Stem Cells Translational Medicine

The human heart lacks cardiac stem cells and the ability to reestablish the loss of ventricular cardiomyocytes (vCMs) after myocardial infarction (MI) resulting in heart dysfunction. vCM replacement using intracardiac transplantation of human induced pluripotent stem cell (iPSC) derived vCMs represent a promising strategy for targeting MI, but low engraftment remains a challenge. Herein, we investigated whether formulation of ventricular iPSC-CMs (iPSC-vCM) as spheres (iPSC-vCMSphere) improves cell retention and cardiac function as directly compared to single-cells (iPSC-vCMSC) after transplantation in a new severely immunocompromised mouse model with MI. Recipient survival immediately after MI and intervention was poor for both Vehicle (50%) and iPSC-vCMSC (36%) groups, whereas 92% of iPSC-vCMSphere treated mice survived, which is comparable to non-immune deficient C57bl/6 mice (91%). All surviving and engrafted animals retained iPSC-vCMs in the infarct border zone at 8-weeks, and heart function remained similar between groups, although minor improvements were observed for iPSC-vCMSphere animals. Surprisingly, iPSC-vCMSpheres were less mature than iPSC-vCMSCs. Thus, our data suggest that iPSC-vCM sphere formulation may offer some benefits for intracardiac delivery as compared with single cell formulated iPSC-vCMs, and thus remains a promising candidate for reestablishing the lost CMs after MI in the future.

BACKGROUND: Peripheral artery disease (PAD) can lead to amputation in advanced cases, making cell therapy using human induced pluripotent stem cells (hiPSCs) a promising therapeutic option. hiPSC-derived endothelial cells (hiPSC-ECs) have shown favorable effects in treating experimental ischemic cardiovascular disease. An autologous approach for PAD patients is preferable to avoid immunological reactions. However, it is yet unknown whether hiPSCs and hiPSC-ECs derived from PAD patients have similar characteristics and potency compared to those derived from non-PAD donors. Therefore, we explored whether there are significant differences in the characteristics and potency of hiPSCs and hiPSC-ECs between non-PAD donors and PAD patients. METHODS: We generated hiPSCs from the blood of non-PAD donors and patients with PAD. We determined the pluripotency of hiPSCs using qRT-PCR, flow cytometry, immunostaining, RNA-seq, and teratoma formation assay. The genetic stability of hiPSCs was confirmed by G-banding karyotyping. We then differentiated hiPSCs into endothelial cells (ECs) in a clinically compatible manner. We compared the characteristics and therapeutic potency of hiPSC-ECs derived from non-PAD and PAD groups using qRT-PCR, flow cytometry, immunostaining, and RNA-seq. In vitro endothelial functions were determined by nitric oxide (NO) production and capillary network formation. Therapeutic potency and incorporation of engrafted hiPSC-ECs were evaluated in a hindlimb ischemia model. RESULTS: We successfully generated hiPSCs from the blood of seven non-PAD donors and eight PAD patients. Both non-PAD and PAD-derived hiPSCs exhibited similar expression levels of pluripotency markers. All hiPSCs, regardless of the group, formed teratomas and showed normal karyotypes. RNA-seq analyses revealed similar gene expression profiles between the groups. hiPSC-ECs derived from both non-PAD and PAD donors exhibited similar expression levels of EC markers at both the gene and protein levels. RNA-seq analyses showed no significant overall differences in gene expression profiles between the groups. Functional analyses demonstrated similar endothelial characteristics and function in hiPSC-ECs from both groups. Both groups showed similar perfusion recovery, limb salvage, and vessel-forming capacity. Engrafted hiPSC-ECs from both groups also exhibited similar angiogenic and vessel-forming capabilities. CONCLUSIONS: Our study demonstrated no significant differences in hiPSCs and hiPSC-ECs derived from non-PAD donors and PAD patients in terms of molecular and cell biological characteristics, therapeutic effects, and vessel-forming capability. Our study indicates that hiPSCs and hiPSC-ECs derived from PAD patients can serve as a novel platform for autologous cell therapy.

Background: For the first time, pluripotent stem cell (PSC)-derived endothelial cells were administered to a human for peripheral artery disease (PAD), in August 2026. This translation is based on a large preclinical literature claiming that donor cells build blood vessels in animal models of limb ischemia, but whether it supports that claim has not been assessed systematically. Methods: In this systematic review and meta-analysis, we searched MEDLINE, Web of Science, Europe PMC, and Embase to August 2026 for studies of PSC-derived vascular cells in animal models of limb ischemia with an acellular control. Limb perfusion at each experiment's primary timepoint was pooled as Hedges' g in a three-level random-effects model with cluster-robust variance. Donor-cell fate and a seven-level ordinal of vascular contribution were graded for every study. This meta-analysis was registered on PROSPERO (CRD420261465308). Results: Sixty-eight studies in 69 reports met the criteria; 51 contributed 114 comparisons at primary timepoints. Cell therapy increased perfusion (Hedges' g 2.26, 95% CI 1.72-2.79; P=4.1x10-11; I2=87.5%; 95% prediction interval -1.47 to 5.98). No prespecified moderator survived Holm-Bonferroni adjustment, including the donor-cell fate grade (P=0.30). A donor cell was resolved in a vessel wall in 35 studies, and flow through such a vessel in 7. Small-study effects were strong (Egger t=6.25, P=9x10-8) but were not reproduced by a sample-size-based test (P=0.80); trim-and-fill moved the estimate to 1.15. On the natural scale, treated limbs recovered about twice the control perfusion (ratio of means 1.96, 95% CI 1.66-2.33). Limb preservation favored cell therapy in 11 studies with countable events (risk ratio 4.88, 95% CI 2.28-10.45), but 43 of 68 studies reported no functional outcome, and only 22 comparisons from 8 studies were both randomized and blindly assessed. In a post hoc analysis that did not survive multiplicity adjustment, effects were largest in studies whose images could not resolve their incorporation claims (g 3.23) versus those that resolved incorporation (g 2.33). Conclusions: PSC-derived vascular cell therapy improved limb perfusion in animal models, consistently across every sensitivity analysis, although the magnitude is uncertain given heterogeneity and small-study effects. Benefit was not associated with the degree of donor-cell incorporation across studies and was largest where incorporation was least resolvable, a pattern more consistent with reporting and measurement bias than with a true mechanistic effect. The first rigorous, blinded test of that benefit may come not from animals but from the patients now receiving these cells.

Background Hypertrophic and dilated cardiomyopathies (HCM and DCM) are the most common inherited cardiomyopathies. However, genotype-specific molecular and functional cardiomyocyte phenotypes and responses to neurohormonal stimulation remain incompletely understood. Here, we investigated whether patient-derived HCM and DCM cardiomyocytes exhibit distinct baseline phenotypes or differential responses to hypertrophic stimulation and pharmacological treatment. Methods Three human-induced pluripotent stem cell (hiPSC) lines were used: a control line, an HCM patient-derived line carrying a MYBPC3 mutation, and a DCM patient-derived line carrying an LMNA mutation. The cells were differentiated into hiPSC-cardiomyocytes, which were exposed to endothelin-1 and the GATA4-targeted compound 3i-1262, followed by transcriptional and protein expression analyses. In addition, engineered heart tissues (EHTs) were generated and cultured for 40 days, with endothelin-1 and 3i-1262 treatment applied during the final 20 days. Lastly, {beta}-adrenergic stimulation with isoprenaline was performed. EHT contractile function was quantified using MUSCLEMOTION. Results Patient-derived hiPSC-cardiomyocytes exhibited genotype-dependent responses to endothelin-1 at the transcriptional and protein levels. DCM-cardiomyocytes failed to maintain structural integrity in the EHTs, resulting in tissue fracture or cessation of beating. Functional analyses demonstrated distinct baseline contractile properties between control and cardiomyopathy EHTs, as well as differential responses to endothelin-1 and isoprenaline. Conclusions Patient-derived hiPSC-cardiomyocytes exhibit genotype-specific molecular and functional phenotypes. EHTs generated from HCM hiPSC-derived cardiomyocytes showed a progressive decline in apparent force, whereas DCM EHTs fractured over time, suggesting mutation-associated phenotypes in 3D cardiac tissue models. These findings highlight the utility of hiPSC-based cardiac models for investigating molecular and functional disease mechanisms and pharmacological responses in inherited cardiomyopathies. Keywords: hypertrophic cardiomyopathy, dilated cardiomyopathy, engineered heart tissues, hiPSC-derived cardiomyocytes, GATA4

The non-receptor protein-tyrosine phosphatases Shp1 (PTPN6) and Shp2 (PTPN11) play critical roles in hematopoietic signaling networks, yet their specific functions in human megakaryopoiesis and thrombopoiesis remain incompletely understood. While Shp2 is recognized in murine models as a positive regulator of thrombopoietin (Tpo)-mediated signaling through the Ras/MAPK and PI3K/AKT pathways, Shp1 has been implicated in RhoA-dependent cytoskeletal remodeling. However, the extent to which these roles translate to human megakaryocyte (MK) development and platelet production is not known. In this study, we systematically investigated the contributions of Shp1 and Shp2 to human MK development and function using CRISPR/Cas9-mediated gene deletion of PTPN6 and PTPN11 in CD34+ hematopoietic stem and progenitor cells (HSPCs), combined with pharmacological inhibition of Shp2 using the structurally-distinct allosteric inhibitors SHP099 and RMC-4550. Efficient gene editing of PTPN6 and PTPN11 resulted in efficient ablation of Shp1 and Shp2 in CD34+ HSPC-derived MKs. Genetic deletion or pharmacological inhibition of Shp2 markedly impaired MK proliferation, polyploidization, maturation, and proplatelet formation, whereas loss of Shp1 expression did not. Further, Shp2 inhibition significantly reduced platelet production in a 3-dimensional human bone marrow tissue model. Deletion and inhibition of Shp2 abrogated Tpo-induced ERK1/2 and AKT phosphorylation, confirming its essential role in Mpl receptor signaling. These findings demonstrate the distinct functional roles of Shp1 and Shp2 in MKs and establish Shp2 as a critical positive regulator of Mpl- mediated megakaryopoiesis and thrombopoiesis. Key PointsO_LIEfficient deletion of Shp1 and Shp2 in human CD34 progenitor cell-derived MKs using CRISPR/Cas9. C_LIO_LILoss of Shp2 expression impairs thrombopoietin-induced human MK maturation, proplatelet formation and Mpl signaling. C_LI

Background aimsThe development of next-generation therapies for sickle cell disease (SCD) and beta thalassemia (beta thal), including fetal globin-inducing small molecules and gene therapy approaches, depends on patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) for discovery and preclinical validation, but commercial vendors stock only healthy donor material and disease-specific banks hold limited inventories. Recent US Food and Drug Administration and National Institutes of Health guidance favoring human cell-based methods over animal testing underscores the value of authentic patient cells. Methods: Over 14 months we recovered, purified, and biobanked CD34+ HSPCs from clinical apheresis product waste and mobilized peripheral blood (PB) otherwise discarded after clinical procedures, using immunomagnetic selection adapted for hemoglobinopathy specimens; a microfluidic technology was evaluated separately. We quantified yield and purity for bead-selected material and cell number and viability for the microfluidic pilot; engraftment was tested in NBSGW mice. Results: Immunomagnetic selection recovered a median of 4.71 x 106 CD34+ cells from just 1 to 2 mL of apheresis product waste, comparable to the 6.0 x 106 cells from a 10 to 40 fold larger volume of PB waste, with similar purity across sources and diagnoses. Because apheresis product waste is far more concentrated, it reaches equivalent yields without the density-gradient steps required for PB waste, approximately halving processing time. Recovered cells engrafted NBSGW mice, confirming preserved repopulating capacity. The microfluidic pilot (two patients, 11 specimens) recovered 2.17 x 106 CD34+ cells per specimen at greater than 90% viability and purity. Conclusions: A center with existing apheresis infrastructure can reproducibly recover, bank, and distribute research-grade patient CD34+ HSPCs, addressing a recognized gap in the hemoglobinopathy pipeline. HighlightsO_LIClinical apheresis waste is used to generate a single-center biobank of high-quality, research-grade CD34+ HSPCs from patients with sickle cell disease and beta-thalassemia. C_LIO_LIConcentrated apheresis waste matches large-volume PB waste in CD34+ yield and purity. C_LIO_LIMicrofluidic enrichment recovers CD34+ cells at >90% viability and purity across 2 patients. C_LIO_LIRecovered CD34+ HSPCs engraft mice and form erythroid cells, preserving function. C_LI

Background: Induced pluripotent stem cell-derived ventricular cardiomyocytes (iPSC-vCMs) hold great promise for replacing ventricular cardiomyocytes lost after myocardial infarction. However, their immature phenotype limits successful engraftment by increasing the risk of post-transplant arrhythmias. In contrast to the atmospheric O2 used during most iPSC-vCM differentiations, O2 levels inside the developing heart remain low, but very little is known on the effect on O2 on iPSC-vCM differentiation. Methods: We used a GMP compliant Quad Physoxia glovebox platform providing continuous stable specified O2 tensions during all processes to simulate the in vivo O2 conditions more closely with the aim of improving iPSC-vCM maturation. Results: We demonstrate by single cell RNA sequencing, and data integration with datasets for human cardiomyocytes from the different heart chambers as well as cell morphology-, ploidy-, and functional studies, that sustained 10% O2 throughout iPSC-CM differentiation promotes atrial- instead of ventricular iPSC-CM subtype specification. Conclusions: While this rejects our original hypothesis and forces some concerns to iPSC-vCM manufacturing by sphere technology, these unexpected data may serve as an attractive and easy approach to refine atrial iPSC-CM specification to benefit their exponentially growing diagnostic-, cytotoxic-, and regenerative use.

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.

Platelets are increasingly recognized as active regulators of tissue repair and liver regeneration beyond their classical roles in hemostasis and thrombosis. Loss of terminal sialic acid from platelet surface glycoproteins, a process known as desialylation, occurs during platelet aging or activation and has been linked to platelet clearance via the asialoglycoprotein receptor (ASGPR) on hepatocytes. However, the mechanisms by which desialylated platelets (D-plts) directly stimulate hepatocyte proliferation remain poorly understood. This study aimed to elucidate the proliferative effects of D-plts on hepatocytes and to identify the underlying signaling mechanisms. D-plts were generated and co-cultured with hepatocyte models exhibiting low or absent levels of asialoglycoprotein receptor 1 (ASGPR1) expression, including HepG2 cells, HuH-7 cells, and human chemically induced liver progenitors. Hepatocyte proliferation was assessed, and the roles of platelet-derived factors and downstream signaling pathways were investigated. Co-culture with D-plts significantly increased hepatocyte proliferation in all three cell models compared with the corresponding controls. Moreover, supernatants derived from stimulated D-plts also significantly enhanced hepatocyte proliferation, suggesting that soluble platelet-derived factors contribute to this effect. Mechanistically, the proliferative effects were mediated predominantly through the ERK1/2 signaling pathway rather than the JAK-STAT pathway in both hepatocytes co-cultured with D-plts and those treated with D-plt-derived supernatants. In conclusion, our findings demonstrate that D-plts directly promote hepatocyte proliferation through an ASGPR-independent pathway, in which ERK1/2 signaling plays a central role. These results highlight a novel mechanism through which platelet desialylation may contribute to liver regeneration. Graphical Abstract(A) Desialylated platelets are readily activated and release increased amounts of EGF, promoting hepatocyte proliferation via the EGF-ERK signaling pathway. (B) Normal platelets show lower reactivity and reduced EGF release than desialylated platelets, resulting in weaker hepatocyte proliferation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/740293v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1cbe93borg.highwire.dtl.DTLVardef@3d57c7org.highwire.dtl.DTLVardef@14dd63forg.highwire.dtl.DTLVardef@12cf508_HPS_FORMAT_FIGEXP M_FIG C_FIG

Endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) are increasingly used to model vascular diseases and test therapeutic strategies. However, the efficiency and reproducibility of differentiation can vary depending on the culture medium and its supplemented factors and stages. Here, we directly compared two defined media, APEL and BPEL, for iPSC-to-ECs differentiation. iPSCs were differentiated over 10 days with sequential growth factor induction, followed by magnetic-activated cell sorting or flow cytometry for CD31+ cells. Both media produced ECs with similar morphology and marker expression, including CD31 and VE-cadherin. Functional assays demonstrated comparable tube formation, indicating equivalent endothelial functionality. Cost analysis indicated that APEL had a higher total reagent cost but generated a higher total cell yield, resulting in a comparable cost per 10 total cells, whereas BPEL was more cost-efficient for producing CD31/VE-cadherin endothelial-specific cells. Our results suggest that APEL and BPEL media are equally effective for generating iPSC-derived ECs, providing flexibility in method selection for vascular disease modeling and drug discovery applications.

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.

BackgroundCardiac fibrosis drives adverse myocardial remodelling through persistent fibroblast activation, ECM deposition, and impaired cardiac function. Current therapies offer limited protection against cardiac fibrosis progression. Elafibranor is a dual PPAR-/{delta} agonist approved for the treatment of liver disease. However, its effects in human models of cardiac fibrosis remain insufficiently explored. MethodsElafibranor was evaluated in complementary human in vitro TGF-{beta}1-induced cardiac fibrosis models: 2D primary fibroblasts, 3D fibroblast spheroids, spontaneously contracting 3D cardiac microtissues, and hiPSC-derived cardiomyocytes. Viability, apoptosis, fibroblast activation, ECM remodelling, mitochondrial respiration, nucleotide and NAD pools, calcium handling, contractility, and transcriptomic profiles were assessed. ResultsAt non-cytotoxic concentrations, elafibranor attenuated TGF-{beta}1-driven cardiac fibrosis responses. In 2D cardiac fibroblasts, it reduced myofibroblast differentiation, procollagen 11 secretion, and partially restored mitochondrial respiratory capacity. In 3D spheroids, it preserved viability, attenuated caspase-3/7 activation, and suppressed procollagen 11 release. In cardiac microtissues, elafibranor reduced ECM accumulation, shifted transcriptomic profiles toward redox-metabolic/cytoprotective pathways, altered adenine nucleotide and NAD pools, and partially recovered contraction parameters. In hiPSC-derived cardiomyocytes, elafibranor modulated calcium handling, contractility, and mitochondrial respiration. ConclusionsElafibranor mitigates TGF-{beta}1-driven cardiac fibrosis by suppressing fibroblast activation and ECM remodelling while promoting adaptive metabolic, redox, and bioenergetic responses, supporting balanced PPAR-/{delta} activation as a potential therapeutic strategy for cardiac fibrosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/745425v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1cbd94eorg.highwire.dtl.DTLVardef@27a44borg.highwire.dtl.DTLVardef@9354baorg.highwire.dtl.DTLVardef@9f9946_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: CAMPs (cellular and acellular matrix products) can facilitate wound closure and improve clinical outcomes in patients with diabetic lower extremity ulcers (DLEU), however, there is a lack of comparative data between different CAMPs. The objective of this study was to use real-world evidence to compare the clinical and economic outcomes of hospital care for DLEU patients treated with a minimally manipulated human umbilical tissue (STRAVIX{lozenge}) or other acellular CAMPs. Methods: This retrospective analysis was conducted on patients with DLEU treated with the CAMPs using data from the Premier PINC AI Healthcare Database. Each patient's earliest inpatient encounter was designated as the index admission. The analysis groups included a cellular human umbilical tissue allograft (STRAVIX) and four different acellular CAMPs (aCAMPs). To balance baseline covariates, inverse probability of treatment weighting (IPTW) was applied. Subsequently, IPTW-adjusted generalized linear models were utilized to estimate healthcare-related outcomes and hospital-related costs. Results: 2,626 DLEUs patients with STRAVIX and 10,002 DLEUs patients with aCAMPs were identified. Compared to the patients treated with aCAMPs, the patients who had STRAVIX in addition to standard of care had significantly lower odds of having an infection reaction, dehiscence, minor and major amputations, after index admission discharge. The number of DLEU related follow-up visits was 17-23% lower, debridement related visits were 19-23% lower and the incidence of all-cause readmission was 22-25% lower in Stravix patients compared to aCAMP patients. The length of hospital stay days was 3% lower and the cost of all-cause visits was 20-25% lower in STRAVIX treated patients compared to aCAMP patients. Conclusion: This large data set demonstrates improved clinical outcomes in patients who had STRAVIX in addition to standard surgical management, when compared to certain acellular CAMPs.

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.

Background: Atrial fibrillation (AF) burden is increasingly recognized as a determinant of clinical risk. Currently, AF burden can only be estimated using long-term rhythm monitoring. Bone morphogenetic protein 10 (BMP10) is a protein secreted from cardiac atria associated with AF and AF-related complications. This study evaluated whether BMP10 concentrations are associated with AF burden in a human atrial model: atrial engineered heart tissue (aEHT). Methods: Human induced pluripotent stem cell-derived atrial cardiomyocytes were cast into atrial engineered heart tissues (aEHTs). To mimic AF burden, mature aEHTs were optogenetically-paced at a high rate of 4 Hz, either intermittently for 4 hours every 2 days (~10% burden) or continuously for 24 hours per day (100% burden). After 18 days of high-rate pacing intervention, 7 days of recovery without pacing followed. BMP10 release was quantified by ELISA and contractile function was assessed by video analysis. EHT transcriptional remodeling in response to mimicked AF burden was assessed by RNA sequencing and the effect of recovery was analyzed by qPCR. Results: High-rate optogenetic pacing mimicking AF lead to a dynamic, burden-dependent BMP10 release: BMP10 concentrations in the medium were increased by intermittent optogenetic pacing (~10% burden) and highest under continuous optogenetic pacing (100% burden). BMP10 release declined toward control levels during recovery. Contractile dysfunction was most impaired after continuous pacing and showed only partial recovery within 7 days after pacing cessation. RNA sequencing revealed distinct burden-dependent transcriptional states. Pacing-regulated transcripts were related to BMP/TGF{beta} signaling, atrial identity, calcium handling, contractile phenotype, and electrophysiological remodeling. After recovery, BMP10 mRNA expression remained elevated despite normalization of BMP10 protein release. Conclusions: AF burden dynamically regulates BMP10 release and functional and molecular remodeling in human aEHTs. BMP10 release depicts a secreted protein-based readout of current or recent atrial high-rate stress, whereas persistent transcriptional changes indicate molecular memory of prior AF burden. These findings support BMP10 release as a burden-sensitive AF biomarker

Mesenchymal stromal cells (MSCs) are promising cell therapeutics, but their development as off-the-shelf products requires cryopreservation, which impairs cell function after thaw. Inflammatory licensing enhances MSC therapeutic attributes, yet protocols vary considerably and little is known about how these variables affect MSC metabolism or post-thaw recovery. We characterized mitochondrial and glycolytic flux in umbilical cord (UC) and bone marrow (BM) MSCs licensed with interferon-{gamma} (IFN-{gamma}), tumor necrosis factor- (TNF-), or both. Brief exposure to either cytokine shifted MSC metabolism toward glycolysis and reduced mitochondrial respiration, whereas prolonged exposure led to heterogeneous responses. Conversely, combined IFN-{gamma} and TNF- increased glycolytic flux and respiratory capacity in most donors, with responses detectable after 2 hours and at concentrations below those conventionally used. Cryopreservation impaired aerobic metabolism, followed by donor-dependent recovery. Preconditioning with combined IFN-{gamma} and TNF- partially improved post-thaw metabolism, particularly in BM MSCs, and enhanced early responsiveness to inflammatory challenge, particularly in UC MSCs, as detected by IDO and candidate biomarkers CTSS and C1S. However, donors with improved post-thaw metabolism did not consistently show increased biomarker responses. These findings demonstrate that metabolic fitness and inducible biomarkers capture complementary aspects of post-thaw MSC quality and may inform preconditioning strategies for cryopreserved MSC products.

Background: Cardiac fibrosis is a hallmark of many cardiovascular diseases, driven by sustained fibroblast activation and excessive extracellular matrix deposition, leading to myocardial stiffening and impaired contractility. Current therapies inadequately address this process. This study evaluated the antifibrotic potential of lanifibranor, a balanced pan-peroxisome proliferator-activated receptors (PPARs) agonist, in TGF-beta1-induced cardiac fibrosis. Methods: Human cardiac microtissues, along with 2D and 3D cardiac fibroblast and cardiomyocyte cultures, were used to assess cell viability, structure, metabolism, contractility, and gene expression. Results: Lanifibranor reduced TGF-beta1-induced fibrosis by limiting fibroblast activation and matrix deposition without affecting viability. In fibroblasts, these effects were associated with partial restoration of mitochondrial respiration and reduced focal adhesion maturation. In cardiac microtissues, lanifibranor improved contraction kinetics, decreased profibrotic transcriptional activity, and preserved bioenergetic homeostasis despite altered nucleotide balance. In cardiomyocytes, treatment normalized contractility and calcium handling while maintaining metabolic stability. Conclusions: Lanifibranor attenuates TGF-beta1-driven cardiac fibrosis by combining antifibrotic effects with metabolic and functional improvements in human models.

Aims: Mature, contractile vascular smooth muscle cells (vSMC) in muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Given the complicated embryonic origins and conflicting views on vSMC heterogeneity, our aim was to establish how closely hPSC-vSMC of a known germ layer lineage and defined phenotype are representative of vSMC obtained from aorta (AoSMC). Methods and Results: We generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines and performed phenotypic and mechanical comparisons with AoSMC. Synthetic hPSC-vSMC and cultured AoSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMC are non-proliferative, capable of phenotype switching and have elevated levels of contractile proteins (CNN1, TAGLN, ACTA2). These contractile proteins also displayed density-dependent, cell-to-cell heterogeneity in both AoSMC and hPSC-vSMC. Single-cell traction force measurements on micropost array detectors (mPADs) yielded comparable forces per micropost in the nN range across cell types. Quantitative 3D force measurements on humanized smooth muscle cell microtissues (SMTs) developed similar forces per cell. Larger forces were generated by MEKi-treated hPSC-vSMC and AoSMC versus controls. Conclusions: We have systematically studied the properties of hPSC-vSMC and performed comprehensive phenotypic and mechanical comparisons with AoSMC. These results demonstrate that synthetic hPSC-vSMC generated in vitro and cultured AoSMC derived from tissues are comparable and mechanically similar, but results with hPSC-vSMC are more reproducible.

Sustained trophic factor delivery via Encapsulated Cell Technology (ECT) is a powerful new class of therapeutics with broad potential for targeted treatment. Intravitreal delivery of ciliary neurotrophic factor (CNTF) via the ECT, NT-501, is a first-in-class therapy that slows the progression of macular telangiectasia type 2 (MacTel). Despite its clinical efficacy, key questions remain regarding its mechanism of action, including whether other implant-derived factors contribute to therapeutic benefit and how optimal dosing should be determined. Resolving these issues is critical for optimizing NT-501 in MacTel and guiding the development of ECT-based therapies for other diseases. We evaluated the biological activity of implant-derived cytokines on retinal tissue, using long-term NT-501 intravitreal implants in rabbits alongside human retinal organoid (hRO) models treated with NT-501-conditioned medium (NT-501-CM). Then, using a MacTel-specific photoreceptor degeneration model in hROs, we showed NT-501-CM significantly reduced photoreceptor cell death, and this protective effect was abolished by either CNTF-neutralizing antibodies or JAK inhibitor. We also established a therapeutic dose-response relationship linking NT-501-derived CNTF levels to JAK/STAT3 activation and photoreceptor protection. These findings directly connect ECT-derived CNTF exposure with JAK/STAT3-mediated photoreceptor protection in human retinal tissue and suggest an optimal concentration range for efficacy.

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.