The Management of Data for the Banking, Qualification, and Distribution of Induced Pluripotent Stem Cells: Lessons Learned from the European Bank for Induced Pluripotent Stem Cells.

The European Bank for induced pluripotent Stem Cells (EBiSC) was established in 2014 as a non-profit project for the banking, quality control, and distribution of human iPSC lines for research around the world. EBiSC iPSCs are deposited from diverse laboratories internationally and, hence, a key activity for EBiSC is standardising not only the iPSC lines themselves but also the data associated with them. This includes enabling unique nomenclature for the cells, as well as applying uniformity to the data provided by the cell line generator versus quality control data generated by EBiSC, and providing mechanisms to share personal data in a secure and GDPR-compliant manner. A joint approach implemented by EBiSC and the human pluripotent stem cell registry (hPSCreg®) has provided a solution that enabled hPSCreg® to improve its registration platform for iPSCs and EBiSC to have a pipeline for the import, standardisation, storage, and management of data associated with EBiSC iPSCs. In this work, we describe the experience of cell line data management for iPSC banking throughout the course of EBiSC's development as a central European banking infrastructure and present a model for how this could be implemented by other iPSC repositories to increase the FAIRness of iPSC research globally.

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction.

BACKGROUND: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. METHODS: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. RESULTS: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA-expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. CONCLUSIONS: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.

ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

Generation of two human induced pluripotent stem cell lines from a patient with Neurofibromatosis type 1 (NF1) and pathogenic NF1 gene variant c.1466 A>G BCRTi011-A as well as a first-degree healthy relative (BCRTi010-A).

We describe the generation of two human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) using a non-integrative episomal reprogramming strategy. The first cell line was derived from a NF1 patient with the genetic variant c.1466A>G (BCRTi011-A) which leads to a cryptic splice site and aberrant splicing. The second one was created from a healthy relative of first-degree (BCRTi010-A). The generated iPSC lines were shown to have tri-lineage differentiation potential, a normal karyotype, and expression of pluripotent markers. Both iPSC lines provide a powerful tool for in vitro disease modeling and therapy development.

An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis.

Transient receptor potential cation channel-6 (TRPC6) gene mutations cause familial focal segmental glomerulosclerosis (FSGS), which is inherited as an autosomal dominant disease. In patients with TRPC6-related FSGS, all mutations map to the N- or C-terminal TRPC6 protein domains. Thus far, the majority of TRPC6 mutations are missense resulting in increased or decreased calcium influx; however, the fundamental molecular mechanisms causing cell injury and kidney pathology are unclear. We report a novel heterozygous TRPC6 mutation (V691Kfs*) in a large kindred with no signs of FSGS despite a largely truncated TRPC6 protein. We studied the molecular effects of V691Kfs* TRPC6 mutant using the tridimensional cryo-EM structure of the tetrameric TRPC6 protein. The results indicated that V691 is localized at the pore-forming transmembrane region affecting the ion conduction pathway, and predicted that V691Kfs* causes closure of the ion-conducting pathway leading to channel inactivation. We assessed the impact of V691Kfs* and two previously reported TRPC6 disease mutants (P112Q and G757D) on calcium influx in cells. Our data show that the V691Kfs* fully inactivated the TRCP6 channel-specific calcium influx consistent with a complete loss-of-function phenotype. Furthermore, the V691Kfs* truncation exerted a dominant negative effect on the full-length TRPC6 proteins. In conclusion, the V691Kfs* non-functional truncated TRPC6 is not sufficient to cause FSGS. Our data corroborate recently characterized TRPC6 loss-of-function and gain-of-function mutants suggesting that one defective TRPC6 gene copy is not sufficient to cause FSGS. We underscore the importance of increased rather than reduced calcium influx through TRPC6 for podocyte cell death.

History and current status of clinical studies using human pluripotent stem cells.

The Human Pluripotent Stem Cell Registry established a database of clinical studies using human pluripotent stem cells (PSCs) as starting material for cell therapies. Since 2018, we have observed a switch toward human induced pluripotent stem cells (iPSCs) from human embryonic stem cells. However, rather than using iPSCs for personalized medicines, allogeneic approaches dominate. Most treatments target ophthalmopathies, and genetically modified iPSCs are used to generate tailored cells. We observe a lack of standardization and transparency about the PSCs lines used, characterization of the PSC-derived cells, and the preclinical models and assays applied to show efficacy and safety.

Generation of human induced pluripotent stem cell line (BCRTi007-A) from urinary cells of a patient with autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder of adults, characterized by uncontrolled cysts formation that causes a gradual impairment of kidney function. We generated a human induced pluripotent stem cell (hiPSC) line from the urinary cells of a patient diagnosed with ADPKD using a non-integrating Epi5™ Episomal iPSC reprogramming strategy. Characterization of the cell line was performed regarding their undifferentiated status, differentiation potential, and quality control for karyotypic integrity, identity, and clearance of reprogramming vectors. The newly derived hiPSC line, namely BCRTi007-A, can be used in vitro for disease modeling of ADPKD as well as testing for novel therapeutic approaches.

Generation of human induced pluripotent stem cell line (BCRTi006-A) from a patient with focal segmental glomerulosclerosis disease.

Individuals with transient reception potential cation channel 6 (TRPC6) mutation have variable phenotypes, ranging from healthy carriers to focal segmental glomerulosclerosis (FSGS). Human induced pluripotent stem cell (hiPSC) line was generated from the urinary cells of a patient with FSGS with a mutant variant of TRPC6. The cells were reprogrammed with Yamanaka factors (OCT3, SOX2, LIN28, L-MYC, and KLF4) using a commercially available Epi5 Reprogramming Kit. The pluripotency of the hiPSC line was confirmed by the expression of common stem cell markers and by their ability to generate all germ layers in vitro. The line is available and registered in the human pluripotent stem cell registry as BCRTi006-A. The generated line represents a valuable tool for disease modeling and drug development for FSGS.

Human pluripotent stem cell registry: Operations, role and current directions.

The human plutiripotent stem cell registry (hPSCreg) is a global database for human embryonic and induced pluripotent stem cells (hESC, hiPSC). The publicly accessible Registry (https://hpscreg.eu) was set up to provide a transparent resource of quality-assessed hPSC lines as well as to increase reproducibility of research and interoperability of data. OBJECTIVES: In this review, we describe the establishment of the Registry and its mission, its development into a knowledgebase for hPSC and the current status of hPSC-focussed databases. The data categories available in hPSCreg are detailed. In addition, sharing and hurdles to data sharing on a global level are described. CONCLUSIONS: An outlook is provided on the establishment of digital representatives of donors using hybrids of data and hPSC-based biological models, and how this can also be used to reposition databases as mediators between donors and researchers.

Human iPSC-Derived Renal Cells Change Their Immunogenic Properties during Maturation: Implications for Regenerative Therapies.

The success of human induced pluripotent stem cell (hiPSC)-based therapy critically depends on understanding and controlling the immunological effects of the hiPSC-derived transplant. While hiPSC-derived cells used for cell therapy are often immature with post-grafting maturation, immunological properties may change, with adverse effects on graft tolerance and control. In the present study, the allogeneic and autologous cellular immunity of hiPSC-derived progenitor and terminally differentiated cells were investigated in vitro. In contrast to allogeneic primary cells, hiPSC-derived early renal progenitors and mature renal epithelial cells are both tolerated not only by autologous but also by allogeneic T cells. These immune-privileged properties result from active immunomodulation and low immune visibility, which decrease during the process of cell maturation. However, autologous and allogeneic natural killer (NK) cell responses are not suppressed by hiPSC-derived renal cells and effectively change NK cell activation status. These findings clearly show a dynamic stage-specific dependency of autologous and allogeneic T and NK cell responses, with consequences for effective cell therapies. The study suggests that hiPSC-derived early progenitors may provide advantageous immune-suppressive properties when applied in cell therapy. The data furthermore indicate a need to suppress NK cell activation in allogeneic as well as autologous settings.

Transposon-mediated glial cell line-derived neurotrophic factor overexpression in human adipose tissue-derived mesenchymal stromal cells: A potential approach for neuroregenerative medicine?

Glial cell line-derived neurotrophic factor (GDNF) has neuroprotective effects and may be a promising candidate for regenerative strategies focusing on neurodegenerative diseases. As GDNF cannot cross the blood-brain barrier to potentially regenerate damaged brain areas, continuous in situ delivery with host cells is desired. Here, a non-viral Sleeping Beauty transposon was used to achieve continuous in vitro overexpression of GDNF in immune-privileged human adipose tissue-derived mesenchymal stromal cells (GDNF-tASCs). In addition, in vivo survival, tolerance, and effectiveness of transfected cells were tested in a very mild 6-hydroxydopamine (6-OHDA)-induced dopamine depletion rat model by means of intrastriatal injection on a sample basis up to 6 months after treatment. GDNF-tASCs showed vast in vitro gene overexpression up to 13 weeks post-transfection. In vivo, GDNF was detectable 4 days following transplantation, but no longer after 1 month, although adipose tissue-derived mesenchymal stromal cells (ASCs) could be visualized histologically even after 6 months. Despite successful long-term in vitro GDNF overexpression and its in vivo detection shortly after cell transplantation, the 6-OHDA model was too mild to enable sufficient evaluation of in vivo disease improvement. Still, in vivo immunocompatibility could be further examined. ASCs initially induced a pronounced microglial accumulation at transplantation site, particularly prominent in GDNF-tASCs. However, 6-OHDA-induced pro-inflammatory immune response was attenuated by ASCs, although delayed in the GDNF-tASCs group. To further test the therapeutic potential of the generated GDNF-overexpressing cells in a disease-related context, a follow-up study using a more appropriate 6-OHDA model is needed.

Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system.

OBJECTIVE: To provide a standardized protocol for large-scale production of proximal tubular epithelial cells (PTEC) generated from human pluripotent stem cells (hPSC). METHODS: The hPSC were expanded and differentiated into PTEC on matrix-coated alginate beads in an automated levitating fluidic platform bioLevitator. Differentiation efficacy was evaluated by immunofluorescence staining and flow cytometry, ultrastructure visualized by electron microscopy. Active reabsorption by PTEC was investigated by glucose, albumin, organic anions and cations uptake assays. Finally, the response to cisplatin-treatment was assessed to check the potential use of PTEC to model drug-induced nephrotoxicity. RESULTS: hPSC expansion and PTEC differentiation could be performed directly on matrix-coated alginate beads in suspension bioreactors. Renal precursors arose 4 days post hPSC differentiation and PTEC after 8 days with 80% efficiency, with a 10-fold expansion from hPSC in 24 days. PTEC on beads, exhibited microvilli and clear apico-basal localization of markers. Functionality of PTECs was confirmed by uptake of glucose, albumin, organic anions and cations and expression of KIM-1 after Cisplatin treatment. CONCLUSION: We demonstrate the efficient expansion of hPSC, controlled differentiation to renal progenitors and further specification to polarized tubular epithelial cells. This is the first report employing biolevitation and matrix-coated beads in a completely defined medium for the scalable and potentially automatable production of functional human PTEC.

3D-SeboSkin Model for Human ex vivo Studies of Hidradenitis Suppurativa/Acne Inversa.

BACKGROUND: Hidradenitis suppurativa/acne inversa (HS) is a chronic, recurrent inflammatory skin disease. Its pivotal pathogenetic event is believed to be the occlusion of the hair follicle generating a perifollicular lympho-histiocytic inflammation. However, knowledge of the exact HS pathogenesis requires further research. OBJECTIVE: To develop a human HS model applicable in preclinical research which could help to understand the pathophysiology of HS and to determine the action of therapeutic candidates. METHODS: The 3D-SeboSkin technology was applied to maintain explants of involved and uninvolved skin of HS patients ex vivo for 3 days. Detection of differential expression of previously detected HS biomarkers was performed by immunohistochemistry in a group of female patients (n = 9, mean age 37.2 ± 8.4 years). RESULTS: The application of the 3D-SeboSkin model preserved the structural integrity of lesional and perilesional HS skin ex vivo, as previously described for healthy skin. Moreover, the HS 3D-SeboSkin setting maintained the differential expression and pattern of several HS biomarkers (S100A9, KRT16, SERPINB3) in epidermal and dermal tissue and the appendages. CONCLUSION: We have validated HS 3D-SeboSkin as a reproducible, human model, which is appropriate for preclinical lesional and perilesional HS skin studies ex vivo.

Generation of 20 human induced pluripotent stem cell lines from patients with focal segmental glomerulosclerosis (FSGS).

Focal segmental glomerulosclerosis (FSGS) is a major cause of familial nephrotic syndrome. We generated 20 induced pluripotent stem cell lines from patients diagnosed with FSGS. The iPSC lines include 8 female and 12 male lines and cover a donor age range from 31 to 78. The lines were generated from peripheral blood mononuclear cells by integration-free reprogramming using Sendai virus vectors. Cell lines were fully characterized regarding their pluripotency and differentiation potential, and quality controlled for karyotypic integrity, identity and clearance of reprogramming vectors. The generated cell lines represent a valuable tool for disease modelling and drug development for FSGS.

Integrated Collection of Stem Cell Bank Data, a Data Portal for Standardized Stem Cell Information.

The past decade has witnessed an extremely rapid increase in the number of newly established stem cell lines. However, due to the lack of a standardized format, data exchange among stem cell line resources has been challenging, and no system can search all stem cell lines across resources worldwide. To solve this problem, we have developed the Integrated Collection of Stem Cell Bank data (ICSCB) (http://icscb.stemcellinformatics.org/), the largest database search portal for stem cell line information, based on the standardized data items and terms of the MIACARM framework. Currently, ICSCB can retrieve >16,000 cell lines from four major data resources in Europe, Japan, and the United States. ICSCB is automatically updated to provide the latest cell line information, and its integrative search helps users collect cell line information for over 1,000 diseases, including many rare diseases worldwide, which has been a formidable task, thereby distinguishing itself from other database search portals.

The Global Alliance for iPSC Therapies (GAiT).

The Global Alliance for iPSC Therapies (GAiT) is a new initiative to support the implementation and clinical application of therapies derived from pluripotent stem cells to the benefit of patients globally. GAiT's mission is to serve as a central, international resource for those organisations developing therapies from clinical-grade induced pluripotent stem cells, and to support the expansion of this nascent field. With the support of its international partners, GAiT already has an early position on manufacturing, regulatory and quality standards. This article details GAiT's development, its mission and structure, as well as how, and by whom, it is funded. The article ends with brief overview of current and upcoming activities.

Human Cell Atlas and cell-type authentication for regenerative medicine.

In modern biology, the correct identification of cell types is required for the developmental study of tissues and organs and the production of functional cells for cell therapies and disease modeling. For decades, cell types have been defined on the basis of morphological and physiological markers and, more recently, immunological markers and molecular properties. Recent advances in single-cell RNA sequencing have opened new doors for the characterization of cells at the individual and spatiotemporal levels on the basis of their RNA profiles, vastly transforming our understanding of cell types. The objective of this review is to survey the current progress in the field of cell-type identification, starting with the Human Cell Atlas project, which aims to sequence every cell in the human body, to molecular marker databases for individual cell types and other sources that address cell-type identification for regenerative medicine based on cell data guidelines.

Mitigating Deficiencies in Evidence during Regulatory Assessments of Advanced Therapies: A Comparative Study with Other Biologicals.

Advanced therapy medicinal products (ATMPs) comprising cell therapy, gene therapy, and tissue-engineered products, offer a multitude of novel therapeutic approaches to a wide range of severe and debilitating diseases. To date, several advanced therapies have received marketing authorization for a variety of indications. However, some products showed disappointing market performance, leading to their withdrawal. The available evidence for quality, safety, and efficacy at product launch can play a crucial rule in their market success. To evaluate the sufficiency of evidence in submissions of advanced therapies for marketing authorization and to benchmark them against more established biological products, we conducted a matched comparison of the regulatory submissions between ATMPs and other biologicals. We applied a quantitative assessment of the regulatory objections and divergence from the expected data requirements as indicators of sufficiency of evidence and regulatory flexibilty, respectively. Our results demonstrated that product manufacturing was challenging regardless of the product type. Advanced therapies displayed critical deficiencies in the submitted clinical data. The submitted non-clinical data packages benefited the most from regulatory flexibility. Additionally, ATMP developers need to comply with more commitments in the post-approval phase, which might add pressure on market performance. Mitigating such observed deficiencies in future product development, may leverage their potential for market success.

A Manually Curated Database on Clinical Studies Involving Cell Products Derived from Human Pluripotent Stem Cells.

The last 5 years have witnessed a significant increase in the number of clinical studies based on human pluripotent stem cells (hPSCs). In parallel, concern is increasing about the proliferation of unregulated stem cell treatments worldwide. Regulated clinical testing is a de facto standard to establish the safety and efficacy of new cell therapies, yet reliable information on clinical studies involving hPSCs is scattered. Our analysis of a multitude of resources found 54 clinical studies involving several types of hPSCs, which are performed in ten countries. While the majority of those studies is based on human embryonic stem cells (hESCs), clinical studies involving human induced pluripotent stem cells increased more strongly in the past 2 years than the number of hESC-based studies. A publicly accessible database was created using the human pluripotent stem cell registry (https://hpscreg.eu) platform, providing a steadily updated comprehensive overview on hPSC-based clinical studies performed worldwide.

Access to stem cell data and registration of pluripotent cell lines: The Human Pluripotent Stem Cell Registry (hPSCreg).

The value of human pluripotent stem cells (hPSC) in regenerative medicine has yet to reach its full potential. The road from basic research tool to clinically validated PSC-derived cell therapy products is a long and winding one, leading researchers, clinicians, industry and regulators alike into undiscovered territory. All stakeholders must work together to ensure the development of safe and effective cell therapies. Similarly, utilization of hPSC in meaningful and controlled disease modeling and drug screening applications requires information on the quality and suitability of the applied cell lines. Central to these common goals is the complete documentation of hPSC data, including the ethical provenance of the source material, the hPSC line derivation, culture conditions and genetic constitution of the lines. Data surrounding hPSC is scattered amongst diverse sources, including publications, supplemental data, researcher lab books, accredited lab reports, certificates of analyses and public data repositories. Not all of these data sources are publicly accessible nor associated with metadata nor stored in a standard manner, such that data can be easily found and retrieved. The Human Pluripotent Stem Cell Registry (hPSCreg; https://hpscreg.eu/) was started in 2007 to impart provenance and transparency towards hPSC research by registering and collecting standard properties of hPSC lines. In this chapter, we present a short primer on the history of stem cell-based products, summarize the ethical and regulatory issues introduced in the course of working with hPSC-derived products and their associated data, and finally present the Human Pluripotent Stem Cell Registry as a valuable resource for all stakeholders in therapies and disease modeling based on hPSC-derived cells.