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.

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.

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.

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.

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.

CD105+CD90+CD13+ identifies a clonogenic subset of adventitial lung fibroblasts.

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.

Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis.

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.

Development of acoustically isolated extracellular plasma vesicles for biomarker discovery in allogeneic hematopoietic stem cell transplantation.

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.

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.

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.

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.

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.

Label-free neuroblastoma cell separation from hematopoietic progenitor cell products using acoustophoresis - towards cell processing of complex biological samples.

Processing of complex cell preparations such as blood and peripheral blood progenitor cell (PBPC) transplants using label-free technologies is challenging. Transplant-contaminating neuroblastoma cells (NBCs) can contribute to relapse, and we therefore aimed to provide proof-of-principle evidence that label-free acoustophoretic separation can be applied for diagnostic NBC enrichment and removal ("purging") from human blood and PBPC products. Neuroblastoma cells spiked into blood and PBPC preparations served as model systems. Acoustophoresis enabled to enrich NBCs from mononuclear peripheral blood cells and PBPC samples with recovery rates of up to 60-97%. When aiming at high purity, NBC purities of up to 90% were realized, however, compromising recovery. Acoustophoretic purging of PBPC products allowed substantial tumour cell depletion of 1.5-2.3 log. PBPC loss under these conditions was considerable (>43%) but could be decreased to less than 10% while still achieving NBC depletion rates of 60-80%. Proliferation of cells was not affected by acoustic separation. These results provide first evidence that NBCs can be acoustically separated from blood and stem cell preparations with high recovery and purity, thus indicating that acoustophoresis is a promising technology for the development of future label-free, non-contact cell processing of complex cell products.

Label-free separation of leukocyte subpopulations using high throughput multiplex acoustophoresis.

Multiplex separation of mixed cell samples is required in a variety of clinical and research applications. Herein, we present an acoustic microchip with multiple outlets and integrated pre-alignment channel to enable high performance and label-free separation of three different cell or particle fractions simultaneously at high sample throughput. By implementing a new cooling system for rigorous temperature control and minimal acoustic energy losses, we were able to operate the system isothermally and sort suspensions of 3, 5 and 7 µm beads with high efficiencies (>95.4%) and purities (>96.3%) at flow rates up to 500 µL min-1 corresponding to a throughput of ∼2.5 × 106 beads per min. Also, human viable white blood cells were successfully fractionated into lymphocytes, monocytes and granulocytes with high purities of 96.5 ± 1.6%, 71.8 ± 10.1% and 98.8 ± 0.5%, respectively, as well as high efficiencies (96.8 ± 3.3%, 66.7 ± 3.2% and 99.0 ± 0.7%) at flow rates up to 100 µL min-1 (∼100 000 cells per min). By increasing the flow rate up to 300 µL min-1 (∼300 000 cells per min) both lymphocytes and granulocytes were still recovered with high purities (92.8 ± 1.9%, 98.2 ± 1 .0%), whereas the monocyte purity decreased to 20.9 ± 10.3%. The proposed isothermal multiplex acoustophoresis platform offers efficient fractionation of complex samples in a label-free and continuous manner at thus far unreached high sample throughput rates.

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.

Acoustofluidic hematocrit determination.

Hematocrit (HCT) measurements of blood from patients, blood donors and athletes are routinely performed on a daily basis. These measurements are often performed in centralized hospital labs by whole blood analyzers, which leads to long time-to-result. On site measurements, based on centrifugation can be done, but these assays require manual handling, are slow and can just measure HCT in contrast to the central lab whole blood analyzers. In this work, we present a microfluidic based method to measure HCT in blood samples by acoustic separation of whole blood into discrete regions of plasma and red blood cells. Comparison of the areas of the red blood cell and plasma regions gives an accurate HCT value, with a linear correlation to the centrifugation-based reference method. A readout can be performed within 2 s of acoustic actuation providing a readout accuracy of approximately 3% points (pp) HCT. Additional accuracy can be achieved by extending the acoustic actuation to 20 s, yielding an error of less than 1 pp HCT. This acoustic tool is optimal for integration into a lab-on-a-chip device with in-line measurements of different clinical parameters.

Rapid and effective enrichment of mononuclear cells from blood using acoustophoresis.

Effective separation methods for fractionating blood components are needed for numerous diagnostic and research applications. This paper presents the use of acoustophoresis, an ultrasound based microfluidic separation technology, for label-free, gentle and continuous separation of mononuclear cells (MNCs) from diluted whole blood. Red blood cells (RBCs) and MNCs behave similar in an acoustic standing wave field, compromising acoustic separation of MNC from RBC in standard buffer systems. However, by optimizing the buffer conditions and thereby changing the acoustophoretic mobility of the cells, we were able to enrich MNCs relative to RBCs by a factor of 2,800 with MNC recoveries up to 88%. The acoustophoretic microchip can perform cell separation at a processing rate of more than 1 × 105 cells/s, corresponding to 5 µl/min undiluted whole blood equivalent. Thus, acoustophoresis can be easily integrated with further down-stream applications such as flow cytometry, making it a superior alternative to existing MNC isolation techniques.

Human Primary Bone Marrow Mesenchymal Stromal Cells and Their in vitro Progenies Display Distinct Transcriptional Profile Signatures.

Bone marrow mesenchymal stromal cells (BM-MSCs) are a rare population of cells that gives rise to skeletal tissues and the hematopoietic stroma in vivo. Recently, we have demonstrated that BM-MSCs fulfill stringent in vivo stem cell criteria when propagated as non-adherent mesenspheres but not as adherent-cultured cells. Motivated by these profound functional differences, the current study aimed to identify potential important MSC regulators by investigating global gene expression profiles of adherent and non-adherent culture-derived BM-MSCs in comparison with primary BM-MSCs. A substantial number of genes were differentially expressed between primary and culture-expanded cells already early upon culture, and numerous genes were found to be different when comparing adherent and non-adherent BM-MSCs. Cluster analysis identified 16 sets of genes of which two displayed comparable gene expression levels in primary and non-adherent cultured cells, but not in adherent cultured cells. This pattern suggested that these clusters contained candidate regulators of BM-MSCs. Gene expression differences were confirmed for selected genes and BM-MSC transcription factors by protein analysis and RT-PCR, respectively. Taken together, these data demonstrated profound gene expression changes upon culture of primary BM-MSCs. Moreover, gene cluster differences provide the basis to uncover the regulatory mechanisms that control primary and cultured BM-MSCs.

Quantitative proteomic characterization of lung-MSC and bone marrow-MSC using DIA-mass spectrometry.

Mesenchymal stromal cells (MSC) are ideal candidates for cell therapies, due to their immune-regulatory and regenerative properties. We have previously reported that lung-derived MSC are tissue-resident cells with lung-specific properties compared to bone marrow-derived MSC. Assessing relevant molecular differences between lung-MSC and bone marrow-MSC is important, given that such differences may impact their behavior and potential therapeutic use. Here, we present an in-depth mass spectrometry (MS) based strategy to investigate the proteomes of lung-MSC and bone marrow-MSC. The MS-strategy relies on label free quantitative data-independent acquisition (DIA) analysis and targeted data analysis using a MSC specific spectral library. We identified several significantly differentially expressed proteins between lung-MSC and bone marrow-MSC within the cell layer (352 proteins) and in the conditioned medium (49 proteins). Bioinformatics analysis revealed differences in regulation of cell proliferation, which was functionally confirmed by decreasing proliferation rate through Cytochrome P450 stimulation. Our study reveals important differences within proteome and matrisome profiles between lung- and bone marrow-derived MSC that may influence their behavior and affect the clinical outcome when used for cell-therapy.