Ames hypopituitary dwarf mice demonstrate imbalanced myelopoiesis between bone marrow and spleen

Ames hypopituitary dwarf mice are deficient in growth hormone, thyroid-stimulating hormone, and prolactin. The phenotype of these mice demonstrates irregularities in the immune system with skewing of the normal cytokine milieu towards a more anti-inflammatory environment. However, the hematopoietic stem and progenitor cell composition of the bone marrow (BM) and spleen in Ames dwarf mice has not been well characterized. We found that there was a significant decrease in overall cell count when comparing the BM and spleen of 4-5 month old dwarf mice to their littermate controls. Upon adjusting counts to differences in body weight between the dwarf and control mice, the number of granulocyte-macrophage progenitors, confirmed by immunophenotyping and colony-formation assay was increased in the BM. In contrast, the numbers of all myeloid progenitor populations in the spleen were greatly reduced, as confirmed by colony-formation assays. This suggests that there is a shift of myelopoiesis from the spleen to the BM of Ames dwarf mice; however, this shift does not appear to involve erythropoiesis. The reasons for this unusual shift in spleen to marrow hematopoiesis in Ames dwarf mice are yet to be determined but may relate to the decreased hormone levels in these mice

Megakaryocytes: Regulators of Bone Mass and Hematopoiesis

poster abstractEmerging evidence demonstrates that megakaryocytes (MK) play a key role in regulating skeletal homeostasis and hematopoiesis. Recent reports show that MK reside in close proximity to hematopoietic stem cells (HSC). Genetic depletion of MK resulted in mitotic activation of HSC suggesting that MK maintain HSC quiescence. Other studies demonstrated that following irradiation, surviving MK migrate to endosteal surfaces where osteoblast (OB) lineage cells dramatically increase and promote engraftment of transplanted HSC. Here we investigated if MK directly impact hematopoiesis or whether they indirectly support HSC function through their interaction with OB-lineage cells. Our data suggests that LSK (Lin-Sca+CD117+, an enriched HSC population) co-cultured with MK and OB generate significantly higher numbers of colony forming cells (HSC function) compared to LSK cocultured with either MK or OB alone. The functionality of this in vitro data was confirmed in vivo with transplantation studies which showed increased engraftment in mice transplanted with LSK cells co-cultured with OB and MK compared to LSK cells co-cultured with OB alone. To test if loss of MK negatively impacts osteoblastogenesis, we generated conditional knockout mice where cMpl, the receptor for the main MK growth factor, thrombopoietin (TPO), was deleted in MK (cMplfl/fl x PF4Cre). Unexpectedly, these mice exhibited a 10-fold increase in platelet numbers, megakaryocytosis, a dramatic expansion of phenotypically defined hematopoietic precursors, and a remarkable 20-fold increase in the bone volume fraction. Collectively, these data indicate that while MK modulate HSC function, this activity is in part mediated through interactions with OB and suggest a complex role for TPO and MK in HSC regulation. While work is needed to further elucidate mechanisms, understanding the coordinated interaction between MK, OB, HSC, and TPO/Mpl should inform the development of novel treatments to enhance HSC recovery following myelosuppressive injuries, as well as bone loss diseases, such as osteoporosis

A novel role for thrombopoietin in regulating osteoclast development in humans and mice

Emerging data suggest that megakaryocytes (MKs) play a significant role in skeletal homeostasis. Indeed, osteosclerosis observed in several MK-related disorders may be a result of increased numbers of MKs. In support of this idea, we have previously demonstrated that MKs increase osteoblast (OB) proliferation by a direct cell-cell contact mechanism and that MKs also inhibit osteoclast (OC) formation. As MKs and OCs are derived from the same hematopoietic precursor, in these osteoclastogenesis studies we examined the role of the main MK growth factor, thrombopoietin (TPO) on OC formation and bone resorption. Here we show that TPO directly increases OC formation and differentiation in vitro. Specifically, we demonstrate the TPO receptor (c-mpl or CD110) is expressed on cells of the OC lineage, c-mpl is required for TPO to enhance OC formation in vitro, and TPO activates the mitogen-activated protein kinases, Janus kinase/signal transducer and activator of transcription, and nuclear factor-kappaB signaling pathways, but does not activate the PI3K/AKT pathway. Further, we found TPO enhances OC resorption in CD14+CD110+ human OC progenitors derived from peripheral blood mononuclear cells, and further separating OC progenitors based on CD110 expression enriches for mature OC development. The regulation of OCs by TPO highlights a novel therapeutic target for bone loss diseases and may be important to consider in the numerous hematologic disorders associated with alterations in TPO/c-mpl signaling as well as in patients suffering from bone disorders

C-Mpl Is Expressed on Osteoblasts and Osteoclasts and Is Important in Regulating Skeletal Homeostasis

C-Mpl is the receptor for thrombopoietin (TPO), the main megakaryocyte (MK) growth factor, and c-Mpl is believed to be expressed on cells of the hematopoietic lineage. As MKs have been shown to enhance bone formation, it may be expected that mice in which c-Mpl was globally knocked out (c-Mpl(-/-) mice) would have decreased bone mass because they have fewer MKs. Instead, c-Mpl(-/-) mice have a higher bone mass than WT controls. Using c-Mpl(-/-) mice we investigated the basis for this discrepancy and discovered that c-Mpl is expressed on both osteoblasts (OBs) and osteoclasts (OCs), an unexpected finding that prompted us to examine further how c-Mpl regulates bone. Static and dynamic bone histomorphometry parameters suggest that c-Mpl deficiency results in a net gain in bone volume with increases in OBs and OCs. In vitro, a higher percentage of c-Mpl(-/-) OBs were in active phases of the cell cycle, leading to an increased number of OBs. No difference in OB differentiation was observed in vitro as examined by real-time PCR and functional assays. In co-culture systems, which allow for the interaction between OBs and OC progenitors, c-Mpl(-/-) OBs enhanced osteoclastogenesis. Two of the major signaling pathways by which OBs regulate osteoclastogenesis, MCSF/OPG/RANKL and EphrinB2-EphB2/B4, were unaffected in c-Mpl(-/-) OBs. These data provide new findings for the role of MKs and c-Mpl expression in bone and may provide insight into the homeostatic regulation of bone mass as well as bone loss diseases such as osteoporosis

Lnk Deficiency Leads to TPO-Mediated Osteoclastogenesis and Increased Bone Mass Phenotype

The Lnk adapter protein negatively regulates the signaling of thrombopoietin (TPO), the main megakaryocyte (MK) growth factor. Lnk-deficient (-/-) mice have increased TPO signaling and increased MK number. Interestingly, several mouse models exist in which increased MK number leads to a high bone mass phenotype. Here we report the bone phenotype of these mice. MicroCT and static histomorphometric analyses at 20 weeks showed the distal femur of Lnk-/- mice to have significantly higher bone volume fraction and trabecular number compared to wild-type (WT) mice. Notably, despite a significant increase in the number of osteoclasts (OC), and decreased bone formation rate in Lnk-/- mice compared to WT mice, Lnk-/- mice demonstrated a 2.5-fold greater BV/TV suggesting impaired OC function in vivo. Additionally, Lnk-/- mouse femurs exhibited non-significant increases in mid-shaft cross-sectional area, yet increased periosteal BFR compared to WT femurs was observed. Lnk-/- femurs also had non-significant increases in polar moment of inertia and decreased cortical bone area and thickness, resulting in reduced bone stiffness, modulus, and strength compared to WT femurs. Of note, Lnk is expressed by OC lineage cells and when Lnk-/- OC progenitors are cultured in the presence of TPO, significantly more OC are observed than in WT cultures. Lnk is also expressed in osteoblast (OB) cells and in vitro reduced alkaline phosphatase activity was observed in Lnk-/- cultures. These data suggest that both direct effects on OB and OC as well as indirect effects of MK in regulating OB contributes to the observed high bone mass. J. Cell. Biochem. 118: 2231-2240, 2017

Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation

Notch-dependent repression of miR-155 in the bone marrow niche regulates hematopoiesis in an NF-κB-dependent manner

The microRNA miR-155 has been implicated in regulating inflammatory responses and tumorigenesis, but its precise role in linking inflammation and cancer has remained elusive. Here, we identify a connection between miR-155 and Notch signaling in this context. Loss of Notch signaling in the bone marrow (BM) niche alters hematopoietic homeostasis and leads to lethal myeloproliferative-like disease. Mechanistically, Notch signaling represses miR-155 expression by promoting binding of RBPJ to the miR-155 promoter. Loss of Notch/RBPJ signaling upregulates miR-155 in BM endothelial cells, leading to miR-155-mediated targeting of the nuclear factor κB (NF-κB) inhibitor κB-Ras1, NF-κB activation, and increased proinflammatory cytokine production. Deletion of miR-155 in the stroma of RBPJ(-/-) mice prevented the development of myeloproliferative-like disease and cytokine induction. Analysis of BM from patients carrying myeloproliferative neoplasia also revealed elevated expression of miR-155. Thus, the Notch/miR-155/κB-Ras1/NF-κB axis regulates the inflammatory state of the BM niche and affects the development of myeloproliferative disorders

Genomic and Proteomic Analysis of the Impact of Mitotic Quiescence on the Engraftment of Human CD34+ Cells

It is well established that in adults, long-term repopulating hematopoietic stem cells (HSC) are mitotically quiescent cells that reside in specialized bone marrow (BM) niches that maintain the dormancy of HSC. Our laboratory demonstrated that the engraftment potential of human HSC (CD34+ cells) from BM and mobilized peripheral blood (MPB) is restricted to cells in the G0 phase of cell cycle but that in the case of umbilical cord blood (UCB) -derived CD34+ cells, cell cycle status is not a determining factor in the ability of these cells to engraft and sustain hematopoiesis. We used this distinct in vivo behavior of CD34+ cells from these tissues to identify genes associated with the engraftment potential of human HSC. CD34+ cells from BM, MPB, and UCB were fractionated into G0 and G1 phases of cell cycle and subjected in parallel to microarray and proteomic analyses. A total of 484 target genes were identified to be associated with engraftment potential of HSC. System biology modeling indicated that the top four signaling pathways associated with these genes are Integrin signaling, p53 signaling, cytotoxic T lymphocyte-mediated apoptosis, and Myc mediated apoptosis signaling. Our data suggest that a continuum of functions of hematopoietic cells directly associated with cell cycle progression may play a major role in governing the engraftment potential of stem cells. While proteomic analysis identified a total of 646 proteins in analyzed samples, a very limited overlap between genomic and proteomic data was observed. These data provide a new insight into the genetic control of engraftment of human HSC from distinct tissues and suggest that mitotic quiescence may not be the requisite characteristic of engrafting stem cells, but instead may be the physiologic status conducive to the expression of genetic elements favoring engraftment

Expansion of prostate epithelial progenitor cells after inflammation of the mouse prostate

Prostatic inflammation is a nearly ubiquitous pathological feature observed in specimens from benign prostate hyperplasia and prostate cancer patients. The microenvironment of the inflamed prostate is highly reactive, and epithelial hyperplasia is a hallmark feature of inflamed prostates. How inflammation orchestrates epithelial proliferation as part of its repair and recovery action is not well understood. Here, we report that a novel epithelial progenitor cell population is induced to expand during inflammation. We used sphere culture assays, immunofluorescence, and flow cytometry to show that this population is increased in bacterially induced inflamed mouse prostates relative to naïve control prostates. We confirmed from previous reports that this population exclusively possesses the ability to regrow entire prostatic structures from single cell culture using renal grafts. In addition, putative progenitor cells harvested from inflamed animals have greater aggregation capacity than those isolated from naïve control prostates. Expansion of this critical cell population requires IL-1 signaling, as IL-1 receptor 1-null mice exhibit inflammation similar to wild-type inflamed animals but exhibit significantly reduced progenitor cell proliferation and hyperplasia. These data demonstrate that inflammation promotes hyperplasia in the mouse prostatic epithelium by inducing the expansion of a selected epithelial progenitor cell population in an IL-1 receptor-dependent manner. These findings may have significant impact on our understanding of how inflammation promotes proliferative diseases such as benign prostatic hyperplasia and prostate cancer, both of which depend on expansion of cells that exhibit a progenitor-like nature

Nonmarrow hematopoiesis occurs in a hyaluronic-acid-rich node and duct system in mice

A hyaluronic-acid-rich node and duct system (HAR-NDS) was found on the surface of internal organs of mice, and inside their blood and lymph vessels. The nodes (HAR-Ns) were filled with immune cells of the innate system and were especially enriched with mast cells and histiocytes. They also contained hematopoietic progenitor cells (HPCs), such as granulocyte-macrophage, erythroid, multipotential progenitors, and mast cell progenitors (MCPs). MCPs were the most abundant among the HPCs in HAR-Ns. Their frequency was fivefold higher than that of the MCPs in bone marrow. In addition, the system contained pluripotent stem cells (PSCs) capable of producing CD45(-)Flk1(+) hemangioblast-like cells, which subsequently generated various types of HPCs and differentiated blood cells. Although HAR-Ns did not appear to harbor enough number of cells capable of long-term reconstitution or short-term radioprotection of lethally irradiated recipients, bone marrow cells were able to engraft in the HAR-NDS and reconstitute hematopoietic potentials of the system. PSCs and HPCs were consistently found in intravenous, intralymphatic, and intestinal HAR-ND. We infer that PSCs and HPCs reside in the HAR-ND and that this novel system may serve as an alternative means to traffic immature and mature blood cells throughout the body