Proinflammatory phenotype of iPS cell-derived JAK2 V617F megakaryocytes induces fibrosis in 3D in vitro bone marrow niche.

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery.

Transformation of primary murine peritoneal mast cells by constitutive KIT activation is accompanied by loss of Cdkn2a/Arf expression.

Mast cells (MCs) are immune cells of the myeloid lineage distributed in tissues throughout the body. Phenotypically, they are a heterogeneous group characterized by different protease repertoires stored in secretory granules and differential presence of receptors. To adequately address aspects of MC biology either primary MCs isolated from human or mouse tissue or different human MC lines, like HMC-1.1 and -1.2, or rodent MC lines like L138.8A or RBL-2H3 are frequently used. Nevertheless, cellular systems to study MC functions are very limited. We have generated a murine connective tissue-like MC line, termed PMC-306, derived from primary peritoneal MCs (PMCs), which spontaneously transformed. We analyzed PMC-306 cells regarding MC surface receptor expression, effector functions and respective signaling pathways, and found that the cells reacted very similar to primary wildtype (WT) PMCs. In this regard, stimulation with MAS-related G-protein-coupled receptor member B2 (MRGPRB2) ligands induced respective signaling and effector functions. Furthermore, PMC-306 cells revealed significantly accelerated cell cycle progression, which however was still dependent on interleukine 3 (IL-3) and stem cell factor (SCF). Phenotypically, PMC-306 cells adopted an immature connective tissue-like MCs appearance. The observation of cellular transformation was accompanied by the loss of Cdkn2a and Arf expression, which are both described as critical cell cycle regulators. The loss of Cdkn2a and Arf expression could be mimicked in primary bone marrow-derived mast cells (BMMCs) by sustained SCF supplementation strongly arguing for an involvement of KIT activation in the regulation of Cdkn2a/Arf expression. Hence, this new cell line might be a useful tool to study further aspects of PMC function and to address tumorigenic processes associated with MC leukemia.

Genetic and Molecular Characterization of the Immortalized Murine Hepatic Stellate Cell Line GRX.

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells.

CRISPR/Cas9-engineered human ES cells harboring heterozygous and homozygous c-KIT knockout.

The receptor tyrosine kinase c-KIT (CD117) has a key role in hematopoiesis and is a marker for endothelial and cardiac progenitor cells. In vivo, deficiency of c-KIT is lethal and therefore using CRISPR/Cas9 editing we generated heterozygous and homozygous c-KIT knockout human embryonic stem cell (ES cell) lines. The c-KIT knockout left ES cell pluripotency unaffected as shown by immunofluorescence and trilineage differentiation potential. Heterozygous and homozygous c-KIT knockouts showed complete loss of exon 17, resulting in ablation of c-KIT protein from the cell surface. c-KIT knockout ES cells provide a valuable tool for further investigating c-KIT biology.

CALR frameshift mutations in MPN patient-derived iPSCs accelerate maturation of megakaryocytes.

Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem cells (iPSCs) to establish an MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in granulocytic cells derived from homozygous CALR mutant iPSCs, rescued by repairing the mutation using CRISPR/Cas9. iPSC-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelet protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated versus unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis dependent on its zygosity, and found novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.

CRISPR/Cas9 mediated CXCL4 knockout in human iPS cells of polycythemia vera patient with JAK2 V617F mutation.

The chemokine CXCL4/platelet factor 4 (PF4) gene, a key player in myelofibrosis, was knocked out by CRISPR/Cas9 in induced pluripotent stem cells (iPS cells) of a polycythemia vera (PV) patient with JAK2 V617F mutation. Two CXCL4KO iPS cell lines with and without JAK2 V617F mutation (UKAi002-B-1 and UKAi002-A-1, respectively) were generated. CXCL4KO iPS cells showed deletion of exon 1 and complete loss of CXCL4 protein. Pluripotency of iPS cells was confirmed by expression of pluripotency markers and trilineage differentiation. CXCL4KO iPS cells are expected to provide a valuable tool for investigating the role of CXCL4 in human diseases.

Nintedanib targets KIT D816V neoplastic cells derived from induced pluripotent stem cells of systemic mastocytosis.

The KIT D816V mutation is found in >80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. Therefore, KIT D816V represents a prime therapeutic target for SM. Here, we generated a panel of patient-specific KIT D816V induced pluripotent stem cells (iPSCs) from patients with aggressive SM and mast cell leukemia to develop a patient-specific SM disease model for mechanistic and drug-discovery studies. KIT D816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineered KIT D816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed for KIT D816V iPSC hematopoiesis. KIT D816V causes constitutive activation of the KIT tyrosine kinase receptor, and we exploited our iPSCs and ESCs to investigate new tyrosine kinase inhibitors targeting KIT D816V. Our study identified nintedanib, a US Food and Drug Administration-approved angiokinase inhibitor that targets vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derived KIT D816V hematopoietic progenitor cells and MCs in the nanomolar range. Nintedanib was also active on primary samples of KIT D816V SM patients. Molecular docking studies show that nintedanib binds to the adenosine triphosphate binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V-targeted therapy of advanced SM.

Prospects and challenges for the genetic counsellor profession in the German-speaking countries: report of a workshop.

The genetic counsellor profession has not yet been established in the German-speaking countries. In 2019 the Medical University of Innsbruck inaugurated the first German-taught Master's degree programme in Genetic and Genomic Counselling. In order to discuss prospects and challenges of the genetic counsellor profession in Germany, Austria and Switzerland (DACH region), the MSc programme team organized a two-day workshop with international speakers and medical geneticists from the DACH region. Day 1 was dedicated to the history, training and international profile of the genetic counsellor profession. Day 2 focused on four specific topics: (i) professional role, (ii) acceptance and job title, (iii) formal requirements and (iv) remuneration concepts for genetic counsellors in the DACH region. The workshop showed that the key factor for the successful implementation of the genetic counsellor profession is acceptance and trust within the medical genetics team. Genetic counsellors complement patient care in aspects that might be underserved considering the increasing demand of counselling in genomic medicine. Successful establishment of the genetic counsellor profession will entail the development of interprofessional teams under medical supervision and in the team of medical geneticists.

Inherited cases of CNOT3-associated intellectual developmental disorder with speech delay, autism, and dysmorphic facies.

De novo pathogenic variants in CNOT3 have recently been reported in a developmental delay disorder (intellectual developmental disorder with speech delay, autism, and dysmorphic facies [IDDSADF, OMIM: #618672]). The patients present with a variable degree of developmental delay and behavioral problems. To date, all reported disease-causing variants occurred de novo and no parent-child transmission was observed. We report for the first time autosomal dominant transmissions of the CNOT3-associated developmental disorder in two unrelated families. The clinical characteristics in our patients match the IDDSADF features reported so far and suggest substantial variability of the phenotype within the same family.

A Newly Established Murine Cell Line as a Model for Hepatocellular Cancer in Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) has become a major risk factor for hepatocellular cancer (HCC) due to the worldwide increasing prevalence of obesity. However, the pathophysiology of NASH and its progression to HCC is incompletely understood. Thus, the aim of this study was to generate a model specific NASH-derived HCC cell line. A murine NASH-HCC model was conducted and the obtained cancer cells (N-HCC25) were investigated towards chromosomal aberrations, the expression of cell type-specific markers, dependency on nutrients, and functional importance of mTOR. N-HCC25 exhibited several chromosomal aberrations as compared to healthy hepatocytes. Hepatocytic (HNF4), EMT (Twist, Snail), and cancer stem cell markers (CD44, EpCAM, CK19, Sox9) were simultaneously expressed in these cells. Proliferation highly depended on the supply of glucose and FBS, but not glutamine. Treatment with a second generation mTOR inhibitor (KU-0063794) resulted in a strong decrease of cell growth in a dose-dependent manner. In contrast, a first generation mTOR inhibitor (Everolimus) only slightly reduced cell proliferation. Cell cycle analyses revealed that the observed growth reduction was most likely due to G1/G0 cell cycle arrest. These results indicate that N-HCC25 is a highly proliferative HCC cell line from a NASH background, which might serve as a suitable in vitro model for future investigations of NASH-derived HCC.

The role of Nav1.7 in human nociceptors: insights from human induced pluripotent stem cell-derived sensory neurons of erythromelalgia patients.

The chronic pain syndrome inherited erythromelalgia (IEM) is attributed to mutations in the voltage-gated sodium channel (NaV) 1.7. Still, recent studies targeting NaV1.7 in clinical trials have provided conflicting results. Here, we differentiated induced pluripotent stem cells from IEM patients with the NaV1.7/I848T mutation into sensory nociceptors. Action potentials in these IEM nociceptors displayed a decreased firing threshold, an enhanced upstroke, and afterhyperpolarization, all of which may explain the increased pain experienced by patients. Subsequently, we investigated the voltage dependence of the tetrodotoxin-sensitive NaV activation in these human sensory neurons using a specific prepulse voltage protocol. The IEM mutation induced a hyperpolarizing shift of NaV activation, which leads to activation of NaV1.7 at more negative potentials. Our results indicate that NaV1.7 is not active during subthreshold depolarizations, but that its activity defines the action potential threshold and contributes significantly to the action potential upstroke. Thus, our model system with induced pluripotent stem cell-derived sensory neurons provides a new rationale for NaV1.7 function and promises to be valuable as a translational tool to profile and develop more efficacious clinical analgesics.

Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells.

Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency. To study IRF8 in human hematopoiesis we generated human IRF8-/- iPS cells and IRF8-/- embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. Upon induction of hematopoietic differentiation, we demonstrate that IRF8 is dispensable for iPS cell and ES cell differentiation into hemogenic endothelium and for endothelial-to-hematopoietic transition, and thus development of hematopoietic progenitors. We differentiated iPS cell and ES cell derived progenitors into CD141+ cross-presenting cDC1 and CD1c+ classical cDC2 and CD303+ plasmacytoid DC (pDC). We found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. Additionally, in an unrestricted differentiation regimen, IRF8-/- iPS cells and ES cells exhibited a clear bias toward granulocytes at the expense of monocytes. IRF8-/- DC showed reduced MHC class II expression and were impaired in cytokine responses, migration, and antigen presentation. Taken together, we engineered a human IRF8 knockout model that allows studying molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC. Stem Cells 2017;35:898-908.

Patient with three euchromatic supernumerary marker chromosomes derived from chromosomes 1, 12, and 18: characterization and evaluation of the aberrations.

The genetic relevance of small supernumerary marker chromosomes (sSMCs) depends on their content of euchromatin. In case of mosaicism, the phenotype of the carrier furthermore is influenced by the distribution of the marker in the body. In the majority of reported cases no correlation of the degree of mosaicism in the tissue(s) analyzed and the phenotype could be detected. In particular, non-acrocentric derived sSMCs show a strong tendency to appear in mosaic state irrespective of the clinical picture. We present a patient with cognitive disability and mild craniofacial dysmorphisms with mosaicism of three different autosomal marker chromosomes. The extra chromosomes were analyzed by a combination of SNP array and a variety of fluorescence in situ hybridization (FISH) probes. All three markers were identified as ring chromosomes containing different amounts of euchromatic material derived from chromosome 1 (1p12 → q21), 12 (12p13.1 → q13.11) and 18 (18p11.21 → q11.2). The size and the frequency of the sSMCs were strikingly different, besides, we observed an unequal combination of the three derivates.

To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture.

Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs - this is of relevance for standardization and automation of cell culture procedures.

Analysis of SYCP3 encoding synaptonemal complex protein 3 in human aneuploidies.

OBJECTIVES: To test the hypothesis that mutations of SYCP3 encoding synaptonemal complex protein 3, result in increased frequency of aneuploidies in humans. METHODS: Mutation analysis of the PCR-amplified 8 coding exons and exon-intron boundaries of the SYCP3 gene was done by direct sequencing of DNA isolated from 35 aneuploid fetuses of women having a potentially increased likelihood for an underlying genetic predisposition for chromosomal non-disjunction. RESULTS: Based on the results of conventional karyotyping, the 35 aneuploid fetuses of 33 women were divided into separate groups: 9 aneuploid conceptuses of couples with recurrent aneuploid conceptions (4 of the women 35 years or younger), 12 conceptuses with double/multiple aneuploidies (5 of the women 35 years or younger), and 14 conceptuses with single aneuploidies of women younger than 35 years (8 trisomies and 6 monosomies). No pathogenic mutations in the SYCP3 coding exons and the immediately flanking intronic sequences were found. CONCLUSIONS: Under the assumption that genetic predisposition for chromosomal non-disjunction leading to aneuploidy is most likely polygenic in nature, our data suggest that SYCP3 mutations are not one of the common causes in humans.

A phenotype map for 14q32.3 terminal deletions.

Detailed molecular-cytogenetic studies combined with thorough clinical characterization are needed to establish genotype-phenotype correlations for specific chromosome deletion syndromes. Although many patients with subtelomeric deletions have been reported, the phenotype maps for many of the corresponding syndromes, including the terminal deletion 14q syndrome, are only slowly emerging. Here, we report on five patients with terminal partial monosomy of 14q32.3 and characteristic features of terminal deletion 14q syndrome. Four of the patients carry de novo terminal deletions of 14q, three of which have not yet been reported. One patient carries an unbalanced translocation der(14)t(9;14)(q34.3;q32.3). Minimum deletion sizes as determined by molecular karyotyping and FISH are 5.82, 5.56, 4.17, 3.54, and 3.29 Mb, respectively. Based on our findings and a comprehensive review of the literature, we refine the phenotype map for typical clinical findings of the terminal deletion 14q syndrome (i.e., intellectual disability/developmental delay, muscular hypotonia, postnatal growth retardation, microcephaly, congenital heart defects, genitourinary malformations, ocular coloboma, and several dysmorphic signs). Combining this phenotype map with benign copy-number variation data available from the Database of Genomic Variants, we propose a small region critical for certain features of the terminal deletion 14q syndrome which contains only seven RefSeq genes.

Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks.

Cells in culture undergo replicative senescence. In this study, we analyzed functional, genetic and epigenetic sequels of long-term culture in human mesenchymal stem cells (MSC). Already within early passages the fibroblastoid colony-forming unit (CFU-f) frequency and the differentiation potential of MSC declined significantly. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow. Notably, all MSC revealed highly consistent senescence-associated modifications at specific CpG sites. These DNA-methylation changes correlated with histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Taken together, culture expansion of MSC has profound functional implications - these are hardly reflected by genomic instability but they are associated with highly reproducible DNA-methylation changes which correlate with repressive histone marks. Therefore replicative senescence seems to be epigenetically controlled.

Pure distal trisomy 2q: a rare chromosomal abnormality with recognizable phenotype.

We present clinical and molecular cytogenetic results of two unrelated patients with isolated distal trisomy of 2q33-qter and 2q35-q37.3 and a remarkable similar facial appearance. Common craniofacial features included a high hairline, broad nasal bridge, prominent nasal tip, thin upper lip vermillion, and large ears. Contrary to patients with duplications proximal to 2q33, the children with pure trisomy distal to 2q35 have normal or increased body measurements and show no major malformations. Moderate psychomotor delay was a constant finding.