Adult Mouse Bone Research Articles

Isolation and Assessment of Single Long-Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow.

Hematopoietic stem cells with long-term repopulating activity can now be routinely obtained at purities of 40% to 50% from suspensions of adult mouse bone marrow. Here we describe robust protocols for both their isolation as CD45(+) EPCR(+) CD150(+) CD48(-) (ESLAM) cells using multiparameter cell sorting and for tracking their clonal growth and differentiation activity in irradiated mice transplanted with single ESLAM cells. The simplicity of these procedures makes them attractive for characterizing the molecular and biological properties of individual hematopoietic stem cells with unprecedented power and precision. © 2016 by John Wiley & Sons, Inc.

Dietary dried plum increases bone mass, suppresses proinflammatory cytokines and promotes attainment of peak bone mass in male mice

Nutrition is an important determinant of bone health and attainment of peak bone mass. Diets containing dried plum (DP) have been shown to increase bone volume and strength. These effects may be linked to the immune system and DP-specific polyphenols. To better understand these relationships, we studied DP in skeletally mature (6-month-old) and growing (1- and 2-month-old) C57Bl/6 male mice. In adult mice, DP rapidly (<2 weeks) increased bone volume (+32%) and trabecular thickness (+24%). These changes were associated with decreased osteoclast surface (Oc.S/BS) and decreased serum CTX, a marker of bone resorption. The reduction in Oc.S/BS was associated with a reduction in the osteoclast precursor pool. Osteoblast surface (Ob.S/BS) and bone formation rate were also decreased suggesting that the gain in bone in adult mice is a consequence of diminished bone resorption and formation, but resorption is reduced more than formation. The effects of DP on bone were accompanied by a decline in interleukins, TNF and MCP-1, suggesting that DP is acting in part through the immune system to suppress inflammatory activity and reduce the size of the osteoclast precursor pool. Feeding DP was accompanied by an increase in plasma phenolics, some of which have been shown to stimulate bone accrual. In growing and young adult mice DP at levels as low as 5% of diet (w/w) increased bone volume. At higher levels (DP 25%), bone volume was increased by as much as 94%. These data demonstrate that DP feeding dramatically increases peak bone mass during growth.

A Simple Method for Isolation, Propagation, Characterization, and Differentiation of Adult Mouse Bone Marrow-Derived Multipotent Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are spindle-shaped, adherent, clonogenic, non-phagocytic, fibroblastic and multipotent in nature with the intrinsic ability of self-renewal and proliferation. Bone marrow is the richest source of MSCs for studying the underlying processes of proliferation, self-renewal, and multiple-lineage differentiation. There have been several studies to improve the method of isolation, propagation, characterization and differentiation of mesenchymal stem cells from the mouse bone marrow, but none are widely acceptable. Owing to unavailability of universally acceptable method of MSCs culture continuous efforts are being made in this direction. Here, we report a simple method with some subtle modifications aiming to improve the overall method of isolation, culture, propagation and differentiation of MSCs in vitro. Following this protocol, we have isolated MSCs with typical spindle-shaped morphology as shown by scanning electron microscopy. These cells also showed expression of MSC-specific markers, CD29 (98.94% ± 0.67%), CD44 (84.27% ± 7.77%), Sca-1 (92.70% ± 3.81%) with negligible expression of HSC-specific markers such as CD45 (0.40% ± 0.10%), CD34 (0.15% ± 0.05%) and CD11b (0.45% ± 0.15%). MSCs were also found to differentiate into mesodermal lineages such as adipocytes, osteocytes and chondrocytes as well as ectodermal neuron-like cells. Moreover, MSCs showed differential basal expression of pluripotency-associated transcription factors such as Oct4, Nanog, Sox2 and Myc. Based on the above findings, we propose a simple protocol that can be used to isolate, culture, propagate and characterize multipotent MSCs from mouse bone marrow for experimental and application purposes.

Identification and Characterization of Lineage(-)CD45(-)Sca-1(+) VSEL Phenotypic Cells Residing in Adult Mouse Bone Tissue.

Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs.

Thrombospondin-2 deficiency in growing mice alters bone collagen ultrastructure and leads to a brittle bone phenotype.

Thrombospondin-2 (TSP2) is a matricellular protein component of the bone extracellular matrix. Long bones of adult TSP2-deficient mice have increased endosteal bone thickness due to expansion of the osteoblast progenitor cell pool, and these cells display deficits in osteoblastic potential. Here, we investigated the effects of TSP2 deficiency on whole bone geometric and mechanical properties in growing 6-wk-old male and female wild-type and TSP2-knockout (KO) mice. Microcomputed tomography and mechanical testing were conducted on femora and L2 vertebrae to assess morphology and whole bone mechanical properties. In a second series of experiments, femoral diaphyses were harvested from wild-type and TSP2-KO mice. Detergent-soluble type I collagen content was determined by Western blot of right femora. Total collagen content was determined by hydroxyproline analysis of left femora. In a third series of experiments, cortical bone was dissected from the anterior and posterior aspects of the femoral middiaphysis and imaged by transmission electron microscopy to visualize collagen fibrils. Microcomputed tomography revealed minimal structural effects of TSP2 deficiency. TSP2 deficiency imparted a brittle phenotype on cortical bone. Femoral tissue mineral density was not affected by TSP2 deficiency. Instead, transmission electron microscopy revealed less intensely stained collagen fibrils with altered morphology in the extracellular matrix assembled by osteoblasts on the anterior surface of TSP2-KO femora. Femoral diaphyseal bone displayed comparable amounts of total collagen, but the TSP2-KO bones had higher levels of detergent-extractable type I collagen. Together, our data suggest that TSP2 is required for optimal collagen fibrillogenesis in bone and thereby contributes to normal skeletal tissue quality.

Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis.

The milk fat globule-EGF-factor 8 protein (MFG-E8) has been identified in various tissues, where it has an important role in intercellular interactions, cellular migration, and neovascularization. Previous studies showed that MFG-E8 is expressed in different cell types under normal and pathophysiological conditions, but its expression in hematopoietic stem cells (HSCs) during hematopoiesis has not been reported. In the present study, we investigated MFG-E8 expression in multiple hematopoietic tissues at different stages of mouse embryogenesis. Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34+ HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis. Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34+ cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34+ cells, but not CD34− cells, highly expressed MFG-E8. We also found that MFG-E8 was not expressed in HSCs in adult mouse bone marrow, and that its expression was confined to F4/80+ macrophages. Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis.

Women Build Long Bones With Less Cortical Mass Relative to Body Size and Bone Size Compared With Men.

The twofold greater lifetime risk of fracturing a bone for white women compared with white men and black women has been attributed in part to differences in how the skeletal system accumulates bone mass during growth. On average, women build more slender long bones with less cortical area compared with men. Although slender bones are known to have a naturally lower cortical area compared with wider bones, it remains unclear whether the relatively lower cortical area of women is consistent with their increased slenderness or is reduced beyond that expected for the sex-specific differences in bone size and body size. Whether this sexual dimorphism is consistent with ethnic background and is recapitulated in the widely used mouse model also remains unclear. We asked (1) do black women build bones with reduced cortical area compared with black men; (2) do white women build bones with reduced cortical area compared with white men; and (3) do female mice build bones with reduced cortical area compared with male mice? Bone strength and cross-sectional morphology of adult human and mouse bone were calculated from quantitative CT images of the femoral midshaft. The data were tested for normality and regression analyses were used to test for differences in cortical area between men and women after adjusting for body size and bone size by general linear model (GLM). Linear regression analysis showed that the femurs of black women had 11% lower cortical area compared with those of black men after adjusting for body size and bone size (women: mean=357.7 mm2; 95% confidence interval [CI], 347.9-367.5 mm2; men: mean=400.1 mm2; 95% CI, 391.5-408.7 mm2; effect size=1.2; p<0.001, GLM). Likewise, the femurs of white women had 12% less cortical area compared with those of white men after adjusting for body size and bone size (women: mean=350.1 mm2; 95% CI, 340.4-359.8 mm2; men: mean=394.3 mm2; 95% CI, 386.5-402.1 mm2; effect size=1.3; p<0.001, GLM). In contrast, female and male femora from recombinant inbred mouse strains showed the opposite trend; femurs from female mice had a 4% larger cortical area compared with those of male mice after adjusting for body size and bone size (female: mean=0.73 mm2; 95% CI, 0.71-0.74 mm2; male: mean=0.70 mm2; 95% CI, 0.68-0.71 mm2; effect size=0.74; p=0.04, GLM). Female femurs are not simply a more slender version of male femurs. Women acquire substantially less mass (cortical area) for their body size and bone size compared with men. Our analysis questions whether mouse long bone is a suitable model to study human sexual dimorphism. Identifying differences in the way bones are constructed may be clinically important for developing sex-specific diagnostics and treatment strategies to reduce fragility fractures.

Increased endoplasmic reticulum stress in mouse osteocytes with aging alters Cox-2 response to mechanical stimuli.

Aging reduces bone mass as well as the anabolic response of bone to mechanical stimuli, resulting in osteopenia. Endoplasmic reticulum (ER) stress impairs the response of myogenic cells to anabolic stimuli, and is involved in sarcopenia, but whether ER stress also contributes to osteopenia is unknown. Therefore, we tested whether ER stress exists in bones of aged mice, and whether this impairs the osteocyte response to mechanical stimulation. Primary osteocytes were obtained from long bones of adult (8 months) and old (24-26 months) mice, treated with or without the pharmacological ER stress inducer tunicamycin, and either or not subjected to mechanical loading by pulsating fluid flow (PFF). The osteocyte response to PFF was assessed by measuring cyclooxygenase-2 (Cox-2) mRNA levels and nitric oxide (NO) production. mRNA levels of ER stress markers were higher in old versus adult osteocytes (+40% for activating transcription factor-4, +120% for C/EBP homologous protein, and +120% for spliced X-box binding protein-1, p < 0.05). The Cox-2 response to PFF was fourfold decreased in cells from old bones (p < 0.001), while tunicamycin decreased PFF-induced Cox-2 expression by threefold in cells from adult bones (p < 0.01). PFF increased NO production by 50% at 60 min in osteocytes from old versus adult bones (p < 0.01). In conclusion, our data indicate that the expression of several ER stress markers was higher in osteocytes from bones of old compared to adult mice. Since ER stress altered the response of osteocytes to mechanical loading, it could be a novel factor contributing to osteopenia.

CCR1-Mediated Formation of Liver Microenvironment for Cancer Metastasis

Accumulating evidence suggests that bone marrow-derived myeloid cells constitute the major components of cancer microenvironment and play significant roles in the progression of cancer. We have previously shown that myeloid cells expressing CCR1, a chemokine receptor, are recruited to the microenvironment of disseminated colon cancer cells in the liver (PNAS 2010). These CCR1+ cells promote metastatic colonization of cancer cells in the liver through producing metalloproteinases MMP9 and MMP2. However, the cell lineage and precise characteristics of these myeloid cells were largely unknown. The aim of this study is to characterize the cell types among these myeloid cells expressing CCR1 and/or MMPs.In order to characterize the CCR1-expressing myeloid cells accumulating at liver metastatic foci, we have established bacterial artificial chromosome (BAC)-based transgenic mouse lines in which membrane-targeted Venus fluorescent protein (mVenus) was expressed under the control of Ccr1cis-regulatory elements. A flow cytometric analysis of adult transgenic mouse bone marrow cells showed that expression of the reporter mVenus fluorescence was restricted to the CD11b+ Gr-1+ cells and the level of endogenous Ccr1 mRNA correlated well with that of mVenus fluorescence, suggesting that expression of the reporter precisely reflected that of the endogenous CCR1.Inoculation of CCL9-expressing mouse colon cancer cell line CMT93 into the spleen of syngeneic hosts causes efficient metastatic colonization of the cancer cells in the liver. Employing this model, we monitored the behavior of the Ccr1-mVenus+ myeloid cells in the cancer metastasis microenvironment, and found that CD45+ CD11b+Ccr1-mVenus+ cells purified from liver metastatic foci were mostly neutrophils. In contrast, within CD45+ CD11b+Ccr1-mVenus– fraction, we identified eosinophils, monocytes and fibrocytes. Notably, the major cell types in the metastatic foci were monocytes and fibrocytes, with the latter producing abundant collagen.Chronologically, accumulation of neutrophils and monocytes preceded that of eosinophils and fibrocytes. In Ccr1−/− mice, early accumulation of Ccr1-mVenus+ Gr-1+ cells was significantly compromised and later recruitment of fibrocytes to the metastatic foci was reduced. Antibody-mediated depletion of neutrophils caused a significant reduction in the fibrocyte number at the metastatic foci. These results suggest that CCR1 plays critical roles in early accumulation of neutrophils and subsequent recruitment of fibrocytes.After having identified four types of myeloid cells in the metastatic foci, we determined the expression levels of CCR1, MMP9 and MMP2 mRNAs in each purified population, and found that CCR1 and MMP9 are expressed preferentially by neutrophils, whereas MMP2 is expressed exclusively by monocytes/fibrocytes. Considering the chronological difference in the appearance of myeloid cells in liver metastatic foci, these results suggest that production of MMP9 and MMP2 in succession by different myeloid cells is necessary for disseminated colon cancer cells to colonize in the liver.In summary, these results suggests that CCR1 is required for recruitment of neutrophils in the early phase of colon cancer dissemination and the subsequent accumulation of fibrocytes, and that MMP9 and MMP2 are produced by neutrophils and fibrocytes, respectively, in a successive manner during the formation of liver metastasis. Further investigation of these cancer-driven host reactions in human colon cancer dissemination may lead to effective preventive measures against cancer metastasis. DisclosuresNo relevant conflicts of interest to declare.

Thyroid hormone interacts with the sympathetic nervous system to modulate bone mass and structure in young adult mice.

To investigate whether thyroid hormone (TH) interacts with the sympathetic nervous system (SNS) to modulate bone mass and structure, we studied the effects of daily T3 treatment in a supraphysiological dose for 12 wk on the bone of young adult mice with chronic sympathetic hyperactivity owing to double-gene disruption of adrenoceptors that negatively regulate norepinephrine release, α(2A)-AR, and α(2C)-AR (α(2A/2C)-AR(-/-) mice). As expected, T3 treatment caused a generalized decrease in the areal bone mineral density (aBMD) of WT mice (determined by DEXA), followed by deleterious effects on the trabecular and cortical bone microstructural parameters (determined by μCT) of the femur and vertebra and on the biomechanical properties (maximum load, ultimate load, and stiffness) of the femur. Surprisingly, α(2A/2C)-AR(-/-) mice were resistant to most of these T3-induced negative effects. Interestingly, the mRNA expression of osteoprotegerin, a protein that limits osteoclast activity, was upregulated and downregulated by T3 in the bone of α(2A/2C)-AR(-/-) and WT mice, respectively. β1-AR mRNA expression and IGF-I serum levels, which exert bone anabolic effects, were increased by T3 treatment only in α(2A/2C)-AR(-/-) mice. As expected, T3 inhibited the cell growth of calvaria-derived osteoblasts isolated from WT mice, but this effect was abolished or reverted in cells isolated from KO mice. Collectively, these findings support the hypothesis of a TH-SNS interaction to control bone mass and structure of young adult mice and suggests that this interaction may involve α2-AR signaling. Finally, the present findings offer new insights into the mechanisms through which TH regulates bone mass, structure, and physiology.

Tibial bone versican content decreases with zoledronate treatment in adult mice

In bone remodeling, the expression and turnover of the proteoglycans versican and aggrecan are poorly understood. We report changes in adult mouse bone contents of versican and aggrecan associated with bothage and treatment with the drug zoledronate. The data may have implications for experimental animal models of osteoporosis and related conditions. Versican and aggrecan are large, aggregating proteoglycans involved in skeletal development, but little is known about their roles in bone remodeling. The purpose of this study was to investigate versican and aggrecan contents in adult mouse bones, and changes in their contents in response to the bisphosphonate zoledronate (ZOL). Mice (9weeks old) were treated with 125μg/kg ZOL or vehicle for 3 or 15weeks. Versican and aggrecan were isolated from tibial bones for Western blotting, automated integrated densitometry, and analysis (two-way ANOVA, α = 0.05). In ZOL-treated mouse bones, compared to vehicle, 340 and 60kDa versican content decreased significantly, and 100 and 60kDa aggrecan content decreased significantly (drug effect). In 24-week-old mouse bones, compared to 12weeks, statistically significant decreases were observed in 340, 80, 60, and 11kDa versican, and in 100, 70, and 40kDa aggrecan (age effect). There was a statistically significant ZOL-age interaction for 330kDa aggrecan. This is the first study to assess physiological versican and aggrecan adaptations in adult mammalian bone tissue, in the presence and absence of ZOL. We observed large decreases in some versican and aggrecan species from 12 to 24weeks. We also observed decreases in several versican and aggrecan species in the presence of ZOL. This indicates that bone proteoglycan expression and turnover may be important in bone remodeling.

Bone marrow Schwann cells induce hematopoietic stem cell hibernation.

Hematopoietic stem cells (HSCs) are clonogenic cells capable of both self-renewal and multilineage differentiation. In adult mouse bone marrow (BM), most HSCs remain in the non-dividing G0-phase of cell cycle, in close contact with supporting cells known as the HSC "niche". In the present study, we focused on signaling mechanisms that regulate stem cell dormancy in the BM niche. We show that TGF-β type II receptor deficiency causes reduced phosphorylation of Smad2/3 and impairs long-term repopulating activity in HSCs, suggesting a significant role for TGF-β/Smad signaling in hematopoiesis. Furthermore, we aimed at defining the candidate BM niche responsible for homeostasis of hematopoiesis, and revealed that non-myelinating Schwann cells sustain HSC hibernation by converting TGF-β from its latent to its active form.

Unique progenitors in adult mouse bone marrow expressing CD127 (IL-7Rα) and high level of cKit efficiently develop into all lymphocytes including T cells and innate lymphoid cells (HEM2P.266)

Abstract All lymphocytes are thought to develop from common lymphoid progenitors (CLPs) that are found in the Lin−Sca-1locKitloCD127+Flt3hi bone marrow (BM) cell population. However, only small numbers of T cells and innate lymphoid cells (ILCs) are generated from CLPs transplanted into irradiated mice, suggesting that they might mainly develop from alternative progenitors such as lymphoid-primed multipotent progenitors (LMPPs). We now have identified novel progenitors in adult mouse BM that efficiently develop into all lymphocytes including T cells and ILC2s. The novel progenitors are similar to LMPPs and found in the cKithiSca-1+Flt3+ BM cell subset, but unlike LMPPs they express the lymphoid lineage marker CD127, thus resembling early thymic progenitors (ETPs). Upon transplantation into irradiated mice, CD127+ LMPP-like cells robustly engraft and develop into T, B, NK cells and ILC2s. They are more efficient than LMPPs and CLPs in generating T cells and ILC2s. Similar to LMPPs, they also retain myeloid potentials. Flt3L-deficient mice have greatly reduced LMPPs and CLPs but have almost normal numbers of CD127+ LMPP-like progenitors, pre-ILC2 in the BM and mature ILC2 in the lung. While the exact relationship between CD127+ LMPP-like progenitors, CD127- LMPPs and CLPs are yet to be clarified, these results suggest that CD127+ LMPP-like BM cells are important progenitors for T cells and ILC2s independent of CLPs.

Cytoskeletal organization during terminal differentiation and enucleation of mammalian erythroblasts (538.3)

Terminal differentiation of red blood cells is a complex morphogenetic process involving chromatin condensation, cell polarization and nuclear expulsion. Microtubules are important for cell polarization, while F‐actin assembly and myosin activity are important for nuclear expulsion. In this study, we used confocal microscopy to examine erythroblasts from mouse fetal liver, adult bone marrow and phlebotomized mouse spleen. In non‐polarized erythroblasts, the F‐actin cytoskeleton is organized into a cage‐like structure surrounding the nucleus. In polarized erythroblasts from fetal liver or spleen, the F‐actin reorganizes to a bright spot at the side of the nucleus. Mitochondria are relocated from the periphery of the cell to this actin spot. This bright spot of F‐actin and the mitochondria travels with the rear of the nucleus as it is ejected. Additional foci of F‐actin are located at the neck separating the expelled nucleus from the reticulocyte. Linear cables of F‐actin are also observed in the nuclear ‘bud’ which are sometimes associated with nuclear pores. Notably, adult mouse bone marrow lacks the bright spot of F‐actin near the nucleus during nuclear expulsion. Arp2/3 and mDia2 are not associated with the bright F‐actin while myosinIIB is associated with these foci. We conclude that the actin cytoskeletal organization of enucleating erythroblasts is not conserved among different erythropoietic tissues.

0311: Role of AMP-activated protein kinase in regulating proliferation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischemic heart disease. Mechanisms underlying control of their proliferation capacity, which is known to be inversely correlated to their differentiation abilities, have not been fully characterized. The AMP-activated protein kinase (AMPK) controls several cell functions including cell proliferation. We hypothesized that AMPK plays a crucial role in the control of MSC proliferation. MSCs isolated from WT or AMPKα1 KO adult mouse bone marrow were stimulated with the specific AMPK activator, A769662. Protein phosphorylation state was determined by immunoblotting, cell viability was quantified by FACS after AnnexinV and Propidium Iodide staining, cell proliferation was quantified by EdU incorporation and gene expression was analyzed by real-time PCR. MSCs isolated from murine bone marrow exclusively expressed the AMPKa1 catalytic subunit. A769662 was able to induce a robust and sustained AMPK activation in WT MSCs but not in AMPKa1 KO MSCs. This A769662-dependent AMPK activation blocks WT MSC proliferation but not in KO MSCs without affecting their viability. Moreover, AMPKa1 deletion induces an increase of basal MSC proliferation. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression prevented the A-769662- dependent inhibition of MSC proliferation. These results suggest that activating AMPK promotes a principal prerequisite for cell differentiation, the arrest of MSC proliferation via p27 regulation.

Stress Hematopoiesis Is Regulated by the Krüppel-Like Transcription Factor ZBP-89

Previous studies have shown that ZBP-89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBP-89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89(-/-) HSPC also resulted in a myeloid-to-B lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89(-/-) HSPC are reproduced in vitro in the ZBP-89-silenced multipotent hematopoietic cell line FDCP-Mix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATA-1 in ZBP89-deficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBP-89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATA-1. These data identify an important role for ZBP-89 in regulating stress hematopoiesis in adult mouse bone marrow.

Mutation in Osteoactivin Decreases Bone Formation in Vivo and Osteoblast Differentiation in Vitro

We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation invitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared to that in wild-type mice [DBA/2J-Gpnmb(+)/SjJ (D2J/Gpnmb(+))]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb(+) mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. Invitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb(+) osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-β receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-β signaling. These data confirm the anabolic role of OA in postnatal bone formation.

Efficacy of Bone Healing in Calvarial Defects Using Piezoelectric Surgical Instruments

This study compared bone healing following the use of 2 piezoelectric surgery units or conventional mechanical cutting with carbide and diamond drills to explore their future applications for bone surgery. Subcritical-size (approximately 1.5-2 mm) calvarial defects were created in the parietal bones of adult mice. Following defect standardization, a full-thickness semicircular defect was created on the parietal bones of 12 mice divided into 4 groups: carbide bur, Surgystar, diamond bur, and Piezoelectric System. Hard tissue healing was assessed using micro-computed tomography at 1 day, 2 weeks, 4 weeks, and 8 weeks after surgery. At 4 weeks, the Surgystar group and Piezoelectric System group showed a significant difference from the carbide group. The Surgystar and Piezoelectric System groups did differ from the diamond group. At 8 weeks, the Surgystar and Piezoelectric System groups differed significantly from the carbide and diamond groups. The fraction of healing results over the 8 weeks demonstrated that the Surgystar group had a significantly higher bone healing percentage than did the carbide group (P = 0.001) and the diamond group (P = 0.026), but it did not differ significantly from the Piezoelectric System group (P = 0.420). The Surgystar and Piezoelectric System are suitable for bone osteotomy and provide faster bone healing in comparison with mechanical instrumentation.

Histone Lysine-specific Demethylase 1 (LSD1) Protein Is Involved in Sal-like Protein 4 (SALL4)-mediated Transcriptional Repression in Hematopoietic Stem Cells

The stem cell protein SALL4 plays a critical role in hematopoiesis by regulating the cell fate. In primitive hematopoietic precursors, it activates or represses important genes via recruitment of various epigenetic factors such as DNA methyltransferases, and histone deacylases. Here, we demonstrate that LSD1, a histone lysine demethylase, also participates in the trans-repressive effects of SALL4. Based on luciferase assays, the amine oxidase domain of LSD1 is important in suppressing SALL4-mediated reporter transcription. In freshly isolated adult mouse bone marrows, both SALL4 and LSD1 proteins are preferentially expressed in undifferentiated progenitor cells and co-localize in the nuclei. Further sequential chromatin immunoprecipitation assay confirmed that these two factors share the same binding sites at the promoter regions of important hematopoietic regulatory genes including EBF1, GATA1, and TNF. In addition, studies from both gain- and loss-of-function models revealed that SALL4 dynamically controls the binding levels of LSD1, which is accompanied by a reversely changed histone 3 dimethylated lysine 4 at the same promoter regions. Finally, shRNA-mediated knockdown of LSD1 in hematopoietic precursor cells resulted in altered SALL4 downstream gene expression and increased cellular activity. Thus, our data revealed that histone demethylase LSD1 may negatively regulate SALL4-mediated transcription, and the dynamic regulation of SALL4-associated epigenetic factors cooperatively modulates early hematopoietic precursor proliferation.

Undifferentiated human adipose‐derived stromal/stem cells loaded onto wet‐spun starch–polycaprolactone scaffolds enhance bone regeneration: Nude mice calvarial defect in vivo study

The repair of large bony defects remains challenging in the clinical setting. Human adipose-derived stromal/stem cells (hASCs) have been reported to differentiate along different cell lineages, including the osteogenic. The objective of the present study was to assess the bone regeneration potential of undifferentiated hASCs loaded in starch-polycaprolactone (SPCL) scaffolds, in a critical-sized nude mice calvarial defect. Human ASCs were isolated from lipoaspirate of five female donors, cryopreserved, and pooled together. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an SPCL scaffold alone, or SPCL scaffold with human ASCs. Histological analysis and Micro-CT imaging of the retrieved implants were performed. Improved new bone deposition and osseointegration was observed in SPCL loaded with hASC engrafted calvarial defects as compared to control groups that showed little healing. Nondifferentiated human ASCs enhance ossification of nonhealing nude mice calvarial defects, and wet-spun SPCL confirmed its suitability for bone tissue engineering. This study supports the potential translation for ASC use in the treatment of human skeletal defects.