Generation of a TMEM43 knockout human induced pluripotent stem cell line (HDZi003-A-1) using CRISPR/Cas9.

TMEM43 (LUMA) is a ubiquitously expressed protein with unknown function. The protein is phylogenetically highly conserved and also found in Drosophila melanogaster (Klinke et al., 2022). TMEM43-p.S358L is a rare, fully penetrant mutation that leads to arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). To understand the function of the ARVC5-associated mutation it is first important to understand the function of the TMEM43 protein. Therefore, a TMEM43 knockout induced pluripotent stem cell (iPSC) line was generated using the CRISPR/Cas9 genome editing system. The resulting cell line had a deficiency of TMEM43 and showed normal morphology and a stable karyotype. The colonies were positive for pluripotency markers and could be differentiated into the three germ layers.

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.

The telomerase inhibitor imetelstat differentially targets JAK2V617F versus CALR mutant myeloproliferative neoplasm cells and inhibits JAK-STAT signaling.

Imetelstat shows activity in patients with myeloproliferative neoplasms, including primary myelofibrosis (PMF) and essential thrombocythemia. Here, we describe a case of prolonged disease stabilization by imetelstat treatment of a high-risk PMF patient enrolled into the clinical study MYF2001. We confirmed continuous shortening of telomere length (TL) by imetelstat treatment but observed emergence and expansion of a KRAST58I mutated clone during the patient's clinical course. In order to investigate the molecular mechanisms involved in the imetelstat treatment response, we generated induced pluripotent stem cells (iPSC) from this patient. TL of iPSC-derived hematopoietic stem and progenitor cells, which was increased after reprogramming, was reduced upon imetelstat treatment for 14 days. However, while imetelstat reduced clonogenic growth of the patient's primary CD34+ cells, clonogenic growth of iPSC-derived CD34+ cells was not affected, suggesting that TL was not critically short in these cells. Also, the propensity of iPSC differentiation toward megakaryocytes and granulocytes was not altered. Using human TF-1MPL and murine 32DMPL cell lines stably expressing JAK2V617F or CALRdel52, imetelstat-induced reduction of viability was significantly more pronounced in CALRdel52 than in JAK2V617F cells. This was associated with an immediate downregulation of JAK2 phosphorylation and downstream signaling as well as a reduction of hTERT and STAT3 mRNA expression. Hence, our data demonstrate that imetelstat reduces TL and targets JAK/STAT signaling, particularly in CALR-mutated cells. Although the exact patient subpopulation who will benefit most from imetelstat needs to be defined, our data propose that CALR-mutated clones are highly vulnerable.

Fully functional monocytic MDSC generation from the murine HoxB8 cell line.

Myeloid-derived suppressor cells (MDSC) play a crucial role in controlling T-cell responses, but their development and suppressor mechanisms are not fully understood. To study the molecular functions of MDSC, a large number of standardized cells are required. Traditionally, bone marrow (BM) has been used to generate myeloid cell types, including MDSC. In this study, we demonstrate that a previously described protocol for generating monocytic MDSC (M-MDSC) from murine BM with GM-CSF can be fully transferred to BM cells that are conditionally transformed with HoxB8 gene (HoxB8 cells). HoxB8 cells have an extended lifespan and efficiently differentiate into MDSC that are quantitatively and qualitatively comparable to M-MDSC from BM cells. Flow cytometric analyses of LPS/IFN-γ activated cultures revealed the same iNOS+ and/or Arg1+ PD-L1high M-MDSC subsets in similar frequencies from BM or HoxB8 cells. In vitro suppression of CD4+ and CD8+ T-cell proliferations was also largely comparable in their efficacy and its iNOS- or Arg1-dependent suppressor mechanisms, which was confirmed by the similar amounts of nitric oxide (NO) secretion measured from the suppressor assay. Therefore, our data suggest that murine M-MDSC generation from HoxB8 cells with GM-CSF can be used to substitute BM cultures.

KIT D816V Mast Cells Derived from Induced Pluripotent Stem Cells Recapitulate Systemic Mastocytosis Transcriptional Profile.

Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.

Intrathymic dendritic cell-biased precursors promote human T cell lineage specification through IRF8-driven transmembrane TNF.

The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.

Capitalizing on paradoxical activation of the mitogen-activated protein kinase pathway for treatment of Imatinib-resistant mast cell leukemia.

Prevention of fatal side effects during cancer therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called "paradoxical activation." In the present work we re-analyzed the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KIT V560G, D816V-expressing human mast cell (MC) leukemia (MCL) cell line HMC-1.2. We observed that low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. The combination of Ponatinib with the MEK inhibitor Trametinib, at nanomolar concentrations, effectively suppressed HMC-1.2 proliferation, metabolic activity, and induced apoptotic cell death. Effectiveness of this drug combination was recapitulated in the human KIT D816V MC line ROSAKIT D816V and in KIT D816V hematopoietic progenitors obtained from patient-derived induced pluripotent stem cells (iPS cells) and systemic mastocytosis patient samples. In conclusion, mutated KIT-driven Imatinib resistance and possible TKI-induced paradoxical activation can be efficiently overcome by a low concentration Ponatinib and Trametinib co-treatment, potentially reducing the negative side effects associated with MCL therapy.

Polyelectrolyte Coating of Ferumoxytol Differentially Impacts the Labeling of Inflammatory and Steady-State Dendritic Cell Subtypes.

Engineered magnetic nanoparticles (MNPs) are emerging as advanced tools for medical applications. The coating of MNPs using polyelectrolytes (PEs) is a versatile means to tailor MNP properties and is used to optimize MNP functionality. Dendritic cells (DCs) are critical regulators of adaptive immune responses. Functionally distinct DC subsets exist, either under steady-state or inflammatory conditions, which are explored for the specific treatment of various diseases, such as cancer, autoimmunity, and transplant rejection. Here, the impact of the PE coating of ferumoxytol for uptake into both inflammatory and steady-state DCs and the cellular responses to MNP labeling is addressed. Labeling efficiency by uncoated and PE-coated ferumoxytol is highly variable in different DC subsets, and PE coating significantly improves the labeling of steady-state DCs. Uncoated ferumoxytol results in increased cytotoxicity of steady-state DCs after labeling, which is abolished by the PE coating, while no increased cell death is observed in inflammatory DCs. Furthermore, uncoated and PE-coated ferumoxytol appear immunologically inert in inflammatory DCs, but they induce activation of steady-state DCs. These results show that the PE coating of MNPs can be applied to endow particles with desired properties for enhanced uptake and cell type-specific responses in distinct target DC populations.

Mast cells as a therapeutic target in myeloproliferative neoplasms.

Mast cells have been implicated as mediators of bone marrow fibrosis and pruritus in myeloproliferative neoplasms (MPNs) with JAK2V617F or calreticulin mutations. We hypothesize that potent KIT inhibitors, already in clinical use for systemic mastocytosis, have therapeutic potential for the treatment of MPNs by directly targeting mast cells.

Cell Cluster Sorting in Automated Differentiation of Patient-specific Induced Pluripotent Stem Cells Towards Blood Cells.

Induced pluripotent stem cells (iPS cells) represent a particularly versatile stem cell type for a large array of applications in biology and medicine. Taking full advantage of iPS cell technology requires high throughput and automated iPS cell culture and differentiation. We present an automated platform for efficient and robust iPS cell culture and differentiation into blood cells. We implemented cell cluster sorting for analysis and sorting of iPS cell clusters in order to establish clonal iPS cell lines with high reproducibility and efficacy. Patient-specific iPS cells were induced to differentiate towards hematopoietic cells via embryoid body (EB) formation. EB size impacts on iPS cell differentiation and we applied cell cluster sorting to obtain EB of defined size for efficient blood cell differentiation. In summary, implementing cell cluster sorting into the workflow of iPS cell cloning, growth and differentiation represent a valuable add-on for standard and automated iPS cell handling.

Hematopoietic differentiation persists in human iPSCs defective in de novo DNA methylation.

BACKGROUND: DNA methylation is involved in the epigenetic regulation of gene expression during developmental processes and is primarily established by the DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B). DNMT3A is one of the most frequently mutated genes in clonal hematopoiesis and leukemia, indicating that it plays a crucial role for hematopoietic differentiation. However, the functional relevance of Dnmt3a for hematopoietic differentiation and hematological malignancies has mostly been analyzed in mice, with the specific role for human hematopoiesis remaining elusive. In this study, we therefore investigated if DNMT3A is essential for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs). RESULTS: We generated iPSC lines with knockout of either exon 2, 19, or 23 and analyzed the impact of different DNMT3A exon knockouts on directed differentiation toward mesenchymal and hematopoietic lineages. Exon 19-/- and 23-/- lines displayed an almost entire absence of de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation efficiency was only slightly reduced in exon 19-/- and rather increased in exon 23-/- lines, while there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A-/- iHPCs recapitulate some DNA methylation patterns of acute myeloid leukemia (AML) with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed growth advantage of exon 23-/- iHPCs in a syngeneic competitive differentiation assay. CONCLUSIONS: Our results demonstrate that iPSCs with homozygous knockout of different exons of DNMT3A remain capable of mesenchymal and hematopoietic differentiation-and exon 23-/- iHPCs even gained growth advantage-despite loss of almost the entire de novo DNA methylation. Partial recapitulation of DNA methylation patterns of AML with DNMT3A mutations by our DNMT3A knockout iHPCs indicates that our model system can help to elucidate mechanisms of clonal hematopoiesis.

Enhancement of proliferation of human umbilical cord blood-derived CD34+ hematopoietic stem cells by a combination of hyper-interleukin-6 and small molecules.

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB-HSCs) in transplantation, however, is the low numbers of HSCs in a unit of cord blood. To overcome this limitation, various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study, we investigated a synergistic effect of the combination of HIL-6, SR1, and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34+ cells. These results suggest that the combination of SR1, UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus, SR1, UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation.

The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates.

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 µm from the outer rim of iPSC colonies. Single-cell RNA-sequencing and spatial reconstruction of gene expression demonstrated that OCT4high subsets, residing at the edge of the colony, have pronounced up-regulation of the TGF-ß pathway, particularly of NODAL and its inhibitor LEFTY. Interestingly, after 5-7 days, iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size without enzymatic or mechanical treatment. Within the early 3D aggregates, radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Early self-detached aggregates revealed up-regulated germline-specific gene expression patterns as compared to conventional EBs. However, there were no marked differences after further directed differentiation toward hematopoietic, mesenchymal, and neuronal lineages. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

PLA/Hydroxyapatite scaffolds exhibit in vitro immunological inertness and promote robust osteogenic differentiation of human mesenchymal stem cells without osteogenic stimuli.

Bone defects stand out as one of the greatest challenges of reconstructive surgery. Fused deposition modelling (FDM) allows for the printing of 3D scaffolds tailored to the morphology and size of bone damage in a patient-specific and high-precision manner. However, FDM still suffers from the lack of materials capable of efficiently supporting osteogenesis. In this study, we developed 3D-printed porous scaffolds composed of polylactic acid/hydroxyapatite (PLA/HA) composites with high ceramic contents (above 20%, w/w) by FDM. The mechanical properties of the PLA/HA scaffolds were compatible with those of trabecular bone. In vitro degradation tests revealed that HA can neutralize the acidification effect caused by PLA degradation, while simultaneously releasing calcium and phosphate ions. Importantly, 3D-printed PLA/HA did not induce the upregulation of activation markers nor the expression of inflammatory cytokines in dendritic cells thus exhibiting no immune-stimulatory properties in vitro. Evaluations using human mesenchymal stem cells (MSC) showed that pure PLA scaffolds exerted an osteoconductive effect, whereas PLA/HA scaffolds efficiently induced osteogenic differentiation of MSC even in the absence of any classical osteogenic stimuli. Our findings indicate that 3D-printed PLA scaffolds loaded with high concentrations of HA are most suitable for future applications in bone tissue engineering.

CRISPR/Cas9 editing in conditionally immortalized HoxB8 cells for studying gene regulation in mouse dendritic cells.

HoxB8 multipotent progenitors (MPP) are obtained by expression of the estrogen receptor hormone binding domain (ERHBD) HoxB8 fusion gene in mouse BM cells. HoxB8 MPP generate (i) the full complement of DC subsets (cDC1, cDC2, and pDC) in vitro and in vivo and (ii) allow CRISPR/Cas9-mediated gene editing, for example, generating homozygous deletions in cis-acting DNA elements at high precision, and (iii) efficient gene repression by dCas9-KRAB for studying gene regulation in DC differentiation.

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.

Curauá-derived carbon dots: Fluorescent probes for effective Fe(III) ion detection, cellular labeling and bioimaging.

This study reports the generation of curauá-derived carbon dots (C-dots) and their suitability for Fe(III) detection, bioimaging and FACS analysis. C-dots were generated from curauá (Ananas erectifolius) fibers by a facile one-step hydrothermal approach. They exhibited graphite-like structure with a mean diameter of 2.4 nm, high water solubility, high levels of carboxyl and hydroxyl functional groups, excitation-dependent multicolor fluorescence emission (in the range 450 nm - 560 nm) and superior photostability. C-dots were highly selective and effective for the detection of ferric Fe(III) ion in an aqueous medium with a detection limit of 0.77 µM in the linear range of 0-30 µM, a value much lower than the guideline limits proposed by the World Health Organization (WHO). In biological cell systems, C-dots were very well tolerated by B16F1 mouse melanoma and J774.A1 mouse macrophages cell lines, both of which effectively internalized C-dots in their cytoplasmic compartment. Finally, C-dots were effective probes for long-term live cell imaging experiments and multi-channel flow cytometry analysis. Collectively, our findings demonstrate that curauá-derived C-dots serve as versatile and effective natural products for Fe(III) ion sensing, labeling and bioimaging of various cell types. This study adds novel C-dots to the library of carbon-based probes and paves the way towards a sustainable conversion of a most abundant biomass waste into value-added products.

Glucocorticoid Effects on Tissue Residing Immune Cells in Giant Cell Arteritis: Importance of GM-CSF.

Giant cell arteritis (GCA) is a systemic granulomatous vasculitis clinically characterized by a prompt response to glucocorticoid therapy. Dendritic cells (DCs) play a central role in the pathogenesis of the disease and are increased in temporal arteries from GCA patients. The aim of this study was to determine the effects of glucocorticoid therapy on granulomatous infiltrates and on peripheral DCs of GCA patients. Immunohistochemical staining of temporal artery specimens from 41 GCA patients revealed a rapid reduction of the number of DCs after initiation of glucocorticoid treatment. TUNEL staining was performed to quantify apoptotic S100+ DC, CD3+ T cells, and CD68+ macrophages in the granulomatous infiltrates. An increase of apoptotic cells up to 9 ± 2% after 4-5 days of glucocorticoid therapy and up to 27 ± 5% (p < 0.001, compared to earlier timepoints) after 6-10 days was detected. A decrease of CCL19 and CCL21 expression was observed after starting glucocorticoid therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression also significantly decreased under glucocorticoid therapy. No GM-CSF expression was detected in the control specimens. Glucocorticoid therapy leads to a rapid, time-dependent reduction of DCs in temporal arteries from GCA patients and reduction of mediators for cell migration. Our data suggest GM-CSF as a novel therapeutic target of GCA.