Stem Cell Reports

Myotonic dystrophy type 1 (DM1) is caused by an expansion of a CTG repeat in the 3' untranslated region of the DMPK gene, leading to accumulation of toxic CUG-repeat RNAs, sequestration of MBNL proteins and widespread splicing dysregulation. Congenital DM1 (CDM), the most severe form of the disease, is associated with profound muscular and neurodevelopmental defects, yet the mechanisms underlying early human brain involvement remain poorly understood. Here, we generated cortical organoids from patient-derived pluripotent stem cells carrying >1000 CTG repeats, an expansion typically associated with CDM, to model early human neurodevelopment. DM1 molecular and cellular hallmarks were detected at early developmental stages, including nuclear DMPK RNA foci in neural progenitor cells and reduced proliferative capacity. As organoids matured, CDM cultures displayed altered cortical composition, with reduced CTIP2 and SATB2 neuronal populations and increased NFIA/GFAP glial cells. In parallel, 120-day-old organoids recapitulated splicing abnormalities previously identified in DM1 patient brain tissue. To assess the contribution of MBNL dysfunction, we analyzed cortical organoids derived from MBNL2 and MBNL1/2/3 knockout induced pluripotent stem cells, which reproduced key neurodevelopmental phenotypes observed in CDM organoids, supporting a central role for MBNL loss of function in impaired corticogenesis. Finally, we evaluated the translational relevance of this model using tideglusib and erythromycin, two compounds currently under clinical evaluation in DM1 patients. Both treatments reduced DMPK RNA foci and restored proliferation defects in SOX2 neural progenitors. Together, these findings establish cortical organoids as a robust human model of CDM-associated neurodevelopmental defects, uncover MBNL-dependent mechanisms underlying early corticogenesis impairment and demonstrate the utility of this platform for translational therapeutic discovery in DM1.

Embryo polarization is critical for the first cell fate segregation. While mechanisms underlying its initiation have been described, the intrinsic signaling pathways that regulate this process remain poorly understood. Here, we show that mouse embryonic stem cells, when aggregated under defined conditions, recapitulate the first lineage segregation to generate trophectoderm (TE)-like cell populations and undergo self-organized morphogenesis into blastocyst-like structures. In the blastoid-forming medium, we identify CHIR99021 is essential for the generation of blastoids from both ESCs and totipotent-like cells. CHIR99021 promotes cell polarization and TE differentiation by activating the WNT/{beta}-catenin pathway and upregulating associated genes. Consistent with this, genetic ablation of {beta}-catenin abolished the cell polarization and disrupted blastoid formation from ESCs, a defect that was restored by {beta}-catenin overexpression. Moreover, {beta}-catenin depletion compromised cell polarization in natural embryos. Collectively, this study establishes the Wnt/{beta}-catenin as a critical regulator initiating polarization in vitro and in mouse early embryo development.

Human pluripotent stem cells serve as a vital model for studying early human lineage specification, yet conventional assessments relying on endpoint canonical markers of the three germ layers may overlook transient intermediate states and broader cellular programs. Here we combined directed differentiation of human induced pluripotent stem cells toward neuroectodermal, cardiac mesodermal, and hepatic endodermal lineages with comparative transcriptomic profiling across timepoints. Our analyses revealed a transient primitive streak-like mesendodermal state shared by mesodermal and endodermal trajectories, followed by lineage-specific divergence characterized by distinct transcriptional, metabolic, proliferative, and chromatin remodeling dynamics. Notably, endodermal differentiation exhibited rapid definitive endoderm commitment with enriched oxidative metabolism, whereas cardiac mesoderm differentiation showed progressive transcriptional remodeling and cardiac progenitor activation. These findings demonstrate that comparative transcriptomics can resolve developmental intermediates and cellular-state dynamics during human germ layer specification, providing a framework for evaluating lineage commitment beyond endpoint canonical marker expression, and to inform strategies for optimizing or redirecting differentiation.

We investigated the role of Netrin-1 (NTN1) in human naive pluripotency using complementary loss- and gain-of-function approaches. In primate embryos and human embryonic stem cells (hESCs), Netrin-1 expression is associated with the naive pluripotent state. Disruption of NTN1 had no detectable effect on hESCs maintained on murine embryonic fibroblasts. However, under sub-optimal culture conditions, NTN1-knockout cells exhibited compromised naive pluripotency, which was rescued with feeder cells overexpressing Netrin-1. Netrin-1 overexpression in hESCs accelerated acquisition of the naive state and markedly increased resistance to differentiation. These effects were accompanied by extensive epigenetic remodeling, including H3K27ac and H2K27me3. Proteomic and phospho-proteomic analyses further revealed rapid Netrin-1-dependent alterations in pathways controlling cell adhesion, signaling, and chromatin regulation. Together, these findings extend the role of Netrin-1 beyond its established functions and identify it as a coordinator of extracellular cues, intracellular signaling, and nuclear regulatory mechanisms that support human naive pluripotency.

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.

This study examined the role of ST3GAL3 as a regulator of excitatory/inhibitory (E/I) synaptic homeostasis using a human iPSC-based model. Neurodevelopmental disorders (NDDs) are increasingly linked to disruptions in the E/I balance, yet the molecular determinants remain poorly defined. ST3GAL3, a sialyltransferase associated with both rare monogenic disorders, including intellectual disability and infantile epilepsy, and complex polygenic conditions, such as ADHD, represents a strong candidate gene for involvement in synaptic regulation. To investigate this, isogenic ST3GAL3 knockout (ST3GAL3 KO) and wildtype (WT) iPSC lines were generated through CRISPR/Cas9 editing and differentiated into cortical neurons using both directed and induced protocols. This dual strategy enabled robust comparisons across cellular contexts and minimised methodological bias. To this end, we conducted functional characterisation using microelectrode array (MEA) technology alongside transcriptomic profiling through RNA sequencing (RNAseq), directly comparing ST3GAL3 KO-derived neurons with their isogenic controls. Functional assays using MEA revealed aberrant bursting patterns, particularly prolonged burst durations and heightened variability in S3GAL3KO neurons. Complementary transcriptomic profiling performed via RNAseq demonstrated downregulation in ST3GAL3 KO lines of genes involved in cognition, memory, as well as glutamatergic and GABAergic synaptic plasticity and functionality, providing molecular evidence for widespread synaptic dysregulation. Together, these findings establish ST3GAL3 as a key regulator of E/I balance in the cortices, advancing current knowledge on the pathophysiological involvement of ST3GAL3 deficiencies in the development of NDDs.

Induced pluripotent stem cells (iPSCs) hold great promise for both allogeneic and autologous cellular therapies. However, broad application and clinical translation is hindered by fragmented, complex and time-intensive workflows, resulting in high manufacturing costs, poor standardization and increased risk of genomic aberrations in derived iPSCs. In this study we developed a standardizable, automatable and time- efficient process for the derivation of monoclonal iPSC lines straight from skin including a comprehensive and cascaded OC strategy. We generated monoclonal iPSC lines derived from human skin punch biopsies of ten donors (age 49-81) via mRNA-based reprogramming that subsequently underwent comprehensive and thorough characterization of phenotypic and genetic properties. The use of a combined mechanical and enzymatic fibroblast isolation protocol and a transient non-integrative reprogramming technology allowed us to obtain 78 monoclonal iPSC lines, ready for banking, molecular characterization and further differentiation within seven weeks from initial sample processing to passage four iPSC lines. The phenotypical characterization via flow cytometry-based pluripotency marker expression and 2D-directed differentiation into the three germ layers showed low intra- and inter-donor variability over all generated lines. A combination of SNP array based CNV analysis followed by whole exome sequencing proved to be the most efficient approach for assessment of genomic integrity. Proof-of-concept experiments for closed system processing revealed that a substantial part of the most error-prone and technically demanding steps can be transferred to semi- automated, closed systems. In conclusion, the described protocol allows for time- efficient, standardizable and automatable generation of high-quality monoclonal iPSC lines from human skin punch biopsies within seven weeks, thus moving the field of autologous iPSC manufacturing one step further towards cost-efficient clinical implementation.

Immune checkpoint inhibitor-associated myocarditis (ICIM) is an uncommon but potentially fatal inflammatory heart disease triggered by cancer immunotherapy, with up to 40% mortality. The underlying mechanisms are still elusive, partly due to the lack of appropriate human disease models. Here, we report the generation of three induced pluripotent stem cell (iPSC) lines derived from an ICIM patient, an ICI-treated patient without myocarditis, and a healthy donor. These lines exhibit typical pluripotent stem cell morphology, express pluripotency markers, maintain normal karyotypes, and differentiate into derivatives of the three germ layers, providing a valuable platform for mechanistic studies and therapeutic discovery.

SYNGAP1 developmental and epileptic encephalopathy (DEE) is a severe neurodevelopmental disorder characterised by intellectual disability, developmental delay, and refractory epilepsy caused by heterozygous variants in SYNGAP1, which encodes Synaptic Ras GTPase-activating protein 1. While SYNGAP1 is best known for its role at the postsynaptic density, increasing evidence indicates that haploinsufficiency also disrupts early neurodevelopment. Here, we used patient-derived induced pluripotent stem cell (iPSC) models to investigate early neurodevelopmental and neuronal phenotypes associated with SYNGAP1 haploinsufficiency. iPSCs derived from a female patient carrying the frameshift variant p.Leu150Valfs*6 were differentiated into two complementary models: micropatterned neural rosettes representing early neuroepithelial organisation and NGN2-induced excitatory neurons representing postmitotic functional development. Patient-derived neural rosettes displayed enlarged, dysmorphic lumens, indicating disrupted neuroepithelial organisation at the earliest stages of brain development. Transcriptomic profiling revealed widespread dysregulation of genes involved in neurodevelopment, cell adhesion and ion channel regulation, including coordinated downregulation of protocadherin family members. Whole-cell patch-clamp electrophysiology demonstrated reduced input resistance, larger action potential amplitudes, and increased inward and outward current densities, consistent with accelerated intrinsic neuronal maturation rather than generalized hyperexcitability. Together, these complementary findings demonstrate that SYNGAP1 haploinsufficiency disrupts early human brain development and accelerates intrinsic neuronal maturation, with pathogenic mechanisms emerging before synaptogenesis and extending beyond SYNGAP1s established synaptic role.

Hematopoietic stem cells(HSCs) maintain their pools by stem-stem division and produce mature blood cells through stem-progenitor or progenitor-progenitor division. A paired daughter cell(PDC) assay combined with single cell transplantation is a powerful method to compare the lineage outputs of two HSC daughter cells. However, single-cell transplantation precludes large-scale analysis of daughter-cell pairs, as only one cell can be transplanted per recipient. Here, we developed a DNA barcoding-based PDC assay to overcome this limitation, enabling simultaneous analysis of 476 daughter pairs from individual HSC divisions and revealing that daughter-cell fates are coordinated and that HSC division patterns are biased toward stem-stem and progenitor-progenitor divisions rather than stem-progenitor divisions. These findings indicate that HSC fate outcomes are directed toward symmetric division outcomes. Integration of single-cell RNA sequencing with DNA barcoding revealed a continuum of HSC states--from balanced HSCs to myeloid-biased HSCs and ultimately to a low-output HSC subset--in which progressively reduced production of mature blood cells relative to stem cell expansion, a proxy for stem-stem division bias, exhibits distinct activities of transcription factors and signaling pathways. Overall, our analysis uncovers characteristic patterns of HSC division and links stem maintenance with distinct molecular features.

Myotonic Dystrophy type 1 (DM1) is an autosomal multisystem disorder manifested due to unstable CTG nucleotide repeat expansion within the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Although progress towards understanding of molecular pathogenesis in muscle and heart has been made, the pathways that affect the brain in DM1 is fundamentally unknown. In addition, the congenital DM1 manifest even more complicated brain abnormalities. Despite the wealth of existing cellular and animal models, iPSCs based studies are being fostered as they replicate the human model more closely to the disease. In view of this context, we set out to characterize the differentiation potential of congenital DM1 patient derived iPSC lines towards neuronal cells. Using neurogenin2 (NGN2) induced direct reprogramming of iPSCs into neurons and chemically defined media-induced neural induction protocol, we find that congenital DM1 mutant iPSC derived neurons exhibited precocious differentiation, as evidenced by their expression of pan-neuronal markers TUJ1 and Map2, along with increased processes extension and neurite length. Moreover, unbiased RNA sequencing analyses and qPCR validation revealed precocious and enhanced expression of several neurogenic transcription factors including, Ascl1, NeuroG2, and NeuroD1. Furthermore, immunofluorescence imaging of MBNL1 and MBNL2, RNA-splicing factors, displayed enhanced nuclear aggregations, a hallmark of the DM1 disease, in the mutant lines. Moreover, investigation of RNA splicing events identified mis-splicing in many important genes/transcripts including RMST, ANK3 and MBD1 during the neural conversion of congenital DM1 lines. These studies reveal novel paradigms that may contribute to neurological pathogenesis in CDM1 patients. These studies also provide a strong foundation for future mechanistic investigation aimed at understanding CDM1 pathology and may open new avenues for the development of gene therapy approaches for individuals with DM1.

Stem cell therapy represents a promising strategy for the replacement and functional restoration of damaged neural tissue in neurodegenerative conditions. Human dental pulp stem cells (hDPSCs) have emerged as potential candidates for neuroregeneration due to their ease of isolation, neural crest origin, neurotrophic and anti-inflammatory capacity, and demonstrated ability to differentiate in vitro into neuronal-like cells exhibiting electrophysiological activity. Although the immunomodulatory and neuroprotective properties of hDPSCs have been reported in multiple models of brain disease, their capacity to functionally integrate into host neuronal circuits remain poorly understood. In this study, we have grafted green fluorescent protein (GFP)-transduced, neural preconditioned hDPSCs into the CA1 region of the hippocampus of C57BL/6J mice. One month after transplantation, GFP+-hDPSCs survived in the brains of non-immunosuppressed mice and remained localized within the grafted area. Notably, the transplanted cells underwent in situ differentiation and exhibited a neuroblast-like phenotype, characterized by positive doublecortin expression and immature neuronal-like electrophysiological properties, like high membrane input resistance, low capacitance, and the ability to generate single action potentials after stimulation. Together, these findings provide the first evidence that hDPSCs can survive and integrate into the hippocampal network of the mouse brain at one-month post graft, supporting their potential use for future therapeutic applications in acute brain lesions and neurodegenerative disorders.

The maintenance of pluripotent stem cells (PSCs) under rapid proliferation requires mechanisms that both suppress replication-driven genome instability and preserve self-renewal capacity. Here, we show that, in contrast to somatic cells where it mainly acts as a backup, the Holliday junction resolvase GEN1 is required in mouse embryonic stem cells (ESCs), where its depletion severely compromises self-renewal and long-term maintenance. Loss of GEN1 induces the accumulation of cells with DNA content greater than 4C and chromosome fusions. Notably, a catalytically inactive GEN1 mutant rescues ESC colony formation, indicating that GEN1 supports ESC maintenance through non-enzymatic functions. In addition, GEN1 depletion increases ESC tolerance to topoisomerase I-mediated replication stress and renders this phenotype dependent on DNA-PK activity, suggesting that GEN1 loss alters how pluripotent cells cope with replication-associated DNA lesions. Together, these findings identify GEN1 as a non-redundant guardian of genome integrity in pluripotent cells, revealing both a catalysis-independent role in self-renewal and a contribution to the replication stress response, with implications for PSC genomic quality control. HighlightsO_LIIn contrast to somatic cells, GEN1 is specifically required for mouse pluripotent cell self-renewal and expansion in vitro. C_LIO_LIGEN1 loss induces accumulation of DNA content greater than 4C and chromosome fusions without loss of core pluripotency markers expression. C_LIO_LICatalytically inactive GEN1 mutant rescues ESC colony-forming capacity. C_LIO_LIGEN1 depletion increases ESC tolerance to topoisomerase I-mediated replication stress in a DNA-PK-dependent manner C_LI eTOCRamos-Lage et al. demonstrate that the resolvase GEN1 is essential for mouse embryonic stem cell self-renewal and genome stability. Strikingly, a catalytically dead mutant rescues colony formation, revealing an unexpected non-enzymatic role for GEN1 in pluripotency maintenance.

Hirschsprung disease (HSCR) is a rare congenital disorder of the enteric nervous system (ENS), marked by the absence of enteric ganglia along variable lengths of the distal gastrointestinal tract, resulting in functional intestinal obstruction. Individuals with Trisomy 21 (Down syndrome) face a 50- to 100-fold increased risk of HSCR relative to the general population, yet the molecular basis of this susceptibility remains poorly understood. Here, we investigated this association using isogenic induced pluripotent stem cells (iPSCs) derived from a mosaic individual with Down syndrome, enabling direct comparison of Trisomy 21 and Disomy 21 cells within an identical genetic background following differentiation into enteric neural crest-derived cells (ENCDCs). Trisomy 21 ENCDCs exhibited reduced proliferative and migratory capacity and an impaired ability to differentiate into enteric neurons relative to Disomy 21 controls. These phenotypes were accompanied by decreased RET expression at both the transcript and protein levels, together with broad downregulation of the RET gene regulatory network, including GDNF, GFRA1, EDNRB, SEMA3C, and NRG1, and of cell cycle and DNA replication pathways. Strikingly, we identified SOD1, a chromosome 21-encoded antioxidant enzyme not previously linked to RET regulation, as a dosage-sensitive driver of this effect: SOD1 overexpression in disomic ENCDCs was sufficient to suppress RET, whereas shRNA-mediated knockdown in Trisomy 21 ENCDCs restored RET expression. Mechanistically, Trisomy 21 ENCDCs displayed markedly elevated catalase and a redox imbalance, and exogenous hydrogen peroxide recapitulated RET suppression in disomic cells, implicating oxidative stress as a mediator of RET downregulation. Collectively, these findings establish Trisomy 21 dosage effects as disruptors of RET-dependent enteric neural crest development and implicate SOD1-driven oxidative stress as a candidate mechanism, providing a framework for understanding the elevated risk of HSCR in Down syndrome.

Proper establishment of the primitive placenta and subsequent tissue homeostasis in the mature placenta are critical for successful pregnancy in humans. Placental insufficiency is associated with adverse pregnancy outcomes, including fetal growth restriction, preeclampsia, and pregnancy loss. Moreover, emerging evidence suggests that placental defects are associated with long-term health challenges that manifest well into adulthood; yet the etiologies of such diseases are largely unknown. Defining the mechanistic basis for placental deficiencies, therefore, has important implications for improving both reproductive health and the lifelong well-being of affected children. Down syndrome is characterized by placental defects of unknown mechanistic origin, and notably, individuals with Down syndrome are at increased risk of developing diseases commonly associated with placental insufficiency later in life. Using induced pluripotent stem cells from Down syndrome patients, we found that stem cell-based embryo models (i.e., blastoids) and directed differentiation systems recapitulate trophoblast cell fate defects observed in placentas affected by Down syndrome. Furthermore, we demonstrate that attenuated estrogen signaling contributes to placental syncytialization defects and identify NRIP1, a transcriptional corepressor of estrogen receptor that is located on chromosome 21, as a key driver of trophoblast cell fate defects. Increased gene dosage of NRIP1 in an otherwise diploid cell line phenocopies cell fate defects observed in trophoblasts affected by Down syndrome. Our study suggests that estrogen signaling is a crucial regulator of trophoblast development and may serve as a potential target for therapeutic intervention. Highlights and eTOC blurbO_LIEstrogen signaling mediates syncytiotrophoblast fusion C_LIO_LIHuman iPS cells provide a tractable model for trophoblast defects in Down syndrome C_LIO_LITrophoblast differentiation and estrogen signaling are disrupted in Down syndrome C_LIO_LIIncreased NRIP1 expression is sufficient to induce trophoblast defects C_LI Logsdon and colleagues apply patient-derived induced pluripotent stem cells to recapitulate placentation defects observed in Down syndrome. The authors demonstrate that attenuated estrogen signaling disrupts trophoblast differentiation and identify NRIP1, a gene found on chromosome 21 that dampens estrogen signaling, as a regulator of trophoblast maturation. NRIP1 and estrogen signaling may represent important therapeutic targets for infertility and Down syndrome.

Cell replacement represents a potential treatment modality for retinal disorders characterized by photoreceptor loss. However, photoreceptor replacement approaches have not been clinically established. To take this forward, the main goal of this study was to systematically compare human photoreceptors of different ages and identify those that enable functional integration into the degenerative retina. Donor cells were isolated from iPSC-derived retinal organoids generated by a GMP-compliant protocol at differentiation days 120, 150, or 200 and transplanted subretinally into cone photoreceptor function loss 1 (Cpfl1) recipients, an inherited mouse model of cone degeneration. While younger photoreceptors showed slightly improved transplantation outcomes, donor photoreceptors of all culture stages displayed long-term survival, cone identity, structural integration into the host retina, and tight interactions with host Mueller glia, including formation of a continuous outer limiting membrane. Transplanted photoreceptors showed signs of advanced maturation, including correct polarization with generation of apical inner- and outer segments, while basal synapses were formed with host bipolar cells. Electrophysiological assessment of host retinal ganglion cells revealed light-evoked responses in transplant-containing regions, providing evidence for functional incorporation of human photoreceptors into the mouse neuro-retinal circuitry. Thus, GMP-compliant human iPSC-derived photoreceptors are stable over a wide range of differentiation stages and constitute a robust cell source for retinal transplantation and functional repair. The findings provide important prerequisites for the development of standardized procedures towards clinical translation of photoreceptor replacement in the retina.

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.

Hematopoietic stem cell (HSC) function declines with age, contributing to immunosenescence and inferior transplantation outcomes. Here, we generated iPSC-derived HSCs (iHSCs) from multiple adult donors and performed integrated epigenetic, transcriptional, telomeric, and functional analyses to see if they retain youthful identity across differentiation and serial transplantation. Longitudinal DNA methylation profiling revealed that, independent of donor age, epigenetic age was reset to near zero in iPSCs and remained under seven years across differentiation and transplantation. In contrast, hematopoietic identity was established through a two-phase process: directional remodeling during in vitro differentiation extinguished pluripotency programs and initiated hematopoietic regulatory networks, while long-term engraftment was associated with a second wave of promoter methylation differences that converged toward primary adult HSCs. Notably, methylation at age-associated sites and global entropy remained stable across both phases, and single-cell telomere analysis demonstrated restoration of telomere length in iHSCs compared to primary adult HSCs. Youthful epigenetic features were maintained through secondary transplantation. These findings demonstrate that long-term HSC identity can be achieved independently of epigenetic aging and establish a framework for evaluating rejuvenated stem cell-derived grafts in regenerative medicine.

STRUCTURED ABSTRACT Purpose of Research: The generation of iPSC lines expressing 21, 56 and 79 glutamine repeats within the HTT protein and homozygous KO of HTT in the KOLF2.1J background as an additional disease series within the iPSC Neurodegenerative Disease Initiative (iNDI) collection. Major Findings: All iPSCs, even those expressing long repeats of 79Q or HTT KO, were capable of differentiating to striatal and cortical neurons, astrocytes and microglia using established protocols. General quality control stains and morphological analyses are described for each differentiation. A selected set of assays were carried out on differentiated cells; expanded repeat expressing astrocytes showed altered expression of astrocyte protein markers and morphological characteristics, and striatal neurons showed altered DARPP-32/CTIP2 colocalization. mRNAseq carried out for striatal neurons showed high similarities in gene expression changes between 79Q and KO lines compared to the unexpanded repeat. Conclusions: The KOLF2.1J isogenic CAG repeat series serves as a community resource to study HD mechanisms with the potential for direct comparison across other neurodegenerative diseases through the iNDI collection.

Immune checkpoint inhibitor-induced myocarditis (ICIM) is a severe immune-related adverse event with heterogeneous clinical presentations and potential genetic susceptibility. Here, we established a human induced pluripotent stem cell (iPSC) line from an ICIM patient with an HLA-type distinct from previously reported line, who developed concurrent type I diabetes following ICI treatment. This line exhibited typical morphology, normal female karyotype, pluripotency, trilineage differentiation into all three germ layers, Sendai virus clearance, and no mycoplasma contamination. Given the fulminant nature and diverse clinical presentations of ICIM, expanding the repertoire of iPSC lines are critical for investigating ICIM heterogeneity and its underlying mechanisms.