Directed differentiation of induced pluripotent stem cells to notochordal-like cells by combinatorial transcription factors activation


Tong, Xiaole (Utrecht University)


"Introduction: Low back pain (LBP) is a leading cause of disability worldwide and intervertebral disc (IVD) degeneration (IVDD) is a major contributor of LBP1. The IVDD is accompanied and often preceded by the replacement of large vacuolated nucleus pulposus cells (NPCs) by non-vacuolated, clustered cells in the notochordal cells (NCs) of the IVD2. The iPSpine project aims to re-populate the degenerated IVD with regenerative iPS-derived NC-like cells (iPS-NLCs). During embryonic development, the determination of cell fates is a result of the combinatorial and concomitant activation of transcription factors. While differentiation of iPS-NLCs can be partially achieved by mRNA transfection of a single gene, such as the notochord-related transcription factor NOTO, such attempts are limited by low and variable differentiation efficiency3. This study aims to achieve optimal notochordal lineage commitment by the concomitant and combinatorial activation of multiple key transcription factors via CRISPR activation (CRISPRa) technology. With CRISPRa, transcription activation complexes are recruited to the endogenous promoters of genes to induce expression.

Methodology: Based on a two-step differentiation protocol, we first established iPS-derived mesodermal progenitor cell by CHIR stimulation to activate the WNT pathway. For notochordal lineage commitment, we focus on NOTO, brachyury (TBXT) and Forkhead Box Protein A2 (FOXA2). Both T and FoxA2 act upstream and are required for the NOTO expression. These three genes were activated at the endogenous gene locus by CRISPRa technology via the synergistic activation mediator (SAM) system, the most efficient dCas9 gene activator4. Gene activation by CRISPRa will be further compared with mRNA transfection to establish the optimal mode of notochordal lineage commitment.

Results: We establish a gene activation and differentiation pipeline in the notochordal cell lineage and show significant activation of all 3 genes by recruitment of CRISPRa to the respective gene promoters in the differentiating iPSCs. Our results indicate better expression levels when transcription is promoted directly via recruitment of transcriptional activation complexes to gene promoters, rather than the introduction of synthetic mRNAs. Time-course analysis of lineage-specific markers shows that combining multiple transcription factors allow for better iPSC-NLC differentiation and commitment toward the notochordal lineage.

Conclusions: We highlight how transcriptional landscapes can be modulated at critical moments of embryonic development to optimize iPSC-NLC differentiation strategies and optimize notochordal lineage commitment.

Funding: iPSpine: Horizon2020(No.825925) and Dutch Arthritis Society (LLP22).

1Clark, S. et al., Lancet. 391(10137):2302 (2018). 2Hunter, C. J. et al., Journal of anatomy. 205(5), 357-362 (2004). 3Colombier, P. et al., Cells. 9(2), 509 (2020). 4Konermann, S. et al., Nature. 517(7536), 583–588 (2015)."

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