Generation of a human induced pluripotent stem cell line from urinary cells of a healthy donor using integration free Sendai virus technology

We have generated a human induced pluripotent stem cell (iPSC) line derived from urinary cells of a 28 year old healthy female donor. The cells were reprogrammed using a non-integrating viral vector and have shown full differentiation potential. Together with the iPSC line, the donor provided blood cells for the study of immunological effects of the iPSC line and its derivatives in autologous and allogeneic settings. The line is available and registered in the human pluripotent stem cell registry as BCRTi005-A

Generation of integration free induced pluripotent stem cells from fibrodysplasia ossificans progressiva (FOP) patients from urine samples

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare, autosomal dominant transmitted genetic disease. Patients experience progressive bone formation replacing tendons, ligaments, muscle and soft tissue. Cause of FOP are gain-of-function mutations in the Bone Morphogenetic Protein (BMP) receptor Activin A receptor type 1 (ACVR1) ( Kaplan et al., 2008). The most common mutation is R206H, which leads to the substitution of codon 206 from arginine to histidine (Shore et al., 2006). Here, we describe the derivation and characterization of two hiPSC lines from two FOP patients, both carrying the mutation R206H. Cells were isolated from urine and reprogrammed using integration free Sendai virus vectors under defined conditions

hPSCreg - the human pluripotent stem cell registry

The human pluripotent stem cell registry (hPSCreg), accessible at http://hpscreg.eu, is a public registry and data portal for human embryonic and induced pluripotent stem cell lines (hESC and hiPSC). Since their first isolation the number of hESC lines has steadily increased to over 3000 and new iPSC lines are generated in a rapidly growing number of laboratories as a result of their potentially broad applicability in biomedicine and drug testing. Many of these lines are deposited in stem cell banks, which are globally established to store tens of thousands of lines from healthy and diseased donors. The Registry provides comprehensive and standardized biological and legal information as well as tools to search and compare information from multiple hPSC sources and hence addresses a translational research need. To facilitate unambiguous identification over different resources, hPSCreg automatically creates a unique standardized name for each cell line registered. In addition to biological information, hPSCreg stores extensive data about ethical standards regarding cell sourcing and conditions for application and privacy protection. hPSCreg is the first global registry that holds both, manually validated scientific and ethical information on hPSC lines, and provides access by means of a user-friendly, mobile-ready web application

Dataset for: Modeling chemotherapy induced neurotoxicity with human induced pluripotent stem cell (iPSC)-derived sensory neurons

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent and potentially irreversible adverse event of cytotoxic chemotherapy. We evaluate whether sensory neurons derived from induced pluripotent stem cells (iPSC-DSN) can serve as human disease model system for chemotherapy induced neurotoxicity. Sensory neurons differentiated from two established induced pluripotent stem cell lines were used (s.c. BIHi005-A https://hpscreg.eu/cell- line/BIHi005-A and BIHi004-B https://hpscreg.eu/cell-line/BIHi004-B, Berlin Institute of Health Stem Cell Core Facility). Cell viability and cytotoxicity assays were performed, comparing susceptibility to four neurotoxic and two non-neurotoxic drugs. RNA sequencing analyses in paclitaxel vs. vehicle (DMSO)treated sensory neurons were performed. Treatment of iPSC-DSN for 24 h with the neurotoxic drugs paclitaxel, bortezomib, vincristine and cisplatin led to a dose dependent decline of cell viability in clinically relevant IC50 ranges, which was not the case for the non-neurotoxic compounds doxorubicin and 5-fluorouracil. RNA sequencing analyses at 24 h, i.e. before paclitaxel-induced cell death occurred, revealed the differential expression of genes of neuronal injury, cellular stress response, and sterol pathways in response to 1 mu M paclitaxel. Neuroprotective effects of lithium chloride co-incubation, which were previously shown in rodent dorsal root ganglia, could be replicated in human iPSC-DSN. Cell lines from the two different donors BIHi005-A and BIHi004-B showed different responses to the neurotoxic treatment in cell viability and cytotoxicity assays

Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7‐related osteopetrosis

Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl-/H+-exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. (c) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)

Somatic mutations and progressive monosomy modify SAMD9-related phenotypes in humans

It is well established that somatic genomic changes can influence phenotypes in cancer, but the role of adaptive changes in developmental disorders is less well understood. Here we have used next-generation sequencing approaches to identify de novo heterozygous mutations in sterile α motif domain–containing protein 9 (SAMD9, located on chromosome 7q21.2) in 8 children with a multisystem disorder termed MIRAGE syndrome that is characterized by intrauterine growth restriction (IUGR) with gonadal, adrenal, and bone marrow failure, predisposition to infections, and high mortality. These mutations result in gain of function of the growth repressor product SAMD9. Progressive loss of mutated SAMD9 through the development of monosomy 7 (–7), deletions of 7q (7q–), and secondary somatic loss-of-function (nonsense and frameshift) mutations in SAMD9 rescued the growth-restricting effects of mutant SAMD9 proteins in bone marrow and was associated with increased length of survival. However, 2 patients with –7 and 7q– developed myelodysplastic syndrome, most likely due to haploinsufficiency of related 7q21.2 genes. Taken together, these findings provide strong evidence that progressive somatic changes can occur in specific tissues and can subsequently modify disease phenotype and influence survival. Such tissue-specific adaptability may be a more common mechanism modifying the expression of human genetic conditions than is currently recognized

Removing critical gaps in chemical test methods by developing new assays for the identification of thyroid hormone system-disrupting chemicals—the athena project

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood–brain and blood–placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation

Spontane und hepatische Differenzierung von humanen embryonalen Stammzellen in 3D Perfusionskulturbioreaktoren

Humane embryonale Stammzellen (hESC) besitzen ein großes Potential als Zellquelle für Anwendungen in der Grundlagenforschung, bei der pharmakologischen Wirkstoffsuche, in der Toxizitätstestung und in zellbasierten Therapien in der regenerativen Medizin. Eine Grundvoraussetzung für solche Anwendungen ist die Verfügbarkeit von Methoden, mit denen sich Zellpräparationen von hoher Reinheit und ausreichender Zellzahl generieren lassen. Das Ziel des Projektes war die Untersuchung des Wachstums und der Differenzierung von hESC in einem Mehrkompartimentbioreaktorsystem für die 3D Zellperfusion. Ein Ziel dieser Studie bestand in der Untersuchung, inwieweit das Bioreaktorsystem die spontane Differenzierung von hESC in verschiedene Zelllinien und die Gewebebildung unterstützt sowie in dem Vergleich dieser Differenzierung mit der Teratombildung aus hESC in Mäusen. Ein weiteres Ziel dieser Studie war die Untersuchung der gerichteten Differenzierung von hESC in die hepatische Zelllinie. Hierzu wurde eine Methode, die ursprünglich zur gerichteten hepatischen Differenzierung von hESC in 2D Kulturen entwickelt worden ist, auf das 3D System übertragen und zwei Pilotexperimente durchgeführt. Die Ergebnisse der Experimente zur spontanen Differenzierung von hESC zeigten, dass die hESC sowohl in Zellen der drei Keimblätter als auch in Zellen der extraembryonalen Linien differenzierten und im Bioreaktor gewebeähnliche Strukturen bildeten. Der Vergleich dieser Gewebestrukturen auf RNA, Protein und histologischer Ebene mit den in Teratomen gebildeten Geweben zeigte eine sehr hohe Ähnlichkeit. In den beiden Pilotexperimenten zur hepatischen Differenzierung im Biorektorsystem ergaben sich keine signifikanten Anzeichen für eine hepatische Reifung, sondern eher für eine spontane Zelldifferenzierung. In zukünftigen Studien müssten deshalb die Konzentrationen der die hepatische Differenzierung beeinflussenden Faktoren in dem benutzten Differenzierungsprotokoll weiter optimiert werden. Die Studie ergab insgesamt, dass 3D-Perfusionsbioreaktoren eine Technologie darstellen, welche die spontane hESC Differenzierung ähnlich wie eine in vivo Umgebung unterstützt. Diese Bioreaktorsysteme könnten daher als eine in vitro Alternative zur in vivo Testung der Teratombildung, welche häufig angewandt wird um das Entwicklungspotential von pluripotenten Zellarten zu untersuchen, dienen. Die Anzahl von Tierversuchen ließe sich so reduzieren. Die definierten und kontrollierbaren Kulturbedingungen in 3D- Perfusionsbioreaktoren könnten auch genutzt werden, um verbleibende undifferenzierte Zellen in aus pluripotenten Zellen gewonnenen Zellpräparationen aufzuspüren, für die Entwicklung von Methoden zur Testung von Embryotoxizität und als ein in vitro System zur Untersuchung bestimmter Aspekte der Gewebeentwicklung.Human embryonic stem cells (hESC) hold great potential as a cell source for applications in basic science, pharmacological drug screening, toxicity testing and cell- based therapies in regenerative medicine. For these, methods for the efficient generation of highly enriched specific cell preparations are key prerequisites. The goal of the project was to investigate growth and differentiation of hESC in a multicompartment bioreactor system for 3D cell perfusion. One aim of this study was to analyze the capacity of the bioreactor system to support spontaneous multilineage differentiation and tissue formation of hESC and to compare to teratoma formation of hESC in mice. Another aim of this study was the evaluation of the directed differentiation of hESC towards the hepatic lineage. For this purpose a method of directed hepatic differentiation that was originally developed for 2D cultures of hESC was translated to the 3D system and two pilot experiments were performed. Results of the experiments on the spontaneous differentiation showed that hESC differentiated into cells of the three germ layers as well as cells of the extraembryonic lineages and formed differentiated tissue-like structures. Comparison of these tissue structures with those formed in teratomas showed a high degree of similarity on the RNA, protein and histological levels. The results of the two pilot experiments on hepatic differentiation in the bioreactor system suggested no significant hepatic maturation but rather spontaneous differentiation. Therefore the concentrations of factors that influence hepatic differentiation used in the applied differentiation protocol have to be further optimized in future studies. The results of this study suggest that 3D perfusion bioreactors provide a technology supporting spontaneous hESC differentiation, similar to an in vivo environment. Therefore the bioreactor system could be used as an in vitro alternative for the in vivo teratoma formation assay that is commonly used to explore the developmental potential of pluripotent cell types and this way can help to reduce animal testing. Furthermore the defined and controllable culture conditions render the system suitable for applications such as the safety testing of remaining undifferentiated cells in cell preparations derived from pluripotent cells, the development of embryotoxicity testing methods and its use as an in vitro system to examine certain aspects of tissue development

Semantic body browser: graphical exploration of an organism and spatially resolved expression data visualization

Advancing technologies generate large amounts of molecular and phenotypic data on cells, tissues and organisms, leading to an ever-growing detail and complexity while information retrieval and analysis becomes increasingly time-consuming. The Semantic Body Browser is a web application for intuitively exploring the body of an organism from the organ to the subcellular level and visualising expression profiles by means of semantically annotated anatomical illustrations. It is used to comprehend biological and medical data related to the different body structures while relying on the strong pattern recognition capabilities of human users.N

Corrigendum to “Generation of a human induced pluripotent stem cell line from urinary cells of a healthy donor using an integration free vector” [Stem Cell Res. 16/2 (2016) 314–317]

We have generated a human induced pluripotent stem cell (iPSC) line derived from urinary cells of a 30 year old healthy female donor. The cells were reprogrammed using a non-integrating viral vector and have shown full differentiation potential. Together with the iPSC-line, the donor provided blood cells for the study of immunological effects of the iPSC line and its derivatives in autologous and allogeneic settings. The line is available and registered in the human pluripotent stem cell registry as BCRTi004-A