Martin Zenke

Background: Neuropathic pain is a burdensome, difficult-to-treat, and highly heterogeneous condition with limited therapeutic options, underscoring the need for robust and reproducible human disease models. Human induced pluripotent stem cell (iPSC)-derived sensory neurons provide a promising platform for patient specific disease modelling and drug screening; however, their translational use is hampered by variability in differentiation efficiency, cellular composition, and functional maturation across protocols and cell lines. Methods: Here, we present a standardized and potentially scalable framework for NGN1 driven differentiation of human iPSCs into sensory neurons. Building on a previously published two step protocol (1), we systematically deconstructed and optimized each stage of differentiation across a large panel of genetically diverse iPSC lines. Results: We identified robust parameters for neural crest like cell (NCLC) generation, established a flow cytometry-based quality control strategy for NCLCs, and defined optimal combinations of seeding density and lentiviral multiplicity of infection to maximize sensory neuron progenitor yield. To improve culture homogeneity, we compared antimitotic selection strategies and demonstrated that tightly timed Ara-C treatment combined with low progenitor seeding density yields consistently pure sensory neuron cultures. We further evaluated maturation under physiologically relevant glucose conditions and performed a systematic review of media compositions to derive two defined maturation media. Morphological, immunocytochemical, transcriptomic, and electrophysiological analyses revealed that time in culture is a major determinant of maturation, while specific supplements such as prostaglandin E2; (PGE2) selectively enhance transcriptional signatures associated with nociceptor identity without substantially altering global network activity. Bulk RNA sequencing demonstrated broad expression of sensory neuron and pain related markers and gene programs across conditions, with long term maturation and PGE2; treatment showing the highest similarity to human dorsal root ganglion reference data. Functional assessment using multi electrode arrays enabled the detection of donor specific electrophysiological phenotypes, including reproducible hyperexcitability in small fiber neuropathy patient derived lines. Conclusions: This study establishes a modular, reproducible NGN1 based differentiation workflow with integrated quality checkpoints that accommodates iPSC line to line variability. The framework provides a practical foundation for translational sensory neuron research, patient specific disease modelling, and scalable drug screening applications.

A drug discovery approach was used to specifically target malignant cells with KIT D816V mutation, which is the predominant disease-causing mutation in clonal mast cell malignancies. To this end, KIT D816V cells derived from induced pluripotent stem cells (iPS cells) of KIT D816V patients were employed to screen a library of FDA approved and experimental drugs for specific killing of KIT D816V cells. We discovered the novel compound LDC 3416, which targets multiple malignant KIT D816V cell types, including hematopoietic stem/progenitor cells and mast cells. Importantly, by exploring the LDC 3416 targeting profile, we identified the phosphodiesterase 3A- Schlafen 12 (PDE3A-SLFN12) molecular glue pathway as a novel approach for specific targeting of malignant KIT D816V cells. We found that the KIT D816V mutant protein leads to increased expression of PDE3A and SLFN12 and thus confers a selective molecular vulnerability to PDE3A-SLFN12 molecular glues. Primary malignant mast cells of KIT D816V patients with indolent and advanced systemic mastocytosis also exhibit increased expression of PDE3A and SLFN12. We extended our study to include additional PDE3A-SLFN12 molecular glues and demonstrate their synergistic action with KIT D816V selective tyrosine kinase inhibitors (TKIs) in killing KIT D816V cells. Furthermore, the PDE3A-SLFN12 molecular glues also target KIT D816V megakaryocytes, a cell type that has been underestimated in malignant mast cell pathophysiology and molecular targeting. The identified molecular glues, along with their synergy with TKIs and their simultaneous targeting of multiple KIT D816V cell types, open novel treatment options for KIT D816V mast cell malignancies and other KIT D816V associated diseases.

Tissue inflammation is shaped by reciprocal interactions between immune cells and stromal niches, yet how leukemic myelopoiesis rewires these circuits remains poorly understood. Here, we identify suppression of interferon regulatory factor 8 (IRF8) in hematopoietic cells as a driver of inflammatory bone marrow niche remodeling in chronic-phase myeloid leukemia (CP-CML). IRF8 expression was reduced in human and murine CML and restored following interferon-α therapy. In mice, hematopoietic IRF8 deficiency induced aberrant neutrophil expansion, extramedullary hematopoiesis, stromal activation and early marrow fibrosis. Single-cell transcriptomics revealed that IRF8-deficient neutrophils engaged perivascular stromal cells, CXCL12-abundant reticular cells and endothelial cells through S100A8/A9–TLR4 and TGF-β–integrin signaling, associated with hypoxia, vascular remodeling and extracellular matrix programs. Mechanistically, IRF8 directly bound the S100a8 locus and restrained the alarmin program in myeloid cells. Conversely, an IRF8-deficient niche amplified BCR–ABL1-driven leukemia and inflammatory alarmin production. These findings identify IRF8 as a hematopoietic regulator of inflammatory neutrophil–stromal communication and reveal how its suppression promotes pathological remodeling of the leukemic bone marrow niche.

Systemic mastocytosis (SM) is characterized by uncontrolled expansion of neoplastic mast cells (MCs) and their accumulation in various tissues and organs, ranging from indolent variants to more advanced forms (advSM). Although several MC- and SM-expressed cell surface antigens have been identified, no immune therapy has been developed for advSM so far. The receptor tyrosine kinase KIT (CD117) is highly expressed on MCs, exceeding the levels of expression on hematopoietic stem and progenitor cells (HSPC). Therefore, targeting CD117 in advSM could be of therapeutic value. In this study, we assessed the therapeutic potential of anti-CD117 chimeric antigen receptor (CAR) T-cells to target neoplastic MCs in SM. In vitro, anti-CD117-CAR T-cells efficiently lysed several SM-related human MC cell lines, MCs differentiated from SM patient-derived induced pluripotent stem (iPS) cells, and neoplastic bone marrow cells obtained from SM patients. Furthermore, in immunocompromised mice engrafted with an advSM-like MC cell line, repetitive applications of anti-CD117-CAR T-cells were able to inhibit MC expansion. These data may pave the way for the development of anti-CD117-CAR T-cell therapies in advSM.

Surface modification is an important tool to tailor the interactive properties of support materials to the desired application. It is commonly applied to design complex systems for applications in bio‐ or electro‐technology. In this work, means of surface structuring and chemical modification are combined to create stable microgel patterned substrates, which can be post‐modified with functional molecules. Poly( N ‐vinylcaprolactam‐ co ‐glycidyl methacrylate) (p(VCL‐ co ‐GMA)) microgels are used as functional colloidal inks to generate surface grafted patterns on glass by microcontact printing. This allows for the fabrication of microgel arrays with controlled geometries (line widths 10–50 µm and spacings 10–50 µm). Preliminary coupling tests in dispersion are conducted to determine the ideal reaction conditions for post‐modifying the printed structures with the fluorescent model‐compound Cyanine3 amine (Cy3 amine) and demonstrating the successful modification with a cell‐adhesive peptide sequence. Surface‐chemical gradients with variable profiles are created on the microgel patterns by dip‐coating the fluorescent model compound Cy3 amine. Eventually, patterned substrates are applied in tissue culture experiments with NIH‐3T3 cells, where peptide‐modified surfaces enable increased control over cell adhesion and motility.

Interferon alfa (IFN-α) is approved for the therapy of patients with polycythemia vera (PV), a subtype of myeloproliferative neoplasm (MPN). Some patients achieve molecular responses (MRs), but clonal factors sensitizing for MRs remain elusive. We integrated colony formation assays with single-cell RNA sequencing (scRNA-seq) and genotyping in PV-derived cells and healthy controls (HCs) to dissect how IFN-α targets diseased clones during erythroid differentiation. IFN-α significantly decreased colony growth in MPNs and HCs with variable transcriptional responses observed in individual colonies. scRNA-seq of colonies demonstrated more mature erythroid colonies in PV than HCs. JAK2V617F-mutant cells exhibited upregulated STAT5A, heme, and G2M checkpoint pathways compared with JAK2WT cells from the same patients. Subgroup analysis revealed that IFN-α significantly decreased immature erythrocytic cells in PV (basophilic erythroblasts P < .05; polychromatic erythroblasts P < .05) but not in HCs. CD71−/CD235a+ cells from HCs (P < .05) but not PV were inhibited by IFN-α, and the number of reticulocytes was less affected in PV. Robust IFN-α responses persisted throughout differentiation, leading to significant apoptosis in PV. Apoptotic cells displayed downregulation of ribosomal genes. This link between apoptosis and ribosomal genes was corroborated through the analysis of mitochondrial variants, demonstrating IFN-α–induced eradication of specific clones, characterized by elevated expression of ribosomal genes. Our findings indicate that PV-derived clones either undergo apoptosis or pass through differentiation, overall reducing the cycling mutant cells over long-term treatment. Furthermore, the significance of ribosomal genes and clonal prerequisites in IFN-α’s therapeutic mechanism is underscored, shedding light on the intricate dynamics of IFN-α treatment in PV.

The vascular niche is a critical regulator of hematopoiesis and disease progression in myeloproliferative neoplasms (MPN). The presence of JAK2V617F+ endothelial cells (EC) in MPN patients and their association with cardiovascular complications highlight the need to understand and therapeutically target this compartment. Using patient-specific induced pluripotent stem cells (iPSC) harboring JAK2 ^WT or the MPN-driver JAK2V617F (heterozygous, JAK2V617F ^HET , or homozygous, JAK2V617F ^HOM ), we identified zygosity-dependent transcriptional profiles in iPSC-derived EC (iEC) at baseline and following interferon-alpha (IFNα) treatment. JAK2V617F ^HET iEC exhibited an endothelial-to-mesenchymal transition (EndMT) signature, while JAK2V617F ^HOM iEC showed suppression of translation and ribosome biogenesis. Leveraging iPSC-based 3D assembloids that mimic the bone marrow (BM) niche, we showed that JAK2V617F-driven EndMT is inhibited by tyrosine kinase inhibitors and IFNα. In both JAK2V617F-driven polycythemia vera and TPO-driven myelofibrosis murine models, scRNA-seq analysis of the BM vascular niche consistently revealed inflammatory and EndMT-associated signatures in arterial and arteriolar EC. Notably, dysregulation of ribosome- and translation-related pathways emerged in the myelofibrosis model and at advanced disease stages in JAK2V617F-driven polycythemia vera, indicating progressive vascular remodeling with disease evolution. Chronic pegylated IFNα treatment in vivo effectively reversed these pathological changes. IFNα’s anti-EndMT activity was further validated in BM biopsies from MPN patients undergoing IFNα therapy. This is the first study to define MPN stage-dependent vascular remodeling and zygosity-specific endothelial effects of JAK2V617F, and to directly link IFNα-mediated EndMT inhibition as a novel antifibrotic mechanism. Our 3D assembloids provide a translational platform for mechanistic studies and therapeutic targeting of the BM microenvironment in MPN. Bullet Points: Arterial vascular remodeling emerges as a novel hallmark of MPN, characterized by TNFα-inflammation, ribosomal dysregulation and EndMT. IFNα restores neoplastic endothelial dysfunction, highlighting its role as a vascular niche-modulating and anti-fibrotic agent in MPN.

Emerging evidence highlights cellular senescence’s pivotal role in chronic kidney disease (CKD). Proximal tubule epithelial cells (PTECs) and fibroblasts are major players in CKD and serve as cellular sources of senescence. The generation of a conditionally immortalized human kidney cell model would allow to better understand the specific mechanisms and factors associated with cellular senescence in a controlled setting, devoid of potential confounding factors such as age and comorbidities. In addition, the availability of human kidney cell lines for preclinical research is sparse and most cell lines do not reflect their in vivo counterparts due to their altered behavior as immortalized cancer-like cells. In this study, PTECs and fibroblasts from human kidneys were isolated and transduced with doxycycline-inducible simian virus 40 large T antigen (SV40LT) vector. By comparing their gene expression with single-cell RNA sequencing data from human kidneys, the newly produced human kidney cell lines demonstrated significant resemblances to their in vivo counterparts. As predicted, PTECs showed functional activity and fibroblasts responded to injury with fibrosis. Withdrawal of the immortalizing factor doxycycline led to p21⁺ cell-cycle arrest and the key hallmarks of senescence. The obtained senescence gene set largely overlapped between both cell lines and with the previously published SenMayo set of senescence-associated genes. Furthermore, crosstalk experiments showed that senescent PTECs can cause a profibrotic response in fibroblasts by paracrine actions. In 76 human kidney sections, the number of p21⁺ cells correlated with the degree of fibrosis, age and reduced glomerular filtration, validating the role of senescence in CKD. In conclusion, we provide a novel cellular ex vivo model to study kidney senescence which can serve as a platform for large scale compounds testing.