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CRISPR activation reveals SOX5/6/9 as key transcriptional regulators directing iPSC-derived cells toward a notochordal lineage

ABSTRACT: Intervertebral disc (IVD) degeneration, a leading cause of chronic lower back pain, is associated with loss of vacuolated notochordal cells (NCs) and fibrotic remodeling of the nucleus pulposus. Emerging therapies increasingly focus on NCs, which are rare but therapeutically relevant cells for regenerating degenerated IVDs. In this study, we used CRISPR-based transactivation (CRISPRa) to direct the differentiation of human induced pluripotent stem cells (iPSCs) into NC lineage. We tested CRISPRa-mediated activation of NOTO, TBXT, FOXA2, SOX5, SOX6, and SOX9, coupled with single-cell sequencing of Aggrecan-2A-mScarlet reporter iPSCs. This approach identified the SOX5/6/9 combination (SOX-trio) as critical for promoting NC lineage commitment. The SOX-trio yielded the largest cell population expressing a range of genes previously associated with NC identity, including SHH, FOXA1, FOXA2, FOXJ1, FN1, ALCAM, KRT8, and KRT18. Our study demonstrates the integration of CRISPRa with single-cell technologies as a powerful platform for investigating and enriching iPSC-derived NCs, supporting future regenerative strategies across various fields.

ORGANISM(S): Homo sapiens

PROVIDER: GSE283877 | GEO | 2026/08/27

REPOSITORIES: GEO

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Project description:Objective: The notochord acts as a central signaling hub during embryonic development. After regression of the notochord, notochordal cells (NCs) reside within the developing intervertebral disc. The NC-secretome, including matrix and extracellular vesicles (NC-EVs), exert regenerative effects in the degenerate intervertebral disc and cartilage. The aim of this study is to unravel the NC-EV associated proteome, and their modulatory role in the context of cartilage and intervertebral disc regeneration. Design: NC-EVs were isolated from conditioned medium from porcine NC-rich tissue (N=7) using differential centrifugation, size exclusion chromatography and sucrose density gradients. To decode the full protein spectrum, the EV fraction was subjected to mass spectrometry (LC-MS/MS). To test bioactivity of selected signaling nodes, functional analysis was done using a chondrocyte-like reporter cell line with transcriptional binding elements (SBE, TCF/LEF and NFκB) driving luciferase reporter gene expression. Hyaluronidase treatment was applied to functionally assess the protein corona on the EV surface. Results: Proteomic data on NC-EVs identified 4,042 unique proteins, including EV-associated markers, with more than 90% overlap between animals. A large number of matrisome proteins and proteins around critical nodes in multiple signaling pathways was identified. Functional validation in chondrocyte-like reporter cells showed that NC-EVs activate SMAD3/4, and inactivate NFκB upon IL1β stimulation. The NC-EVs effects were comparable to NC-EV depleted controls, indicating that the soluble proteins play a profound role in modulating signaling. This was further supported by the fact that treatment of NC-EVs with hyaluronidase resulted in a significant functional differences between NC-EVs and NC-EV depleted controls. Conclusions: This study uncovers the first NC-EV proteome and illustrates that the adsorbed proteins on the NC-EV surface contribute to their functionality in NFκB inhibition and SMAD3/4 activation.

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CRISPR activation reveals SOX5/6/9 as key transcriptional regulators directing iPSC-derived cells toward a notochordal lineage

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