Oral Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction: In vitro derivation of notochordal cells (NCs) constitute an active and promising research avenue for the restoration and repair of degenerated disc. Understanding the regulation at the genetic and epigenetic levels during notochordal derivation will help identifying conditions for an effective cell derivation. Epigenetics regulation involving chromatin-related mechanisms play critical role in cell differentiation¹, however, their role during notochordal differentiation of human induced pluripotent stem cells (hiPSCs) remains completely unknown. Here, we studied molecular understanding of hiPSCs differentiation into notochordal cells and explored the epigenetic regulation during differentiation process. Using global transcriptome analysis, we identified histone lysine demethylases 2,4,7 (KDM2, 4, 7) as negative regulators of notochord differentiation and regulate the expression of notochordal markers.

Methods: We used CRISPR-Cas9 based targeted genome editing approach to insert mCherry reporter into 3’UTR of the specific notochordal marker NOTO gene using human fibroblast iPSC lines (HDFa-YK27). We used our previously reported stepwise method using cocktail of four factors (LDN, AGN, bFGF and CHIR99021)) for NCs derivation of hiPSC² for 5 days (Fig.1). To define the molecular pathways involving differentiation of hiPSCs into NCs, we performed an unbiased genome wide transcriptome analysis at different stages of differentiation (Day 0, 3 and 5) (Fig. 2). Differentially expressed genes (DEG) were analyzed using DESeq2 method. We selected KDM 2, 4 and 7 for further analyses (Fig.3). KDMs activity was regulated with specific inhibitors at the day 3 and 5 of the notochordal differentiation. Gene expression, Western immunoblotting and immunofluorescence staining was utilized to validate notochordal commitment (Fig. 4 &5). 

Results:

Using cocktail of four-factor (LAFC) for 5 days, we can efficiently differentiate hiPSCs into notochordal cells as demonstrated by  induction of mCherry fluorescent reporter, indicating the expression of NOTO gene. We further  characterized NCs using various methods (Fig. 2&3).The global transcriptome analysis during differentiation process using three independent hiPSC lines further showed induction of a plethora of genes related to notochord and NP like cells. Interestingly, gene ontology (GO) analysis showed the enrichment of various chromatin marks related to the histone lysine demethylation (H3K) activity suggesting the potential involvement of epigenetic regulators during notochordal differentiation. The analysis of >800 epigenetic regulators showed that 61 epigenetic genes were down-regulated, and 36 factors were up-regulated during notochord differentiation. The gene expression analysis during differentiation process showed an inverse correlation between histone demethylases (KDM2A, KDM4A, KDM7A) and marker of notochordal genes indicating that inhibition of KDM activity may promotes notochordal differentiation. Interestingly, supplementation with KDM inhibitors during the stepwise differentiation protocol resulted in the enhancement NOTO gene expression and NCs markers.

Discussion: Our studies discovered histone demethylases (KDM2A, KDM4A and KDM7A) as negative regulator of notochordal differentiation and treatment with inhibitors of KDM2A and KDM4A promoted the differentiation of hiPSCs into notochordal cells indicating the new strategies to improve the cell-based therapy in IDD. Our study further provides an additional layer of epigenetic regulation of notochordal derivation which will help in devising an effective cell derivation strategy required to maintain mature NP cells.