Retinal Development and Disorders · Journal article

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

Stem Cell Reports · August 27, 2026

Preliminary Why this matters to you Expert discussion

Early or partial results. Treat as a signal, not a conclusion.

AI Summary

This study demonstrates that CRISPRa-mediated activation of SOX5, SOX6, and SOX9 (SOX-trio) promotes expression of notochordal cell-associated genes in iPSC-derived populations, as assessed by single-cell sequencing. The work establishes a proof-of-concept for using CRISPR-based transactivation coupled with single-cell technologies to identify and enrich notochordal lineage cells for potential regenerative applications, but lacks functional validation, in vivo data, or mechanistic confirmation that these cells possess authentic notochordal identity and regenerative capacity.

What was studied

Exploratory in vitro study with CRISPR-based transactivation screening and single-cell sequencing. Human induced pluripotent stem cells (iPSCs) with Aggrecan-2A-mScarlet reporter engineered for tracking notochordal lineage commitment. Intervention: CRISPR-based transactivation (CRISPRa) of notochordal lineage-associated transcription factors (NOTO, TBXT, FOXA2, SOX5, SOX6, SOX9).

Key findings

SOX5/6/9 combination (SOX-trio) identified as critical for promoting notochordal cell lineage commitment from iPSCs SOX-trio yielded largest cell population expressing notochordal-associated genes: SHH, FOXA1, FOXA2, FOXJ1, FN1, ALCAM, KRT8, KRT18 CRISPRa screening tested activation of NOTO, TBXT, FOXA2, SOX5, SOX6, and SOX9 individually or in combination

Safety

Safety was not reported in the material analysed. Check the source before drawing any conclusion about harm.

Who this may help

This work is at the discovery stage and does not yet support clinical application. Researchers developing iPSC-based regenerative therapies for intervertebral disc degeneration should note the SOX-trio as a candidate transcriptional program for notochordal cell enrichment, pending functional and in vivo validation.

Plain-language summary

This is a proof-of-concept study using CRISPR-based gene activation to direct iPSC differentiation toward notochordal cells, with single-cell sequencing validation but no functional in vivo data, clinical outcomes, or comparison to standard differentiation methods.

Study design

As stated by the source record.

MethodsCRISPR-based transactivation (CRISPRa) was applied to activate individual or combined transcription factors (NOTO, TBXT, FOXA2, SOX5, SOX6, SOX9) in human iPSCs carrying an Aggrecan-2A-mScarlet reporter. Differentiated cells were analyzed by single-cell sequencing to identify which activation conditions produced the largest population expressing notochordal cell-associated genes.

DesignExploratory in vitro study with CRISPR-based transactivation screening and single-cell sequencing

PopulationHuman induced pluripotent stem cells (iPSCs) with Aggrecan-2A-mScarlet reporter engineered for tracking notochordal lineage commitment

InterventionCRISPR-based transactivation (CRISPRa) of notochordal lineage-associated transcription factors (NOTO, TBXT, FOXA2, SOX5, SOX6, SOX9)

Clinical bottom line

Caveats

No sample size, replication number, or statistical analysis reported
No functional assays (proliferation, migration, differentiation capacity) or in vivo transplantation data
No comparison to alternative notochordal differentiation methods or to primary notochordal cells
Gene expression patterns reported only as gene names; no quantitative effect sizes, fold changes, or p-values provided

Evidence assessment

Graded across the dimensions that decide whether you should act, each from what the source actually supports. There is no single score, and where a dimension was not assessed it says so.

Study design

limited

Uncontrolled, mechanistic proof-of-concept using CRISPRa screening without randomization or comparator arms.

Directness

limited

iPSC-derived cells in vitro; no evidence of in vivo function, transplantation efficacy, or clinical utility.

Consistency

insufficient

Single study; no independent replication or comparison to other NC differentiation protocols reported.

Risk of bias

limited

Single-arm design without blinding; reporter-based enrichment may select for marker expression rather than true identity.

Sample adequacy

insufficient

No sample size, statistical power calculation, or number of replicates stated in abstract.

What is missing. This record has no reported figures. That is a gap in the analysis, not a judgement about the study.

Source 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 the 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.

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Retinal Development and DisordersDeepani W. Poramba‐LiyanageNiels GeijsenDanielle VersluisFrank M. RiemersXiaole TongMarieke Visscher