Bone Marrow-derived Progenitor Cells Research Articles

Mechanical loading intensities affect the release of extracellular vesicles from mouse bone marrow-derived hematopoietic progenitor cells and change their osteoclast-modulating effect.

Low-intensity loading maintains or increases bone mass, whereas lack of mechanical loading and high-intensity loading decreases bone mass, possibly via the release of extracellular vesicles by mechanosensitive bone cells. How different loading intensities alter the biological effect of these vesicles is not fully understood. Dynamic fluid shear stress at low intensity (0.7 ± 0.3 Pa, 5 Hz) or high intensity (2.9 ± 0.2 Pa, 1 Hz) was used on mouse hematopoietic progenitor cells for 2 min in the presence or absence of chemical compounds that inhibit release or biogenesis of extracellular vesicles. We used a Receptor activator of nuclear factor kappa-Β ligand-induced osteoclastogenesis assay to evaluate the biological effect of different fractions of extracellular vesicles obtained through centrifugation of medium from hematopoietic stem cells. Osteoclast formation was reduced by microvesicles (10 000× g) obtained after low-intensity loading and induced by exosomes (100 000× g) obtained after high-intensity loading. These osteoclast-modulating effects could be diminished or eliminated by depletion of extracellular vesicles from the conditioned medium, inhibition of general extracellular vesicle release, inhibition of microvesicle biogenesis (low intensity), inhibition of ESCRT-independent exosome biogenesis (high intensity), as well as by inhibition of dynamin-dependent vesicle uptake in osteoclast progenitor cells. Taken together, the intensity of mechanical loading affects the release of extracellular vesicles and change their osteoclast-modulating effect.

The Role of Hematopoietic Stem Cells in Lung Cancer Response to Radiation Therapy

Non-small cell lung carcinoma (NSCLC) comprises approximately 85% of all lung cancer cases in the United States. We demonstrated that after radiation therapy (RT), bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) are recruited to the NSCLC tumor microenvironment (TME). These HSPCs seemed to positively affect tumor regrowth as their numbers directly correlated with regrowth rates. The TME is comprised of tumor tissue, stromal cells, immune cells, bone marrow-derived cells, and extra cellular matrix (ECM) proteins and has emerged as one of the predominant defensive weapons with which cancer can resist various forms of therapeutic intervention. The ability of tumor-associated HSPCs to maintain hematopoietic potency in tumors suggested a prominent role of these cells in tumor physiology, however; what was missing was a mechanistic understanding of: 1) how HSPCs participate in tumor growth post-RT and 2) how HSPCs are maintained within tumors. To answer these questions, we established NSCLC tumors in mice using Lewis Lung Carcinoma (LLC) cells and assessed tumor-associated HSPCs activity and molecular changes in the TME following RT. We observed that RT induces the TME to produce colony stimulating factor (CSF)-1, a factor that promotes the differentiation of HSPCs into tumor supportive M2-macrophages. Adoptive transplantation of dsRed labeled HSPCs into tumor bearing mice followed by RT directly showed the differentiation of HSPCs into M2-macrophages and a concomitant increase in the regrowth of tumors post-RT. Use of a therapeutic inhibitor of the CSF-1 receptor (GW2580) prevented HSPC differentiation in to M2-macrophages and significantly decreasing regrowth rates. We next assessed how tumor-associated HSPCs maintain their hematopoietic potency within the TME. Analysis of tumors demonstrated high expression levels of the laminin 5-1-1 isoform, which is known to bind CD49f (integrin alpha 6), a marker of functional HSPCs. Tumors exposed to RT demonstrated significantly reduced expression of both laminin 5-1-1 and CD49f. When bound, CD49f inhibits phosphorylation of focal adhesion kinase (FAK) resulting in maintenance of pluripotent stem cells. Analysis of tumor-associated HSPCs demonstrated significantly higher levels of phosphorylated FAK following tumor exposure to RT. Since RT induces HSPC differentiation in to M2-macophages, this observation suggests that tumor-associated HSPC functionality is maintained through the interaction between laminin 5-1-1- and CD49f. The identification of single HSPCs that differentiated in to M2-macrophages and which expressed phosphorylated FAK supports this conclusion. Taken together, this data province's evidence of a mechanism wherein RT disrupts laminin 5-1-1/CD49f interactions releasing tumor-associated HSPCs from maintenance signals. Concomitantly, RT induces the production of CSF-1 within the TME, which induces these “released” HSPCs to differentiate into tumor supportive M2-macrophages and promote tumor regrowth post-RT. Overall, these studies define a new understanding of tumor biology in which HSPCs are significant mediators of tumor response to RT. These findings also identify new cellular and molecular pathways to target in the treatment of NSCLC to achieve more optimal clinical outcomes.

Interleukin-17A Directly Signals to Bone Marrow-Derived Hematopoietic Stem and Progenitor Cells to Promote Their Expansion and Differentiation in Vitro

Rationale: IL-17 is a family of six pro-inflammatory cytokines (named from A to F) with distinct patterns of cellular expression. Among these, IL-17A is secreted by lymphoid subsets such as T-helper 17 cells and innate lymphoid cells type 3. Through its action on many tissues, IL-17A is recognized as a key mediator of response to infection and pathogenic states such as cancer and auto-immunity. Until now, the main effect of IL-17A on hematopoiesis has been attributed to an indirect loop through IL-17RA signaling on bone marrow stromal cells, which stimulates secretion of hematopoietic factors such as G-CSF. However, we hypothesized that IL-17A can directly signal to hematopoietic stem and progenitor cells (HSPCs) to determine their self-renewal and differentiation. Methodology: We analyzed the expression of the IL-17 signaling pathway in publicly available human and mouse hematopoietic single-cell RNAseq datasets. Mouse HSPCs were isolated using FACS to perform clonogenic and differentiation assays in Methocult (StemCell Technologies) and StemPro-34 (Gibco). Spectral flow cytometry (SONY ID7000) was used for analysing IL-17RA expression and differentiation of mouse HSPCs. IL-17A levels were measured in blood and bone marrow serum using ELISA (Invitrogen). Results: IL-17RA is expressed at the surface of mouse HSPCs, with greatest levels in common lymphoid progenitors and granulocyte-monocyte progenitors. mRNA expression of IL-17RA signaling pathway is also detectable in human HSPCs, with greatest levels in young individuals. IL-17A supplementation leads to an increase in clonogenic capacity of mouse HSPCs in methylcellulose colony-formation assays. In liquid culture, supplementation with IL-17A leads to a greater expansion of sorted Lin -/s-kit +/Sca-1 + progenitors, accompanied by an increased proportion of myeloid-committed cells.IL-17A is also detected in the bone marrow serum, with possible production from subsets of cells expressing RORγt, a master regulator in development of T helper 17 (Th17) cells. Conclusions: Our work suggests the ability of IL-17A to directly promote clonogenic potential of HSPCs and drive a myeloid-biased lineage commitment in progenitor subsets. Additional studies are required to characterize the response of HSPCs to direct IL-17A signaling in vivo. Considering the important role of IL-17A as a mediator of immune response, and tissue repair, further studies are warranted to elucidate whether modulation of this pathway may be therapeutic in the context of hematopoietic failure or malignancy.

Effective Prognostic Model for Therapy Response Prediction in Acute Myeloid Leukemia Patients.

Acute myeloid leukemia (AML) is a hematopoietic disorder characterized by the malignant transformation of bone marrow-derived myeloid progenitor cells with extremely short survival. To select the optimal treatment options and predict the response to therapy, the stratification of AML patients into risk groups based on genetic factors along with clinical characteristics is carried out. Despite this thorough approach, the therapy response and disease outcome for a particular patient with AML depends on several patient- and tumor-associated factors. Among these, tumor cell resistance to chemotherapeutic agents represents one of the main obstacles for improving survival outcomes in AML patients. In our study, a new prognostic scale for the risk stratification of AML patients based on the detection of the sensitivity or resistance of tumor cells to chemotherapeutic drugs in vitro as well as MDR1 mRNA/P-glycoprotein expression, tumor origin (primary or secondary), cytogenetic abnormalities, and aberrant immunophenotype was developed. This study included 53 patients diagnosed with AML. Patients who received intensive or non-intensive induction therapy were analyzed separately. Using correlation, ROC, and Cox regression analyses, we show that the risk stratification of AML patients in accordance with the developed prognostic scale correlates well with the response to therapy and represents an independent predictive factor for the overall survival of patients with newly diagnosed AML.

Abstract 5136: Surgical stress promotes long-lasting protumorigenic changes in bone marrow derived progenitor cells

Abstract Introduction: Surgery is a crucial intervention for cancer patients. However, the perioperative period is characterized by an increased risk for accelerated growth of metastatic disease. Our recent work shows that the protumorigenic systemic inflammatory changes and the formation of neutrophil extracellular traps (NETs) persist months after surgery in both mice and humans, and parallel micrometastatic tumor growth and recurrence. Here we hypothesized that surgery may induce long-term sustained epigenetic and transcriptomic changes in bone marrow derived granulocytes-monocytes progenitor cells (GMPs) leading to functional reprogramming of mature neutrophils and persistent systemic release of these protumorigenic NET-forming neutrophils. Methods: Eight-week-old mice were subjected to non-lethal surgical stress (laparotomy) for 30 mins. Seven days after, mice were inoculated with 1x106 MC38 (murine colorectal cancer cells) subcutaneously. For bone marrow (BM) transplant model, a group of B6 congenic (CD45.1) mice were subjected to laparotomy. BM derived cells were harvested 7 days after the procedure and orthotopically transplanted into B6 CD45.2 irradiated (1000 rads) mice. After the establishment of hematopoiesis (6 weeks), MC38 cancer cells were implanted into the recipient mice. Results: Mice that underwent laparotomy had significantly increased tumor volume at 3 weeks compared to control mice, that only underwent anesthesia (Laparotomy vs control **p<0.01). While the intratumoral flowcytometry analysis showed no difference in the frequency of infiltrating immune cells, quantitative (q) PCR analysis revealed significant plasticity in the tumor infiltrating neutrophils (TANs) observed by an increase in the expression of genes (MCP1, Arg1) associated with pro-inflammatory (N2) type neutrophils compared to control. A persistent increased level of NETs was also observed within the tumors and circulation of mice several weeks after laparotomy. ATAC-seq and Bulk-RNA-seq analysis further revealed major epigenetic and transcriptomic changes in TANs, circulating neutrophils and GMPs. To substantiate long-lasting surgical effects, tumor burden was assessed in the BM recipient mice and showed to be significantly increased in mice that had received BM cells from laparotomy mice versus BM cells from sham group. Conclusion: Surgery promotes long-term rewiring of the bone marrow derived cells resulting in increased tumor growth. Understanding the underlying molecular mechanism may help with therapeutic interventions to prevent protumorigenic surgical effects and improve patient outcomes. Citation Format: Hamza O. Yazdani, Tony Haykal, Ruiqi Yang, Celine Tohme, David A. Geller, Samer Tohme. Surgical stress promotes long-lasting protumorigenic changes in bone marrow derived progenitor cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5136.

CD34+ progenitor cells as diagnostic and therapeutic targets in Alzheimer's disease.

CD34+ progenitor cells as diagnostic and therapeutic targets in Alzheimer's disease.

Dysregulated transforming growth factor-beta mediates early bone marrow dysfunction in diabetes

Diabetes affects select organs such as the eyes, kidney, heart, and brain. Our recent studies show that diabetes also enhances adipogenesis in the bone marrow and reduces the number of marrow-resident vascular regenerative stem cells. In the current study, we have performed a detailed spatio-temporal examination to identify the early changes that are induced by diabetes in the bone marrow. Here we show that short-term diabetes causes structural and molecular changes in the marrow, including enhanced adipogenesis in tibiae of mice, prior to stem cell depletion. This enhanced adipogenesis was associated with suppressed transforming growth factor-beta (TGFB) signaling. Using human bone marrow-derived mesenchymal progenitor cells, we show that TGFB pathway suppresses adipogenic differentiation through TGFB-activated kinase 1 (TAK1). These findings may inform the development of novel therapeutic targets for patients with diabetes to restore regenerative stem cell function.

Stable human cartilage progenitor cell line stimulates healing of meniscal tears and attenuates post-traumatic osteoarthritis.

Meniscal tearing in the knee increases the risk of post-traumatic osteoarthritis (OA) in patients. The therapeutic application of tissue-specific mesenchymal progenitor cells is currently being investigated as an emerging biologic strategy to help improve healing of musculoskeletal tissues like meniscal fibrocartilage and articular hyaline cartilage. However, many of these approaches involve isolating cells from healthy tissues, and the low yield of rare progenitor populations (< 1% of total cells residing in tissues) can make finding a readily available cell source for therapeutic use a significant logistical challenge. In the present study, we investigated the therapeutic efficacy of using expanded cartilage-derived and bone marrow-derived progenitor cell lines, which were stabilized using retroviral SV40, for repair of meniscus injury in a rodent model. Our findings indicate that these cell lines express the same cell surface marker phenotype of primary cells (CD54+, CD90+, CD105+, CD166+), and that they exhibit improved proliferative capacity that is suitable for extensive expansion. Skeletally mature male athymic rats treated with 3.2 million cartilage-derived progenitor cell line exhibited approximately 79% greater meniscal tear reintegration/healing, compared to injured animals that left untreated, and 76% greater compared to animals treated with the same number of marrow-derived stromal cells. Histological analysis of articular surfaces also showed that cartilage-derived progenitor cell line treated animals exhibited reduced post-traumatic OA associated articular cartilage degeneration. Stable cell line treatment did not cause tumor formation or off-target engraftment in animals. Taken together, we present a proof-of-concept study demonstrating, for the first time, that intra-articular injection of a stable human cartilage-derived progenitor cell line stimulates meniscus tear healing and provide chondroprotection in an animal model. These outcomes suggest that the use of stable cell lines may help overcome cell source limitations for cell-based medicine.

Post-transcriptional control of T-cell development in the thymus

T-cell development in the thymus is dependent on the continual colonization by bone-marrow derived progenitor cells. Once inside the thymus, progenitors undergo a series of well-defined differentiation events, including lineage commitment, somatic recombination of T-cell receptor (TCR) gene loci, and selection of clones with productively recombined yet non-autoreactive TCRs. Cell-cell interactions, cytokine signals, transcriptional as well as epigenetic programs controlling T-cell development are comparatively well-characterized. In contrast, the contribution of post-transcriptional control and its underlying mechanisms remain largely elusive. Here, we summarize recent advances in our understanding of post-transcriptional regulation of T-cell development, focussing on microRNAs (miRNAs) and RNA-binding proteins (RBPs). We highlight the current challenges, and how they can potentially be overcome with evolving sophisticated methodology to enable a thorough mechanistic understanding and decipher the regulatory networks operating in the gene expression programs of T-cell development.

Bone Marrow-Derived Stem Cell Factor Regulates Prostate Cancer-Induced Shifts in Pre-Metastatic Niche Composition.

Skeletal metastasis is the leading cause of morbidity and mortality in prostate cancer, with 80% of advanced prostate cancer patients developing bone metastases. Before metastasis, bone remodeling occurs, stimulating pre-metastatic niche formation and bone turnover, and platelets govern this process. Stem cell factor (SCF, Kit Ligand) is increased in advanced prostate cancer patient platelet releasates. Further, SCF and its receptor, CD117/c-kit, correlate with metastatic prostate cancer severity. We hypothesized that bone-derived SCF plays an important role in prostate cancer tumor communication with the bone inducing pre-metastatic niche formation. We generated two cell-specific SCF knockout mouse models deleting SCF in either mature osteoblasts or megakaryocytes and platelets. Using two syngeneic androgen-insensitive murine prostate cancer cell lines, RM1 (Ras and Myc co-activation) and mPC3 (Pten and Trp53 deletion), we examined the role of bone marrow-derived SCF in primary tumor growth and bone microenvironment alterations. Platelet-derived SCF was required for mPC3, but not RM1, tumor growth, while osteoblast-derived SCF played no role in tumor size in either cell line. While exogenous SCF induced proangiogenic protein secretion by RM1 and mPC3 prostate cancer cells, no significant changes in tumor angiogenesis were measured by immunohistochemistry. Like our previous studies, tumor-induced bone formation occurred in mice bearing RM1 or mPC3 neoplasms, demonstrated by bone histomorphometry. RM1 tumor-bearing osteoblast SCF knockout mice did not display tumor-induced bone formation. Bone stromal cell composition analysis by flow cytometry showed significant shifts in hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and osteoblast cell percentages in mice bearing RM1 or mPC3 tumors. There were no significant changes in the percentage of macrophages, osteoclasts, or osteocytes. Our study demonstrates that megakaryocyte/platelet-derived SCF regulates primary mPC3 tumor growth, while SCF originating from osteoblasts plays a role in bone marrow-derived progenitor cell composition and pre-metastatic niche formation. Further, we show that both the source of SCF and the genetic profile of prostate cancer determine the effects of SCF. Thus, targeting the SCF/CD117 signaling axis with tyrosine kinase inhibitors could affect primary prostate carcinomas or play a role in reducing bone metastasis dependent on the gene deletions or mutations driving the patients’ prostate cancer.

Chemokine C-X-C receptor 4 mediates recruitment of bone marrow-derived nonhematopoietic and immune cells to the pregnant uterus†.

Bone marrow-derived progenitor cells (BMDPCs) are mobilized to the circulation in pregnancy and get recruited to the pregnant decidua where they contribute functionally to decidualization and successful implantation. However, the molecular mechanisms underlying BMDPCs recruitment to the decidua are unknown. CXCL12 ligand and its CXCR4 receptor play crucial roles in the mobilization and homing of stem/progenitor cells to various tissues. To investigate the role of CXCL12-CXCR4 axis in BMDPCs recruitment to decidua, we created transgenic GFP mice harboring CXCR4 gene susceptible to tamoxifen-inducible Cre-mediated ablation. These mice served as BM donors into wild-type C57BL/6J female recipients using a 5-fluorouracil-based nongonadotoxic submyeloablation to achieve BM-specific CXCR4 knockout (CXCR4KO). Successful CXCR4 ablation was confirmed by RT-PCR and in vitro cell migration assays. Flow cytometry and immunohistochemistry showed a significant increase in GFP+ BM-derived cells (BMDCs) in the implantation site as compared to the nonpregnant uterus of control (2.7-fold) and CXCR4KO (1.8-fold) mice. This increase was uterus-specific and was not observed in other organs. This pregnancy-induced increase occurred in both hematopoietic (CD45+) and nonhematopoietic (CD45-) uterine BMDCs in control mice. In contrast, in CXCR4KO mice there was no increase in nonhematopoietic BMDCs in the pregnant uterus. Moreover, decidual recruitment of myeloid cells but not NK cells was diminished by BM CXCR4 deletion. Immunofluorescence showed the presence of nonhematopoietic GFP+ cells that were negative for CD45 (panleukocyte) and DBA (NK) markers in control but not CXCR4KO decidua. In conclusion, we report that CXCR4 expression in nonhematopoietic BMDPCs is essential for their recruitment to the pregnant decidua.

Beneficial Role of Vitamin D on Endothelial Progenitor Cells (EPCs) in Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the leading cause of death in the world. Endothelial progenitor cells (EPCs) are currently being explored in the context of CVD risk. EPCs are bone marrow derived progenitor cells involved in postnatal endothelial repair and neovascularization. A large body of evidence from clinical, animal, and in vitro studies have shown that EPC numbers in circulation and their functionality reflect endogenous vascular regenerative capacity. Traditionally vitamin D is known to be beneficial for bone health and calcium metabolism and in the last two decades, its role in influencing CVD and cancer risk has generated significant interest. Observational studies have shown that low vitamin D levels are associated with an adverse cardiovascular risk profile. Still, Mendelian randomization studies and randomized control trials (RCTs) have not shown significant effects of vitamin D on cardiovascular events. The criticism regarding the RCTs on vitamin D and CVD is that they were not designed to investigate cardiovascular outcomes in vitamin D-deficient individuals. Overall, the association between vitamin D and CVD remains inconclusive. Recent clinical and experimental studies have demonstrated the beneficial role of vitamin D in increasing the circulatory level of EPC as well as their functionality. In this review we present evidence supporting the beneficial role of vitamin D in CVD through its modulation of EPC homeostasis.

Rosiglitazone induces adipogenesis of both marrow and periosteum derived mesenchymal stem cells during endochondral fracture healing

BackgroundType 2 diabetes mellitus (T2DM) afflicts about six percent of the global population, and these patients suffer from a two-fold increased fracture risk. Thiazolidinediones (TZDs), including rosiglitazone, are commonly used medications in T2DM because they have a low incidence of monotherapy failure. It is known that rosiglitazone is associated with secondary osteoporosis, further increasing the fracture risk in an already susceptible population. However, it is not yet understood how rosiglitazone impacts endochondral bone healing after fracture. The aim of this study is to elucidate how rosiglitazone treatment impacts endochondral fracture healing, and how rosiglitazone influences the differentiation of skeletal stem and progenitor cells from the bone marrow and the periosteum. MethodsAn in-vivo mouse femur fracture model was employed to evaluate differences in fracture healing between mice treated with and without rosiglitazone chow. Fracture healing was assessed with histology and micro computed tomography (μCT). In-vitro assays utilized isolated mouse bone marrow stromal cells and periosteal cells to investigate how rosiglitazone impacts the osteogenic capability and adipogenicity of these cells. ResultsThe in-vivo mouse femur fracture model showed that fracture callus in mice treated with rosiglitazone had significantly more adipose content than those of control mice that did not receive rosiglitazone. In addition, μCT analysis showed that rosiglitazone treated mice had significantly greater bone volume, but overall greater porosity when compared to control mice. In-vitro experimentation showed significantly less osteogenesis and more adipogenesis in bone marrow derived progenitor cells that were cultured in osteogenic media. In addition, rosiglitazone treatment alone caused significant increases in adipogenesis in both bone marrow and periosteum derived cells. ConclusionRosiglitazone impairs endochondral fracture healing in mice by increasing adipogenesis and decreasing osteogenesis of both bone marrow and periosteum derived skeletal progenitor cells.

Circulating fibrocyte levels correlate with infarct size in patients with ST elevation myocardial infarction treated with primary percutaneous coronary intervention

Study objectiveInfarct size is a strong predictor of outcomes after ST elevation myocardial infarction (STEMI). Circulating fibrocytes are bone marrow-derived progenitor cells associated with fibrotic processes. We tested whether fibrocytes correlate with infarct size in STEMI patients treated with primary percutaneous coronary intervention (PCI). DesignProspective observational study. SettingAcademic medical center. ParticipantsSubjects with STEMI treated with primary PCI. InterventionsPeripheral blood draw and cardiac magnetic resonance imaging (CMR). Main outcome measureCorrelation of fibrocyte levels with infarct size. MethodsPeripheral blood fibrocytes were quantified at discharge from STEMI hospitalization and at 6 months follow-up using flow cytometry. Infarct size was determined within 2 weeks of discharge and at 6 months follow-up using late gadolinium enhancement on CMR. ResultsAmong 14 patients (median age 54 years, 79% men) with STEMI, there was a statistically significant positive correlation between fibrocyte levels at 6 months and 6-month infarct size on CMR (r = 0.58, p = 0.031). In addition, there was positive correlation between peak troponin I level (r = 0.85, p < 0.001), and white blood cell count (r = 0.55, p = 0.042) during the hospital stay and 6-month infarct size on CMR. ConclusionsCirculating fibrocytes measured 6 months after STEMI positively correlate with 6-month infarct size assessed by CMR.

Systemic cytology. A novel diagnostic approach for assessment of early systemic disease

Recognition of low grade or asymptomatic systemic diseases suggests prevention of the worst, yet has been proven challenging ever since. Biomarker-based liquid biopsy has emerged in recent years as a practical platform for the assessment of systemic diseases yet, technical realizations were mainly focused on cancer, faced challenges in accuracy at early stage and are lacking provision of sufficient evidence of disease. In particular in cell-based cancer liquid biopsy, obstacles are rarity and heterogeneity of circulating tumor and tumor-associated rare cells. Evidence is mounting about an entire spectrum of distinct circulating rare cell types that denotes the systemic component of a certain physiological state. Therefore, circulating rare cells in combination may arise from yet, also account for systemic diseases, which we denote as multi-rare cell association and involves foremost bone marrow-derived progenitor and stem cells yet, also matured somatic cell types. One would expect immense diagnostic value in the read-out of the so called rare cell population which represents cytological evidence of abnormality. We hypothesize that comprehensive rare cell population profiling as contrasted to the biomarker screening approach may realize the premise of a biopsy as to confirm, characterize, grade, stage or predict a systemic disease. This novel approach represents the “missing link” in diagnostic care of in particular early or residual systemic disease and presumes a steady gain in knowledge about the clinical interpretation of rare cell population profiles thus, expecting the knowledge-driven transformation of cell-based liquid biopsy from suggestion to confirmation. We support our hypothesis by past findings made by others and us and provide insights how to interpret a certain rare cell population profile.

Dysregulated zinc and sphingosine-1-phosphate signaling in pulmonary hypertension: Potential effects by targeting of bone morphogenetic protein receptor type 2 in pulmonary microvessels.

Recently identified molecular targets in pulmonary artery hypertension (PAH) include sphingosine-1-phosphate (S1P) and zinc transporter ZIP12 signaling. This study sought to determine linkages between these pathways, and with BMPR2 signaling. Lung tissues from a rat model of monocrotaline-induced PAH and therapeutic treatment with bone marrow-derived endothelial-like progenitor cells transduced to overexpress BMPR2 were studied. Multifluorescence quantitative confocal microscopy (MQCM) was applied for analysis of protein expression and localization of markers of vascular remodeling (αSMA and BMPR2), parameters of zinc homeostasis (zinc transporter SLC39A/ZIP family members 1, 10, 12 and 14; and metallothionein MT3) and S1P extracellular signaling (SPHK1, SPNS2, S1P receptor isoforms 1, 2, 3, 5) in 20-200 µm pulmonary microvessels. ZIP12 expression in whole lung tissue lysates was assessed by western blot. Spearman nonparametric correlations between MQCM readouts and hemodynamic parameters, Fulton index (FI), and right ventricular systolic pressure (RVSP) were measured. In line with PAH status, pulmonary microvessels in monocrotaline-treated animals demonstrated significant (p < .05, n = 6 per group) upregulation of αSMA (twofold) and downregulation of BMPR2 (20%). Upregulated ZIP12 (92%), MT3 (57.7%), S1PR2 (54.8%), and S1PR3 (30.3%) were also observed. Significant positive and negative correlations were demonstrated between parameters of zinc homeostasis (ZIP12, MT3), S1P signaling (S1PRs, SPNS2), and vascular remodeling (αSMA, FI, RVSP). MQCM and western blot analysis showed that monocrotaline-induced ZIP12 upregulation could be partially negated by BMPR2-targeted therapy. Our results indicate that altered zinc transport/storage and S1P signaling in the monocrotaline-induced PAH rat model are linked to each other, and could be alleviated by BMPR2-targeted therapy.

Bone marrow-derived progenitor cells contribute to remodeling of the postpartum uterus.

Endometrial stem/progenitor cells play a role in postpartum uterine tissue regeneration, but the underlying mechanisms are poorly understood. While circulating bone marrow (BM)-derived cells (BMDCs) contribute to nonhematopoietic endometrial cells, the contribution of BMDCs to postpartum uterus remodeling is unknown. We investigated the contribution of BMDCs to the postpartum uterus using 5-fluorouracil-based nongonadotoxic BM transplant from green fluorescent protein (GFP) donors into wild-type C57BL/6J female mice. Flow cytometry showed an influx of GFP+ cells to the uterus immediately postpartum accounting for 28.7% of total uterine cells, followed by a rapid decrease to prepregnancy levels. The majority of uterine GFP+ cells were CD45+ leukocytes, and the proportion of nonhematopoietic CD45-GFP+ cells peaked on postpartum day (PPD) 1 (17.5%). Immunofluorescence colocalization of GFP with CD45 pan-leukocyte and F4/80 macrophage markers corroborated these findings. GFP+ cells were found mostly in subepithelial stromal location. Importantly, GFP+ cytokeratin-positive epithelial cells were found within the luminal epithelium exclusively on PPD1, demonstrating direct contribution to postpartum re-epithelialization. A subset (3.2%) of GFP+ cells were CD31+CD45- endothelial cells, and found integrated within blood vessel endothelium. Notably, BM-derived GFP+ cells demonstrated preferential proliferation (PCNA+) and apoptosis (TUNEL+) on PPD1 vs resident GFP- cells, suggesting an active role for BMDCs in rapid tissue turnover. Moreover, GFP+ cells gradually acquired cell senescence together with decreased proliferation throughout the postpartum. In conclusion, BM-derived progenitors were found to have a novel nonhematopoietic cellular contribution to postpartum uterus remodeling. This contribution may have an important functional role in physiological as well as pathological postpartum endometrial regeneration.

The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Chromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

Tumor Suppressor, NORE1A (RASSF5), Is an Icsbp-Dependent Inhibitor of Aberrant Stress Granulopoiesis

Within the innate immune system, emergency (stress) granulopoiesis serves the specific and essential function of periodic granulocyte production in response to an infectious or inflammatory challenge. Emergency granulopoiesis occurs in four phases (immediate release of mature granulocytes from the bone marrow, expansion of the common granulocyte/monocyte progenitor pool, accelerated differentiation, and termination of the response) and requires IL1β, an IL1β-dependent increase in G-CSF and the transcription factors Stat3 and C/EPBβ. Our group has previously shown that interferon consensus sequence binding protein (Icsbp) is required for the termination of emergency granulopoiesis. Icsbp deficiency is associated with increased and sustained production of Fap1 and Gas2, aberrantly sustained granulocytosis and promotion of myeloid leukemia. We have identified a novel Icsbp target gene, NORE1A (RASSF5), that is likewise required for the termination of emergency granulopoiesis. Nore1A is a tumor suppressor that is down-regulated in many human cancers. We found that Icsbp acts as a NORE1A transcriptional activator and that Nore1A expression is decreased in bone marrow-derived myeloid progenitor cells from ICSBP -/- mice compared to wild-type (WT), and does not appropriately increase during ex vivo differentiation with G-CSF or IL1β. In cell culture, ICSBP -/- murine bone marrow cells were resistant to intrinsic or Fas-induced apoptosis compared to WT. However, transduction with a Nore1A expression vector rescued Fas-induced apoptosis in ICSBP -/- murine bone marrow-derived myeloid progenitor cells, associated with activation of the pro-apoptotic Mst1 kinase. We injected WT or NORE1 -/- mice intraperitoneally every 4 weeks with an ovalbumin/alum mixture (to stimulate emergency granulopoiesis) or saline control. NORE1 -/- mice demonstrated aberrantly exaggerated granulocytosis in response to repeated cycles of ovalbumin/alum stimulation compared to WT mice. NORE1 -/- mice also demonstrated a tendency to develop myeloid leukemia, even in the absence of overt immune stimulation. These findings suggest that Nore1A inhibits aberrant stress granulopoiesis by promoting apoptosis of myeloid cells in an Icsbp-dependent fashion. Our data implicate Nore1A deficiency in the deregulation of granulopoiesis and promotion of leukemogenesis that accompanies clinical scenarios associated with decreased Icsbp activity, such as human chronic myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Eosinophil Lineage-Committed Progenitors as a Therapeutic Target for Asthma.

Eosinophilic asthma is the most prevalent phenotype of asthma. Although most asthmatics are adequately controlled by corticosteroid therapy, a subset (5–10%) remain uncontrolled with significant therapy-related side effects. This indicates the need for a consideration of alternative treatment strategies that target airway eosinophilia with corticosteroid-sparing benefits. A growing body of evidence shows that a balance between systemic differentiation and local tissue eosinophilopoietic processes driven by traffic and lung homing of bone marrow-derived hemopoietic progenitor cells (HPCs) are important components for the development of airway eosinophilia in asthma. Interleukin (IL)-5 is considered a critical and selective driver of terminal differentiation of eosinophils. Studies targeting IL-5 or IL-5R show that although mature and immature eosinophils are decreased within the airways, there is incomplete ablation, particularly within the bronchial tissue. Eotaxin is a chemoattractant for mature eosinophils and eosinophil-lineage committed progenitor cells (EoP), yet anti-CCR3 studies did not yield meaningful clinical outcomes. Recent studies highlight the role of epithelial cell-derived alarmin cytokines, IL-33 and TSLP, (Thymic stromal lymphopoietin) in progenitor cell traffic and local differentiative processes. This review provides an overview of the role of EoP in asthma and discusses findings from clinical trials with various therapeutic targets. We will show that targeting single mediators downstream of the inflammatory cascade may not fully attenuate tissue eosinophilia due to the multiplicity of factors that can promote tissue eosinophilia. Blocking lung homing and local eosinophilopoiesis through mediators upstream of this cascade may yield greater improvement in clinical outcomes.