IntroductionThe discovery of induced pluripotent stem cells (iPSCs) revolutionized the field of translational medicine by enabling the reprogramming of adult somatic cells into a pluripotent state. From personalized disease models to innovative cell therapies, iPSCs are poised to play a central role in the future of clinical medicine. iPSCs hold enormous promises due to their ability to self-renew indefinitely and differentiate into all somatic cell types, thus offering patient-specific cellular models and therapeutic options without the ethical constraints of embryonic stem cells (ESCs). iPSCs, which exhibit pluripotency similar to embryonic stem cells, are generated by introducing specific factors into terminally differentiated cells, inducing a shift in their epigenetic and transcriptional landscape, which leads to the reactivation of the pluripotency program of the cells. Nevertheless, the mechanisms underlying successful reprogramming remain poorly understood.MethodsIn this study we performed a statistical evaluation of reprogramming efficiencies of 150 iPSC lines generated in our lab, comparing factors such as the starting somatic cell type, passage number, donor´s health status, donor age and sex, reprogramming methodology, and growth conditions.Results/DiscussionWe found that the most relevant factor influencing reprogramming efficiency is the developmental status of the starting cells. While other parameters may exert minor effects, inherent donor-specific biological characteristics appear to play the strongest role in determining reprogramming outcomes.

Drug development is hindered by high attrition rates, with clinical trial failures accounting for 90% of unsuccessful candidates and 60% of R&D costs, often due to unanticipated cardiotoxicity. Existing models lack physiological relevance, particularly the vascular component critical for drug distribution and cardioprotection. To address this, we developed a heart-on-a-chip (HoC) platform integrating human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells from a single cell line, ensuring genetic uniformity and native-like cell-cell interactions. The tri-culture system maintained >90% cell viability under perfusion for 7 days and exhibited functional maturity, as demonstrated by expected chronotropic responses to the β-agonist isoproterenol. Crucially, the inclusion of endothelial cells mitigated doxorubicin-induced cardiotoxicity, a protective effect absent in conventional models, highlighting the endothelial layer's role in replicating in vivo drug responses. By combining physiological mimicry with scalability, this HoC platform offers a transformative tool for improving preclinical cardiotoxicity assessment and reducing reliance on animal models.

Introduction Diabetic cardiomyopathy (DCM) arises from the interplay of metabolic overload, inflammation, and structural remodeling that ultimately impair cardiac function. However, the transcriptional mechanisms coordinating these pathogenic processes remain incompletely defined. This study aimed to reconstruct and functionally characterize the transcriptional regulatory architecture associated with DCM.Methods We combined differential expression data from human hiPSC-derived cardiomyocytes exposed to diabetic conditions with literature-curated transcription factor-target interactions to reconstruct a comprehensive transcriptional regulatory subnetwork for DCM. Network topology and functional enrichment analyses were performed to identify regulatory modules and hierarchical organization. Selected network-derived predictions were experimentally evaluated in a type 2 diabetes mouse model.Results Transcriptional reprogramming was organized into six functional modules encompassing metabolic, inflammatory, hypoxic, fibrotic, and hormonal pathways. Established DCM-associated transcription factors, including FOS, JUN, STAT3, and MYC, ranked among the highest-centrality hubs. Notably, ESR1, a key regulator of estrogen signaling, emerged as a previously unrecognized high-centrality node within the DCM network. In contrast, TRPS1, HBP1, and NFIA showed lower centrality and operated as locally acting regulators, consistent with a multilayered regulatory architecture. Experimental validation in diabetic mice demonstrated significant downregulation of ESR1 and STAT6, together with upregulation of TRPS1 and HBP1, supporting cross-species concordance of selected regulatory signatures.Discussion These findings define the modular organization of a curated transcriptional regulatory subnetwork underlying DCM and highlight candidate regulators that warrant future functional perturbation studies and biomarker-oriented validation. This integrative network-based framework provides mechanistic insight into transcriptional coordination in diabetic cardiac disease.

Ewing sarcoma (ES) is an aggressive bone and soft tissue neoplasm characterized by EWSR1/ETS rearrangements and whose cellular origin remains unclear. EWS-FLI1 expression in human pediatric mesenchymal stem cells (hpMSCs) induces a quantitatively and qualitatively different transcriptional response than its expression in human adult MSCs (haMSCs), but fails to form tumors in vivo. ES cells have early developmental lineage signatures distinct from postnatal MSCs. Here, we have generated MSCs from experimental teratomas out of human embryonic stem cells (heSCs). Transduction of these human embryonic mesenchymal stem cells (heMSCs) with EWS-FLI1 results in the acquisition of an ES transcriptome, although the oncogene does not preferentially bind to promoters, but to intronic and intergenic microsatellites with >10 CA dinucleotides and GGAA repeats, respectively. In heMSCs, EWS-FLI1 directly regulates BRCA1 expression, although EWS-FLI1-expressing cells show defects in DNA damage repair. Xenografting of EWS-FLI1-transduced heMSCs resulted in the formation of tumors expressing characteristic ES markers. In summary, EWS-FLI1 enforces an aberrant transcriptome and endows in vivo transforming capacity when expressed in an undifferentiated early heMSC. Our approach represents an innovative experimental method for understanding critical aspects of the biology of developmental tumors, from leukemia to sarcomas, in which few (even single) genetic alterations are able to transform a fetal stem cell. ### Competing Interest Statement The authors have declared no competing interest.

Cardiac hypertrophy is a cellular process characterized by the increased size of cardiomyocytes in response to a high workload or stress. 17-beta estradiol (E2) has cardioprotective and anti-hypertrophic effects by maintaining mitochondrial network and function. MUL1 is a mitochondrial ubiquitin ligase directly involved in the control of mitochondrial fission and mitophagy. Studies from our group and others have previously shown that cardiomyocyte hypertrophy is associated with mitochondrial fission and dysfunction. These findings led us to study in vitro whether E2 regulates MUL1 to prevent cardiac hypertrophy, mitochondrial fission, and dysfunction induced by the catecholamine norepinephrine (NE). Our results showed that NE induces hypertrophy in cultured rat cardiomyocytes. Pre-treatment with E2 (10-100 nM) prevented the NE-dependent increases in cell perimeter and the hypertrophic stress markers ANP and BNP at both the protein and mRNA levels. NE induced the fragmentation of the mitochondrial network and reduced ATP levels, effects that were both prevented by E2. In silico analysis suggested a putative binding site for estrogen receptors on the MUL1 gene promoter. In accordance with this finding, E2 prevented increases in MUL1 mRNA and protein levels induced by NE. Our data also showed that a siRNA MUL1 knockdown counteracted NE-induced cardiomyocyte hypertrophy and mitochondrial dysfunction, mirroring the protective effect triggered by E2. In contrast, a MUL1 adenovirus did not prevent the E2 protection from cardiomyocyte hypertrophy. Further, in vivo analysis in a transgenic mouse model overexpressing MUL1 revealed that only young male mice overexpressed the protein. Consequently, they exhibited increased levels of the hypertrophic marker ANP, an elevated heart weight, and larger cardiomyocyte size. Therefore, our data demonstrate that 17-beta estradiol prevents cardiac myocyte hypertrophy by regulating MUL1.

Cardiac tissue engineering is a rapidly growing field that holds great promise for the development of new therapies for heart disease. While significant progress has been made in the field over the past two decades, engineering functional myocardium of clinically relevant size and thickness remains an unmet challenge. A major roadblock in this respect is the current difficulty in incorporating efficient vascularization into engineered constructs. One potential solution involves the use of microvascular fragments from adipose tissue, which have demonstrated encouraging results in improving vascularization and graft survival following transplantation. However, this method lacks precise control over the vascular architecture within the constructs. Here, we set out to investigate the use of 3D bioprinting for the fabrication of human cardiac tissue constructs composed of human induced pluripotent stem cell derivatives, while allowing for the precise control of the distribution and density of microvessel fragments within the bioprinted constructs. We carefully selected and optimized bioink compositions based on their printability, biocompatibility, and construct stability. Following transplantation into immunodeficient mice, 3D bioprinted cardiac constructs containing microvessel fragments exhibited rapid and efficient vascularization, resulting in prolonged graft survival. Overall, our studies underscore the advantages of employing engineering design and self-assembly across different scales to address current limitations of tissue engineering, and highlight the usefulness of 3D bioprinting in this context.

Astrocytes and microglia carrying the LRRK2-G2019S mutation contribute to non-cell- autonomous dopaminergic neuron (DAn) degeneration in Parkinson’s disease (PD), but the mechanisms underlying their interplay remain unclear. Here, we developed a novel induced pluripotent stem cell (iPSC)-derived tri-culture system comprising healthy DAn and either LRRK2-mutant or isogenic control iPSC-derived astrocytes and microglia. Using integrated functional assays and transcriptomic profiling, we found that mutant astrocytes adopt a hyperreactive state, driving microglial activation and subsequent DAn degeneration. Mechanistically, we identified a selective downregulation of ceruloplasmin (CP), a copper-dependent ferroxidase, in mutant astrocytes, leading to disrupted iron homeostasis with accumulation of Fe2+ and ROS. This iron dysregulation mediated both microglial reactivity and neurodegeneration. Notably, pharmacological restoration of CP re-established iron homeostasis, reduced microglial activation, and protected DAn from degeneration. Our findings uncover a novel astrocyte-microglia-neuron axis driving PD pathogenesis and showcase the power of our unique stem cell tri-culture platform for dissecting disease mechanisms and discovering therapeutic targets. ### Competing Interest Statement The authors have declared no competing interest.

Parkinson's disease (PD) is a progressive, incurable neurodegenerative disorder characterized by the loss of neuromelanin (NM)-containing dopamine neurons (DAn) in the substantia nigra of the midbrain. Non-neuronal cells are increasingly recognized as contributors to PD. We generated human microglia-like cells (hMG) from induced pluripotent stem cells (iPSC) derived from patients with LRRK2 PD-causing mutations, gene-corrected isogenic controls, and healthy donors. While neither genotype induced neurodegeneration in healthy DAn, LRRK2 hMG become hyperreactive to LPS stimulation, exhibiting increased cytokine expression, reactive oxygen species, and phagocytosis. When exposed to NM-containing particles, but not α-synuclein fibrils, LRRK2 hMG trigger DAn degeneration, in a process that is prevented by pre-treatment with the immunomodulatory drug ivermectin. Finally, post-mortem analysis of midbrain tissue of LRRK2-PD patients show increased microglia activation around NM-containing neurons, confirming our in vitro findings. Overall, our work highlights NM-activated microglia's role in PD progression, and provides a model for testing therapeutic targets.

Biomechanical alterations contribute to the decreased regenerative capacity of hematopoietic stem cells (HSCs) upon aging. RhoA is a key regulator of mechano-signaling but its role for mechanotransduction in stem cell aging has not been investigated yet. Here, we show that murine HSCs respond to increased nuclear envelope (NE) tension by inducing NE translocation of P-cPLA2, which cell intrinsically activates RhoA. Interestingly, aged HSCs experience physiologically higher intrinsic NE tension, associated with increased NE P-cPLA2 and RhoA activity. Reducing RhoA activity lowers NE tension in aged HSCs. Feature image analysis of HSC nuclei reveals that chromatin remodeling is associated to RhoA inhibition, which includes the restoration of youthful levels of the heterochromatin marker H3K9me2 and a decrease in chromatin accessibility and transcription at retrotransposons. Eventually, we demonstrate that RhoA inhibition upregulates Klf4 expression and transcriptional activity, improving aged HSCs regenerative capacity and lympho/myeloid skewing in vivo . Overall, our data support that an intrinsic mechano-signaling axis dependent on RhoA can be pharmacologically targeted to rejuvenate stem cell function upon aging.

Ewing sarcoma (ES) is an aggressive bone and soft tissue neoplasm characterized by EWSR::ETS rearrangements whose cellular origin remains unclear. EWS::FLI1 expression in human pediatric mesenchymal stem cells (MSCs) induces a transcriptional response distinct from that of human adult MSCs, but fails to form tumors. Here we show that EWS::FLI1 expression in human embryonic mesenchymal stem cells (heMSCs) results in the acquisition of an ES transcriptome, with the oncogene not preferentially binding to gene promoters, but to intronic and intergenic microsatellites. In heMSCs, EWS::FLI1 directly regulates the expression of the DNA repair protein BRCA1, although cells expressing EWS::FLI1 show DNA damage. Xenografting of EWS::FLI1-transduced heMSCs results in the formation of tumors expressing characteristic ES markers. In summary, we show that EWS::FLI1 enforces an aberrant transcriptome and solely is able to endow transforming capacity when expressed in undifferentiated, early heMSCs.

Alternative end-joining (alt-EJ) is an error-prone DNA repair pathway that cancer cells deficient in homologous recombination rely on, making them vulnerable to synthetic lethality via inhibition of poly(ADP-ribose) polymerase (PARP). Targeting alt-EJ effector DNA polymerase theta (POLθ), which synergizes with PARP inhibitors and can overcome resistance, is of significant preclinical and clinical interest. However, the transcriptional regulation of alt-EJ and its interactions with processes driving cancer progression remain poorly understood. Here, we show that alt-EJ is suppressed by hypoxia while positively associated with MYC (myelocytomatosis oncogene) transcriptional activity. Hypoxia reduces PARP1 and POLQ expression, decreases MYC binding at their promoters, and lowers PARylation and alt-EJ-mediated DNA repair in cancer cells. Tumors with HIF1A mutations overexpress the alt-EJ gene signature. Inhibition of hypoxia-inducible factor 1α or HIF1A expression depletion, combined with PARP or POLθ inhibition, synergistically reduces the colony-forming capacity of cancer cells. Deep learning reveals the anticorrelation between alt-EJ and hypoxia across regions in tumor images, and the predictions for these and MYC activity achieve area under the curve values between 0.70 and 0.86. These findings further highlight the critical role of hypoxia in modulating DNA repair and present a strategy for predicting and improving outcomes centered on targeting alt-EJ.

Parkinson's disease (PD) is a progressive and yet incurable neurodegenerative condition characterized by loss of neuromelanin-containing dopamine neurons in the substantia nigra of the midbrain. The contribution of non-neuronal cells to neuron degeneration in PD is receiving increasing attention. Here, we generated functional microglia-like cells from induced pluripotent stem cells (iPSC) from patients with PD associated to LRRK2 mutations, the most common cause of genetic PD, along with their gene-corrected isogenic controls and with iPSC from healthy donors. Microglia-like cells of either genotype did not induce neurodegeneration of healthy dopamine neurons in co-culture experiments. However, LRRK2-mutant microglia became hyperreactive upon LPS stimulation when compared with controls, as judged by cytokine expression profile, production of reactive oxygen species. We then tested a-synuclein and neuromelanin as potential endogenous stimuli for activating mutant microglia. Upon exposure to neuromelanin-containing particles, but not to preformed a-synuclein fibrils, LRRK2-mutant microglia induced the degeneration of healthy dopamine neurons, in a process that could be prevented by pre-treatment with the immunomodulatory drug ivermectin. Finally, the analysis of post-mortem midbrain tissue of LRRK2-PD patients found increased numbers of activated microglia cells in close contact with neuromelanin-containing neurons. Taken together, our findings uncover a potential critical role of neuromelanin-activated microglia in the context of PD progression, and provide an experimental model of PD to test new therapeutic targets. ### Competing Interest Statement The authors have declared no competing interest.

Parkinson's disease (PD) is a neurodegenerative disease associated with progressive death of midbrain dopamine (DAn) neurons in the substantia nigra (SN). Since it has been proposed that patients with PD exhibit an overall proinflammatory state, and since astrocytes are key mediators of the inflammation response in the brain, here we sought to address whether astrocyte-mediated inflammatory signaling could contribute to PD neuropathology. For this purpose, we generated astrocytes from induced pluripotent stem cells (iPSCs) representing patients with PD and healthy controls. Transcriptomic analyses identified a unique inflammatory gene expression signature in PD astrocytes compared with controls. In particular, the proinflammatory cytokine IL-6 was found to be highly expressed and released by PD astrocytes and was found to induce toxicity in DAn. Mechanistically, neuronal cell death was mediated by IL-6 receptor (IL-6R) expressed in human PD neurons, leading to downstream activation of STAT3. Blockage of IL-6R by the addition of the FDA-approved anti-IL-6R antibody, Tocilizumab, prevented PD neuronal death. SN neurons overexpressing IL-6R and reactive astrocytes expressing IL-6 were detected in postmortem brain tissue of patients at early stages of PD. Our findings highlight the potential role of astrocyte-mediated inflammatory signaling in neuronal loss in PD and pave the way for the design of future therapeutics.

Background: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the MYBPC3 gene (p.Lys600Asnfs*2), but who exhibited highly divergent clinical manifestations. Methods: We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant. Results: Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant. Conclusions: Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3 . Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.

Background Induced pluripotent stem cell (iPSC)-derived cell therapies are an interesting new area in the field of regenerative medicine. One of the approaches to decrease the costs of iPSC-derived therapies is the use of allogenic homozygous human leukocyte antigen (HLA)-matched donors to generate iPSC lines and to build a clinical-grade iPSC bank covering a high percentage of the Spanish population. Methods The Spanish Stem Cell Transplantation Registry was screened for cord blood units (CBUs) homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes. Seven donors were selected with haplotypes covering 21.37% of the haplotypes of the Spanish population. CD34-positive hematopoietic progenitors were isolated from the mononuclear cell fraction of frozen cord blood units from each donor by density gradient centrifugation and further by immune magnetic labeling and separation using purification columns. Purified CD34 + cells were reprogrammed to iPSCs by transduction with the CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit. Results The iPSCs generated from the 7 donors were expanded, characterized, banked and registered. Master cell banks (MCBs) and working cell banks (WCBs) from the iPSCs of each donor were produced under GMP conditions in qualified clean rooms. Conclusions Here, we present the first clinical-grade, iPSC haplobank in Spain made from CD34 + cells from seven cord blood units homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes within the Spanish population. We describe their generation by transduction with Sendai viral vectors and their GMP-compliant expansion and banking. These haplolines will constitute starting materials for advanced therapy medicinal product development (ATMP).

Tyrosine hydroxylase deficiency (THD) is a rare genetic disorder leading to dopaminergic depletion and early-onset parkinsonism. Affected children present with either a severe form that does not respond to L-Dopa treatment (THD-B), or a milder L-Dopa responsive form (THD-A). We generated induced pluripotent stem cells (iPSCs) from THD patients that were differentiated into dopaminergic neurons (DAn) and compared with control-DAn from healthy individuals and gene-corrected isogenic controls. Consistent with patients, THD iPSC-DAn displayed lower levels of DA metabolites and reduced TH expression, when compared to controls. Moreover, THD iPSC-DAn showed abnormal morphology, including reduced total neurite length and either an abnormal TH proximodistal gradient (THDA), or neurite arborization defects (THDB). Treatment of THD-iPSC-DAn with L-Dopa rescued the neuronal defects and disease phenotype only in THDA-DAn. Interestingly, L-Dopa treatment at the stage of neuronal precursors could prevent the alterations in THDB-iPSC-DAn, thus suggesting the existence of a critical developmental window in THD. Our iPSC-based model recapitulates THD disease phenotypes and response to treatment, representing a promising tool for investigating pathogenic mechanisms, drug screening, and personalized management.