osteoarthritis. However, the inflammatory environment of the joint inhibits the repair process. Previously, it has been shown that signalling via transforming growth factor β family members is crucial for cartilage homeostasis. Therefore we evaluated the effect of inflammatory mediators on downstream mediators of TGFβ signalling: the R-SMAD transcription factors. Materials and methods Human mesenchymal stem cells were differentiated in monolayer or pellet cultures in chondrogenic differentiation medium. Differentiation was assessed at day 7 using GAG measurements. On day 3 of differentiation inflammatory stimuli were added. To determine the effect on TGFβ signalling the CAGA-luciferase reporter assay and immuno blotting for SMAD phospho-isoforms were used. A human phospho-kinase antibody array was performed to identify kinases activated by inflammation. Results Addition of IL1β and OAC-med to differentiating MSC at day 3, inhibited chondrogenic differentiation as measured by GAG deposition at day 7. Transcriptome analyses at 8 h post stimulation with inflammatory mediators revealed deregulation of TGFβ-related and regulated genes. TGFβ signalling was inhibited by inflammation as evidenced by inhibition of the SMAD3 dependant CAGA-luciferase reporter. SMAD2/3 activation by TGFβ is largely determined by C-terminal phosphorylation through the ALK5 receptor. However C-terminal phosphorylation was not affected after addition of inflammatory stimuli. SMAD proteins contain a highly variable linker region that can be phosphorylated by various kinases. These linker modifications are known to modulate SMAD activation and degradation. Stimulation with either IL1β or OAC-med resulted in serine phosphorylation of the SMAD2 linker region within 1 h. To determine which kinase might be responsible for SMAD linker modification a human phospho-kinase antibody array was performed. The MAP kinase pathway was strongly activated within 30 min as evidenced by a > 1.5 fold increase in pERK1/2 and pP38 compared to control pellets. Conclusions Inhibition of TGFβ signalling in differentiating MSC by IL1β and OAC-med appears to be mediated through SMAD serine linker modifications. The hot favourite kinases for phosphorylation of these serines are members of the MAP kinase family.

A6.04 CRISPR/CAS9 mediated genome engineering of human mesenchymal stem cells

Background and objectives CRISPR/Cas9 is a novel technique for genomic inactivation of genes. In this study we assessed the feasibility of generating knock-out cell populations from human mesenchymal stem cells, without sub-cloning of cells. Materials and methods CRISPR guide RNAs were designed and cloned into a lentiviral Cas9 expression system. HEK293 cells or MSC were transduced with lentivirus and selected with puromycin. CRISPR efficiency for genomic modification was determined by genomic PCR, surveyor nuclease digestion while protein expression was assessed by immunoblotting. Results Transforming Growth Factor β (TGFβ) signalling is crucial for chondrogenic differentiation of MSC and the maintenance of the articular chondrocyte phenotype. Currently the exact role of individual downstream SMAD transcription factors is unknown. Efficient RNA interference mediated knock-down of SMAD proteins might still allow signal transduction via the remaining SMAD protein. To generate knock-out cell populations, CRISPRs (guide RNAs) were designed to target the second coding exon of the five R-SMAD genes. The CRISPR efficiency was assessed using the surveyor nuclease assay on HEK293 cells and MSC. The evaluated CRISPRs had a variable efficiency in HEK293 cells: 20–90% genomic modification of the target gene was observed. Increasing the viral load (MOI) clearly improved this efficiency in MSC, while increased antibiotic selection pressure did not. To further improve the on-target efficiency we used a double guide RNA targeting strategy to excise a 100 base pair fragment. By regular PCR it was estimated that 25% of the total cell population carried the truncated form of the gene. The surveyor nuclease assay showed an even higher percentage of genomic modification. Importantly, this double targeting strategy decreased SMAD protein expression in HEK293T by 90%. MSC were expected to be more vulnerable to CRISPR/Cas9 genome engineering and/or high viral loads. Following careful optimisation of lentiviral transduction and antibiotic selection protocols for MSC, we evaluated the effect of four control CRISPR/Cas9 constructs targeting GFP. Our preliminary findings indicate that MSC stably expressing CRISPR/Cas9 exhibit normal differentiation characteristics. Conclusions We show that efficient targeting of genes using CRISPR/Cas9 in MSC cell populations, leading to strongly decreased protein expression, is feasible without clonal selection.