Stem Cell Reports

Human pluripotent stem cells (hPSCs) rely predominantly on glycolysis and exhibit relatively low mitochondrial respiration. Under these conditions, the mitochondrial F1Fo ATP synthase tends to operate in reverse mode, hydrolyzing ATP. ATP synthase inhibitory factor subunit 1 (IF1) inhibits this F1Fo ATP hydrolysis, but its role in hPSCs remains unclear. Here, we generated human induced pluripotent stem cells (hiPSCs) with stable IF1 knockdown (IF1-KD). IF1-KD enhanced F1Fo ATP hydrolysis and elevated the mitochondrial membrane potential (MMP). Although core pluripotency transcription factors were maintained, IF1-KD cells exhibited a partial epithelial-mesenchymal transition (EMT)-like state and biased trilineage differentiation. Mechanistically, the elevated MMP was accompanied by enhanced store-operated Ca{superscript 2} entry (SOCE) and nuclear translocation of NFATc3. Moreover, lowering the MMP attenuated SOCE, and NFATc3 overexpression reproduced the EMT-like gene expression. These results support a model in which IF1, by inhibiting F1Fo ATP hydrolysis, prevents excessive elevation of the MMP and thereby suppresses the transition to a partial EMT-like state via the MMP-SOCE-NFAT axis, contributing to the maintenance of the epithelial state associated with hiPSC pluripotency.

The neural crest (NC) is a transient stem cell population which migrates throughout the developing embryo to contribute to diverse tissues dependent on axial origin. For example, cranial NC can give rise to bone and cartilage, while more posterior NC populations give rise to peripheral nervous system and neuroendocrine tissues. Perturbations in neural crest development can lead to severe congenital anomalies and cancers, with over 700 neurocristopathies reported. In humans, early NC development remains poorly understood due to the inaccessibility of tissue samples, thus necessitating the development of in vitro models. Currently, a limited number of NC organoid protocols are available, but these mainly focus on cranial NC and lack relevant tissue architecture. Here, we describe a novel bioengineered pipeline to derive human pluripotent stem cell (hPSC)-derived neuroepithelial organoids, "neurocrestoids" featuring physiologically-relevant tissue architecture. We show that neurocrestoids recapitulate the dynamics of induction, delamination, and migration of human neural crest cells (NCCs), and can be directly compared to murine NC explants for cross-species validation. Organoids express an array of HOX genes indicating the successful generation of cranial, vagal and trunk NCCs. Moreover, we have integrated our neurocrestoids with a customised micropatterned substrate suitable for live visualisation and guided separation of SOX10-positive migratory human NCCs. Our "NCC migration on-chip" are reproducible across multiple hPSC lines and should be scalable for future diagnostic and therapeutic applications, significantly improving our ability to study human NC pathologies.

The rapid rise of stem cell-based human embryo models has reignited interest in studying early human development while offering a promising platform to de-risk drugs. Among these, three-dimensional human gastruloids provide a tractable system to model symmetry breaking, germ layer specification and axial organization. However, existing gastruloid protocols remain expensive, specialized, variable and evaluated in a limited number of human pluripotent stem cell (hPSC) lines, restricting broader adoption. Here, we present a simple, robust, standardized gastruloid protocol achieving greater than 90% elongation efficiency with low inter- and intra-experimental variability, developed primarily in BJNhem20, a well-characterized Indian-origin human embryonic stem cell line. Further, we show that the protocol is applicable in a diverse set of hPSC lines. Using a TBXT (Brachyury)-GFP reporter in BJNhem20, we optimized cell seeding density, induction medium and Wnt activation strength, guided by real-time, quantitative assessment of mesoderm induction and symmetry breaking, allowing precise titration of CHIR99021. Comparative testing identified an in-house "Essential 6" medium formulation as the most consistent condition for robust TBXT induction. Optimization of aggregation density produced reproducible gastruloids with polarized TBXT expression and consistent axial elongation, within 72 hours. Single-cell RNA sequencing of individual gastruloids confirmed high transcriptional reproducibility and conserved lineage clusters, aligned with developmental trajectories. Cell line-specific CHIR99021 titration was sufficient to successfully transfer the optimized protocol to two additional lines, BJNhem19 and RUES2-GLR. This simplified and robust protocol reduces costs and improves accessibility, enabling broader application of stem cell-based human embryo models.

Genetically diverse panels of human pluripotent stem cells enable genetic dissection of cellular phenotypes, but comparable induced pluripotent stem cell (iPSC) resources in model organisms remain limited. We generated a panel of iPSCs from the Diversity Outbred (DO) mouse population and established 288 genetically unique lines that retain the allele frequency distribution, heterozygosity, and low population structure of the source population. The lines exhibit consistent growth, pluripotent gene expression profiles, and capacity to form embryoid bodies. Transcriptomic profiling of the lines revealed significant variation in gene expression driven in part by genetic background. We used expression quantitative trait locus (eQTL) mapping to identify over 10,000 regulatory loci that influence gene expression variation, including multiple distal eQTL hotspots that are key gene regulatory hubs and are shared with DO embryonic stem cells (ESCs). The largest hotspot, mediated by Lifr, showed a shift in founder allele effects relative to ESCs, consistent with differences in cellular state and culture conditions. These results establish the DO iPSC panel as a genetically diverse, publicly accessible platform derived from a laboratory mouse genetic reference population, enabling integration of in vitro cellular phenotypes with in vivo traits within a closed, outbred population.

Photoreceptor transplantation is considered a disease-agnostic therapeutic strategy for retinal degenerative diseases with highly heterogenous genetic, molecular, and cellular pathologies. While integration of human photoreceptors enriched from stem cell-derived retinal organoids was noted in previous preclinical studies, the potential influence of retinal degeneration severity on transplantation efficiency has not been systematically assessed. Here, we employed mice presenting mild or severe retinal degeneration as recipients for human induced pluripotent stem cell-derived photoreceptors. Donor cells formed multi-cellular clusters that structurally integrated from 3 weeks post-transplantation (wpt) in mildly degenerated retinas, closely interacting with host Muller glia, resulting in proper maturation characterized by inner/outer segment and synapse formation by 26 wpt. In contrast, in severely degenerated hosts, donor photoreceptors remained mainly singularized and scattered in the subretinal space, showing limited structural integration or signs of maturation. Differential maturation of donor cells in mild vs. severe hosts was confirmed by single-cell RNA-sequencing analysis. However, transplantation at the beginning of the degeneration process of the severe model allowed structural integration and maturation of donor photoreceptors, despite complete loss of endogenous photoreceptors over time. The study thus shows that survival, integration, and maturation of donor photoreceptors depend on the degenerative retinal microenvironment shaping significantly transplantation efficiency.

Advances in transcriptome profiling have revealed transcriptomic differences across different cellular states. However, functional interpretation requires precise perturbation tools and experimental frameworks. This study benchmarked two widely used modalities: CRISPR interference (CRISPRi) and Cas13d/CasRx. A standardized workflow was established to generate human pluripotent stem cells (PSCs) with inducible ZIM3-dCas9 or CasRx expression. The cell lines were subjected to flow cytometry, copy number, and immunocytochemical analyses. The knockdown performance was validated via robust OCT4 suppression and the expected downstream effects on pluripotency genes. Time-course measurements indicated that CRISPRi produced faster and stronger repression but slower recovery after inducer withdrawal. In contrast, CasRx yielded slower and typically weaker knockdown with rapid reversibility. Furthermore, a key limitation of CRISPRi was demonstrated using the ATF5-NUP62 locus, wherein CRISPRi could co-repress genes with overlapping promoter regions. In contrast, CasRx avoids these limitations and supports isoform-resolved targeting of circular and alternatively spliced transcripts, albeit with variable efficiency. These results provide practical guidance for selecting complementary knockdown tools to improve the interpretability of transcriptomic function studies. MOTIVATIONAdvances in transcriptome profiling have enabled the detection of subtle cell type-specific differences. However, mechanistic interpretation still depends on perturbation tools that can modulate transcripts with high precision and efficiency. Recent CRISPR-based modalities, CRISPRi and Cas13/CasRx, function as robust and orthogonal methods to achieve the knockdown of specific gene targets. However, a standardized approach for cell line preparation and comparative studies on their relative performances and limitations remains unclear. Consequently, this study presents a standardized workflow for generating cell lines that support high-efficiency knockdown using CRISPRi and CasRx. Moreover, it compares the trade-offs in potency, reversibility, and isoform resolution, along with a practical overview of method-specific pitfalls to guide tool selection and data interpretation in future studies. HIGHLIGHTSO_LIDoxycycline-inducible AAVS1 knock-in human PSC platforms for CRISPRi (ZIM3-dCas9) and CasRx (RfxCas13d) were generated to enable standardized RNA perturbation experiments. C_LIO_LIThe prepared cell lines demonstrated strong OCT4 knockdown, with expected downstream effects on the expression of another pluripotency gene, NANOG. C_LIO_LIA comparison of knockdown characteristics and their reversibility revealed rapid and sustained repression with CRISPRi, whereas slow but rapid recovery was observed with CasRx. C_LIO_LIA CRISPRi-specific off-target effect arising from TSS proximity/overlap (ATF5-NUP62) was identified, whereas CasRx achieved ATF5 knockdown without collateral repression of the neighboring NUP62 gene. C_LIO_LICasRx enables isoform-resolved knockdown of structural isoforms (circHIPK3 vs. linear HIPK3 mRNA) and splice isoforms (RAB6A-iso1 vs. RAB6A-iso2). C_LI

Human pluripotent stem cells (hPSCs) provide a powerful platform for modeling early human embryonic development. Here, we investigate the mechanisms underlying mesodermal heterogeneity using a minimal directed differentiation system that simultaneously generates paraxial (PXM), intermediate (IM) and lateral plate mesoderm (LPM) populations. Single-cell RNA sequencing across defined time points during hPSC differentiation revealed a temporal sequence of lineage specification with LPM emerging first, followed by PXM and IM differentiation. Ligand-receptor and differential gene expression analyses identified BMP4 as a key regulator enriched in LPM-associated clusters versus mesoderm progenitors (MPs) that hold PXM and IM precursors. Whereas LPM cells cluster with an early BMP4 signal, IM clusters are associated with later BMP4. Moreover, these early and late BMP4 signals regulate this lineage specification potentially through distinct downstream pathways. Leveraging this insight, we established a stepwise protocol combining early BMP inhibition with subsequent BMP4 supplementation, suppressing initial LPM fate to efficiently induce IM from a mixed MP population. Longer culture of these selective IM progenitors promotes more mature nephrogenesis. Moreover, we demonstrate that during early differentiation high levels of BMP4 can still redirect MPs to more lateroventral fates, illustrating a degree of plasticity within the mesoderm lineage. Together, our results define a temporal framework for BMP4 signaling in mesoderm fate determination and provide a strategy for selective mesoderm differentiation from hPSCs. HIGHLIGHTSO_LIDevelopment of a minimal 2D differentiation platform allows for heterogenous mesoderm formation. C_LIO_LITemporal BMP4 signaling differentially directs mesoderm fates, with early exposure favoring LPM and late exposure promoting IM identity. C_LIO_LILPM cells arise first while later mesoderm progenitors hold both IM and PXM-fated cells. C_LIO_LISequential BMP modulation promotes IM and enhances nephrogenesis. C_LI

IntroductionReliable generation of hepatocyte-like cells (HLCs) from pluripotent stem cells remains limited by heterogeneity and incomplete maturation of the cells. Derivation of induced pluripotent- and embryonic stem cells into hepatocytes typically relies on complex, and costly reagent-intensive protocols, with inconsistent reporting of differentiation efficiencies and functional maturation criteria. Variability in protocol designs highlights the need for optimisation, particularly in mouse embryonic stem cells (mESCs) systems that can be more comparable with mouse models for underpinning translational and toxicological studies. Here, we developed and evaluated two cytokine-based strategies: an advanced hepatic-inducing cocktail (A-HIC) and a simplified hepatic-inducing cocktail (HIC), both designed to reduce complexity while increasing functional maturation. MethodsHepatic differentiation and maturation were assessed by morphology, immunofluorescence, flow cytometry, and qRT-PCR. Functional competence was evaluated via urea production, glutathione synthesis, indocyanine green handling, cytochrome P450 inducibility, and impedance-based cell layer integrity monitoring. ResultsMorphological, molecular and phenotypic analyses confirmed that both protocols supported hepatic lineage progression, generating heterogeneous populations of hepatoblast-like and more mature HLCs. Gene expression confirmed the loss of pluripotency, transient endoderm induction, and subsequent hepatic specification. Functionally, cells exhibited glycogen storage, inducible urea production, glutathione depletion, and active ICG uptake and clearance, with stable monolayer formation by day 21. A-HIC-derived HLCs demonstrated enhanced maturation, with higher ASGR1 expression and stronger Cyp1a1 induction. DiscussionThese findings suggest that both protocols generate functional HLCs; however, A-HIC yields a higher proportion of functionally mature cells with reduced variability. This approach enables a simple, cost-effective, and time-efficient generation of HLCs, supported by improved functional characterisation with potential applicability to more complex pluripotent systems, including human iPSC-based models for disease modelling and toxicology.

Dissecting polygenic disease mechanisms requires human cell models that harbour multiple targeted genetic modifications in a defined background. However, generating and rigorously validating such models remains difficult. We developed a cytosine base editing workflow to generate multiplex-knockout (KO) human induced pluripotent stem cell (hiPSC) lines. First, we assessed six cytosine base editor (CBE) variants and selected evoBE4max. We then combined sgRNA-guided introduction of premature termination codons and splice-site mutations with fluorescence-based enrichment. This yielded a median on-target C-to-T editing efficiency of 77.5% (range, 27.0-86.5%) across six loci. We generated single-, double-, and triple-KO hiPSC lines for endolysosomal Ca{superscript 2} signalling components (OCaR2, TPC1, TPC2) and confirmed loss-of-function at transcript and protein levels. We performed extensive quality control, including pluripotency assessment, SNP-array karyotyping, and whole-genome sequencing, which indicated minimal guide-directed off-target editing. We further extended multiplex editing to ORAI Ca{superscript 2} channel paralogs. This framework supports scalable production of quality-controlled multiplex-KO hiPSC lines.

The protracted timing required for oligodendrocyte differentiation from human pluripotent stem cells (hPSCs) has limited their use in disease modeling, drug screening, and cell therapy. In particular, the signals that drive oligodendrocyte specification and maturation after neural induction and ventral patterning remain poorly understood. Here, we present a protocol to derive human oligodendrocytes from hPSCs that is based solely on extrinsic cues, and we identify dual inhibition of BMP and Notch signaling as critical drivers of oligodendrocyte commitment and maturation. By day 42 of differentiation, up to 70% of the cells are positive for the oligodendrocyte marker O4, with minimal astrocyte contamination, and show robust expression of mature myelin markers including MBP, MOG, and MAG. These hPSC-derived oligodendrocytes closely match the molecular identity of primary fetal human oligodendrocytes as assessed by single-cell RNA sequencing and are functional as shown by in vitro myelination assays. In addition to the rapid generation of myelinating oligodendrocytes, the new protocol can be modularly adapted for the efficient production of PDGFR+ oligodendrocyte precursors or mixed glial populations containing AQP4+ astrocytes, thereby providing a cellular toolbox for the study of human glial lineages in translational applications.

Hematopoietic stem cells (HSCs) in the adult mouse can be prospectively isolated to near-purity through phenotypic markers, enabling detailed analysis of stem cell function. Homeobox B5 (Hoxb5) was previously identified as a definitive marker of long-term (LT) HSCs in adult bone marrow1. In contrast, fetal HSCs have not been purified to the same extent. Here, we show that Hoxb5 is expressed in fetal liver (FL) HSCs at embryonic day (E) 12.5-16.5 using a single-color tri-mCherry reporter driven by endogenous Hoxb5 regulation. Prospective purification by stringent multiparameter flow cytometry revealed Hoxb5 FL-HSCs to exhibit robust, multilineage reconstitution upon serial transplantation. Quantitative assays reveal that Hoxb5 enriches FL-HSCs to near-single-cell purity, analogous to its role in the adult bone marrow, underscoring its reliability in distinguishing LT-HSCs throughout hematopoietic ontogeny. Notably, Hoxb5 expression is not exclusive to FL-HSCs, as it is also detected across the fetal liver hematopoietic hierarchy and in fetal liver endothelial cells, suggesting developmental stage-specific regulation of its expression. In addition, single-cell RNA sequencing of FL-HSCs identified distinct transcriptional states defined by Hoxb5 expression. These findings establish Hoxb5 as a robust marker for enhancing the purification of fetal liver phenotypic HSCs (pHSC) and provide a framework for dissecting the molecular regulation of HSC ontogeny.

Human pluripotent stem cell-derived islets (SC-islets) offer an excellent medium for human pancreatic disease modelling and mechanistic studies into diabetes. While substantial progress has been made in differentiation protocols, their implementation in different laboratories result in variable {beta}-cell proportions with contaminant non-endocrine and proliferative cell types. To date, no facility-level implementation exists for producing SC-islets that can be shipped and benchmarked across multiple sites. Here, we describe the scalable optimisation, standardization, and facility-level implementation of an established human stem cell differentiation strategy that consistently results in a high proportion of {beta}-cells, with up to 75% of cells co-expressing C-peptide and the pancreatic endocrine marker, ISL1. Functionally, SC-islets exhibit glucose-responsive calcium influx and insulin secretion, recapitulating key physiological {beta}-cell functions. Single-cell transcriptomic profiling reveals a simplified endocrine landscape dominated by {beta}-cells, with a striking transcriptional similarity to human primary {beta}-cells (Pearsons r2[~]0.9). We observe smaller fractions of - and enterochromaffin-like cells with very low levels of poly-hormonal or proliferating cell types (<3%). Taken together, we provide a well-defined, reproducible and accessible in vitro SC-islet platform benchmarked for functionality at multiple recipient sites.

BackgroundInduced pluripotent stem cells (iPSCs) are an important model for studying human diseases in vitro. However, previous studies have shown that iPSC reprogramming and extended cell culture can introduce genomic structural variants (SVs). Technologies like karyotyping, CNV microarrays, and whole-genome sequencing have limitations in resolution, sensitivity, or the ability to detect large and complex structural variants compared to optical genome mapping (OGM). OGM is a genome-wide structural variant detection method that analyzes fluorescently labeled ultra-high-molecular-weight DNA molecules to identify copy-number and balanced rearrangements. At sufficient coverage, OGM can detect SVs at approximately [&ge;]2 kbp and identify mosaic events supported by molecule-level evidence, offering higher resolution than conventional karyotyping or SNP-array-based QC. Here, we compared iPSC clones derived from peripheral blood mononuclear cells (PBMCs) and fibroblasts (FBCs) to determine whether starting somatic cell source is associated with differences in structural variant burden and SV-type profiles after nuclear reprogramming into iPSCs. ResultsWe analyzed 73 low-passage iPSC clones generated from 25 parental lines using OGM. Compared with PBMC-iPSCs, FBC-iPSCs showed higher SV burden with the enrichment of duplications [&ge;]100 kbp, more frequent overlap with protein-coding genes, fragile sites, and recurrent chromosomal hotspot regions. In contrast, PBMC-iPSCs showed fewer SVs overall, and a higher proportion of clones without detectable clone-specific SVs. ConclusionsOGM provides a high-resolution approach for post-reprogramming genomic quality control by detecting clone-specific structural variants at approximately [&ge;]2 kbp, including events below the resolution of conventional cytogenetic and SNP-array-based assays. In these early passage iPSCs, SVs overlapped protein-coding genes, fragile sites, and recurrent culture-associated chromosomal regions, underscoring the need for clone-level genomic assessment before downstream applications. FBC-derived iPSCs showed a higher SV burden, including more frequent and larger duplications, whereas PBMC-derived iPSCs more often lacked detectable clone-specific SVs. These findings suggest that PBMC-iPSCs and FBC-iPSCs can differ in post-reprogramming SV profiles and support the use of OGM as a QC strategy during iPSC generation and selection.

The ability to revascularize target tissues and organs through cell-based therapy would provide a novel approach for the treatment of a range of ischemic disorders including cardiovascular diseases, stroke and peripheral artery disease. Towards this goal, we have identified a human pluripotent stem cell (hPSC)-derived vascular progenitor (VP) population generated via an epicardial intermediate with functional engraftment properties. VP cells efficiently engraft the mammary fat pad and hind limb skeletal muscle of NSG recipient mice and form vessel-like structures that integrate with the host vasculature. In an ischemic hind limb mouse model, VPs generate extensive vascular grafts that improve perfusion, restore some function and preserve muscle integrity over a three-month period post-transplant. Single-cell transcriptomic and flow cytometric analyses show that the VP population, initially identified by the co-expression of CD140b, CD13 and KDR, displays an epicardial lineage signature and expresses a spectrum of genes and proteins indicative of vascular progenitor stage cells. Together, these findings demonstrate that it is possible to revascularize both normal and ischemic tissue through the transplantation of an appropriate hPSC-derived progenitor and in doing so, lay the foundation for developing cell-based therapy approaches to treat ischemic diseases. Graphical Abstract LegendHuman pluripotent stem cells are differentiated through an epicardial intermediate to generate vascular progenitor (VP) cells characterized by expression of CD140b, CD13 and KDR. These VP cells demonstrate the capacity to engraft both mammary fat pad and skeletal muscle tissue where they form stable perfused vascular networks. In a hindlimb ischemia model, VP cell transplantation restores blood flow and improves functional outcomes. eTOC BlurbFernandes et al. develop a protocol to generate engraftable vascular progenitors from human pluripotent stem cells through an epicardial intermediate. These cells form functional vessels in vivo, restore perfusion in ischemic tissue, and demonstrate tissue-specific adaptation while maintaining endothelial identity, providing a foundation for therapeutic revascularization. HighlightsO_LIA staged differentiation protocol generates vascular progenitors (VPs) from hPSCs via an epicardial intermediate. C_LIO_LIVP cells form stable, perfused vascular networks following transplantation into multiple tissue sites. C_LIO_LIVP cell therapy with or without VEGF nanoparticles restores perfusion and improves functional outcomes in hindlimb ischemia. C_LIO_LISingle-cell analysis reveals tissue-specific adaptation while maintaining endothelial identity. C_LI

Leukemia inhibitory factor (LIF)/STAT3 signaling is central to maintaining naive pluripotency in mouse embryonic stem cells (mESCs). We identify Asgard, a previously uncharacterized long non-coding RNA, as a direct STAT3 target required for efficient self-renewal. Asgard is rapidly induced by LIF, enriched in the epiblast, and its depletion reduces alkaline phosphatase-positive colony formation while enhancing differentiation. Mechanistically, Asgard fulfils a dual role: it acts as the primary transcript for the differentiation-promoting microRNA Odin, while also functioning as a sponge to sequester Odin and related miRNAs. This dual mechanism enables Asgard to both generate and buffer pro-differentiation signals, thereby stabilizing the pluripotent state while preserving responsiveness to lineage cues. Our work reveals a new paradigm in RNA-mediated control of stem cell identity, where a single STAT3-regulated lncRNA couples microRNA production with competitive inhibition to safeguard naive pluripotency.

Induced pluripotent stem cells (iPSCs) are widely used as patient-specific disease models, yet substantial unexplained variability in molecular and functional readouts limits their reliability. Here, we systematically investigated the sources of variation in iPSC-derived neurons for three rare genetic disorders: Myotonic Dystrophy Type 1, chromodomain-DNA-helicase-binding protein 2-related disorder and N-acetylneuraminic acid synthase deficiency. This was performed by profiling multi-omics layers: genomics, epigenomics, transcriptomics, proteomics, metabolomics and lipidomics. Our study found that clonal variability was comparable to inter-patient differences and that neuronal differentiation state and nutrient-driven metabolic activity emerged as dominant contributors to variability observed across omics layers. Clonal differences could partly be attributed to stochastic differences in DNA methylation established during reprogramming. By modeling and correcting the observed variation, we improved the detection of disease-associated molecular signatures. Our study provides guidelines for improved study design and data analysis to minimize variability, enabling robust biomarker discovery and reliable iPSC-based disease modeling.

Human pluripotent stem cell (hPSC)-derived therapies are advancing rapidly toward clinical application, yet heterogeneity of transplanted cell populations remains a major barrier to safety, predictability and scalability. Existing strategies to mitigate this risk either incompletely eliminate proliferative cells or ablate the entire graft, thereby compromising therapeutic benefit. Here we present NeuroGuard, a lineage-selective suicide gene platform that decouples safety from efficacy by preserving functional neurons while enabling inducible elimination of all other cell types after transplantation. NeuroGuard integrates an inducible caspase-9 system with NEUROD1-driven Cre recombination, protecting post-mitotic neurons from apoptosis while rendering non-neuronal and proliferative populations susceptible to ablation. In vitro, activation of the system enriched neuronal content to >90% and increased dopaminergic neuron proportion >3-fold. Following transplantation of ventral midbrain progenitors, timed activation eliminated proliferative and glial populations, resulting in compact, neuron-enriched grafts without loss of dopaminergic neuron number, target innervation or behavioural recovery in Parkinsonian rodents. Single-cell transcriptomics confirmed selective removal of non-neuronal lineages while preserving neuronal identity and maturation programs. This work establishes a generalizable framework for post-engraftment editing of cell therapy composition, providing a versatile strategy to enhance the safety and functional predictability of regenerative therapies.

Axonal degeneration and insufficient neuronal survival remain major barriers to central nervous system repair. Stem cells from human exfoliated deciduous teeth (SHED) represent an accessible, developmentally immature, neural crest-derived mesenchymal stem cell population with potential relevance for neuroregenerative medicine. Here, we show that SHED display enhanced proliferative stability, preserved mesenchymal identity, and more sustained expansion capacity than adult dental pulp stem cells, supporting their suitability for scalable regenerative applications. Using embryonic chick retinal explants at neurogenic and post-neurogenic stages, we demonstrate that SHED robustly promote retinal ganglion cell axonogenesis, axonal regeneration, and neuronal survival. At embryonic day 5, SHED enhanced axonal outgrowth in both newly generated EdU/TUJ1 neurons and pre-existing EdU-/TUJ1 retinal ganglion cells. At embryonic day 13, when retinal neurons are post-mitotic and intrinsically less regenerative, SHED still significantly increased regenerative axonal extension and reduced developmental cell death. To investigate the molecular mechanisms underlying the neuroprotective and axogenic effects of SHED, proteomic profiling of SHED-retina co-culture secretomes was performed, revealing a highly enriched extracellular environment containing matrix-associated and neurodevelopmental proteins, including thrombospondin-1 (THBS1), galectin1 and 3, and multiple proteins associated with IGF2 pathway. Proteomic analysis of the SHED secretome, together with prior evidence implicating thrombospondin signaling in neuronal development and synaptogenesis, identified THBS1 as a strong candidate mediator of SHED-induced effects in chick retinal co-culture systems. Neutralization of THBS1, particularly in combination with gabapentin-mediated blockade of 2{delta}-1-dependent thrombospondin signaling, markedly reduced SHED-induced axonal growth and induced neuritic swellings consistent with impaired axonal integrity. In contrast, inhibition of THBS1 signaling did not significantly abolish the neuroprotective effect of SHED on neuronal survival, suggesting that distinct paracrine mechanisms independently regulate axonal regeneration and cell survival. Together, these findings demonstrate that SHED-derived combined secreted factors promote neuronal survival and axonal regeneration through partially divergent extracellular matrix-associated developmental pathways, positioning SHED and their secretome as promising candidates for cell-based and cell-free neuroregenerative strategies.

Hematopoietic stem cells (HSCs) generated from induced pluripotent stem cells (iPSCs) offer a promising patient-specific alternative to allogeneic transplantation, yet current differentiation protocols fail to fully recapitulate in vivo HSC maturation. During mouse development, yolk sac (YS)-derived macrophages populate the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence, but the mechanisms by which they support ex vivo hematopoietic stem and progenitor cell (HSPC) generation remain poorly defined. Bulk RNA sequencing revealed that mature AGM CD206 macrophages upregulate pro-inflammatory cytokines and the adhesion molecule F4/80. Using F4/80 knockout embryos, we identify a previously unreported, niche-specific role for F4/80 in restraining the frequency and colony-forming activity of HSPC subsets in the AGM, while supporting endothelial cell maintenance; this effect was absent in the YS. Lineage-tracing with a Cdh5-CreERT2;Rosa26LSL-tdTomato pulse-chase system confirmed that both CD206 and CD206- AGM cells originate from early YS-derived endothelial precursors, with no evidence of local macrophage generation within the AGM. Functional co-culture assays further demonstrated that the ability of CD206 macrophages to enhance the progenitor potential of hemogenic endothelium is AGM-specific and not an intrinsic, ontogeny-determined property, as YS macrophages failed to confer the same benefit even when paired with AGM endothelial cells, and AGM macrophages were ineffective with YS endothelium. Differential expression and NicheNet ligand-receptor interaction analyses identified a small set of AGM-restricted macrophage genes - including Mmp2, Nrep, Ccl2, and Cxcl16 - which are predicted to interact with both endothelial and cluster cells during endothelial-to-hematopoietic transition. Together, these findings establish that AGM macrophages acquire niche-specific transcriptional and functional properties upon entry into the aortic microenvironment, independent of their YS origin, and identify candidate macrophage-derived factors and a novel regulatory role for F4/80 in shaping HSPC output. These insights may guide the refinement of iPSC-based HSC differentiation protocols through the targeted, temporally controlled addition of macrophage-associated signals.

Astrocytes play essential roles in neuronal development, function, and disease, yet existing methods to derive astrocytes from human pluripotent stem cells (hPSCs) are complex and can involve months of in vitro maturation. We developed a genomic safe-harbor knock-in system for inducible expression of the astrogenic transcription factors NFIA, NFIB, and SOX9, enabling rapid and robust generation of functional induced astrocytes (iAstrocytes). Across five hPSC lines, NFIB-SOX9 and NFIA-NFIB-SOX9 combinations efficiently generated highly pure populations expressing astrocyte-specific and synaptogenic genes. iAstrocytes displayed cytokine-induced expression of complement factors C3 and C4 and were amenable to CRISPR interference (CRISPRi) gene expression knockdown. Optimization of culture conditions enabled survival of NFIB-SOX9 iAstrocytes in co-culture with human induced neurons (iNeurons). Through pharmacological and genetic perturbations, we uncovered a previously undescribed phenomenon in which co-culture with iAstrocytes promoted the development of synchronized iNeuron network calcium activity mediated by specific gap junction proteins. This rapid and genetically tractable iAstrocyte platform provides a robust model to dissect human genetic and environmental effects on astrocyte-neuron interactions.