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.

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.

Testosterone and Cortisol Salivary Samples Are Stable Across Multiple Freeze-Thaw Cycles.

ABSTRACT: Sontag, SA, Cabarkapa, D, and Fry, AC. Testosterone and cortisol salivary samples are stable across multiple freeze-thaw cycles. J Strength Cond Res 37(4): 915-918, 2023-When processing salivary samples for biomarker analysis, avoiding multiple freeze-thaw cycles is generally recommended. However, confusing tissue handling instructions or challenges with collections in the field sometimes makes this problematic. Thus, the purpose of this study was to examine if the stability of salivary testosterone (T) and cortisol (C) hormones remains unchanged when exposed to multiple freeze-thaw cycles. Seven healthy recreationally active adults provided salivary samples at rest (i.e., 1600 hours) for analysis of T and C. Samples were separated into 4 aliquots for each hormone and underwent 4 freeze-thaw cycles (T1-T4 and C1-C4) before being analyzed by enzyme-linked immunosorbent assay. The overall analysis of variance model was significant for T ( p = 0.008) and nonsignificant for C ( p = 0.820). A follow-up post hoc comparison indicated significant differences in salivary hormonal concentrations between T1 and T4 ( p = 0.029), T2 and T4 ( p = 0.007), and T3 and T4 ( p = 0.032). The findings of this study indicate that salivary steroid hormones seem to be relatively stable following multiple freeze-thaw cycles. However, C seems to be more stable when exposed to multiple freeze-thaw cycles, as T concentrations did reveal a significant decrease by the fourth thaw cycle.

Toward Clinical Application of Leukocyte Counts Based on Targeted DNA Methylation Analysis.

BACKGROUND: Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type-specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated. METHODS: We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR). RESULTS: Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number. CONCLUSIONS: Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization.

The influence of chronic training status on the mechanical behavior of the vastus lateralis during repetitive trapezoidal contractions.

OBJECTIVES: To examine maximal strength and fatigability of the knee extensors, and mechanomyographic amplitude (MMGRMS)-force relationships of the vastus lateralis (VL) during repetitive muscle actions for 5 aerobically-(AT), 5 resistance-trained-(RT), and 5 sedentary (SED) individuals. METHODS: Participants performed maximal voluntary contractions before (MVCPRE) and after (MVCPOST) attempting 20 isometric trapezoidal muscle actions at 50% MVCPRE. MMG was recorded from the VL. b terms (slopes) were calculated from the natural log-transformed MMGRMS-force relationships for each participant (increasing and decreasing segments). MMGRMS was averaged during steady force. RESULTS: RT had greater MVCPRE (P<0.001) and MVCPOST (P=0.001-0.004) than AT and SED. Only AT completed 20 muscle actions and exhibited no decrease in MVCPOST (P=0.149). The b terms were greater for RT than AT during the increasing segment of the first contraction (P=0.001) and decreasing segment of the last contraction (P=0.033). The b terms were also greater for RT (P=0.006) during the increasing than decreasing segment for the first contraction. MMGRMS during steady force was greater during the last contraction when collapsed across training status (P=0.021). CONCLUSION: Knee extensor MVC and fatigability, and motor unit control strategies for the VL during a series of repetitive contractions were influenced by chronic training status.

Endurance training alters motor unit activation strategies for the vastus lateralis, yet sex-related differences and relationships with muscle size remain.

PURPOSE: To examine the effects of 10 weeks of endurance cycling training on mechanomyographic amplitude (MMGRMS)-torque relationships and muscle cross-sectional area (mCSA) of the vastus lateralis (VL) for 10 sedentary males (Age ± SD; 20.2 ± 1.9 years) and 14 sedentary females (21.9 ± 5.3 years). METHODS: Participants performed maximal voluntary contractions (MVCs) and an isometric ramp up muscle action to 70% MVC of the knee extensors before (PRE) and after training at the same absolute pre-treatment submaximal torque (POSTABS). MMG was recorded from the VL and b terms were calculated from the natural log-transformed MMGRMS-torque relationships for each subject. mCSA was determined with ultrasonography. RESULTS: Cycling decreased MVCs from pre- (168.10 ± 58.49 Nm) to post-training (160.78 ± 58.39 Nm; p = 0.005) without changes in mCSA. The b terms were greater for POSTABS (0.623 ± 0.204) than PRE (0.540 ± 0.226; p = 0.012) and for males (0.717 ± 0.171) than females (0.484 ± 0.168; p = 0.003). mCSA was correlated with the b terms for PRE (p < 0.001, r = 0.674) and POSTABS (p = 0.020, r = 0.471). CONCLUSION: The decrease in MVC and increase in MMGRMS (b terms) post-training suggests increased motor unit (MU) recruitment to match pre-training torques. The greater acceleration in the b terms by males may reflect sex-related differences in fiber-type area. MMGRMS-torque relationships during a high-intensity contraction provided insight on MU activation strategies following endurance training and between sexes. Furthermore, the findings suggest a relationship between MMGRMS and muscle size.

MAPK, androgen, and glucocorticoid receptor phosphorylation following high-frequency resistance exercise non-functional overreaching.

PURPOSE: Stressful training with insufficient recovery can impair muscle performance. Expression of mitogen-activated protein kinases (MAPK) has been reported at rest following overreaching and overtraining. The acute myocellular exercise response to stressful training with insufficient recovery has not been investigated. We investigated MAPK, androgen, and glucocorticoid receptor phosphorylation following a period of stressful training. METHODS: Sixteen resistance-trained men were matched on barbell squat 1 repetition maximum strength and randomized into a group that performed normal training or stressful training with insufficient recovery. The control group (CON) performed three speed-squat training sessions on non-consecutive days, while the stressful training group (NFOR) performed 15 training sessions over 7.5 days. Resting and post-exercise skeletal muscle biopsies were obtained prior to (T1) and after the training period (T2). Samples were analyzed for total and phosphorylated androgen receptor (AR), glucocorticoid receptor (GR), and MAPKs (ERK, JNK, and p38). RESULTS: Total AR were down-regulated post-exercise at T2 in NFOR only. Phospho-AR at ser515 increased in both groups post-exercise at T1; however, ser515 only increased at T2 in NFOR. Phosphorylated ERK, JNK, and p38 increased post-exercise in CON and NFOR at T1 and T2. Post-exercise phospho-p38 was blunted in NFOR at T2 compared to T1. After the training intervention, resting phospho-p38 was higher in NFOR compared to T1. At T2, post-exercise phospho-GR at ser226 was lower compared to T1, and resting levels increased in NFOR. CONCLUSION: Steroid receptors are phosphorylated after acute resistance exercise, and in addition to MAPKs, are differentially regulated after stressful training with insufficient recovery.

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.

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.

Chronic training status affects muscle excitation of the vastus lateralis during repeated contractions.

This study examined electromyographic amplitude (EMGRMS)-force relationships during repeated submaximal knee extensor muscle actions among chronic aerobically-(AT), resistance-trained (RT), and sedentary (SED) individuals. Fifteen adults (5/group) attempted 20 isometric trapezoidal muscle actions at 50% of maximal strength. Surface electromyography (EMG) was recorded from vastus lateralis (VL) during the muscle actions. For the first and last successfully completed contractions, linear regression models were fit to the log-transformed EMGRMS-force relationships during the linearly increasing and decreasing segments, and the b terms (slope) and a terms (antilog of y-intercept) were calculated. EMGRMS was averaged during steady force. Only the AT completed all 20 muscle actions. During the first contraction, the b terms for RT (1.301 â€‹± â€‹0.197) were greater than AT (0.910 â€‹± â€‹0.123; p â€‹= â€‹0.008) and SED (0.912 â€‹± â€‹0.162; p â€‹= â€‹0.008) during the linearly increasing segment, and in comparison to the linearly decreasing segment (1.018 â€‹± â€‹0.139; p â€‹= â€‹0.014), respectively. For the last contraction, the b terms for RT were greater than AT during the linearly increasing (RT â€‹= â€‹1.373 â€‹± â€‹0.353; AT â€‹= â€‹0.883 â€‹± â€‹0.129; p â€‹= â€‹0.018) and decreasing (RT â€‹= â€‹1.526 â€‹± â€‹0.328; AT â€‹= â€‹0.970 â€‹± â€‹0.223; p â€‹= â€‹0.010) segments. In addition, the b terms for SED increased from the linearly increasing (0.968 â€‹± â€‹0.144) to decreasing segment (1.268 â€‹± â€‹0.126; p â€‹= â€‹0.015). There were no training, segment, or contraction differences for the a terms. EMGRMS during steady force increased from the first- ([64.08 â€‹± â€‹51.68] â€‹µV) to last-contraction ([86.73 â€‹± â€‹49.55] â€‹µV; p â€‹= â€‹0.001) collapsed across training statuses. The b terms differentiated the rate of change for EMGRMS with increments in force among training groups, indicating greater muscle excitation to the motoneuron pool was necessary for the RT than AT during the linearly increasing and decreasing segments of a repetitive task.

Influence of Pennation Angle and Muscle Thickness on Mechanomyographic Amplitude-Torque Relationships and Sex-Related Differences in the Vastus Lateralis.

This study examined potential sex-related differences and correlations among the pennation angle (PA), muscle thickness (MT), and mechanomyographic amplitude (MMGRMS)-torque relationships of the vastus lateralis (VL) in 11 healthy males and 12 healthy females. The PA and MT of the VL were quantified with ultrasound. Participants performed an isometric muscle action of the knee extensors that linearly increased to 70% of maximal strength followed by a 12 s plateau. MMG was recorded from the VL. Linear regression models were fit to the log-transformed MMGRMS-torque relationships to calculate b terms (slopes) for the linearly increasing segment. MMGRMS was averaged during the plateau. Males exhibited greater PA (p < 0.001), MT (p = 0.027), b terms (p = 0.005), and MMGRMS (p = 0.016). The b terms were strongly (p < 0.001, r = 0.772) and moderately correlated (p = 0.004, r = 0.571) with PA and MT, respectively, while MMGRMS was moderately correlated with PA (p = 0.018, r = 0.500) and MT (p = 0.014, r = 0.515). The greater mechanical behavior of individuals possessing a larger PA and MT of the VL may reflect increased cross-bridge activity within the muscle fibers. Additionally, PA may help explain sex-related differences in MMGRMS between sexes.

A noninvasive test for estimating myosin heavy chain of the vastus lateralis in females with mechanomyography.

This study examined relationships between percent myosin heavy chain (%MHC) expression and mechanomyographic amplitude (MMGRMS). Fifteen females (age ± SD=21.3 ± 5.3 yrs) completed isometric trapezoidal contractions at 30% and 70% maximal voluntary contraction (MVC). MMG was recorded from the vastus lateralis (VL). Participants gave a muscle biopsy of the VL post-testing. MMGRMS-torque relationships during the linearly varying segments were log-transformed and linear regressions were applied to calculate b terms (slopes). For the steady torque segment, MMGRMS was averaged. Correlations were performed for type I%MHC with the MMG variables. Multiple regression was utilized to examine prediction equations for type I%MHC. Type I%MHC was significantly correlated with the b terms during the increasing segment of the 70% MVC (p = 0.003; r = -0.718), and MMGRMS during steady torque at 30% (p = 0.008; r = -0.652) and 70% MVC (p = 0.040; r = -0.535). Type I%MHC reduced the linearity of the MMGRMS-torque relationship during the high-intensity linearly increasing segment, and MMGRMS at a low- and high-intensity steady torque. A combination of MMG variables estimated type I%MHC expression with 81.2% accuracy. MMG recorded during a low- and high-intensity isometric trapezoidal contraction may offer a simple, noninvasive test for estimating type I%MHC expression of the VL in sedentary females.

Sex-related differences in motor unit behavior are influenced by myosin heavy chain during high- but not moderate-intensity contractions.

AIMS: Motor unit recruitment and firing rate patterns of the vastus lateralis (VL) have not been compared between sexes during moderate- and high-intensity contraction intensities. Additionally, the influence of fiber composition on potential sex-related differences remains unquantified. METHODS: Eleven males and 11 females performed 40% and 70% maximal voluntary contractions (MVCs). Surface electromyographic (EMG) signals recorded from the VL were decomposed. Recruitment thresholds (RTs), MU action potential amplitudes (MUAPAMP ), initial firing rates (IFRs), mean firing rates (MFRs), and normalized EMG amplitude (N-EMGRMS ) at steady torque were analyzed. Y-intercepts and slopes were calculated for MUAPAMP , IFR, and MFR versus RT relationships. Type I myosin heavy chain isoform (MHC) was determined with muscle biopsies. RESULTS: There were no sex-related differences in MU characteristics at 40% MVC. At 70% MVC, males exhibited greater slopes (p = 0.002) for the MUAPAMP , whereas females displayed greater slopes (p = 0.001-0.007) for the IFR and MFR versus RT relationships. N-EMGRMS at 70% MVC was greater for females (p < 0.001). Type I %MHC was greater for females (p = 0.006), and was correlated (p = 0.018-0.031) with the slopes for the MUAPAMP , IFR, and MFR versus RT relationships at 70% MVC (r = -0.599-0.585). CONCLUSION: Both sexes exhibited an inverse relationship between MU firing rates and recruitment thresholds. However, the sex-related differences in MU recruitment and firing rate patterns and N-EMGRMS at 70% MVC were likely due to greater type I% MHC and smaller twitch forces of the higher threshold MUs for the females. Evidence is provided that muscle fiber composition may explain divergent MU behavior between sexes.

High-Intensity Cycling Training Necessitates Increased Neuromuscular Demand of the Vastus Lateralis During a Fatiguing Contraction.

Purpose: To examine the effects of a 5-week continuous cycling training intervention on electromyographic amplitude (EMGRMS)- and mechanomyographic amplitude (MMGRMS)-torque relationships of the vastus lateralis (VL) during a prolonged contraction. Methods: Twenty-four sedentary, young adults performed maximal voluntary contractions (MVCs) and a prolonged isometric trapezoidal contraction at the same absolute 40% MVC for the knee extensors before (PRE) and after training (POSTABS). Individual b- (slopes) and a-terms (y-intercepts) were calculated from the log-transformed electromyographic amplitude (EMGRMS)- and mechanomyographic amplitude (MMGRMS)-torque relationships during the increasing and decreasing segments of the trapezoid. EMGRMS and MMGRMS was normalized for the 45-s steady torque segment. Results: At PRE, b-terms for the EMGRMS-torque relationships during the linearly decreasing segment were greater than the increasing segment (p < .001), and decreased from PRE to POSTABS (p = .027). a-terms were greater during the linearly increasing than decreasing segment at PRE, while the a-terms for the linearly decreasing segment increased from PRE to POSTABS (p = .027). For the MMGRMS-torque relationships, b-terms during the linearly decreasing segment decreased from PRE to POSTABS (p = .013), while a-terms increased from PRE to POSTABS when collapsed across segments (p = .022). Steady torque EMGRMS increased for POSTABS (p < .001). Conclusion: Although cycling training increased aerobic endurance, incorporating resistance training may benefit athletes/individuals as the alterations in neuromuscular parameters post-training suggest a greater neural cost (EMGRMS) and mechanical output (MMGRMS) to complete the same pre-training fatiguing contraction.

Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy.

Physical activity impacts immune homeostasis and leads to rapid and marked increase in cell-free DNA (cfDNA). However, the origin of cfDNA during exercise remains elusive and it is unknown if physical activity could improve or interfere with methylation based liquid biopsy. We analyzed the methylation levels of four validated CpGs representing cfDNA from granulocytes, lymphocytes, monocytes, and non-hematopoietic cells, in healthy individuals in response to exercise, and in patients with hematological malignancies under resting conditions. The analysis revealed that physical activity almost exclusively triggered DNA release from granulocytes, highlighting the relevance as a pre-analytical variable which could compromise diagnostic accuracy.

Human DC3 Antigen Presenting Dendritic Cells From Induced Pluripotent Stem Cells.

Dendritic cells (DC) are professional antigen-presenting cells that develop from hematopoietic stem cells. Different DC subsets exist based on ontogeny, location and function, including the recently identified proinflammatory DC3 subset. DC3 have the prominent activity to polarize CD8+ T cells into CD8+ CD103+ tissue resident T cells. Here we describe human DC3 differentiated from induced pluripotent stem cells (iPS cells). iPS cell-derived DC3 have the gene expression and surface marker make-up of blood DC3 and polarize CD8+ T cells into CD8+ CD103+ tissue-resident memory T cells in vitro. To test the impact of malignant JAK2 V617F mutation on DC3, we differentiated patient-specific iPS cells with JAK2 V617Fhet and JAK2 V617Fhom mutations into JAK2 V617Fhet and JAK2 V617Fhom DC3. The JAK2 V617F mutation enhanced DC3 production and caused a bias toward erythrocytes and megakaryocytes. The patient-specific iPS cell-derived DC3 are expected to allow studying DC3 in human diseases and developing novel therapeutics.

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.

Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.

Tracking of epigenetic changes during hematopoietic differentiation of induced pluripotent stem cells.

BACKGROUND: Differentiation of induced pluripotent stem cells (iPSCs) toward hematopoietic progenitor cells (HPCs) raises high hopes for disease modeling, drug screening, and cellular therapy. Various differentiation protocols have been established to generate iPSC-derived HPCs (iHPCs) that resemble their primary counterparts in morphology and immunophenotype, whereas a systematic epigenetic comparison was yet elusive. RESULTS: In this study, we compared genome-wide DNA methylation (DNAm) patterns of iHPCs with various different hematopoietic subsets. After 20 days of in vitro differentiation, cells revealed typical hematopoietic morphology, CD45 expression, and colony-forming unit (CFU) potential. DNAm changes were particularly observed in genes that are associated with hematopoietic differentiation. On the other hand, the epigenetic profiles of iHPCs remained overall distinct from natural HPCs. Furthermore, we analyzed if additional co-culture for 2 weeks with syngenic primary mesenchymal stromal cells (MSCs) or iPSC-derived MSCs (iMSCs) further supports epigenetic maturation toward the hematopoietic lineage. Proliferation of iHPCs and maintenance of CFU potential was enhanced upon co-culture. However, DNAm profiles support the notion that additional culture expansion with stromal support did not increase epigenetic maturation of iHPCs toward natural HPCs. CONCLUSION: Differentiation of iPSCs toward the hematopoietic lineage remains epigenetically incomplete. These results substantiate the need to elaborate advanced differentiation regimen while DNAm profiles provide a suitable measure to track this process.

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.