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.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by adrenergically induced ventricular arrhythmias that cause sudden cardiac death. Using non-integrative episomal plasmids we reprogrammed skin fibroblasts of three heterozygous and two homozygous carriers of a mutation in the gene that encodes the ryanodine receptor type 2 (RYR2), RYR2_c.G1069A/p.G357S, previously associated to CPVT in a large family of the Gran Canaria Island. The resulting hiPSC cell lines have normal karyotype, differentiate into cells of the 3 germ layers, and express pluripotency markers and genes.

Despite rapid clinical translation, induced pluripotent stem cell (iPSC)-derived therapies face limited global adoption. Harmonized quality control (QC) remains absent, with even fundamental parameters evaluated inconsistently across laboratories. To address this, we conducted two international Quality Assessment Rounds (QARs): QAR 2019 (18 sites, 11 countries) and QAR 2023 (23 sites, 12 countries), evaluating flow cytometry-based assessment of the undifferentiated state and qPCR-based genomic integrity testing. QAR 2019 showed high consistency in genomic integrity testing, while uncovering substantial variability in flow cytometry, prompting QAR 2023 to introduce standardized workflows. These improvements enabled systematic, cross-site evaluation of marker performance across cell states, identifying OCT3/4, TRA-1-60, and SSEA5 as consistently robust pluripotency-associated markers. This global benchmarking effort provides the first empirical multi-site evidence for reproducible iPSC QC and marker-level reliability. Together, these findings establish a foundation for harmonized QC supporting interoperable iPSC banks, regulatory alignment, and scalable manufacturing of globally accessible regenerative therapies.

Patient-derived induced pluripotent stem cells (hiPSC) are a valuable approach to model cardiovascular diseases. We nucleofected non-integrating episomal vectors in skin fibroblasts of four family members. Two of them carried the single nucleotide variant (SNV) SCN5A_c.287 T > C, leading to NaV1.5_p.L96P, and two were non-carrier family members. The resulting hiPSC cell lines differentiate into cells of the 3 germ layers, display normal karyotypes and express markers of the undifferentiated hPSC state. Thus, they are a reliable source to study the effect of the identified mutation in a physiologically relevant environment.

mRNA reprogramming is a technology for generating iPSCs with high efficiency and safety. However, it is not suitable for reprogramming non-adherent cells, the primary cell type in blood. An alternative is to obtain adherent cells from blood for this technology. To validate this approach, we generated a human iPSC line from blood outgrowth endothelial cells (BOECs) using mRNA-based reprogramming. The resulting line, CTRL EiPS J9 mR6F-8, meets iPSC criteria, including an undifferentiated state, pluripotency, genome integrity. This line is available upon request.

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).

Tissue-specific cells differentiated from patient-derived human induced pluripotent stem cells (hiPSC) are a relevant cellular model to study several diseases. We obtained a hiPSC line from skin fibroblasts of a patient affected by familial atrial fibrillation by nucleofection of non-integrating episomal vectors. The resulting hiPSC line displays a normal karyotype, expresses pluripotency surface markers and pluripotency genes, and differentiates into cells of the 3 germ layers. Therefore, it represents a reliable model to study the disease in a physiologically relevant cellular environment.

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease caused by the abnormal production of misfolded TTR protein by liver cells, which is then released systemically. Its amyloid deposition in the heart is linked to cardiac toxicity and progression toward heart failure. A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) from a patient suffering familial transthyretin amyloid cardiomyopathy carrying a c.128G>A (p.Ser43Asn) mutation in the TTR gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for therapeutic discovery.

NF2-related schwannomatosis is an autosomal dominant syndrome that predisposes to the development of benign tumors of the nervous system. Schwannomas, particularly bilateral vestibular schwannomas (VS), are the most characteristic features of the disease. These tumors are caused by the bi-allelic inactivation of the NF2 gene in a cell of the Schwann cell lineage. Our current understanding of the molecular pathogenesis of the NF2 gene, as well as the development of new effective therapies is hampered by the absence of human non-perishable cell-based bearing distinct NF2 pathogenic variants. With this aim, we generated and characterized three isogenic paired induced pluripotent stem cell (iPSC) lines with single or bi-allelic inactivation of NF2 by combining the direct reprogramming of VS cells with the use of CRISPR/Cas9 editing. Our results show a critical function of NF2 for the maintenance of a stable pluripotent state. However, we were able to nudge them towards the Neural Crest-Schwann Cell (NC-SC) axis by applying a 3D Schwann cell differentiation protocol. NF2 (+/−) and NF2 (−/−) spheroids homogeneously expressed classical markers of the NC-SC lineage. In addition, NF2 (−/−) SC-like spheroids showed dysregulation of multiple signaling pathways already described for merlin-deficient SC, and altered in human schwannomas. Therefore, NF2 (+/−) and NF2 (−/−) SC-like spheroids can represent a bona fide human in vitro cellular model to study the role of NF2 pathogenesis.### Competing Interest StatementThe authors have declared no competing interest.

The effects of genetic mutations on protein function can be studied in a physiologically relevant environment using tissue-specific cells differentiated from patient-derived induced pluripotent stem cells (iPSC). However, it is crucial to use iPSC derived from healthy individuals as control. We generated an iPS cell line from skin fibroblasts of a healthy Caucasian male by nucleofection of non-integrating episomal vectors. This cell line has normal karyotype, expresses pluripotency surface markers and pluripotency genes, and successfully differentiates into cells of the 3 germ layers. Therefore, it can be used as control for any disease of interest that is modelled using iPSC.

Patient-derived induced pluripotent stem cells (iPSC) are a valuable approach to model cardiovascular diseases. We nucleofected non-integrating episomal vectors in skin fibroblasts of three family members carrying a single nucleotide variant (SNV) in SCN5A, which encodes the cardiac-type sodium channel, and of a related healthy control. The SNV SCN5A_c.4573G > A had been previously identified in a Brugada Syndrome patient. The resulting iPS cell lines differentiate into cells of the 3 germ layers, display normal karyotypes and express pluripotency surface markers and genes. Thus, they are a reliable source to study the effect of the identified mutation in a physiologically relevant environment.

ABSTRACTNF2-related schwannomatosis is an autosomal dominant syndrome that predisposes to the development of benign tumors of the nervous system. Schwannomas, particularly bilateral vestibular schwannomas (VS), are the most characteristic features of the disease. These tumors are caused by the bi-allelic inactivation of theNF2gene in a cell of the Schwann cell lineage. Our current understanding of the molecular pathogenesis of theNF2gene, as well as the development of new effective therapies is hampered by the absence of human non-perishable cell-based bearing distinctNF2pathogenic variants. With this aim, we generated and characterized three isogenic paired induced pluripotent stem cell (iPSC) lines with single or bi-allelic inactivation ofNF2by combining the direct reprogramming of VS cells with the use of CRISPR/Cas9 editing. Our results show a critical function ofNF2for the maintenance of a stable pluripotent state. However, we were able to nudge them towards the Neural Crest-Schwann Cell (NC-SC) axis by applying a 3D Schwann cell differentiation protocol.NF2(+/−) andNF2(−/−) spheroids homogeneously expressed classical markers of the NC-SC lineage. In addition,NF2(−/−) SC-like spheroids showed dysregulation of multiple signaling pathways already described for merlin-deficient SC, and altered in human schwannomas. Therefore,NF2(+/−) andNF2(−/−) SC-like spheroids can represent a bona fide humanin vitrocellular model to study the role ofNF2pathogenesis.

Background: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). Methods: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. Results: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. Conclusions: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA.

Skin fibroblasts were obtained from four patients with 7q11.23 microduplication syndrome carrying the reciprocal rearrangement of Williams-Beuren syndrome at the 7q11.23 genomic region. Induced pluripotent stem cells (iPSCs) were generated by retroviral infection of fibroblasts with polycystronic vectors. The generated iPSC clones ESi058B, ESi057B, ESi070A and ESi071A had the 7q11.23 duplication with no additional genomic alterations, a stable karyotype, expressed pluripotency markers and could differentiate towards the three germ layers in vitro via embryoid body formation and in vivo by teratoma formation. Patient's derived iPSCs are a valuable resource for in vitro modeling of 7q11.23 microduplication syndrome. Resource Table.

A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

A skin biopsy was obtained from a 25-year-old female patient with autosomal recessive Alport syndrome (ARAS) with the homozygous COL4A3 mutation c.345delG, p.(P166Lfs*37). Dermal fibroblasts were derived and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53shRNA. The generated induced Pluripotent Stem Cell (iPSC) clone AS FiPS1 Ep6F-2 was free of genomically integrated reprogramming genes, had the specific homozygous mutation, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. This iPSC line offers a useful resource to study Alport syndrome pathomechanisms and drug testing.