IU Indianapolis ScholarWorks :: Browsing by Author "Mohamad, Safa F."

Browsing by Author "Mohamad, Safa F."

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Aging negatively impacts the ability of megakaryocytes to stimulate osteoblast proliferation and bone mass
(Elsevier, 2019) Maupin, Kevin A.; Himes, Evan R.; Plett, Artur P.; Chua, Hui Lin; Singh, Pratibha; Ghosh, Joydeep; Mohamad, Safa F.; Abeysekera, Irushi; Fisher, Alexa; Sampson, Carol; Hong, Jung-Min; Childress, Paul; Alvarez, Marta; Srour, Edward F.; Bruzzaniti, Angela; Pelus, Louis M.; Orschell, Christie M.; Kacena, Melissa A.; Orthopaedic Surgery, School of Medicine
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.
CD166 Engagement Augments Mouse and Human Hematopoietic Progenitor Function via Activation of Stemness and Cell Cycle Pathways
(Oxford University Press, 2019) Zhang, Jing; Ghosh, Joydeep; Mohamad, Safa F.; Zhang, Chi; Huang, Xinxin; Capitano, Maegan L.; Gunawan, Andrea M.; Cooper, Scott; Guo, Bin; Cai, Qingchun; Broxmeyer, Hal E.; Srour, Edward F.; Microbiology and Immunology, School of Medicine
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.
Cellular components of the hematopoietic niche and their regulation of hematopoietic stem cell function
(Wolters Kluwer, 2021) Ghosh, Joydeep; El Koussa, Roy; Mohamad, Safa F.; Liu, Jianyun; Kacena, Melissa A.; Srour, Edward F.; Medicine, School of Medicine
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.
Megakaryocyte and Osteoblast Interactions Modulate Bone Mass and Hematopoiesis
(Mary Ann Liebert, 2018-05-15) Alvarez, Marta B.; Xu, LinLin; Childress, Paul J.; Maupin, Kevin A.; Mohamad, Safa F.; Chitteti, Brahmananda R.; Himes, Evan; Olivos, David J.; Cheng, Ying-Hua; Conway, Simon J.; Srour, Edward F.; Kacena, Melissa A.; Orthopaedic Surgery, School of Medicine
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.
Megakaryocytes promote osteoclastogenesis in aging
(Impact Journals, 2020-07-07) Kanagasabapathy, Deepa; Blosser, Rachel J.; Maupin, Kevin A.; Hong, Jung Min; Alvarez, Marta; Ghosh, Joydeep; Mohamad, Safa F.; Aguilar-Perez, Alexandra; Srour, Edward F.; Kacena, Melissa A.; Bruzzaniti, Angela; Orthopaedic Surgery, School of Medicine
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.
Mitigating oxygen stress enhances aged mouse hematopoietic stem cell numbers and function
(American Society for Clinical Investigation, 2021-01-04) Capitano, Maegan L.; Mohamad, Safa F.; Cooper, Scott; Guo, Bin; Huang, Xinxin; Gunawan, Andrea M.; Sampson, Carol; Ropa, James; Srour, Edward F.; Orschell, Christie M.; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
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.
Neonatal Osteomacs and Bone Marrow Macrophages Differ in Phenotypic Marker Expression and Function
(Wiley, 2021) Mohamad, Safa F.; Gunawan, Andrea; Blosser, Rachel; Childress, Paul; Aguilar-Perez, Alexandra; Ghosh, Joydeep; Hong, Jung Min; Liu, Jianyun; Kanagasabapathy, Deepa; Kacena, Melissa A.; Srour, Edward F.; Bruzzaniti, Angela; Medicine, School of Medicine
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.
Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function
(ASH, 2017-12) Mohamad, Safa F.; Xu, Linlin; Ghosh, Joydeep; Childress, Paul J.; Abeysekera, Irushi; Himes, Evan R.; Wu, Hao; Alvarez, Marta B.; Davis, Korbin M.; Aguilar-Perez, Alexandra; Hong, Jung Min; Bruzzaniti, Angela; Kacena, Melissa A.; Srour, Edward F.; Biomedical Sciences and Comprehensive Care, School of Dentistry
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.
Osteomacs promote maintenance of murine hematopoiesis through megakaryocyte-induced upregulation of Embigin and CD166
(Elsevier, 2024) Mohamad, Safa F.; El Koussa, Roy; Ghosh, Joydeep; Blosser, Rachel; Gunawan, Andrea; Layer, Justin; Zhang, Chi; Karnik, Sonali; Davé, Utpal; Kacena, Melissa A.; Srour, Edward F.; Microbiology and Immunology, School of Medicine
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.
The Role of Osteomacs in Regulating Stem Cell Function and the Hematopoietic Niche
(2020-02) Mohamad, Safa F.; Srour, Edward F.; Bruzzaniti, Angela; Haneline, Laura S.; Pelus, Louis M.; Kacena, Melissa A.
Maintenance of hematopoietic stem cell (HSC) function is an orchestrated event requiring the participation of multiple cell types within the hematopoietic niche. Among the key cellular components of the niche are a group of specialized bone-resident macrophages known as osteomacs (OM). Reported here is a detailed characterization of OM and description of discriminating phenotypic and functional properties that clearly distinguish OM from bone marrow-derived macrophages (BM Mφ). Furthermore, it was established that OM support hematopoiesis enhancing activity of osteoblasts and that this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing OM, osteoblasts and megakaryocytes. Interestingly, BM Mφ were unable to mediate the same hematopoiesis enhancing activity regardless of whether megakaryocytes were present in co-culture or not. Subsequently, to understand the importance of networking between the residents of the niche, 3D tissue cytometry was performed on fixed and stained unperturbed bone marrow sections. This approach identified the spatial relationships between OM, osteoblasts, megakaryocytes and HSC within the niche and defined parameters, under which these cell types coexist in undamaged bone marrow. In addition, single cell mRNAseq and CyTOF was performed to assess genetic and proteomic expression changes in OM following their interaction with megakaryocytes. These studies revealed the upregulation of CD166 and embigin on OM via osteoblast and megakaryocyte interactions. Clonogenic assays were conducted to examine the impact of these molecules in hematopoietic function. When these assays were initiated with CD166 KO OM or shRNA-mediated embigin knockdown OM, it was established that loss of these surface molecules on OM caused a decline in the normal OM-mediated hematopoietic enhancing activity. Conversely, recombinant CD166 and embigin partially substituted for OM activity thus identifying potential mediators through which OM maintain hematopoietic function. This data, for the first time, reveal intimate spatial interactions between OM, osteoblasts, megakaryocytes and HSC in the hematopoietic niche. They also illustrate the importance of crosstalk between OM, osteoblasts and megakaryocytes and reveal novel mediators such as CD166 and embigin that cooperate with other elements of the niche to support HSC function.

Browsing by Author "Mohamad, Safa F."

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