Aug 2026· Stem Cell Reports· Vol 21, pp. 103060· 0 citations· 48 references
Medicine
TL;DR
This 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.
Abstract
Summary 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.
SOX2+ pituitary stem cells (PSCs) are a critical source of signalling cues during pituitary development, yet the molecular orchestrators of this paracrine environment remain poorly defined. In this study, we identify ZFP36L1 as a key regulator of secreted factor expression within the PSC niche and demonstrate that its precise control is essential for terminal differentiation during pituitary organogenesis.
Using a Sox2-CreERT2 model to induce constitutive active ZFP36L1 (ZFP36L1-CA) in PSCs from E12.5, we observed severe pituitary hypoplasia and a profound lack of terminal differentiation across POU1F1+ and NR5A1+ hormone-producing lineages by E18.5. To determine the nature of this developmental arrest, we performed mosaic lineage tracing via tamoxifen titration. Notably, wild-type cells neighbouring ZFP36L1-CA cells also failed to undergo hormone commitment, establishing a non-cell autonomous defect and suggesting a disruption of the extrinsic signalling environment.
This requirement for regulated ZFP36L1 expression was further validated using a Hesx1-Cre model, where early developmental ZFP36L1-CA expression resulted in dwarfism, perinatal lethality, and failure of POU1F1+ and NR5A1+ endocrine cell commitment. Crucially, this differentiation defect was rescued when Hesx1-Cre mutant pituitaries were co-cultured with normal PSCs, providing functional confirmation that the phenotype is driven by the loss of essential stem-cell-derived paracrine signals.
Mechanistic interrogation through scRNA-seq and mRNA in situ hybridisation identified secreted factors regulated by ZFP36L1 that co-localise with PSCs in vivo. ELISAs confirmed that these factors are actively secreted by PSCs and are significantly downregulated upon ZFP36L1-CA expression.
Collectively, these findings establish that ZFP36L1 regulation in PSCs is indispensable for pituitary organogenesis. These experiments identify specific paracrine mediators required for terminal differentiation in the anterior pituitary, offering new molecular targets for efficient regenerative approaches.
O. Sherwin, S. Manshaei, T. Willis et al.· European Journal of Endocrin...· 0 citations
Utilizing key developmental cues and refining their orchestrating role in degeneration represents a promising strategy for understanding and treating intervertebral disc (IVD) degeneration, a major cause of chronic lower back pain. Here, we focus on notochordal cells (NCs), which originate from the embryonic notochord and reside in the developing nucleus pulposus. These distinctly vacuolated cells exhibit robust regenerative effects and hold promise for new therapeutic approaches. Dogs, like humans, suffer from the consequences of IVD degeneration. As the IVD matures and degenerates, NCs are replaced by smaller non-vacuolated NP cells (NPCs). The dog was employed as a model to capture, at the single-cell level, the heterogeneity of resident cells by studying the nucleus pulposus tissue at three stages (i.e., juvenile, young adult and degenerate adult). Here, we integrated transcriptomic data with repressive histone H3 lysine 27 trimethylation (H3K27me3) profiles at the single-cell level to assess changes in chromatin states and gene expression across this IVD degeneration-associated cell phenotypic transition. H3K27me3 enrichment on key genes involved in IVD development and homeostasis, such as Brachyury (TBXT), aligns with the observed attenuation during ageing and degeneration seen in both dog and human IVDs. This study further demonstrates that eliminating repressive histone marks, together with CRISPR-mediated gene transactivation, enhances TBXT gene expression in human NPCs derived from degenerated aged discs. Our findings underscore how extensive insights gained through single-cell omics can lead to the identification of crucial cellular cues that may enable degenerate NPCs to regain a healthier phenotype.
Deepani W. Poramba-Liyanage, Xiao-Le Tong, F. Riemers et al.· Bone Research· 0 citations
Highlights What are the main findings? MBD3 knockdown during bovine iPSC reprogramming significantly enhances the differentiation efficiency of primordial germ cell-like cells (PGCLCs) under a 2D monolayer culture system. MBD3-knockdown iPSCs exhibit elevated bivalent histone modifications (H3K4me3/H3K27me3) at TGF-β-related gene loci and upregulated DPPA4/DNMT3L expression, which primes enhanced NODAL signaling to drive PGCLC formation. What are the implications of the main findings? MBD3 functions as an epigenetic gatekeeper that restricts germline competence during reprogramming, and its depletion predisposes iPSCs toward a PGCLC fate. These findings provide a mechanistic basis for optimizing bovine iPSC-derived PGCLC protocols through targeted epigenetic modulation, with potential applications in livestock breeding and germline conservation. Abstract Methyl-CpG binding domain protein 3 (MBD3) is a member of the nucleosome remodeling and deacetylase (NuRD) corepressor protein complex, which plays a pivotal role in embryonic development, pluripotent stem cell (PSC) differentiation, and induced pluripotent stem cell (iPSC) reprogramming. However, whether MBD3 is also involved in iPSC differentiation into primordial germ cell-like cells (PGCLCs) is unclear. In this study, bovine iPSCs generated by MBD3 knockdown (MBD3 KD) during reprogramming were subjected to PGCLC differentiation using a monolayer cell culture method. Our results revealed that MBD3 KD enhanced the ability of bovine iPSCs to differentiate into PGCLCs. MBD3-KD bovine iPSC-derived PGCLCs exhibited the characteristics of in vivo primordial germ cells (PGCs) in a migratory state. MBD3 KD increased NODAL signal transduction during the induction of PGCLCs and was associated with increased expression of PRDM1 and SOX15. Moreover, MBD3 KD increased the bivalent modification of transforming growth factor beta (TGF-β)-related genes concomitant with the upregulation of DPPA4 and DNMT3L expression in bovine iPSCs, and this epigenetic change was associated with enhanced NODAL signaling. Our results indicate that MBD3 KD during reprogramming favors the differentiation of bovine iPSCs into PGCLCs, further highlighting the impact of MBD3 on histone modifications during germ cell development.
Wen Yuan, Jing Wang, Wei-Qi Li et al.· Cells· 0 citations
Neuromesodermal progenitors (NMPs) produce the spinal cord and musculoskeleton in the elongating anterior-posterior axis. In vivo, NMPs possess dual potency, coinciding with regions coexpressing SOX2 and Brachyury (TBXT). In vitro, SOX2/TBXT co-expressing cells can be produced from pluripotent cells and, like their in vivo counterparts, can produce neural tube and somitic mesoderm. However, the functional characteristics of in vitro SOX2/TBXT co-expressing cells remain unclear, confounding comparisons with in vivo data. To address this, we developed a dual Sox2/Tbxt reporter mouse ESC line. SOX2/TBXT reporter-positive cells emerge in vitro from pluripotent populations with dynamics that mirror their appearance in the embryo. Purified SOX2/TBXT co-expressing populations can differentiate towards neurectoderm or mesoderm, including lateral mesoderm upon BMP stimulation. In gastruloids, quantitative live imaging shows that WNT or NOTCH inhibition rapidly leads to downregulation of TBXT expression and diminished axial extension. We show that clonally plated SOX2/TBXT co-expressing cells are bipotent NMPs that can also self-propagate. By combining clonal analysis with mathematical modelling, we identify two thresholds of SOX2/TBXT expression, switching clonal output from neural- to mesoderm-biased, and from mesoderm-biased to mesoderm-specified. Media and substrate composition alter the lineage outcomes of in vitro derived mouse NMPs. Thus, this Sox2/Tbxt double reporter cell line provides support for unsuspected heterogeneity in NMPs, together with evidence for a role of these transcription factors in directing cell fate to drive axis elongation.
A. Binagui-Casas, Anna Granés, A. Ceccarelli et al.· bioRxiv· 1 citation
Seminoma, which is the most frequent testicular germ cell tumor, has stem cell-like features related to treatment resistance and recurrence, yet the molecular mechanisms that preserve its stemness are not well understood. This study aimed to discover the tumorigenesis and stemness regulation of seminoma. In vivo CRISPR/Cas9 knockout library screening was performed using Tcam-2 seminoma cells implanted into NSG mice to identify tumorigenesis drivers. Parallel FACS-based screening enriched CD117/CD133 double-negative cells to assess the regulators of seminoma stemness. Functional validation included clonogenicity assays, sphere formation tests, and tumor initiation experiments in vivo. RNA sequencing analyzed downstream pathways. In vivo and FACS-based CRISPR/Cas9 screening uncovers VENTX as a crucial element in maintaining seminoma stemness. Functional validation showed that VENTX knockout decreased clonogenicity, sphere formation, and CD117+/CD133+ populations in Tcam-2 cells, and also hindered tumor initiation in vivo. RNA sequencing identified a connection between VENTX and pluripotency pathways, demonstrating a downregulation of ID1/2/3 (essential differentiation inhibitors) and an imbalance in Wnt/β-catenin and TGF-β signaling. VENTX expression showed a significant association with DNA methylation-based stemness scores in testicular germ cell tumors. In conclusion, VENTX is a key regulator of seminoma stemness, presenting a promising target for therapy to address recurrence and resistance in seminoma.
Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor’s genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.
D. Skoczek, J. Hohendorff, Maciej T. Małecki et al.· Human Genetics· 0 citations
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