Neonatal Calvarial Cells Research Articles

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.

Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages.

SUMMARYThe transcriptional regulator Runx2 (runt-related transcription factor 2) has essential but distinct roles in osteoblasts and chondrocytes in skeletal development. However, Runx2-mediated regulatory mechanisms underlying the distinctive programming of osteoblasts and chondrocytes are not well understood. Here, we perform an integrative analysis to investigate Runx2-DNA binding and chromatin accessibility ex vivo using neonatal osteoblasts and chondrocytes. We find that Runx2 engages with cell-type-distinct chromatin-accessible regions, potentially interacting with different combinations of transcriptional regulators, forming cell-type-specific hotspots, and potentiating chromatin accessibility. Genetic analysis and direct cellular reprogramming studies suggest that Runx2 is essential for establishment of chromatin accessibility in osteoblasts. Functional enhancer studies identify an Sp7 distal enhancer driven by Runx2-dependent binding and osteoblast-specific chromatin accessibility, contributing to normal osteoblast differentiation. Our findings provide a framework for understanding the regulatory landscape encompassing Runx2-mediated and cell-type-distinct enhancer networks that underlie the specification of osteoblasts.

Osteal Macrophages and Megakaryocytes Increase Adipogenesis

Bone marrow adipose tissue (MAT) increases with aging and contributes to low bone density and skeletal fractures. However, the cells and factors within the bone marrow (BM) that regulate adipogenesis remain poorly understood. In the current study, we examined the role of osteal macrophages (OMs) and megakaryocytes (MKs) on the regulation of adipogenesis. We cultured murine osteoblasts/osteoblast progenitors (OBs from hereon) derived from neonatal calvarial cells (CCs, a combination of OBs and OMs) or OBs isolated by fluorescence activated cell sorting (FACS) in the presence or absence of fetal liver derived murine MK. The cells underwent induced adipogenesis for 5-7 days by supplementation of media with insulin, indomethacin, and dexamethasone, and then the number of adipocytes was quantified. We found that co-culturing MKs and OMs with OBs results in up to a 7.8-fold and 11.7-fold increase in adipocytes, respectively. We also elucidated that thrombopoietin (TPO), the major growth factor for MKs, inhibits adipogenesis in both OBs and CCs by approximately 60%. Similarly, we found that CCs and OBs derived from mice deficient in the TPO receptor, Mpl, had approximately 30% more adipocytes than their wild-type (WT) counterparts. Finally, in vitro findings were corroborated in vivo through quantification of MKs and adipocytes in mice in which MK number was elevated or reduced. Mice with significantly higher numbers of BM-residing MKs also had significantly higher numbers of BM-residing adipocytes. Because there is typically an inverse relationship between adipogenesis and osteogenesis, understanding ways to inhibit adipogenesis could lead to an increase in OB number and bone formation, which in turn could lead to new treatments for bone loss diseases such as osteoporosis.

Increased Osteoclast and Decreased Osteoblast Activity Causes Reduced Bone Mineral Density and Quality in Genetic Hypercalciuric Stone-Forming Rats.

ABSTRACTTo study human idiopathic hypercalciuria (IH), we developed an animal model, genetic hypercalciuric stone‐forming (GHS) rats, whose pathophysiology parallels that in IH. All GHS rats form kidney stones and have decreased BMD and bone quality compared with the founder Sprague–Dawley (SD) rats. To understand the bone defect, we characterized osteoclast and osteoblast activity in the GHS compared with SD rats. Bone marrow cells were isolated from femurs of GHS and SD rats and cultured to optimize differentiation into osteoclasts or osteoblasts. Osteoclasts were stained for TRAcP (tartrate resistant acid phosphatase), cultured to assess resorptive activity, and analyzed for specific gene expression. Marrow stromal cells or primary neonatal calvarial cells were differentiated to osteoblasts, and osteoblastic gene expression as well as mineralization was analyzed. There was increased osteoclastogenesis and increased resorption pit formation in GHS compared with SD cultures. Osteoclasts had increased expression of cathepsin K, Tracp, and MMP9 in cells from GHS compared with SD rats. Osteoblastic gene expression and mineralization was significantly decreased. Thus, alterations in baseline activity of both osteoclasts and osteoblasts in GHS rats, led to decreased BMD and bone quality, perhaps because of their known increase in vitamin D receptors. Better understanding of the role of GHS bone cells in decreased BMD and quality may provide new strategies to mitigate the low BMD and increased fracture risk found in patients with IH. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Defining Parameters Attributing to the Role of Osteomacs in Regulating Stem Cell Function and the Hematopoietic Niche

Networking between hematopoietic stem cells (HSC) and cells of the hematopoietic niche is critical for the maintenance of stem cell renewal and function. HSC maintenance in the hematopoietic niche is considered to be the product of intimate interactions between cellular and soluble elements of the niche and stem cells. Among the cellular components of the niche participating in this function are a group of specialized bone-resident macrophages known as osteomacs (OM). Previously, we established the importance of osteoblasts (OB) in hematopoiesis and quite recently, we described the importance of OM and their interactions with OB and megakaryocytes (MK) in sustaining HSC function. We have also illustrated that CD166 is a critical functional marker of stem cell function and competence of the hematopoietic niche. Interestingly, immature OB which are CD166+ mediate the highest level of hematopoietic enhancing activity. We report here the importance of CD166 on calvarie-resident OM (identified as CD45+F4/80+ cells) and outline how these cells require cooperation from MK to increase CD166 expression and sustain HSC function.Bone resident-osteomacs, which are phenotypically similar but functionally different from bone marrow-derived macrophages, were collected by the enzymatic digestion of neonatal calvarial cells (NCC) or long bones of adult mice. Transplantation assays indicated that OM are relatively radioresistant and survive several weeks post lethal radiation. However, they eventually deplete and are replenished by progeny of donor HSC. To understand the importance of OM-OB-MK interactions in maintaining HSC function in the niche, we performed 3D cytometry on fixed and stained bone marrow sections that revealed intimate spatial interactions between OM, OB, MK and HSC. To assess changes in gene expression observed due to these interactions, we cultured NCC for 16hr in the absence or presence of MK prepared from fetal liver followed by sorting out OM from each group. These cells were then captured as single cells and sequenced to identify potential targets through which OM enhanced hematopoietic activity. Strikingly, several genes involved in the hematopoietic stem cell differentiation pathway including lmo2, fli1 and ikzf1 were upregulated in OM cultured in the presence of MK. Other genes that were upregulated were embigin and PF-4, both of which have been implicated in the maintenance of HSC function. Interestingly, OM express embigin, angiogenin and IL-18 mRNA similar to proximal osteolineage cells which we previously described as HSC regulators. To investigate changes at the translational level, we performed single cell proteomics using CyTOF. NCC were cultured for 2 days in the absence and presence of MK followed by staining for a panel of 29 surface and intracellular markers. Expression of markers such as CD166, embigin, mac-2 and STAT3 amongst others was elevated on OM cultured with MK compared to OM cultured without. These data informed our decision to focus our future investigations on CD166 and embigin.Next CD166+OM and CD166-OM were isolated by cell sorting and used in co-culture assays with OB to support the production of clonogenic cells in vitro. Only the CD166+ fraction of OM maintained hematopoietic activity similar to unsorted OM, implicating CD166 as one of the mediators of OM function. These results were validated using recombinant CD166 protein to substitute for OM function. Under these conditions, recombinant CD166 supported the hematopoietic enhancing activity of OB in the absence of OM. Recombinant Angiogenin and IL-18 were unable to augment the CD166-mediated support of hematopoiesis. Interestingly, CD166 knockout OM were unable to mediate the same hematopoietic enhancing activity observed with WT OM regardless of the presence or absence of MK in culture. In vivo transplantation studies to corroborate these findings have been initiated and are very early to yield meaningful conclusions.These data demonstrate that CD166 is one of the important mediators through which OM maintain HSC function. However, CD166-OM mediated HSC function is only maintained in conjunction with OB-MK interactions. Our data indicate the importance of crosstalk between OM, OB and MK which leads to the expression of novel mediators such as CD166 to support HSC function. DisclosuresNo relevant conflicts of interest to declare.

Activation of β-catenin signaling in MLO-Y4 osteocytic cells versus 2T3 osteoblastic cells by fluid flow shear stress and PGE 2: Implications for the study of mechanosensation in bone

The osteocyte is hypothesized to be the mechanosensory cell in bone. However, osteoblastic cell models have been most commonly used to investigate mechanisms of mechanosensation in bone. Therefore, we sought to determine if differences might exist between osteocytic and osteoblastic cell models relative to the activation of β-catenin signaling in MLO-Y4 osteocytic, 2T3 osteoblastic and primary neonatal calvarial cells (NCCs) in response to pulsatile fluid flow shear stress (PFFSS). β-catenin nuclear translocation was observed in the MLO-Y4 cells at 2 and 16 dynes/cm 2 PFFSS, but only at 16 dynes/cm 2 in the 2T3 or NCC cultures. The MLO-Y4 cells released high amounts of PGE 2 into the media at all levels of PFFSS (2–24 dynes/cm 2) and we observed a biphasic pattern relative to the level of PFFSS. In contrast PGE 2 release by 2T3 cells was only detected during 16 and 24 dynes/cm 2 PFFSS starting at > 1 h and never reached the levels produced by the MLO-Y4 cells. Exogenously added PGE 2 was able to induce β-catenin nuclear translocation in all cells suggesting that the differences between the cell lines observed for β-catenin nuclear translocation were associated with the differences in PGE 2 production. To investigate a possible mechanism for the differences in PGE 2 release by the MLO-Y4 and 2T3 cells we examined the regulation of Ptgs2 (Cox-2) gene expression by PFFSS. 2T3 cell Ptgs2 mRNA levels at both 0 and 24 h after 2 h of PFFSS showed biphasic increases with peaks at 4 and 24 dynes/cm 2 and 24-hour levels were higher than zero-hour levels. MLO-Y4 cell Ptgs2 expression was similarly biphasic; however at 24-hour post-flow Ptgs2 mRNA levels were lower. Our data suggest significant differences in the sensitivity and kinetics of the response mechanisms of the 2T3 and neonatal calvarial osteoblastic versus MLO-Y4 osteocytic cells to PFFSS. Furthermore our data support a role for PGE 2 in mediating the activation of β-catenin signaling in response to the fluid flow shear stress.

Stimulation of Ectopic Bone Formation in Response to BMP-2 by Rho Kinase Inhibitor: A Pilot Study

The small GTPase Rho and Rho-associated protein kinase (Rho kinase, ROCK) signal participates in a variety of biological functions including vascular contraction, tumor invasion, and penile erection. Evidence also suggests Rho-ROCK is involved in signaling for mesenchymal cellular differentiation. However, whether it is involved in osteoblastic differentiation is unknown. We therefore asked whether Rho-ROCK signaling participates in recombinant human bone morphogenetic protein (rhBMP-2)-induced osteogenesis both in vitro and in vivo. Continuous delivery of a specific ROCK inhibitor (Y-27632) enhanced ectopic bone formation induced by rhBMP-2 impregnated into an atelocollagen carrier in mice without affecting systemic bone metabolism. Treatment with Y-27632 also enhanced the osteoblastic differentiation of cultured murine neonatal calvarial cells. These effects were associated with increased expression of BMP-4 gene. Expression of a dominant negative mutant of ROCK in ST2 cells promoted osteoblastic differentiation, while a constitutively active mutant of ROCK attenuated osteoblastic differentiation and the ROCK inhibitor reversed this phenotype. Thus, ROCK inhibits osteogenesis, and a ROCK inhibitor in combination with the local delivery of rhBMP/collagen composite may be clinically applicable for stimulating bone formation.

Comparison of the Action of Transient and Continuous PTH on Primary Osteoblast Cultures Expressing Differentiation Stage-Specific GFP

Primary calvarial osteoblast cultures derived from type I collagen promoter-GFP reporter transgenic mice were used to examine progression of the osteoblast lineage. This system was validated by assessing the effect of PTH on osteoblast growth in real time. The anabolic effect of PTH seemed to be the result of enhanced osteoblast differentiation rather than expansion of a progenitor population. Activation of green fluorescent protein (GFP) marker genes driven by Col1a1 promoter fragments has been associated with the level of osteoblast differentiation. GFP-marked cultures provide an approach to continuously monitor the level of osteoblast differentiation in real time without the termination of cultures. Neonatal calvarial cells transgenic for pOBCol2.3GFP and pOBCol3.6GFP were used to establish calvarial osteoblast cultures. Parathyroid hormone (PTH) was added either continuous (days 1-21) or transient (days 1-7) to examine its diverse effect on osteoblast differentiation in cultures for 21 days. Three fluorescent markers were used: (1) pOBCol3.6GFP, which is activated in preosteoblastic cells; (2) pOBCol2.3GFP, which is restricted to differentiated osteoblasts; and (3) xylenol orange (XO), which stains the mineralized nodules. Progression of osteoblast differentiation indicated by fluorescent markers was documented throughout the entire period of culture. Recorded fluorescent images were analyzed in the patterns of expression and quantitated in the area of expression. Continuous PTH blocked osteoblast differentiation, which was evident by the attenuation of pOBCol3.6GFP and an absence of pOBCol2.3GFP. In contrast, transient PTH inhibited the initial osteoblast differentiation but ultimately resulted in a culture with more mineralized nodules and enhanced osteoblast differentiation expressing strong levels of pOBCol3.6GFP and pOBCol2.3GFP. Quantitative analysis showed that transient PTH first decreased then later increased areas of GFP expression and XO staining, which correlated with results of Northern blot and alkaline phosphatase activity. Transient PTH caused a decrease in DNA content during the treatment and after the removal of PTH. GFP-marked cultures combined with fluorescent image analysis have the advantage to assess the effect of PTH on osteoblast differentiation in real time. Results suggest that the anabolic effect of transient PTH is caused by an enhancement in osteoblast differentiation rather than an increase in the population of progenitor cells.

Use of VSV-G pseudotyped retroviral vectors to target murine osteoprogenitor cells.

Marrow stromal cells (MSC) and neonatal calvarial cells have the potential to differentiate and express markers of mature osteoblasts. Furthermore, MSCs can generate multiple differentiated connective tissue phenotypes. These properties and their ability to be expanded ex vivo make them good models for ex vivo gene therapy. In this study we examined the ability of vesicular stomatitis virus (VSV-G) pseudotyped retroviral vectors to transduce osteoprogenitor cells derived from bone marrow and from neonatal calvaria. Retrovectors encoding either beta-galactosidase or green fluorescent protein (eGFP) were used for transduction of primary murine marrow stromal and primary neonatal calvarial cell cultures. High infection efficiency was demonstrated by fluorescence-activated cell analysis when GFP was used as a marker or by estimating the number of beta-galactosidase-positive cells. Expression of markers of differentiated bone cells, including Col1a1, bone sialoprotein, and osteocalcin mRNA and alkaline phosphatase activity was not impaired by retroviral transduction. Our data suggest that VSV-G pseudotypes retroviral vectors are suitable for introducing genes into osteoprogenitor cells without affecting osteoprogenitor lineage progression.

Different responsiveness of cells from adult and neonatal mouse bone to mechanical and biochemical challenge.

Neonatal rodent calvarial bone cell cultures are often used to study bone cell responsiveness to biochemical and mechanical signals. However, mechanical strains in the skull are low compared to the axial and appendicular skeleton, while neonatal, rapidly growing bone has a more immature cell composition than adult bone. In the present study, we tested the hypothesis that bone cell cultures from neonatal and adult mouse calvariae, as well as adult mouse long bones, respond similarly to treatment with mechanical stress or 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3). Treatment with pulsating fluid shear stress (0.6 +/- 0.3 Pa, 5 Hz) caused a rapid (within 5 min) 2-4-fold increase in NO production in all cases, without significant differences between the three cell preparations. However, basal NO release was significantly higher in neonatal calvarial cells than adult calvarial and long bone cells. The response to 1,25(OH)2 D3), measured as increased alkaline phosphatase activity, was about three times higher in the neonatal cells than the adult cell cultures. We conclude that all three types of primary bone cell cultures responded similarly to fluid shear stress, by rapid production of NO. However, the neonatal cell cultures were different in basal metabolism and vitamin D3 responsiveness, suggesting that cell cultures from adult bone are best used for in vitro studies on bone cell biology.

The osteocyte working group

The osteocyte is hypothesized to be the mechanosensory cell in bone. However, osteoblastic cell models have been most commonly used to investigate mechanisms of mechanosensation in bone. Therefore, we sought to determine if differences might exist between osteocytic and osteoblastic cell models relative to the activation of β-catenin signaling in MLO-Y4 osteocytic, 2T3 osteoblastic and primary neonatal calvarial cells (NCCs) in response to pulsatile fluid flow shear stress (PFFSS). β-catenin nuclear translocation was observed in the MLO-Y4 cells at 2 and 16 dynes/cm2 PFFSS, but only at 16 dynes/cm2 in the 2T3 or NCC cultures. The MLO-Y4 cells released high amounts of PGE2 into the media at all levels of PFFSS (2–24 dynes/cm2) and we observed a biphasic pattern relative to the level of PFFSS. In contrast PGE2 release by 2T3 cells was only detected during 16 and 24 dynes/cm2 PFFSS starting at > 1 h and never reached the levels produced by the MLO-Y4 cells. Exogenously added PGE2 was able to induce β-catenin nuclear translocation in all cells suggesting that the differences between the cell lines observed for β-catenin nuclear translocation were associated with the differences in PGE2 production. To investigate a possible mechanism for the differences in PGE2 release by the MLO-Y4 and 2T3 cells we examined the regulation of Ptgs2 (Cox-2) gene expression by PFFSS. 2T3 cell Ptgs2 mRNA levels at both 0 and 24 h after 2 h of PFFSS showed biphasic increases with peaks at 4 and 24 dynes/cm2 and 24-hour levels were higher than zero-hour levels. MLO-Y4 cell Ptgs2 expression was similarly biphasic; however at 24-hour post-flow Ptgs2 mRNA levels were lower. Our data suggest significant differences in the sensitivity and kinetics of the response mechanisms of the 2T3 and neonatal calvarial osteoblastic versus MLO-Y4 osteocytic cells to PFFSS. Furthermore our data support a role for PGE2 in mediating the activation of β-catenin signaling in response to the fluid flow shear stress.

Osteoblast culture on bioerodible polymers: studies of initial cell adhesion and spread

AbstractThe development of systems for the growth of osteoblasts on bioerodible polymeric matrices was explored. Three classes of bioerodible polymers were studied as possible matrix supports for osteoblast growth: the poly(anhydrides), poly(phosphazenes) and poly(lactic acid/glycolic acid) copolymers. Neonatal calvarial cells from Sprague–Dawley rats were seeded onto polymer disks at a density of 1 × 104 cells/cm2. Initial attachment and spreading, rate of growth and morphology were determined, and retention of osteoblast‐like phenotype was assessed through measurements of alkaline phosphatase activity in the presence and absence of 1,25(OH)2 vitamin D3. All results were considered relative to tissue culture polystyrene.Cells were found to attach to all polymers at 8 hr post‐seeding. By 24 hr, cell numbers on all polymers were found to be decreased, except for poly(lactic acid/glycolic acid). Rat calvarial osteoblasts seeded on poly‐(lactic acid/glycolic acid) reached confluency and retained their phenotype.Successful construction of viable osteoblast–bioerodible polymer composite materials, as presented in our study, may find their usefulness as grafts for atrophic non‐unions of bone, for healing craniofacial and other defects and for use as prosthetic implants or coatings. Composite systems of osteoblast cultures may also find their usefulness in furthering our understanding of bone differentiation, maturation and metabolism in a matrix environment.