Stem Cells

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.

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.

Satellite Cells (SCs) and Fibro-Adipogenic Progenitors (FAPs) are muscle-resident cell populations crucial for maintaining skeletal muscle homeostasis and coordinating regeneration after injuries. However, primary human SCs and FAPs are difficult to co-isolate, and their broad use in translational research has been limited by a lack of standardized biobanking protocols. Recently, we published a protocol for efficient co-isolation of SCs and FAPs from human skeletal muscle. Here, we extend those efforts to establish a comprehensive pipeline for the cryopreservation, cold-chain transport, and independent-site utilization of human SCs and FAPs. Cells taken through this pipeline maintained lineage-specific markers, including Pax7, MyoD and CD56 for SCs, and PDGFR and TE7 for FAPs, indicating retention of their pre-biobanking phenotype. Furthermore, SCs demonstrate robust myogenic differentiation capacity, and FAPs demonstrate both fibrogenic and adipogenic differentiation capacity post-transport. Finally, previously biobanked SCs were incorporated into in vitro 3D muscle constructs, demonstrating their utility for human-based New Approach Methodologies (NAMs). This framework for multi-site collaboration facilitates broader access to human primary muscle cells, which will improve the scalability and translatability of human-based NAMs for skeletal muscle research.

Background: Human induced pluripotent stem cells (hiPSCs) hold promise for vascular regeneration, but preliminary research often relies on neonatal donors, whereas clinical applications will use cells derived from aged individuals. Although the impact of donor age on reprogramming efficiency has been studied, its effect on the functionality of hiPSC-derived endothelial progenitors (hiPSC-EPs) remains unclear. This question is the focus of the current study. Methods and Results: We derived EPs from iPSCs sourced from three neonatal donors (ND) and three mature donors (MD) matched 1:1 for sex and somatic cell origin. We assessed their functional, epigenetic, and transcriptomic characteristics. Despite higher CD34? yields from MD-iPSCs, MD-hiPSC-EPs formed poorly interconnected and non-lumenized vascular structures in 3D hydrogels, compared to neonatal donor (ND) lines. In 2D culture, MD-hiPSC-EPs exhibited reduced cell density and aberrant VE-Cadherin localization. DNA methylation analysis revealed that somatic cell origin was the dominant driver of variance, but consistent differences in methylation of mesoderm commitment, angiogenesis, ECM remodeling, and cytoskeleton-related genes were observed between age groups. Epigenetic age prediction showed MD-hiPSC-EPs had more developmentally advanced signatures, potentially explaining their shift away from vasculogenic competence. Our RNA-sequencing findings confirm trends seen in the DNA methylation data and show differential expression of pathways linked to mitochondrial regulation and nitric oxide signaling. Conclusions: Donor age significantly alters the vasculogenic function of hiPSC-EPs. These findings underscore the necessity of donor-specific considerations in hiPSC-based vascular engineering and highlight potential barriers to translating hiPSC-derived therapeutics into aged patient populations.

Background & Aims Dynamic cell cycle control is critical for intestinal crypt maintenance and injury responses, yet genetic regulators driving these changes remain poorly defined. As reserve intestinal stem cells (rISCs) are often considered to be slowly-cycling and can resist replication-dependent injury, factors that restrain proliferation may confer cytoprotection. Here, we define SOX9 as a regulator of intestinal stem cell (ISC) cycling and injury resistance. Methods Primary human ISCs were engineered to tune SOX9 levels, visualize cell cycle state, and manipulate cell cycle regulators. Using this system, we tested how SOX9 dosage impacts stemness, differentiation, proliferative recovery after SOX9 washout, and survival after 5-FU-mediated injury. Transcriptional analyses identified candidate links between SOX9 levels and cell cycle control, which were functionally tested using inducible INK4A (CDKN2A) and Cyclin D2 (CCND2) ISC lines. Results SOX9 induction lengthens the cell cycle in a dose-dependent manner largely by elongating G1 phase through the INK4A-Rb pathway. The effects of high SOX9 levels repressing proliferation and stem cell activity are reversible. SOX9 induction protects against 5-FU toxicity. This protection is mimicked by INK4A overexpression or pharmacological G1 phase arrest and repressed by CCND2 induction. Conclusions These findings identify SOX9-mediated G1 elongation as a reversible cytoprotective program that confers key functional properties associated with rISCs: proliferative restraint, retained stem cell potential, and resistance to replication-dependent injury. This positions G1 length as a potential determinant of which crypt cells survive injury to act as reserve stem cells.

BackgroundPremature ovarian insufficiency (POI) affects approximately 1% of women under 40 and is characterized by elevated levels of gonadotropins, reduced estradiol, impaired folliculogenesis, and infertility. Bone marrow-derived mesenchymal stem cell (BM-MSC)-based therapy has emerged as a promising regenerative strategy in preclinical POI models. This systematic review and meta-analysis evaluated BM-MSC-based interventions, including cell transplantation and secretome/extracellular vesicle administration, in animal models of POI. MethodsA systematic review and meta-analysis was conducted following PRISMA guidelines. PubMed, Web of Science, Scopus, ScienceDirect, and the Cochrane Library were searched from inception to February 19, 2025. Preclinical studies assessing BM-MSC-based interventions in animal models of POI were included. ResultsThirty-four studies comprising 1,357 animals were included. Compared with controls, BM-MSC-based therapy increased serum estradiol (standardized mean difference [SMD] 3.11; 95% confidence interval [CI] 2.38-3.84) and anti-Mullerian hormone (SMD 1.86; 95% CI 1.03-2.69), while reducing follicle-stimulating hormone (SMD -3.54; 95% CI -4.37 to -2.71) and luteinizing hormone (SMD -3.44; 95% CI -5.17 to -1.70). Follicular counts increased across developmental stages, with fewer atretic follicles. Reproductive outcomes improved, including normal estrous cycles (risk ratio [RR] 7.80; 95% CI 3.15-19.34), pregnancy occurrence (RR 3.72; 95% CI 2.14-6.44), and offspring number (SMD 1.57; 95% CI 1.04-2.09). ConclusionBM-MSC-based therapy consistently improved hormonal, follicular, and reproductive outcomes in preclinical POI models. More well-designed, standardized, and adequately controlled studies to confirm these findings are warranted. Systematic review registration: CRD42023449053

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.

In this manuscript, we used an Id4-eGfp mouse line to produce the first proteomic and phosphoproteomic database for mouse spermatogonial stem cells (SSCs) and progenitor spermatogonia. Our proteomic analyses identified 8,464 proteins in spermatogonia, superseding the depth of previously published spermatogonia datasets by >2000 proteins. While the comparison of SSCs and progenitors revealed few unique proteins (18 and 3, respectively), 532 proteins exhibited significantly different abundance (FC {+/-} 1.5, p-value [≤] 0.05) between these sub-populations. Interestingly, in overlaying the proteome with transcriptomic data, correlation was poor (R2 = 0.236), re-iterating discordance between transcript and protein abundance in the testis. In our phosphoproteomic analyses, phosphosites were identified in 19.5% of proteins (3,604 total phosphosites). Unique protein phosphorylation was substantially more common than unique protein expression when comparing SSCs and progenitors, with 38 and 191 unique phosphosites identified, respectively, in addition to significant differences in abundance at 60 and 257 phosphosites. In identifying a wave of phosphorylation that accompanies the progenitor transition, we performed predictive analyses to identify three potential master kinases for follow up validation studies (PAK1, BUB1, ABL2). The inhibition of these kinases resulted in a significant reduction in the capacity for progenitor spermatogonia to differentiate, and caused elevated apoptosis and DNA damage. Finally, we explored the testis phenotype of 42 knockout mouse lines for proteins that were either differentially expressed (26) or differentially phosphorylated (15) in our dataset. Of these lines, 21 exhibited a testis phenotype in at least one of two biological replicates observed (severity score [≥] 1, compared to 0.15 for controls), with increased numbers of Sertoli-only tubules being evident in four of these lines. This manuscript provides a comprehensive roadmap to understand the multifaceted layers of regulation over fate decisions in undifferentiated spermatogonia. These data have been provided in an accessible platform via our ShinySpermatogoniaCells app to encourage future progress in the field.

Mesenchymal Stromal Cells (MSC) face several heterogeneity challenges hindering clinical and commercial success. Employing a multiple response model, interplay between donor heterogeneity, and critical processing parameters (CPPs), effects on MSC potency and cell expansion attributes were investigated through computed composite attribute scores. Twelve unique CPP combinations were tested in thirteen marrow-derived MSC(M) and five adipose-tissue MSC(AT) training and test datasets, respectively. Donor heterogeneity and select CPP conditions affected a curated gene panel (surrogate for MSC potency); while MSC expansion was primarily influenced by CPPs. Model performances were evaluated against clinical effectiveness data from a previously deployed clinical trial; top-performing model predicted donor rankings coincided with clinical effectiveness data, validating the modeling approach used. Our model predicted that only 8% of tested donors were agnostic to CPPs; a majority (62%) of donors showed CPP-dependent optimal composite quality attributes, with MSC seeding density as a key driver; medium supplementation and oxygen preferences were highly donor dependent. Approximately 30% of donors performed poorly at all conditions tested and may be prospectively identified using a subset of genes (TGFB, VEGF, PDCD1LG1, PDCD1LG2, IDO). Model predicted optimal parameters worked for 69% of tested donors, while sub-optimal parameters worked for only 23% of donors and were confirmed in an independent CD14+ macrophage assay. Our integrated computational and experimental framework predictably identified interactive effects of donor heterogeneity and CPP conditions to optimize MSC potency attributes.

Astrocytes assume multiple phenotypes in the brain in response to stress, injury, inflammation, and aging. Given the complexity of this critical cell type in the CNS, it is important to gain a greater understanding of the differences between these phenotypes and to potentially identify therapeutic approaches to modifying astrocyte function in the context of disease and aging. We compared senescent and reactive astrocytes using a strictly defined paradigm to induce these phenotypes in human astrocytes. Gene expression profiling reveals overlapping but distinct expression profiles. Reactive astrocytes predominantly express genes involved in inflammatory responses while senescent astrocytes express genes and a secretome that suggests a role in synaptic pruning. Unexpectedly, functional analysis in a simplified neurite outgrowth assay suggests that senescent astrocytes retain the ability to support neurite outgrowth while reactive astrocytes lose this capacity. The data suggests that senescent and reactive astrocytes play distinct functional roles in the physiology of the aging brain. However, the overlapping inflammatory nature of senescent and reactive astrocytes makes it difficult to discriminate between them using existing toolsets designed to identify senescent cells.

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.

BackgroundiPSCs generated from healthy individuals constitute an important control resource for disease modelling applications but existing biobanks are highly skewed towards populations of European ancestry while well characterized control lines from Indian populations remain limited. Given the extensive genetic diversity of the Indian subcontinent, the availability of ethnically relevant healthy control lines is important for developing accurate disease models and reducing population specific confounding effects. MethodologyWe used peripheral blood mononuclear cells (PBMNCs) of a healthy female donor of Eastern Indian origin for the generation a wild type iPSC line using non-integrating episomal reprogramming vectors. Established colonies were expanded and characterized through morphological assessment, expression of pluripotency and trilineage markers, episomal vector clearance analysis, and chromosomal stability evaluation and mycoplasma contamination analysis. ResultsThe line generated exhibited characteristic pluripotent stem cell morphology and also showed strong expression of pluripotency markers, was free from any contamination and free from the reprogramming vectors confirming an integration free system. The cells maintained a normal diploidy number during characterization. Expression of lineage specific markers associated with ectoderm, mesoderm and endoderm confirmed the developed iPSCs functional capacity to undergo trilineage differentiation. ConclusionWe have developed and validated an iPSC line from an underrepresented Indian population. This well characterized, ethnicity specific iPSC line provides a valuable cell line for establishing a high quality, well characterized control baseline, which is a major missing element in South Asian stem cell repositories and thus will provide a solid foundation for future disease specific modelling and screening.

The complexity of stem cell differentiation programs remains incompletely understood across stem cell types, including for human bone marrow mesenchymal stromal/stem (BM-MSCs) cells, a heterogeneous cell population orchestrating bone formation and establishing a functional hematopoietic niche in the bone marrow. BM-MSCs form and repair bone through the evolutionarily conserved process of endochondral ossification (EO), initiated by deposition of a transient cartilage template subsequently remodeled into bone and bone marrow tissues. Despite their considerable potential for skeletal regeneration, the early molecular and cellular events underlying BM-MSCs commitment to endochondral ossification remain elusive. To overcome donor-dependent variability in chondrogenic potential that limits mechanistic studies, we here exploit OssiGel as a potent chondro-inductive extracellular matrix offering robust recapitulation of endochondral ossification by BM-MSCs. Through multi-omics profiling of OssiGel-primed BM-MSCs, we identify rapid chromatin remodeling at chondrogenic enhancer regions as a prerequisite for lineage commitment. The emergence of a chondro-progenitor population is detected as early as 3 days in vivo, and correlates with successful EO recapitulation. Mechanistically, we identify LINC02511 as a novel enhancer-associated element involved in the onset of EO. We confirm presence of LINC02511 in human skeletal atlases, and its CRISPR-mediated silencing was shown to significantly impair EO. By integrating tissue engineering with single cell multi-omics profiling, our study provides a framework for deciphering BM-MSCs fate decisions, highlighting the role of enhancers and non-coding elements as key determinants of early lineage specification. These findings advance our understanding of BM-MSCs biology and will prompt their translational exploitation in regenerative medicine.

Proliferation of cytotrophoblasts (CTBs) and their differentiation into invasive extravillous trophoblasts (EVTs) are critical processes in early placental development. Defects in these processes are associated with adverse pregnancy outcomes, including recurrent pregnancy loss (RPL). There is evidence that reduced expression of the maintenance DNA methyltransferase DNMT1 in the placenta occurs in pregnancy loss and that RPL is associated with aberrant DNA methylation patterns. Therefore, we investigated the role of DNMT1 in human trophoblast growth and differentiation. Using human trophoblast stem cells (hTSCs), an in vitro analog to CTBs, we found that shRNA-mediated knockdown of DNMT1 led to decreased hTSC proliferation, genome-wide reductions in methylation, broad changes in gene expression, and impaired EVT differentiation. Transcriptome profiling of DNMT1-deficient hTSCs and hTSC-derived EVTs highlighted aberrant cytokine expression, drawing a connection to prior reports of immunological dysfunction in RPL. Finally, using a catalytic DNMT1 chemical inhibitor, we demonstrate the canonical methyltransferase activity of DNMT1 is essential for EVT differentiation and invasion. This study identifies new roles for DNMT1 in trophoblasts and addresses the molecular basis of the associations between DNMT1 expression, altered DNA methylation profiles, and RPL.

Human liver organoids (HLOs) and liver-on-a-chip (LoC) systems are emerging as physiologically relevant human models for studying liver function, disease, and drug metabolism, often in combination with human induced pluripotent stem cell (hiPSC)-derived tissues. However, hiPSC-derived models often display batch-to-batch variation and incomplete maturation, and the contribution of microfluidic flow to hepatic maturation remains insufficiently characterized. Here, we developed a cryopreservable and scalable workflow to generate hiPSC-derived hepatic organoids that can be directly matured to either static HLOs or LoC systems, enabling matched comparison of both platforms. Transcriptomic and functional characterization revealed progressive hepatic maturation during organoid differentiation, including increased expression of liver-specific metabolic pathways, enhanced albumin secretion, and increased CYP3A4 activity. Compared to mature HLOs, LoCs exposed to continuous microfluidic flow exhibited transcriptomic profiles suggesting further maturation, with increased enrichment of pathways related to lipid metabolism, xenobiotic metabolism, transport, and tissue organization. These findings demonstrate that microfluidic perfusion promotes hepatic metabolic specialization compared to static organoid culture while maintaining donor-specific characteristics. Together, this study establishes a robust hiPSC-derived LoC platform and highlights the potential of flow-based systems for improved modeling of human liver physiology, disease mechanisms, and drug responses.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for cardiac regenerative medicine and disease modeling. However, hiPSC-CMs generated through conventional in vitro differentiation exhibit immature, fetal-like phenotypes. While in vivo bioincubation in neonatal rodent hearts promotes hiPSC-CM maturation toward adult-like phenotypes, studies in large animal models remain limited, particularly with detailed morphological characterization. In this study, we investigated bioincubation of fluorescently labeled hiPSC-CMs in both neonatal rat and pig hearts. Human iPSCs were differentiated into cardiomyocytes expressing GFP or RFP reporters and subsequently injected intramyocardially into neonatal rats (GFP-labeled) and pigs (RFP-labeled). After 4-8 weeks of bioincubation, fluorescent hiPSC-CMs were isolated using large-particle fluorescence-activated cell sorting (COPAS), which preserves cellular morphology of adult-like cardiomyocytes. Immunostaining for cardiac troponin T revealed well-organized sarcomeric structures in multinucleated hiPSC-CMs. Bioincubated hiPSC-CMs displayed rod-shaped morphology with binucleation, characteristic features of mature adult cardiomyocytes. Quantitative analysis demonstrated that bioincubated hiPSC-CMs from rat hearts exhibited sarcomere length and cell circularity comparable to native rat adult cardiomyocytes, though with higher intra-cellular variability in sarcomere organization. Histological examination confirmed successful engraftment of RFP-positive hiPSC-CMs within pig myocardium, with engrafted cells also displaying mature adult-like features. These findings provide critical proof-of-concept data for bioincubation in large animal models and support further investigation for disease modeling, drug screening, and regenerative cell therapies. SIGNIFICANCE STATEMENTHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer tremendous potential for cardiac disease modeling and regenerative therapies, but their clinical application is limited by their immature characteristics. Here we show that in vivo bioincubation in neonatal rat hearts enables hiPSC-CMs to achieve structural maturity, exhibiting features of adult cardiomyocytes, including organized sarcomeres, rod-shaped morphology, and multinucleation. We further provided proof-of-concept evidence for engraftment in neonatal pig hearts for maturation, supporting feasibility in large animal models. The use of large-particle cell sorting enables recovery of intact, adult-sized cardiomyocytes for subsequent analysis. These findings establish a practical and scalable platform for generating structurally mature human cardiomyocytes through in vivo bioincubation.

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.

Clinical obesity, defined as the presence of excess adiposity in conjunction with the presence of at least one clinical presentation of disease, remains a significant social and economic burden. Pharmacological weight loss agents based on glucagon-like peptide-1 receptor (GLP-1R) targeting are effective but exhibit significant side-effects leading to cessation of treatment, weight regain and, importantly, re-development of co-morbidities. Adipose tissue, now established as a central mediator of energy balance, endocrine signalling and inflammation, plays a significant role in the protection against metabolic disease onset. Loss of adipose tissue expandability, insulin sensitivity and function is thought to be a pivotal event in the transition to clinical obesity. Targeting adipose tissue dysfunction prior to or following development of metabolic disease remains a key therapeutic strategy either in conjunction with incretin-based weight loss therapy, or as a stand-alone therapy. The recent confirmation of functional glucose-dependent insulinotropic polypeptide receptor (GIPR) in mature adipocytes has led to a significant shift in our mechanistic understanding of dual GLP-1R/GIPR agonists such as tirzepatide, with the addition of adipocyte-specific targeting thought to underpin its clinical superiority to GLP-1R agonism alone. However, the expression, regulation and mechanistic targets of GIPR, and indeed many G protein-coupled receptors (GPCRs), in human adipocytes remains unclear, with adipocyte development being particularly under-studied in this regard. Here we use unbiased transcriptomic analyses of human adipocyte development with high temporal resolution to identify the onset of human GPCR expression, uncovering a previously undocumented surge in expression following adipogenic induction and elegant gene waves throughout adipocyte development that may provide attractive pharmacological targets for adipose tissue dysfunction with or without weight loss. We functionally characterise GIPR, GLP-1 and CALCR/RAMP (Amylin) receptor activation at key differentiation timepoints, and identify a novel amylin response in early adipogenesis, presenting committed adipogenic precursors as a primary target of amylin signalling. HighlightsO_LIComprehensive transcriptomic analysis of human adipogenesis with improved temporal resolution and depth C_LIO_LIIdentification and classification of >150 GPCRs differentially regulated across adipocyte differentiation C_LIO_LIFunctional validation highlighting targeting of adipose stem cells and adipocytes using clinically approved receptor agonists C_LIO_LIGeneration of novel human adipocyte stem cell lines from healthy and obese individuals to drive early target validation and exploration of adipocyte biology with increased pre-clinical power. C_LI SummaryComprehensive temporal RNA sequencing across human adipocyte development with a focus on G-protein coupled receptor expression and activity.

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.

Senescence human bone marrow mesenchymal stem cells (BMSCs), vulnerable to age-related defects, is poor in tissue regeneration. Cells in bone marrow accumulated senescent contributing to the development of metabolic energy regulation hold prospects for therapeutic advances. This study aimed to evaluate energy metabolic changes in male bone marrow mesenchymal stem cells senescence process. Our research established cell specific surface marker and enzymes expression level changes, as well as ECAR and OCR resonance. Notably, CD14, HLA-DRB1 and CD90 upregulated, glycolysis-related genes are increased, tricarboxylic acid cycle-related genes are decreased. We firstly identified links between time-dependent cell aging process and energy metabolism in BMSCs.