Osteomacs promote maintenance of murine hematopoiesis through megakaryocyte-induced upregulation of Embigin and CD166.

Maintenance of hematopoietic stem cell (HSC) function in the niche is an orchestrated event. Osteomacs (OM) are key cellular components of the niche. Previously, we documented that osteoblasts, OM, and megakaryocytes interact to promote hematopoiesis. Here, we further characterize OM and identify megakaryocyte-induced mediators that augment the role of OM in the niche. Single-cell mRNA-seq, mass spectrometry, and CyTOF examination of megakaryocyte-stimulated OM suggested that upregulation of CD166 and Embigin on OM augment their hematopoiesis maintenance function. CD166 knockout OM or shRNA-Embigin knockdown OM confirmed that the loss of these molecules significantly reduced the ability of OM to augment the osteoblast-mediated hematopoietic-enhancing activity. Recombinant CD166 and Embigin partially substituted for OM function, characterizing both proteins as critical mediators of OM hematopoietic function. Our data identify Embigin and CD166 as OM-regulated critical components of HSC function in the niche and potential participants in various in vitro manipulations of stem cells.

Isolation of Murine Neonatal and Adult Osteomacs to Examine Their Role in the Hematopoietic Niche.

Maintenance of hematopoietic stem cell (HSC) function is an orchestrated event between multiple cell types, and crosstalk between these cell types is an essential part of HSC regulation. Among the cell groups of the niche involved in this process are a group of bone-resident macrophages known as osteomacs (OM). Previously, it was demonstrated that OM and osteoblasts contained within neonatal calvarial cells are critical to maintain hematopoietic function. Additionally, interactions between neonatal calvarial cells and megakaryocytes further enhance this hematopoietic activity. In this chapter, we explore one such interaction involving OM and osteoblasts in the hematopoietic niche. We describe a protocol to isolate OM from both neonatal and adult mice, and subsequently use colony-forming assays to demonstrate their interaction with osteoblasts in maintaining HSC function.

Utilizing CyTOF to Examine Hematopoietic Stem and Progenitor Phenotype.

Regulation of hematopoiesis is dependent upon interactions between hematopoietic stem/progenitor cells and niche components, requiring a highly diverse array of different cell-cell interactions and cell signaling events. The overwhelming diversity of the components that can regulate hematopoiesis, especially when factoring in how the cell surface and intracellular protein expression profiles of hematopoietic stem/progenitor cells and niche components differ between homeostatic conditions and stressed conditions such as aging and irradiation, can make utilizing techniques like flow cytometry daunting, particularly while examining small cell populations such as hematopoietic stem cells (HSCs). Due to the complexity of the hematopoietic system, high-dimensional single-cell genomics and proteomics are constantly performed to understand the heterogeneity and expression profiles within this system. This chapter describes one such single-cell assay, which utilizes mass cytometry Time of Flight (CyTOF) technology to determine differences in expression profile within HSC, using changes in HSC populations due to gender and aging.

Cellular components of the hematopoietic niche and their regulation of hematopoietic stem cell function.

PURPOSE OF REVIEW: Development and functions of hematopoietic stem cells (HSC) are regulated by multiple cellular components of the hematopoietic niche. Here we review the recent advances in studying the role of three such components -- osteoblasts, osteomacs, and megakaryocytes and how they interact with each other in the hematopoietic niche to regulate HSC. RECENT FINDINGS: Recent advances in transgenic mice models, scRNA-seq, transcriptome profile, proteomics, and live animal imaging have revealed the location of HSC within the bone and signaling molecules required for the maintenance of the niche. Interaction between megakaryocytes, osteoblasts and osteomacs enhances hematopoietic stem and progenitor cells (HSPC) function. Studies also revealed the niche as a dynamic entity that undergoes cellular and molecular changes in response to stress. Aging, which results in reduced HSC function, is associated with a decrease in endosteal niches and osteomacs as well as reduced HSC--megakaryocyte interactions. SUMMARY: Novel approaches to study the cellular components of the niche and their interactions to regulate HSC development and functions provided key insights about molecules involved in the maintenance of the hematopoietic system. Furthermore, these studies began to build a more comprehensive model of cellular interactions and dynamics in the hematopoietic niche.

Neonatal Osteomacs and Bone Marrow Macrophages Differ in Phenotypic Marker Expression and Function.

Osteomacs (OM) are specialized bone-resident macrophages that are a component of the hematopoietic niche and support bone formation. Also located in the niche are a second subset of macrophages, namely bone marrow-derived macrophages (BM Mφ). We previously reported that a subpopulation of OM co-express both CD166 and CSF1R, the receptor for macrophage colony-stimulating factor (MCSF), and that OM form more bone-resorbing osteoclasts than BM Mφ. Reported here are single-cell quantitative RT-PCR (qRT-PCR), mass cytometry (CyTOF), and marker-specific functional studies that further identify differences between OM and BM Mφ from neonatal C57Bl/6 mice. Although OM express higher levels of CSF1R and MCSF, they do not respond to MCSF-induced proliferation, in contrast to BM Mφ. Moreover, receptor activator of NF-κB ligand (RANKL), without the addition of MCSF, was sufficient to induce osteoclast formation in OM but not BM Mφ cultures. OM express higher levels of CD166 than BM Mφ, and we found that osteoclast formation by CD166-/- OM was reduced compared with wild-type (WT) OM, whereas CD166-/- BM Mφ showed enhanced osteoclast formation. CD110/c-Mpl, the receptor for thrombopoietin (TPO), was also higher in OM, but TPO did not alter OM-derived osteoclast formation, whereas TPO stimulated BM Mφ osteoclast formation. CyTOF analyses demonstrated OM uniquely co-express CD86 and CD206, markers of M1 and M2 polarized macrophages, respectively. OM performed equivalent phagocytosis in response to LPS or IL-4/IL-10, which induce polarization to M1 and M2 subtypes, respectively, whereas BM Mφ were less competent at phagocytosis when polarized to the M2 subtype. Moreover, in contrast to BM Mφ, LPS treatment of OM led to the upregulation of CD80, an M1 marker, as well as IL-10 and IL-6, known anti-inflammatory cytokines. Overall, these data reveal that OM and BM Mφ are distinct subgroups of macrophages, whose phenotypic and functional differences in proliferation, phagocytosis, and osteoclast formation may contribute physiological specificity during health and disease. © 2021 American Society for Bone and Mineral Research (ASBMR).

Mitigating oxygen stress enhances aged mouse hematopoietic stem cell numbers and function.

Bone marrow (BM) hematopoietic stem cells (HSCs) become dysfunctional during aging (i.e., they are increased in number but have an overall reduction in long-term repopulation potential and increased myeloid differentiation) compared with young HSCs, suggesting limited use of old donor BM cells for hematopoietic cell transplantation (HCT). BM cells reside in an in vivo hypoxic environment yet are evaluated after collection and processing in ambient air. We detected an increase in the number of both young and aged mouse BM HSCs collected and processed in 3% O2 compared with the number of young BM HSCs collected and processed in ambient air (~21% O2). Aged BM collected and processed under hypoxic conditions demonstrated enhanced engraftment capability during competitive transplantation analysis and contained more functional HSCs as determined by limiting dilution analysis. Importantly, the myeloid-to-lymphoid differentiation ratio of aged BM collected in 3% O2 was similar to that detected in young BM collected in ambient air or hypoxic conditions, consistent with the increased number of common lymphoid progenitors following collection under hypoxia. Enhanced functional activity and differentiation of old BM collected and processed in hypoxia correlated with reduced "stress" associated with ambient air BM collection and suggests that aged BM may be better and more efficiently used for HCT if collected and processed under hypoxia so that it is never exposed to ambient air O2.

Megakaryocytes promote osteoclastogenesis in aging.

Megakaryocytes (MKs) support bone formation by stimulating osteoblasts (OBs) and inhibiting osteoclasts (OCs). Aging results in higher bone resorption, leading to bone loss. Whereas previous studies showed the effects of aging on MK-mediated bone formation, the effects of aging on MK-mediated OC formation is poorly understood. Here we examined the effect of thrombopoietin (TPO) and MK-derived conditioned media (CM) from young (3-4 months) and aged (22-25 months) mice on OC precursors. Our findings showed that aging significantly increased OC formation in vitro. Moreover, the expression of the TPO receptor, Mpl, and circulating TPO levels were elevated in the bone marrow cavity. We previously showed that MKs from young mice secrete factors that inhibit OC differentiation. However, rather than inhibiting OC development, we found that MKs from aged mice promote OC formation. Interestingly, these age-related changes in MK functionality were only observed using female MKs, potentially implicating the sex steroid, estrogen, in signaling. Further, RANKL expression was highly elevated in aged MKs suggesting MK-derived RANKL signaling may promote osteoclastogenesis in aging. Taken together, these data suggest that modulation in TPO-Mpl expression in bone marrow and age-related changes in the MK secretome promote osteoclastogenesis to impact skeletal aging.

CD166 Engagement Augments Mouse and Human Hematopoietic Progenitor Function via Activation of Stemness and Cell Cycle Pathways.

Hematopoietic stem (HSC) and progenitor (HPC) cells are regulated by interacting signals and cellular and noncellular elements of the hematopoietic niche. We previously showed that CD166 is a functional marker of murine and human HSC and of cellular components of the murine niche. Selection of murine CD166+ engrafting HSC enriched for marrow repopulating cells. Here, we demonstrate that CD166-CD166 homophilic interactions enhance generation of murine and human HPC in vitro and augment hematopoietic function of these cells. Interactions between cultured CD166+ Lineage- Sca-1+ c-Kit+ (LSK) cells and CD166+ osteoblasts (OBs) significantly enhanced the expansion of colony-forming units (CFUs). Interactions between CD166+ LSK cells and immobilized CD166 protein generated more CFU in short-term cultures than between these cells and bovine serum albumin (BSA) or in cultures initiated with CD166- LSK cells. Similar results were obtained when LSK cells from wildtype (WT) or CD166 knockout (KO) (CD166-/- ) mice were used with immobilized CD166. Human cord blood CD34+ cells expressing CD166 produced significantly higher numbers of CFUs following interaction with immobilized CD166 than their CD166- counterparts. These data demonstrate the positive effects of CD166 homophilic interactions involving CD166 on the surface of murine and human HPCs. Single-cell RNA-seq analysis of CD150+ CD48- (signaling lymphocyte activation molecule (SLAM)) LSK cells from WT and CD166-/- mice incubated with immobilized CD166 protein revealed that engagement of CD166 on these cells activates cytokine, growth factor and hormone signaling, epigenetic pathways, and other genes implicated in maintenance of stem cell pluripotency-related and mitochondria-related signaling pathways. These studies provide tangible evidence implicating CD166 engagement in the maintenance of stem/progenitor cell function. Stem Cells 2019;37:1319-1330.

Aging negatively impacts the ability of megakaryocytes to stimulate osteoblast proliferation and bone mass.

Osteoblast number and activity decreases with aging, contributing to the age-associated decline of bone mass, but the mechanisms underlying changes in osteoblast activity are not well understood. Here, we show that the age-associated bone loss critically depends on impairment of the ability of megakaryocytes (MKs) to support osteoblast proliferation. Co-culture of osteoblast precursors with young MKs is known to increase osteoblast proliferation and bone formation. However, co-culture of osteoblast precursors with aged MKs resulted in significantly fewer osteoblasts compared to co-culture with young MKs, and this was associated with the downregulation of transforming growth factor beta. In addition, the ability of MKs to increase bone mass was attenuated during aging as transplantation of GATA1low/low hematopoietic donor cells (which have elevated MKs/MK precursors) from young mice resulted in an increase in bone mass of recipient mice compared to transplantation of young wild-type donor cells, whereas transplantation of GATA1low/low donor cells from old mice failed to enhance bone mass in recipient mice compared to transplantation of old wild-type donor cells. These findings suggest that the preservation or restoration of the MK-mediated induction of osteoblast proliferation during aging may hold the potential to prevent age-associated bone loss and resulting fractures.

Isolation and Identification of Murine Bone Marrow-Derived Macrophages and Osteomacs from Neonatal and Adult Mice.

Hematopoietic stem cells (HSCs) are regulated by multiple components of the hematopoietic niche, including bone marrow-derived macrophages and osteomacs. However, both macrophages and osteomacs are phenotypically similar. Thus, specific phenotypic markers are required to differentially identify the effects of osteomacs and bone marrow macrophages on different physiological processes, including hematopoiesis and bone remodeling. Here, we describe a protocol for isolation of murine bone marrow-derived macrophages and osteomacs from neonatal and adult mice and subsequent identification by multi-parametric flow cytometry using an 8-color antibody panel.

Megakaryocyte and Osteoblast Interactions Modulate Bone Mass and Hematopoiesis.

Emerging evidence demonstrates that megakaryocytes (MK) play key roles in regulating skeletal homeostasis and hematopoiesis. To test if the loss of MK negatively impacts osteoblastogenesis and hematopoiesis, we generated conditional knockout mice where Mpl, the receptor for the main MK growth factor, thrombopoietin, was deleted specifically in MK (Mplf/f;PF4cre). Unexpectedly, at 12 weeks of age, these mice exhibited a 10-fold increase in platelets, a significant expansion of hematopoietic/mesenchymal precursors, and a remarkable 20-fold increase in femoral midshaft bone volume. We then investigated whether MK support hematopoietic stem cell (HSC) function through the interaction of MK with osteoblasts (OB). LSK cells (Lin-Sca1+CD117+, enriched HSC population) were co-cultured with OB+MK for 1 week (1wk OB+MK+LSK) or OB alone (1wk OB+LSK). A significant increase in colony-forming units was observed with cells from 1wk OB+MK cultures. Competitive repopulation studies demonstrated significantly higher engraftment in mice transplanted with cells from 1wk OB+MK+LSK cultures compared to 1wk OB+LSK or LSK cultured alone for 1 week. Furthermore, single-cell expression analysis of OB cultured±MK revealed adiponectin as the most significantly upregulated MK-induced gene, which is required for optimal long-term hematopoietic reconstitution. Understanding the interactions between MK, OB, and HSC can inform the development of novel treatments to enhance both HSC recovery following myelosuppressive injuries, as well as bone loss diseases, such as osteoporosis.

Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function.

Networking between hematopoietic stem cells (HSCs) and cells of the hematopoietic niche is critical for stem cell function and maintenance of the stem cell pool. We characterized calvariae-resident osteomacs (OMs) and their interaction with megakaryocytes to sustain HSC function and identified distinguishing properties between OMs and bone marrow (BM)-derived macrophages. OMs, identified as CD45+F4/80+ cells, were easily detectable (3%-5%) in neonatal calvarial cells. Coculture of neonatal calvarial cells with megakaryocytes for 7 days increased OM three- to sixfold, demonstrating that megakaryocytes regulate OM proliferation. OMs were required for the hematopoiesis-enhancing activity of osteoblasts, and this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing osteoblasts, OMs, and megakaryocytes. With or without megakaryocytes, BM-derived macrophages were unable to functionally substitute for neonatal calvarial cell-associated OMs. In addition, OMs differentiated into multinucleated, tartrate resistant acid phosphatase-positive osteoclasts capable of bone resorption. Nine-color flow cytometric analysis revealed that although BM-derived macrophages and OMs share many cell surface phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and Gr-1), only a subgroup of OMs coexpressed M-CSFR and CD166, thus providing a unique profile for OMs. CD169 was expressed by both OMs and BM-derived macrophages and therefore was not a distinguishing marker between these 2 cell types. These results demonstrate that OMs support HSC function and illustrate that megakaryocytes significantly augment the synergistic activity of osteoblasts and OMs. Furthermore, this report establishes for the first time that the crosstalk between OMs, osteoblasts, and megakaryocytes is a novel network supporting HSC function.

Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and ß-Globin Expression in Human Fetal Erythroblasts.

In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult ß- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal ß-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and ß-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the ß-globin locus control region (LCR), and the ß-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the ß- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with ß-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the ß-hemoglobinopathies.

Krüppel-like transcription factors KLF1 and KLF2 have unique and coordinate roles in regulating embryonic erythroid precursor maturation.

The Krüppel-like transcription factors KLF1 and KLF2 are essential for embryonic erythropoiesis. They can partially compensate for each other during mouse development, and coordinately regulate numerous erythroid genes, including the ß-like globins. Simultaneous ablation of KLF1 and KLF2 results in earlier embryonic lethality and severe anemia. In this study, we determine that this anemia is caused by a paucity of blood cells, and exacerbated by diminished ß-like globin gene expression. The anemia phenotype is dose-dependent, and, interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. Mechanistic studies led to the discovery that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous loss of KLF1 and KLF2 than with loss of a single factor. KLF1 and KLF2 coordinately regulate several proliferation-associated genes, including Foxm1. Differential expression of FoxM1, in particular, correlates with the observed KLF1 and KLF2 gene dosage effects on anemia. Furthermore, KLF1 binds to the FoxM1 gene promoter in blood cells. Thus KLF1 and KLF2 coordinately regulate embryonic erythroid precursor maturation through the regulation of multiple homeostasis-associated genes, and KLF2 has a novel and essential role in this process.