Three-dimensional spatial mapping of the human hematopoietic microenvironment in healthy and diseased bone marrow.

The bone marrow hematopoietic microenvironment (HME) plays a pivotal role in regulating normal and diseased hematopoiesis. However, the spatial organization of the human HME has not been thoroughly investigated yet. Therefore, we developed a three-dimensional (3D) immunofluorescence model to analyze changes in the cellular architecture in control and diseased bone marrows (BMs). BM biopsies from patients with myeloproliferative neoplasms (MPNs) were stained sequentially for CD31, CD34, CD45, and CD271 with repetitive bleaching steps to realize five color images with DAPI as a nuclear stain. Hematopoietically normal age-matched BM biopsies served as controls. Twelve subsequent slides per sample were stacked to create three-dimensional bone marrow reconstructions with the imaging program Arivis Visions 4D. Iso-surfaces for niche cells and structures were created and exported as mesh objects for spatial distribution analysis in the 3D creation suite Blender. We recapitulated the bone marrow architecture using this approach and produced comprehensive 3D models of endosteal and perivascular BM niches. MPN bone marrows displayed apparent differences compared to the controls, especially concerning CD271 staining density, megakaryocyte (MK) morphology, and distribution. Furthermore, measurements of the spatial relationships of MKs and hematopoietic stem and progenitor cells with vessels and bone structures in their corresponding niche environments revealed the most pronounced differences in the vascular nice in polycythemia vera. Taken together, using a repetitive staining and bleaching approach allowed us to establish a 5-color analysis of human BM biopsies, which is difficult to achieve with conventional staining approaches. Based on this, we generated 3D BM models which recapitulated key pathological features and, importantly, allowed us to define the spatial relationships between different bone marrow cell types. We, therefore, believe that our method can provide new and valuable insights into bone marrow cellular interaction research.

Survival and risk of vascular complications in myelofibrosis-A population-based study from the Swedish MPN group.

OBJECTIVE: To gain knowledge of underlying risk factors for vascular complications and their impact on life expectancy in myelofibrosis. METHODS: From a cohort of 392 myelofibrosis patients registered in the Swedish MPN registry 58 patients with vascular complications during follow-up were identified. Patients with vascular complications were compared with both 1:1 matched controls and the entire myelofibrosis cohort to explore potential risk factors for vascular complications and their impact on survival. RESULTS: Incidence of vascular complications was 2.8 events per 100 patient-years and the majority of complications were thrombotic. Patients with complications were significantly older and had lower hemoglobin when compared to the entire cohort. In the case-control analysis, no significant risk factor differences were observed. The major cause of death was vascular complications and median survival was significantly impaired in patients with vascular complications (48 months) compared to controls (92 months). Inferior survival in patients with vascular complications was found to be dependent on IPSS risk category in a Cox regression model. CONCLUSION: Vascular complications have a considerable impact on survival in MF. At diagnosis, risk assessment by IPSS does not only predict survival but is also associated with the risk of vascular complications.

Acoustophoresis Enables the Label-Free Separation of Functionally Different Subsets of Cultured Bone Marrow Stromal Cells.

Culture-expanded mesenchymal stromal cells (MSCs) are promising candidates for clinical cell-based therapies. MSC products are heterogeneous and we therefore investigated whether acoustophoresis, an ultrasound-based separation technology, could be used for the label-free enrichment of functionally different MSC populations. Acoustophoresis uses an ultrasonic standing wave field in a microchannel that differentially affects the movement of cells depending on their acoustophysical properties, such as size, density, and compressibility. Human bone marrow (BM) MSCs were generated by standard adherent culture in xeno-free medium and separated by microchip acoustophoresis. MSCs with up to 20% higher proliferation and 1.7-fold increased clonogenic potential were enriched in the side outlet of the chip compared to the input sample. These cells were significantly smaller (average diameter 14.5 ± 0.4 µm) compared to the center outlet fraction (average diameter 17.1 ± 0.6 µm) and expressed higher levels of genes related to proliferation and stem cell properties (i.e., Ki-67 [1.9-fold], Nanog1 [6.65-fold], Oct4 [2.9-fold], and CXCL12 [1.8-fold], n = 3) in the side outlet compared to input. Fractions of MSCs in G0 /G1 cell cycle phase were significantly enriched in the side fraction and an up to 2.8-fold increase of cells in S/G2 /M phases were observed in center fractions compared to side fractions and 1.3-fold increased compared to the input sample. Acoustophoresis did not compromise MSC phenotype, proliferation, clonogenic capacity, and viability (generally 87-98%), nor did it affect differentiation or immunomodulatory capacities. These results demonstrate that label-free acoustic separation can enrich functionally different MSC subsets which can potentially be employed to produce better-defined stromal cell products from cultured MSCs. Hence, acoustophoresis is a potentially promising separation technology to provide improved cell products for research and possible future clinical use. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Early growth response 1 regulates hematopoietic support and proliferation in human primary bone marrow stromal cells.

Human bone marrow stromal cells (BMSC) are key elements of the hematopoietic environment and they play a central role in bone and bone marrow physiology. However, how key stromal cell functions are regulated is largely unknown. We analyzed the role of the immediate early response transcription factor EGR1 as key stromal cell regulator and found that EGR1 was highly expressed in prospectively-isolated primary BMSC, down-regulated upon culture, and low in non-colony-forming CD45neg stromal cells. Furthermore, EGR1 expression was lower in proliferative regenerating adult and fetal primary cells compared to adult steady-state BMSC. Overexpression of EGR1 in stromal cells induced potent hematopoietic stroma support as indicated by an increased production of transplantable CD34+CD90+ hematopoietic stem cells in expansion co-cultures. The improvement in bone marrow stroma support function was mediated by increased expression of hematopoietic supporting genes, such as VCAM1 and CCL28 Furthermore, EGR1 overexpression markedly decreased stromal cell proliferation whereas EGR1 knockdown caused the opposite effects. These findings thus show that EGR1 is a key stromal transcription factor with a dual role in regulating proliferation and hematopoietic stroma support function that is controlling a genetic program to co-ordinate the specific functions of BMSC in their different biological contexts.

A high-content cytokine screen identifies myostatin propeptide as a positive regulator of primitive chronic myeloid leukemia cells.

Aberrantly expressed cytokines in the bone marrow (BM) niche are increasingly recognized as critical mediators of survival and expansion of leukemic stem cells. To identify regulators of primitive chronic myeloid leukemia (CML) cells, we performed a high-content cytokine screen using primary CD34+ CD38low chronic phase CML cells. Out of the 313 unique human cytokines evaluated, 11 were found to expand cell numbers ≥2-fold in a 7-day culture. Focusing on novel positive regulators of primitive CML cells, the myostatin antagonist myostatin propeptide gave the largest increase in cell expansion and was chosen for further studies. Herein, we demonstrate that myostatin propeptide expands primitive CML and normal BM cells, as shown by increased colony-forming capacity. For primary CML samples, retention of CD34-expression was also seen after culture. Furthermore, we show expression of MSTN by CML mesenchymal stromal cells, and that myostatin propeptide has a direct and instant effect on CML cells, independent of myostatin, by demonstrating binding of myostatin propeptide to the cell surface and increased phosphorylation of STAT5 and SMAD2/3. In summary, we identify myostatin propeptide as a novel positive regulator of primitive CML cells and corresponding normal hematopoietic cells.

Highly reduced survival in essential thrombocythemia and polycythemia vera patients with vascular complications during follow-up.

OBJECTIVE: To explore the relative importance of risk factors, treatments, and blood counts for the occurrence of vascular complications and their impact on life expectancy in essential thrombocythemia (ET) and polycythemia vera (PV). METHODS: Nested case-control study within the Swedish MPN registry. From a cohort of 922 ET patients and 763 PV patients, 71 ET and 81 PV cases with vascular complications were compared with matched controls. RESULTS: Incidence of vascular complications was 2.0 and 3.4 events per 100 patient-years in ET and PV, respectively. At diagnosis, no significant risk factor differences were observed between cases and controls in neither of the diseases. At the time of vascular event, ET complication cases did not differ significantly from controls but in PV, cases had significantly higher WBCs and were to a lesser extent treated with anti-thrombotic and cytoreductive therapy. Life expectancy was significantly decreased in both ET and PV cases compared with controls. CONCLUSIONS: The risk of vascular complications is high in both ET and PV, and these complications have a considerable impact on life expectancy. The protective effect of anti-thrombotic and cytoreductive therapy for vascular complications in PV underscores the importance of avoiding undertreatment.

Acoustic Enrichment of Extracellular Vesicles from Biological Fluids.

Extracellular vesicles (EVs) have emerged as a rich source of biomarkers providing diagnostic and prognostic information in diseases such as cancer. Large-scale investigations into the contents of EVs in clinical cohorts are warranted, but a major obstacle is the lack of a rapid, reproducible, efficient, and low-cost methodology to enrich EVs. Here, we demonstrate the applicability of an automated acoustic-based technique to enrich EVs, termed acoustic trapping. Using this technology, we have successfully enriched EVs from cell culture conditioned media and urine and blood plasma from healthy volunteers. The acoustically trapped samples contained EVs ranging from exosomes to microvesicles in size and contained detectable levels of intravesicular microRNAs. Importantly, this method showed high reproducibility and yielded sufficient quantities of vesicles for downstream analysis. The enrichment could be obtained from a sample volume of 300 µL or less, an equivalent to 30 min of enrichment time, depending on the sensitivity of downstream analysis. Taken together, acoustic trapping provides a rapid, automated, low-volume compatible, and robust method to enrich EVs from biofluids. Thus, it may serve as a novel tool for EV enrichment from large number of samples in a clinical setting with minimum sample preparation.

Effects of JAK1/2 inhibition on bone marrow stromal cells of myeloproliferative neoplasm (MPN) patients and healthy individuals.

OBJECTIVE: Philadelphia-negative myeloproliferative neoplasms (MPNs) commonly share hyperactive JAK-STAT signaling affecting hematopoietic stem cells (HSC) and their progeny. The JAK1/2 inhibitor Ruxolitinib has remarkable clinical efficacy, including spleen reduction, improvement of constitutional symptoms, and bone marrow (BM) fibrosis reversal. Whether this is due to inhibition of JAK2-mutated HSC only, or whether Ruxolitinib also affects BM stroma is not known. METHODS: This study investigated potential effects of Ruxolitinib on BM mesenchymal stromal cells (MSC), which are not only major regulators of hematopoiesis but also contribute to fibrosis, from 10 healthy donors and 7 JAK2V617F -positive MPN patients. RESULTS: Ruxolitinib moderately inhibited the growth of healthy donor MSC (HD-MSC) and MSC from JAK2V617F+ MPN patients (P-MSC) in short- and long-term assays. The clonogenic potential of HD-MSC was not affected by Ruxolitinib. JAK-STAT signaling, however, was markedly inhibited in both HD-MSC and P-MSC, the latter of which showed higher expression of fibrosis-associated and hematopoiesis-maintenance genes. Moreover, Ruxolitinib reduced MSC secretion of MCP-1 and IL-6. CONCLUSION: Ruxolitinib affected JAK2 signaling in MSC at clinically relevant doses, which is likely to contribute to the normalization of the inflammatory milieu in MPNs. Thus, combined HSC and stroma-directed interventions have the potential to improve constitutional symptoms and reduce stromal proliferation in MPNs.

Identification of two distinct mesenchymal stromal cell populations in human malignant glioma.

Gene profiling has revealed that malignant gliomas can be divided into four distinct molecular subtypes, where tumors with a mesenchymal gene expression are correlated with short survival. The present investigation was undertaken to clarify whether human malignant gliomas contain endogenous mesenchymal stromal cells (MSC), fulfilling consensus criteria defined by The International Society for Cellular Therapy, recruited from the host. We found that MSC-like cells can be isolated from primary human malignant gliomas. Two distinct MSC-like cell populations, differing in their expression of the CD90 surface marker, were discovered after cell sorting. RNA sequencing revealed further genetic differences between these two cell populations and MSC-like cells lacking CD90 produced higher amounts of VEGF and PGE2 compared to cells with the true MSC phenotype, implying that the CD90- MSC-like cells most probably are more active in tumor vascularization and immunosuppression than their CD90+ counterpart. The results highlight the CD90- subpopulation as an important tumor component, however, its functional effects in glioma remains to be resolved. Using the protocols presented here, it will be possible to isolate, characterize and analyze brain tumor-derived MSC-like cells in more detail and to further test their functions in vitro and in in vivo xenograft models of glioma.

Genomic profiling and directed ex vivo drug analysis of an unclassifiable myelodysplastic/myeloproliferative neoplasm progressing into acute myeloid leukemia.

Myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are rare genetically heterogeneous hematologic diseases associated with older age and a poor prognosis. If the disease progresses into acute myeloid leukemia (AML), it is often refractory to treatment. To gain insight into genetic alterations associated with disease progression, whole exome sequencing and single nucleotide polymorphism arrays were used to characterize the bone marrow and blood samples from a 39-year-old woman at MDS/MPN-U diagnosis and at AML progression, in which routine genetic diagnostics had not identified any genetic alterations. The data revealed the presence of a partial tandem duplication of the MLL gene as the only detectable copy number change and 11 non-silent somatic mutations, including DNMT3A R882H and NRAS G13D. All somatic lesions were present both at initial MDS/MPN-U diagnosis and at AML presentation at similar mutant allele frequencies. The patient has since had two extramedullary relapses and is at high risk of a future bone marrow relapse. A directed ex vivo drug sensitivity analysis showed that the patient's AML cells are sensitive to, for example, the MEK inhibitor trametinib and the proteasome inhibitor bortezomib, indicating that she may benefit from treatment with these drugs. © 2016 Wiley Periodicals, Inc.

Integrated Acoustic Separation, Enrichment, and Microchip Polymerase Chain Reaction Detection of Bacteria from Blood for Rapid Sepsis Diagnostics.

This paper describes an integrated microsystem for rapid separation, enrichment, and detection of bacteria from blood, addressing the unmet clinical need for rapid sepsis diagnostics. The blood sample is first processed in an acoustophoresis chip, where red blood cells are focused to the center of the channel by an acoustic standing wave and sequentially removed. The bacteria-containing plasma proceeds to a glass capillary with a localized acoustic standing wave field where the bacteria are trapped onto suspended polystyrene particles. The trapped bacteria are subsequently washed while held in the acoustic trap and released into a polymer microchip containing dried polymerase chain reaction (PCR) reagents, followed by thermocycling for target sequence amplification. The entire process is completed in less than 2 h. Testing with Pseudomonas putida spiked into whole blood revealed a detection limit of 1000 bacteria/mL for this first-generation analysis system. In samples from septic patients, the system was able to detect Escherichia coli in half of the cases identified by blood culture. This indicates that the current system detects bacteria in patient samples in the upper part of the of clinically relevant bacteria concentration range and that a further developed acoustic sample preparation system may open the door for a new and faster automated method to diagnose sepsis.

Efficient purification of CD4+ lymphocytes from peripheral blood progenitor cell products using affinity bead acoustophoresis.

Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations, utilizing primarily magnetic or fluorescence activated sorting methods. Here, we investigated the performance of microfluidic acoustophoresis for the separation of lymphocyte subsets from PBPC, and present a novel method for affinity-bead-mediated acoustic separation of cells which can otherwise not be acoustically discriminated. As the acoustic force on a particle depends on particle size, density and compressibility, targeting of cells by affinity specific beads will generate cell-bead complexes that exhibit distinct acoustic properties relative to nontargeted cells and are, thus, possible to isolate. To demonstrate this, PBPC samples (n = 22) were obtained from patients and healthy donors. Following density gradient centrifugation, cells were labeled with anti-CD4-coated magnetic beads (Dynal) and isolated by acoustophoresis and, for comparison, standard magnetic cell sorting technique in parallel. Targeted CD4+ lymphocytes were acoustically isolated with a mean (±SD) purity of 87 ± 12%, compared with 96 ± 3% for control magnetic sorting. Viability of sorted cells was 95 ± 4% (acoustic) and 97 ± 3% (magnetic), respectively. The mean acoustic separation efficiency of CD4+ lymphocytes to the target fraction was 65 ± 22%, compared with a mean CD4+ lymphocyte recovery of 56 ± 15% for magnetic sorting. Functional testing of targeted CD4+ lymphocytes demonstrated unimpaired mitogen-mediated proliferation capacity and cytokine production. Hematopoietic progenitor cell assays revealed a preserved colony forming ability of nontarget cells post sorting. We conclude that the acoustophoresis platform can be utilized to efficiently isolate bead-labeled CD4+ lymphocytes from PBPC samples in a continuous flow format, with preserved functional capacity of both target and nontarget cells. These results open up for simultaneous affinity-bead-mediated separation of multiple cell populations, something which is not possible with current standard magnetic cell separation technology. © 2014 International Society for Advancement of Cytometry.

Regulation of advanced therapy medicinal products in Europe and the role of academia.

BACKGROUND AIMS: Advanced therapy medicinal products (ATMP) are gene therapy, somatic cell therapy or tissue-engineered products regulated under (EC) No. 1394/2007 to ensure their free movement within the European Union while guaranteeing the highest level of health protection for patients. Academic good manufacturing practice (GMP) centers are major contributors in the development of ATMPs and this study assessed the impact of regulations on them. METHODS: European academic and non-industrial facilities (n = 747) were contacted, and a representative sample of 50 replied to a detailed questionnaire. Experienced centres were further selected in every Member State (MS) for semi-structured interviews. Indicators of ATMP production and development success were statistically assessed, and opinions about directive implementation were documented. RESULTS: Facilities experienced in manufacturing cell therapy transplant products are the most successful in developing ATMPs. New centres lacking this background struggle to enter the field, and there remains a shortage of facilities in academia participating in translational research. This is compounded by heterogeneous implementation of the regulations across MS. CONCLUSIONS: GMP facilities successfully developing ATMPs are present in all MS. However, the implementation of regulations is heterogeneous between MS, with substantial differences in the definition of ATMPs and in the approved manufacturing environment. The cost of GMP compliance is underestimated by research funding bodies. This is detrimental to development of new ATMPs and commercialization of any that are successful in early clinical trials. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective European Union legislation in this field.

Specific subsets of mesenchymal stroma cells to treat lung disorders--finding the Holy Grail.

Accumulating studies, both in animals and human clinical trials with mesenchymal stroma cells (MSC) support the hypothesis of therapeutic effects of these cells in various disorders. However, despite success in immune-mediated disorders such as Crohns' disease, lung disorders such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary disease (IPF) treated with MSC have so far not yielded a revolutionary effect on clinical symptoms. Promising data on immunomodulatory effects in COPD have kept nourishing the research into finding specific traits of MSC beneficial in disease. A heterogeneous population of injected cells might drown a potential therapeutic role of a specific group of MSC. Thus careful analysis of MSC regarding their molecular capabilities such as delivering specific therapeutic vesicles to the environment, or plain cytokine/chemokine fingerprinting might prove useful in augmenting therapies against lung diseases.

Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing.

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.

A temporal developmental map separates human NK cells from noncytotoxic ILCs through clonal and single-cell analysis.

ABSTRACT: Natural killer (NK) cells represent the cytotoxic member within the innate lymphoid cell (ILC) family that are important against viral infections and cancer. Although the NK cell emergence from hematopoietic stem and progenitor cells through multiple intermediate stages and the underlying regulatory gene network has been extensively studied in mice, this process is not well characterized in humans. Here, using a temporal in vitro model to reconstruct the developmental trajectory of NK lineage, we identified an ILC-restricted oligopotent stage 3a CD34-CD117+CD161+CD45RA+CD56- progenitor population, that exclusively gave rise to CD56-expressing ILCs in vitro. We also further investigated a previously nonappreciated heterogeneity within the CD56+CD94-NKp44+ subset, phenotypically equivalent to stage 3b population containing both group-1 ILC and RORγt+ ILC3 cells, that could be further separated based on their differential expression of DNAM-1 and CD161 receptors. We confirmed that DNAM-1hi S3b and CD161hiCD117hi ILC3 populations distinctively differed in their expression of effector molecules, cytokine secretion, and cytotoxic activity. Furthermore, analysis of lineage output using DNA-barcode tracing across these stages supported a close developmental relationship between S3b-NK and S4-NK (CD56+CD94+) cells, whereas distant to the ILC3 subset. Cross-referencing gene signatures of culture-derived NK cells and other noncytotoxic ILCs with publicly available data sets validated that these in vitro stages highly resemble transcriptional profiles of respective in vivo ILC counterparts. Finally, by integrating RNA velocity and gene network analysis through single-cell regulatory network inference and clustering we unravel a network of coordinated and highly dynamic regulons driving the cytotoxic NK cell program, as a guide map for future studies on NK cell regulation.

CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization.

Nonhematopoietic bone marrow mesenchymal stem cells (BM-MSCs) are of central importance for bone marrow stroma and the hematopoietic environment. However, the exact phenotype and anatomical distribution of specified MSC populations in the marrow are unknown. We characterized the phenotype of primary human BM-MSCs and found that all assayable colony-forming units-fibroblast (CFU-Fs) were highly and exclusively enriched not only in the lin⁻/CD271⁺/CD45⁻/CD146⁺ stem-cell fraction, but also in lin⁻/CD271⁺/CD45⁻/CD146(⁻/low) cells. Both populations, regardless of CD146 expression, shared a similar phenotype and genotype, gave rise to typical cultured stromal cells, and formed bone and hematopoietic stroma in vivo. Interestingly, CD146 was up-regulated in normoxia and down-regulated in hypoxia. This was correlated with in situ localization differences, with CD146 coexpressing reticular cells located in perivascular regions, whereas bone-lining MSCs expressed CD271 alone. In both regions, CD34⁺ hematopoietic stem/progenitor cells were located in close proximity to MSCs. These novel findings show that the expression of CD146 differentiates between perivascular versus endosteal localization of non-hematopoietic BM-MSC populations, which may be useful for the study of the hematopoietic environment.

Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing.

Hematopoiesis is regulated by the bone marrow (BM) stroma. However, cellular identities and functions of the different BM stromal elements in humans remain poorly defined. Based on single-cell RNA sequencing (scRNAseq), we systematically characterized the human non-hematopoietic BM stromal compartment and we investigated stromal cell regulation principles based on the RNA velocity analysis using scVelo and studied the interactions between the human BM stromal cells and hematopoietic cells based on ligand-receptor (LR) expression using CellPhoneDB. scRNAseq led to the identification of six transcriptionally and functionally distinct stromal cell populations. Stromal cell differentiation hierarchy was recapitulated based on RNA velocity analysis and in vitro proliferation capacities and differentiation potentials. Potential key factors that might govern the transition from stem and progenitor cells to fate-committed cells were identified. In situ localization analysis demonstrated that different stromal cells were localized in different niches in the bone marrow. In silico cell-cell communication analysis further predicted that different stromal cell types might regulate hematopoiesis through distinct mechanisms. These findings provide the basis for a comprehensive understanding of the cellular complexity of the human BM microenvironment and the intricate stroma-hematopoiesis crosstalk mechanisms, thus refining our current view on human hematopoietic niche organization.

A novel immunohistochemical sequential multi-labelling and erasing technique enables epitope characterization of bone marrow pericytes in primary myelofibrosis.

AIM: In Philadelphia (Ph)-negative chronic myeloproliferative neoplasms, increased microvascular density, bizarre vessel architecture and increased number of pericytes are among the distinct histopathological features. The aim of this study was to characterize bone marrow pericytes in primary myelofibrosis (PMF) using a novel multi-labelling immunohistochemical technique. METHODS AND RESULTS: Bone marrow biopsies from a normal donor (n = 1) and patients with PMF (n = 3) were subjected to an immunohistochemical sequential multi-labelling and erasing technique (SE-technique). Antigens of interest in the first and/or second layer were detected with an immunoperoxidase system and visualized with aminoethylcarbazole. After imaging, erasing and blocking of immunoreagents, the slides were stained with a traditional double immunolabelling procedure. In addition, we applied a Photoshop® colour palette, creating a single composite image of the sequential staining procedures. We successfully applied four layers of antibodies on one slide using CD146, smooth muscle actin, CD34, CD271 and Ki67 in different combinations. The SE-technique significantly improves morphological and phenotypical studies in bone marrow specimens. CONCLUSIONS: To our knowledge, the SE-technique is the first to multi-label antigens, identifying vessel and pericyte architecture in bone marrow by light microscopy. This technique may unravel novel aspects of the composition of the microvessel structures in patients with PMF and related neoplasms.

Single-cell transcriptional profiling informs efficient reprogramming of human somatic cells to cross-presenting dendritic cells.

Type 1 conventional dendritic cells (cDC1s) are rare immune cells critical for the induction of antigen-specific cytotoxic CD8+ T cells, although the genetic program driving human cDC1 specification remains largely unexplored. We previously identified PU.1, IRF8, and BATF3 transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts, but reprogramming of human somatic cells was limited by low efficiency. Here, we investigated single-cell transcriptional dynamics during human cDC1 reprogramming. Human induced cDC1s (hiDC1s) generated from embryonic fibroblasts gradually acquired a global cDC1 transcriptional profile and expressed antigen presentation signatures, whereas other DC subsets were not induced at the single-cell level during the reprogramming process. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the cDC1-inducing transcription factor network as key drivers of the process. Combining IFN-γ, IFN-ß, and TNF-α with constitutive expression of cDC1-inducing transcription factors led to improvement of reprogramming efficiency by 190-fold. hiDC1s engulfed dead cells, secreted inflammatory cytokines, and performed antigen cross-presentation, key cDC1 functions. This approach allowed efficient hiDC1 generation from adult fibroblasts and mesenchymal stromal cells. Mechanistically, PU.1 showed dominant and independent chromatin targeting at early phases of reprogramming, recruiting IRF8 and BATF3 to shared binding sites. The cooperative binding at open enhancers and promoters led to silencing of fibroblast genes and activation of a cDC1 program. These findings provide mechanistic insights into human cDC1 specification and reprogramming and represent a platform for generating patient-tailored cDC1s, a long-sought DC subset for vaccination strategies in cancer immunotherapy.