Abstract Myelofibrosis (MF) is the most severe myeloproliferative neoplasm. Current therapies – except for allogeneic stem cell transplantation – are largely supportive which highlights the need for improved disease models and novel therapeutic targets. Here, we established a humanized MF model by transplanting thrombopoietin (THPO)-overexpressing human bone marrow CD34□ cells into humanized bone marrow ossicles generated in immunodeficient NSG mice. THPO overexpression induced progressive reticulin fibrosis in vivo, accompanied by myeloid skewing, increased megakaryocyte clustering, and redistribution of human hematopoietic cells to murine spleen and femur, consistent with extramedullary hematopoiesis. THPO-driven ossicles also exhibited features of osteosclerosis, including increased trabecular bone and osteoid formation, indicating active pathological remodeling of the niche. Mechanistically, fibrosis was associated with increased SPP1/OPN expression, which was also observed in bone marrow biopsies from MF patients. Importantly, in vivo neutralization of SPP1 attenuated myeloid skewing, reduced megakaryocyte expansion, and decreased fibrosis severity, highlighting SPP1-driven niche remodeling as a potential therapeutic target in MF. This humanized MF model thus provides a translationally relevant platform to dissect microenvironment-driven MF pathogenesis and evaluate targeted therapies.

Abstract Background The variable clinical outcomes of mesenchymal stromal cell (MSC)-based therapy in acute respiratory distress syndrome (ARDS) are attributed to a variety of factors, including host microenvironmental factors. Interleukin-1β (IL-1β) has been linked to the development and progression of ARDS, and we have previously found that IL-1β could be used to predict MSC activation in vitro. However, the exact mechanisms through which IL-1β alters the MSC function and its interaction with the host immune cells remains unknown. Therefore, the aim of this study was to assess how IL-1β alters MSC function, with a specific focus on MSC-neutrophil interaction. Methods Human bone marrow-derived MSCs were exposed to 20 ng/ml IL-1β for 1 or 24 h. Following exposure, MSCs were analyzed using bulk RNA sequencing and key secretome proteins were measured in their conditioned medium. A transwell culture system was used to evaluate the neutrophil recruitment capacity of IL-1β-exposed MSCs, with or without NF-kB inhibition. MSCs exposed to serum free medium were used as controls in all experiments. Results The sequencing data revealed that genes involved in response to biotic stimuli and immune response were altered in MSCs exposed to IL-1β compared to control cells. In particular, genes essential for neutrophil recruitment were significantly upregulated after IL-1β exposure. The functional in vitro studies further validated these results, demonstrating that MSCs exposed to IL-1β had a significantly higher neutrophil recruitment capacity compared to unstimulated MSCs. Finally, inhibition of the NF-kB pathway resulted in a significant decrease of the MSC’s capacity to recruit neutrophils to levels similar as to the unstimulated control MSCs. Conclusion These data provide mechanistic insight into how inflammatory factors present in the host microenvironment might affect the interaction between MSCs and immune cells. This further highlights the need to understand the MSC mode of action, and to map out how the MSC fate might change in different host environments after administration.

Fluorescence in situ hybridization (FISH) can be employed to study the expression and subcellular localization of nucleic acids by using labeled antisense strands that hybridize with the target RNA or DNA molecules. Likewise, immunofluorescence antibody staining (IF) takes advantage of the specific interaction between a fluorophore-labeled antibody and its corresponding antigen. This protocol reports the combination of RNA-FISH and IF antibody staining for simultaneous detection of both RNA transcripts and proteins of interest in routine formalin-fixed paraffin-embedded (FFPE) bone marrow biopsy samples. Herein, we provide a detailed description of the methodology that we have developed and optimized to study the spatial expression of two transcripts-TGFB1 and PDGFA1-in human hematopoietic (CD45+) and non-hematopoietic (CD271+) cells in the bone marrow of patients with acute lymphoblastic leukemia (ALL). Key features • The protocol describes the simultaneous visualization of RNA target transcripts and protein expression. • In situ RNA and surface marker analysis was established for routine human bone marrow biopsies.

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer. Bone marrow (BM) fibrosis in ALL has been associated with adverse outcomes, however, little is known about the mechanisms that cause fibrosis in ALL. Therefore, we established a novel and advanced analysis method by combining multi-color immunofluorescence staining with in-situ RNA expression analysis (RNAscope®) investigate the spatial expression of putative fibrotic drivers in ALL bone marrows. We analyzed standard BM biopsies from pediatric ALL patients. Sequential 5-color immunofluorescence (IF) staining with CD45, CD271, CD31, CD34 and DAPI was used to identify different BM cell types. Combined RNAscope® and IF staining was established for spatial mRNA expression analysis of transforming growth factor beta 1 ( TGFB1 ) and platelet-derived growth factor alpha 1 ( PDGFA1 ), which are known to play major roles in primary myelofibrosis (PMF). PMF and normal BM samples served as controls. As expected, ALL bone marrows showed high cellularities and prominent populations of blast cells. CD271+ MSC density was increased in ALL and was associated with fibrosis in a similar manner as observed for PMF. TGFB1 and PDGFA1 expression was considerably increased in ALL megakaryocytes (MKs) compared to PMF patients and normal controls. Furthermore, MK TGFB1 and PDGFA1 expression intensities in fibrotic ALL correlated with fibrosis grade. TGFB1 and PDGFA1 were also expressed in leukemic blasts, however at lower intensities compared to ALL MKs. Taken together, advanced in-situ RNA and IF staining not only revealed increased expression of TGFB1 and PDGFA1 in fibrotic pediatric ALL, but also identified ALL blasts and MKs as their cellular origin at the single cell level. These novel data strongly suggest a role of these cytokines as potential fibrosis drivers in ALL. More broadly, our findings demonstrate that combined RNA and surface marker analysis is a powerful tool to provide new and valuable insights into bone marrow pathophysiology. ### Competing Interest Statement The authors have declared no competing interest. Swedish Childhood Cancer Foundation, PR2023-0062 Swedish Cancer Foundation, 23 2957 Pj 01 H Swedish Bloodcancer Association (Blodcancerförbundet) Foundation Siv-Inger and Per-Erik Anderssons minnesfond John Persson Foundation ALF (Government Public Health Grant)

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer. Bone marrow (BM) fibrosis in ALL has been associated with adverse outcomes; however, little is known about the mechanisms that cause fibrosis in ALL. Therefore, we established a novel and advanced analysis method by combining multicolor immunofluorescence (IF) staining with in situ RNA expression analysis (RNAscope) to investigate the spatial expression of putative fibrotic drivers in ALL BMs. We analyzed standard BM biopsies from pediatric patients with ALL. Sequential 5-color IF staining with CD45, CD271, CD31, CD34, and DAPI was used to identify different BM cell types. Combined RNAscope and IF staining was established for spatial messenger RNA expression analysis of transforming growth factor beta 1 (TGFB1) and platelet-derived growth factor alpha 1 (PDGFA1), which are known to play major roles in primary myelofibrosis (PMF). PMF and normal BM samples served as controls. As expected, ALL BMs showed high cellularities and prominent populations of blast cells. CD271+ mesenchymal stromal cell density was increased in ALL and was associated with fibrosis in a similar manner as observed for PMF. TGFB1 and PDGFA1 expression was considerably increased in ALL megakaryocytes (MKs) compared with patients with PMF and normal controls. Furthermore, MK TGFB1 and PDGFA1 expression intensities in fibrotic ALL correlated with fibrosis grade. TGFB1 and PDGFA1 were also expressed in leukemic blasts, however, at lower intensities compared with ALL MKs. Taken together, advanced in situ RNA and IF staining not only revealed increased expression of TGFB1 and PDGFA1 in fibrotic pediatric ALL but also identified ALL blasts and MKs as their cellular origin at the single-cell level. These novel data strongly suggest a role of these cytokines as potential fibrosis drivers in ALL. More broadly, our findings demonstrate that combined RNA and surface marker analysis is a powerful tool to provide new and valuable insights into BM pathophysiology.

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 ROR gamma t(+) ILC3 cells, that could be further separated based on their differential expression of DNAM-1 and CD161 receptors. We confirmed that DNAM-1(hi) S3b and CD161(hi)CD117(hi) 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.

Growing evidence suggests that changes in the hematopoietic microenvironment (HME) can influence treatment response in leukemia. Among HME disturbances, bone marrow (BM) fibrosis stands out as the most severe and recognizable form, characterized by excessive deposition of reticulin fibers. In acute lymphoblastic leukemia (ALL), BM fibrosis has been linked to unfavorable outcome and high reticulin fiber density at diagnosis has been associated with elevated levels of therapy-surviving leukemia cells. However, the mechanisms that cause fibrosis in ALL have not been thoroughly investigated thus far. In contrast, transforming growth factor beta 1 (TGFB1) and platelet-derived growth factor alpha 1 (PDGFA1) have been identified to play major roles in classical primary myelofibrosis (PMF). We therefore aimed to investigate potential fibrosis mechanisms in ALL by comparing the cellular composition and spatial expression of TGFB1 and PDGFA1 in ALL bone marrows compared with classical primary myelofibrosis (PMF). We analyzed formalin-fixed paraffin-embedded BM biopsies from pediatric ALL patients (n=9) compared with PMF samples (n=3) and hematologically normal controls (n=3). Sequential immunofluorescence (IF) staining with CD45, CD271, CD31, and CD34 antibodies and DAPI as nuclear stain was realized by repetitive rounds of staining, scanning, bleaching and restaining resulting in 5-color images (Bräunig et al., Cytometry A. 2023 (10):763-776). Slides were scanned with an Olympus VS120 slide scanner. The chosen markers were sufficient to identify hematopoietic stem and progenitor cells (HSPCs) (CD271-/ CD31-/CD34/45+), megakaryocytes (MKs) (CD45-/low/ CD271-/ CD34low/+/ CD31+), mesenchymal stromal cells (MSCs) (CD45 -/low/ CD271+/ CD31/34-), and endothelial cells (ECs) (CD45-/ CD271-/ CD34low/+/ CD31+). CD271+ mesenchymal stromal cell (MSC) density was assessed by volumetric analysis using the image analysis platform Arivis. Combined RNAscope® and IF staining was established for spatial analysis of TGFB1 and PDGFA1 mRNA expression. As expected, ALL bone marrows showed high cellularities and prominent populations of blast cells expressing typical surface markers. The density of MSCs in ALL was seemingly increased, especially around vessels, with increasing degrees of fibrosis in comparison to the control samples. CD271+ MSC densities as calculated by normalized CD271+ volumes were significantly higher in ALL with grade 2 fibrosis compared to non-fibrotic ALL bone marrows and controls. Megakaryocyte (MK) numbers in ALL samples were generally reduced. Interestingly, however, TGFB1 and PDGFA1 expression was considerably increased in ALL MKs compared to MKs from PMF patients and normal controls. This was unexpected as MKs in ALL do not belong to the malignant clone. Furthermore, TGFB1 and PDGFA1 expression intensities in MKs in fibrotic ALL correlated with fibrosis grade and fibrotic pathway activity. In addition, TGFB1 and PDGFA1 were also expressed in leukemic blasts, however at lower intensities compared to ALL MKs. Expression of TGFB1 and PDGFA1 in ALL blasts was furthermore confirmed by reanalysis of publically available single-cell RNA sequencing data (Witkowski et al., Cancer Cell, 2020 37(6): 867-882). Taken together, simultaneous in-situ RNA expression and IF staining identified - for the first time - increased TGFB1 and PDGFA1 expression in pediatric ALL, thus suggesting a role of these driver cytokines in ALL fibrosis. Hence, our findings provide novel insights into fibrosis pathomechanisms in ALL that point to potential therapeutic targets to improve treatment outcomes in fibrotic ALL.

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 HMEforming 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.

Myelofibrosis (MF) is the most severe form of the myeloproliferative neoplasms (MPN). Current standard MF therapies - except for allogeneic stem cell transplantation - are not curative and have only a limited effect on fibrosis, leaving patients with an urgent need for more effective and targeted treatments. While murine models have successfully captured some aspects of MF, the true challenge lies in creating an in vivo model that faithfully mirrors human MF pathophysiology. We therefore aimed to establish the first humanized MF model based on transplantation of thrombopoietin (THPO)-overexpressing human CD34+ cells into humanized bone marrow tissues established in immunodeficient NSG mice. We first generated humanized bone marrow (BM) tissues (ossicles) by subcutaneous implantation of 2×106 human BM-derived stromal cells (BMSCs) into immunodeficient NSG using a modified matrix preparation. This resulted in the formation of subcutaneous ossicles containing functional BM after 8 weeks (8-week ossicles). To mimic human MF development in vivo, we adopted a strategy to overexpress THPO in hematopoietic stem and progenitors (HSPCs). We generated THPO-overexpressing (THPO-oe) CD34+ HSPCs by lentiviral transduction of human CD34-enriched (MACS) bone marrow cells using THPO-GFP lentivirus. Control cells were transduced with control lentivirus vector (GFP). THPO-oe CD34+ cells and controls, respectively, were transplanted into 8-week ossicles by direct intraossicle injection (3×105 cells/ossicle). FACS analysis of ossicles showed robust engraftment (up to > 80%) of viable human hematopoietic cells as early as 4 weeks after transplantation of THPO-oe CD34+ cells. The cellularity of THPO-oe CD34+ injected ossicles was 1.4 ± 0.6 ×107 cells/ossicle which was comparable to controls. Engrafted human CD45+ cells demonstrated a significantly higher myeloid to lymphoid ratio of 0.14 in the THPO group (n=17) compared with 0.08 for GFP controls (n=14) (p = 0.0147). THPO overexpression in human BM CD34+ cells furthermore resulted in a notable increase in human ossicle megakaryocytes as indicated by a 1.67-fold increase (n=11) of CD41a+/CD42b+ megakaryocytes compared with controls. An increase in megakaryocyte production from THPO-oe CD34+ cells was furthermore confirmed by in vitro culture experiments. Additionally and importantly, histological analysis of ossicles showed marked reticulin fibrosis in 8-week THPO-oe CD34+ cell transplanted ossicles compared to controls, which corresponded to grade 2 MF. Time course experiments are in progress to evaluate the kinetics of fibrosis development in this model. Interestingly, human hematopoietic cells were clearly detected in murine spleens and femurs as early as 4 weeks following intra-ossicle injection, indicating that transplanted human cells migrated to murine hematopoietic tissues. However, spleen size was not increased in mice transplanted with THPO-oe CD34+ cells and preliminary data showed no increased formation of reticulin fibers in murine spleens and bone marrows. In summary, our study demonstrates that THPO overexpression in human BM CD34+ cells is sufficient to induce BM myelofibrosis in a humanized xenotransplantation model (MF ossicles). This novel model, which is the first of its kind, corresponds to one of the established murine MF models and faithfully recapitulates important MF disease hallmarks. The MF ossicle model will therefore not only provide a platform for comprehensive mechanistic studies in human MF but will also serve as a valuable tool for drug testing and identification of novel targets for therapy.

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.

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.

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.

The bone marrow hematopoietic microenvironment (HME) plays a pivotal role in regulating normal and diseased hematopoiesis. Important cellular components of the human (HME) include the bone marrow (BM) stromal cells (Li, BioRxive 2022) and other cell types, such as vascular endothelial cells (ECs), and megakaryocytes (MKs). Perturbations of the HME can affect normal and diseased hematopoiesis. In Philadelphia-negative myeloproliferative neoplasms (MPNs), which include Essential Thrombocythemia (ET), Polycythemia Vera (PV), Primary Myelofibrosis (PMF), and its precursor, pre-fibrotic MF (pre-PMF), the HME is of critical importance for disease development and progression (Mead, Blood 2017). However, despite this important role, little is known about the spatial organization of the different HME elements in MPNs. Therefore, we aimed to develop a novel method of immunofluorescence (IF) multiplexing with markers for HME and hematopoietic cells based on sequential staining and bleaching in combination with scanning IF microscopy and standard imaging analysis (Fig.1). Herein we show that this approach allowed us to generate 3D reconstitutions of standard biobank-stored BM samples from MPN patients and age-matched hematopoietically normal controls. Furthermore, we demonstrate how the free and open-source 3D computer graphics software toolset Blender (Stichting Blender Foundation, Amsterdam) can be utilized to analyze cell distributions and spatial relationships in 3D. BM biopsies from patients with MPN and controls were stained sequentially for CD31, CD34, CD45, and CD271 with repetitive bleaching steps to realize five color images with DAPI as a nuclear stain. Twelve subsequent slides per sample were stacked to create three-dimensional BM reconstructions with the imaging program Arivis Vision 4D. Iso-surfaces for niche cells and structures were created and exported as mesh objects for spatial distribution analysis in Blender. We recapitulated the BM architecture using this approach and produced comprehensive 3D models of endosteal and perivascular BM niches. MPN bone marrows displayed considerable differences between the different MPN types, especially concerning megakaryocyte (MK) morphology and distribution, as well as CD271 staining density and CD271+ cell numbers. Whereas ET and pre-PMF were comparable to their paired normal control, we found that PMF and especially PV samples showed considerably increased numbers and densities of CD271+ cells. In PMF the generated MSC objects formed a thick fibrillary structure reminiscent of classic fibrosis staining that was particularly dense close to vessels, bone surfaces, and MKs. Measurements of the spatial relationships of MKs and hematopoietic stem and progenitor cells (HSPC) to vessels and bone structures, respectively, revealed the most pronounced differences in PV, with median distances of PV-MKs to vessels being significantly lower than in controls (PV 7.62 µm vs 8.20 µm control, p<0.05). Also, the median distance in PV of HSPCs to vessels (2,88 µm) and bones (52.23 µm) was less than in the controls (46.31 µm and 236.7 µm, respectively; p<0.01). Taken together, we report a novel sequential staining and bleaching technique for multi-color analysis of normal and diseased BMs. The three-dimensional reconstruction technique developed herein revealed important aspects of the spatial HME composition in MPNs and normal BMs and provides a platform for a deeper investigation of BM pathophysiology, for example by increasing numbers of antibodies and including visualization of the expression of relevant genes and proteins. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background Infection and graft-versus-host disease (GvHD) are the major causes for mortality and morbidity of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Plasma-derived extracellular vesicles (EVs) contain disease-related proteins, DNAs and RNAs, and have recently been suggested as potential biomarker candidates for transplantation complications. However, EV isolation from small plasma volumes in clinical biomarker studies using conventional methods is challenging. We therefore investigated if EVs isolated by novel automated acoustic trapping could be developed as potential biomarkers for allo-HSCT complications by performing a clinical proof-of-principle study. Results Plasma samples were collected from twenty consecutive patients with high-risk/relapsed hematologic malignancies undergoing allo-HSCT before transplantation and post-transplant up to 12 weeks. EVs were isolated from small plasma sample volumes (150 μl) by an automated, acoustofluidic-based particle trapping device, which utilizes a local λ/2 ultrasonic standing wave in a borosilicate glass capillary to capture plasma EVs among pre-seeded polystyrene microbeads through sound scatter interactions. We found that EVs could be reliably isolated from all plasma samples ( n = 173) and that EV numbers increased more than 2-fold in the majority of patients after transplantation. Also, sufficient quantities of RNA for downstream microRNA (miRNA) analysis were obtained from all samples and EV miRNA profiles were found to differ from whole plasma profiles. As a proof of principle, expression of platelet-specific miR-142-3p in EVs was shown to correlate with platelet count kinetics after transplantation as expected. Importantly, we identified plasma EV miRNAs that were consistently positively correlated with infection and GvHD, respectively, as well as miRNAs that were consistently negatively correlated with these complications. Conclusions This study demonstrates that acoustic enrichment of EVs in a clinical biomarker study setting is feasible and that downstream analysis of acoustically-enriched EVs presents a promising tool for biomarker development in allo-HSCT. Certainly, these findings warrant further exploration in larger studies, which will have significant implications not only for biomarker studies in transplantation but also for the broad field of EV-based biomarker discovery.

Mesenchymal cells are important components of specified niches in the lung, and can mediate a wide range of processes including tissue regeneration and repair. Dysregulation of these processes can lead to improper remodeling of tissue as observed in several lung diseases. The mesenchymal cells responsible remain poorly described, partially due to the heterogenic nature of the mesenchymal compartment and the absence of appropriate markers. Here, we describe that CD105+CD90+ mesenchymal cells can be divided into two populations based on their expression of CD13/aminopeptidase N (CD105+CD90+CD13− and CD105+CD90+CD13+). By prospective isolation using FACS, we show that both these populations give rise to clonogenic fibroblast-like cells, but with an increased clonogenic and proliferative capacity of CD105+CD90+CD13+ cells. Transcriptomic and spatial analysis pinpoints an adventitial fibroblast subset as the origin of CD105+CD90+CD13+ clonogenic mesenchymal cells in human lung.

Background Graft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products. Methods Neuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture. Results PDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34 + stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B , TH and DDC genes, while > 85% of CD34 + cells could be retained in the graft. Conclusion These results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.

Background: It is hypothesized that different populations of mesenchymal cells, including mesenchymal stromal cells (MSC), have specific involvement in chronic lung disease, however some populations have a suggested regenerative function. The limited description of this mesenchymal cell heterogeneity and lack of specific markers makes it difficult to understand their endogenous function, further impeding development of therapeutic approaches targeting aberrant mesenchymal cell activity. Aim: To explore the heterogeneity of MSCs in lung tissue and to identify functionally different subpopulations. Methods: Mesenchymal cells were isolated from human donor lungs and healthy regions from lung tumor resections, and characterized using fluorescence-activated cell sorting (FACS) in combination with in vitro assays for colony formation and VEGF production (ELISA). Results: We identified Aminopeptidase N/CD13 as a marker that distinguished two distinct cell populations (CD13neg/low and CD13high) in a MSC enriched population (CD45neg/CD31neg/CD105pos/CD90pos). By FACS we observed that colony-forming MSCs were highly enriched in the CD45neg/CD31neg/EpCAMneg/CD105pos fraction and that they derived from both CD90pos/CD13neg/low and CD90pos/CD13high populations. Interestingly, preliminary data showed that cells from the CD90pos/CD13neg/low population had an increased production of VEGF and lower proliferation compared to cells from the CD90pos/CD13high population. Conclusion: Our data suggests that CD90pos/CD13neg/low and CD90pos/CD13high lung-derived MSC are functionally different. This highlights the need to understand the complexity within the mesenchymal cell populations in lung.