Bone Marrow Stromal Cell Cultures Research Articles

Anabolic effects of IGF-1 signaling on the skeleton

This review focuses on the anabolic effects of IGF-1 signaling on the skeleton, emphasizing the requirement for IGF-1 signaling in normal bone formation and remodeling. We first discuss the genomic context, splicing variants, and species conservation of the IGF-1 locus. The modulation of IGF-1 action by growth hormone (GH) is then reviewed while also discussing the current model which takes into account the GH-independent actions of IGF-1. Next, the skeletal phenotypes of IGF-1-deficient animals are described in both embryonic and postnatal stages of development, which include severe dwarfism and an undermineralized skeleton. We then highlight two mechanisms by which IGF-1 exerts its anabolic action on the skeleton. Firstly, the role of IGF-1 signaling in the modulation of anabolic effects of parathyroid hormone (PTH) on bone will be discussed, presenting in vitro and in vivo studies that establish this concept and the proposed underlying molecular mechanisms involving Indian hedgehog (Ihh) and the ephrins. Secondly, the crosstalk of IGF-1 signaling with mechanosensing pathways will be discussed, beginning with the observation that animals subjected to skeletal unloading by hindlimb elevation are unable to mitigate cessation of bone growth despite infusion with IGF-1 and the failure of IGF-1 to activate its receptor in bone marrow stromal cell cultures from unloaded bone. Disrupted crosstalk between IGF-1 signaling and the integrin mechanotransduction pathways is discussed as one of the potential mechanisms for this IGF-1 resistance. Next, emerging paradigms on bone-muscle crosstalk are examined, focusing on the potential role of IGF-1 signaling in modulating such interactions. Finally, we present a future outlook on IGF research.

Evidence of a Hematopoietic Origin of Human Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) have been considered to be of an ontological lineage distinct from hematopoietic cells in the bone marrow. Numerous studies have shown that BMSCs in culture exhibit an immunophenotype negative for hematopoietic markers such as CD45, CD34 and CD11b. In this brief report, we discover that human BMSCs can be isolated by positive selection from CD45+ or CD34+ cells of whole bone marrow aspirates, but not CD11b+ fractions. Adherent cells from the CD34+ and CD45+ fractions displayed growth, morphology, surface markers, and in vitro multipotency consistent with BMSCs from whole marrow. These cells were then transplanted subcutaneously in mice and were found to support the formation of hematopoietic tissue in ectopic sites. These results suggest an unidentified link between human hematopoietic cells and BMSCs and may point to the existence of a common progenitor.

Conditional Mesenchymal Disruption of Pkd1 Results in Osteopenia and Polycystic Kidney Disease

Conditional deletion of Pkd1 in osteoblasts using either Osteocalcin(Oc)-Cre or Dmp1-Cre results in defective osteoblast-mediated postnatal bone formation and osteopenia. Pkd1 is also expressed in undifferentiated mesenchyme that gives rise to the osteoblast lineage. To examine the effects of Pkd1 on prenatal osteoblast development, we crossed Pkd1 flox/flox and Col1a1(3.6)-Cre mice, which has been used to achieve selective inactivation of Pkd1 earlier in the osteoblast lineage. Control Pkd1 flox/flox and Pkd1 flox/+, heterozygous Col1a1(3.6)-Cre;Pkd1 flox/+ and Pkd1 flox/null, and homozygous Col1a1(3.6)-Cre;Pkd1 flox/flox and Col1a1(3.6)-Cre;Pkd1 flox/null mice were analyzed at ages ranging from E14.5 to 8-weeks-old. Newborn Col1a1(3.6)-Cre;Pkd1 flox/null mice exhibited defective skeletogenesis in association with a greater reduction in Pkd1 expression in bone. Conditional Col1a1(3.6)-Cre;Pkd1 flox/+ and Col1a1(3.6)-Cre;Pkd1 flox/flox mice displayed a gene dose-dependent decrease in bone formation and increase in marrow fat at 6 weeks of age. Bone marrow stromal cell and primary osteoblast cultures from homozygous Col1a1(3.6)-Cre;Pkd1 flox/flox mice showed increased proliferation, impaired osteoblast development and enhanced adipogenesis ex vivo. Unexpectedly, we found evidence for Col1a1(3.6)-Cre mediated deletion of Pkd1 in extraskeletal tissues in Col1a1(3.6)-Cre;Pkd1 flox/flox mice. Deletion of Pkd1 in mesenchymal precursors resulted in pancreatic and renal, but not hepatic, cyst formation. The non-lethality of Col1a1(3.6)-Cre;Pkd1 flox/flox mice establishes a new model to study abnormalities in bone development and cyst formation in pancreas and kidney caused by Pkd1 gene inactivation.

Nuclear fibroblast growth factor 2 (FGF2) isoforms inhibit bone marrow stromal cell mineralization through FGF23/FGFR/MAPK in vitro

Fibroblast growth factor 23 (FGF23) is responsible for phosphate wasting and the phenotypic changes observed in human diseases such as X-linked hypophosphatemia (XLH). Targeted overexpression of nuclear high-molecular weight fibroblast growth factor 2 isoforms (HMW isoforms) in osteoblasts resulted in a transgenic mouse with phenotypic changes similar to XLH, including increased FGF23, hypophosphatemia, and rickets/osteomalacia. The goal of this study was to assess whether HMW isoforms also reduced mineralized bone formation via phosphate-independent effects in bone marrow stromal cells (BMSCs) by modulating FGF23/FGF receptor (FGFR)/extracellular signal-regulated kinase (ERK) signaling. To determine if decreased bone formation in BMSC cultures from HMW transgenic mice could be rescued by blocking this pathway, an FGF23 neutralizing antibody, the FGFR tyrosine kinase inhibitor SU5402 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059 were used. FGF23 levels in the conditioned medium of HMW BMSC cultures were dramatically increased compared to BMSC from control (Vector) mice. Mineralized nodule formation was significantly decreased in HMW BMSC cultures compared with control cultures. The decreased nodule formation in HMW cultures was partially rescued by the FGF23 neutralizing antibody, SU5402 and PD98059. mRNA levels for the osteoblast-related genes, osteocalcin, Runt-related transcription factor 2 (Runx2), and osterix, and the osteocyte-related gene dentin matrix acidic phosphoprotein 1 (Dmp1) were significantly decreased in HMW cultures compared with control cultures, and the decreases were partially rescued by SU5402 or PD98059 treatment. Matrix-gla-protein (Mgp) mRNA was significantly higher in HMW cultures compared with control cultures, reduced by SU5402, but further increased by PD98059. Our results suggest that phosphate-independent effects of HMW isoforms in vitro may be directly mediated in part via FGF23 and that HMW isoforms signal via FGF23/FGFR/MAPK to inhibit bone formation in vitro.

Identification of a Candidate Proteomic Signature to Discriminate Multipotent and Non-Multipotent Stromal Cells

Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105−) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

Abstract LB-307: Modeling the breast cancer metastatic niche

Abstract Mortality of breast cancer patients is due overwhelmingly to the metastatic spread of the disease. There is compelling evidence that dissemination of breast cancer cells at distant sites is an early event. At the time of detection and diagnosis, patients have disseminated breast cancer cells in the bone marrow (BM). In some of these patients the disseminated cells proliferate and generate metastases, typically in 3-5 years for ER negative tumors and 10-15 years for ER positive tumors. In other patients metastases never develop. These clinical characteristics offer the opportunity for intervention aimed to prevent metastasis development, provided the mechanisms underlying growth at the metastatic site are better understood. A challenge in this undertaking has been the lack of models for disseminated cells survival, dormancy and proliferation. We have established novel experimental systems that model the bone microenvironment of the breast cancer metastatic niche in vitro and in vivo. These models are based on 3D complex cultures of human BM stromal cells and breast cancer cell lines (BCCL) in biomatrices. In one of these systems all of the cell components are defined, represented by mesenchymal cell lines (HS-5), osteoblasts (hFOB) & endothelial cells (HUVEC) co-cultivated in a collagen 3D matrix (BM Niche Model I). The second system uses primary human BM stromal cells (BM Niche Model II). Small numbers of BCCLs are added to matrix pre-seeded to sub-confluency with niche cells in order to model conditions of disseminated tumor cells. The cell lines used in the niche models differentiate along osteogenic and adipogenic lineages, and support survival of hematopoietic stem cells. Our studies demonstrate that in BM Niche Model I, BCCLs remain dormant, but retain their ability to divide when re-plated in standard culture conditions. Whereas in BM Niche Model II, BCCLs proliferate. Importantly the proliferation of BCCLs in this model is similar to that observed in intratibial inoculation models in vivo. These matrices pre-seeded with cells can also be implanted subcutaneously, where they are readily vascularised. Comparing the profile of secreted cytokines and growth factors in these two BM niche models, we identified twenty factors (growth factors, cytokines, cognate receptors and metalloproteinases) necessary for survival and proliferation of disseminated breast cancer cells in the bone marrow microenvironment. Some of these factors have been previously associated with higher risk of metastasis development. We are currently validating these candidates in our in vitro culture systems, and using subcutaneous implantation and intratibial injection in vivo. These BM Niche model systems will be instrumental in understanding the biology of disseminated tumour cells and metastasis formation. They also have the potential application for in vitro drug screening to identify anti-metastatic treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-307. doi:1538-7445.AM2012-LB-307

Abstract 3206: Three-dimensional hyaluronan hydrogel culture systems facilitate study of the complex microenvironment of bone metastatic prostate cancer cells

Abstract The bone marrow microenvironment is a complex milieu, containing various stromal and endothelial cell populations as well as a unique extracellular matrix. To better understand cellular interactions that occur in potentially lethal stage IV prostate cancer (PCa), it is helpful to recreate the bone marrow microenvironment. Traditional two dimensional (2D) cell culture systems do not accurately portray this environment, since culture on plastic activates atypical signaling pathways and makes co-culture of various cell types difficult. To overcome these limitations, we developed novel methods of three-dimensional (3D) culture to study the biology of bone metastatic PCa cells. These 3D culture systems utilize ECM-modified hyaluronan (HA) hydrogels to culture PCa cells and bone marrow stromal cells (BMSC) in a variety of well controlled configurations. The HA hydrogel reflects the low modulus environment of the bone marrow as well as HA's prevalence within the tissue. Various types of PCa cells, including cells derived from patient bone metastases, were cultured in HA hydrogels and viability was maintained as assessed by live/dead assays over time. Because HA is a bio-active signaling molecule that can interact with PCa cells through its receptors, cluster of differentiation 44 (CD44) or receptor for hyaluronan mediated motility (RHAMM), changes in morphology and signaling pathway activation were observed in response to the HA hydrogel culture. To investigate how PCa and BMSC interact, we encapsulated BMSC (HS27a) within a collagen-modified HA hydrogel. These cells formed a reticular network in the 3D matrix, reminiscent of the architecture of bone marrow stroma. When co-cultured with BMSC, bone metastatic PCa cells adhered to the reticular network of stromal cells and exhibited phenotypic changes suggesting neuroendocrine differentiation. We are developing a bilayered HA-based system to model, in 3D, the interface between PCa and the bone marrow stroma. Our model system includes 6 mm diameter disks of HA composed of two well-defined layers, one encapsulating BMSCs and the other containing PCa cells; each cell line can be labeled with a different fluorescent protein. These bilayered disks can be imaged to study the means of invasion of PCa into bone marrow stroma mimicking metastasis. Changes in cell-cell interactions and in cell morphologies in 3D can be imaged without disturbing the dynamic system, allowing for experiments to be monitored over time. These structured 3D units provide a novel means to study cancer and stroma invasiveness, cell-cell interactions and phenotypic changes in a 3D biomimetic system that simulates the bone microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3206. doi:1538-7445.AM2012-3206

Fibroblast Growth Factor 2 Stimulation of Osteoblast Differentiation and Bone Formation Is Mediated by Modulation of the Wnt Signaling Pathway

Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/β-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and β-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and β-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3β, a negative regulator of Wnt/β-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted β-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.

Metastatic breast cancer cells inhibit osteoblast differentiation through the Runx2/CBFβ-dependent expression of the Wnt antagonist, sclerostin

IntroductionBreast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction by stimulating osteoclasts to resorb bone and by preventing osteoblasts from producing new bone. The Runt-related transcription factor 2, Runx2, is an important determinant of bone metastasis in breast cancer. Runx2 is known to mediate activation of osteoclast activity and inhibition of osteoblast differentiation by metastatic breast cancer cells. However, while Runx2-regulated genes that mediate osteoclast activation have been identified, how Runx2 determines inhibition of osteoblasts is unknown.MethodsThe aim of this study was to determine how Runx2 mediates the ability of metastatic breast cancer cells to modulate the activity of bone cells. We have previously demonstrated that Runx2 requires the co-activator core binding factor beta (CBFβ) to regulate gene expression in breast cancer cells. We, therefore, performed independent microarray analyses to identify target genes whose expression is dependent upon both Runx2 and CBFβ. Common target genes, with a role in modulating bone-cell function, were confirmed using a combination of siRNA, quantitative reverse transcriptase PCR (qRT-PCR), ELISA, promoter reporter analysis, Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The function of Runx2/CBFβ-regulated genes in mediating the ability of MDA-MB-231 to inhibit osteoblast differentiation was subsequently established in primary bone marrow stromal cell cultures and MC-3T3 osteoblast cells.ResultsWe show that Runx2/CBFβ mediates inhibition of osteoblast differentiation by MDA-MB-231 cells through induction of the Wnt signaling antagonist, sclerostin. We demonstrate that MDA-MB-231 cells secrete sclerostin and that sclerostin-expression is critically dependent on both Runx2 and CBFβ. We also identified the osteoclast activators IL-11 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as new target genes of Runx2/CBFβ in metastatic breast cancer cells.ConclusionsThis study demonstrates that Runx2 and CBFβ are required for the expression of genes that mediate the ability of metastatic breast cancer cells to directly modulate both osteoclast and osteoblast function. We also show that Runx2-dependent inhibition of osteoblast differentiation by breast cancer cells is mediated through the Wnt antagonist, sclerostin.

Nicotinamide phosphoribosyltransferase (Nampt) affects the lineage fate determination of mesenchymal stem cells: A possible cause for reduced osteogenesis and increased adipogenesis in older individuals

Human aging is associated with a progressive decline in bone mass and an accumulation of marrow fat. We found that osteoblast differentiation was reduced and adipocyte formation increased in bone marrow stromal cells derived from aged mice compared with young controls. The increased adipogenesis correlated with a relatively lower Sirt1 activity and a lower intracellular NAD(+) concentration. We suppose that these effects were caused by age-related reduction of nicotinamide phosphoribosyltransferase (Nampt), the enzyme catalyzing NAD resynthesis from nicotinamide (NAM). In support of this hypothesis, treatment with Nampt inhibitor FK866 increased adipocyte formation and reduced mineralization in primary cultured bone marrow stromal cells. In addition, knockdown of Nampt in the mouse mesenchymal cell line C3H10T1/2 cells resulted in decreased Sirt1 activity and enhanced adipogenesis. Interestingly, although Nampt deficiency resulted in both decreased intracellular NAD(+) and increased NAM, the cell differentiation could be controlled only by regulation of NAM. These results indicate that the lineage fate determination of mesenchymal stem cells (MSCs) is influenced by cell energy metabolism and points to a possible mechanism for the development of senile osteoporosis. Furthermore, we suggest that side effects on bone should be considered when evaluating the long-term safety of NAD-interfering pharmaceuticals.

Phenotypic and functional characterization of human bone marrow stromal cells in hollow-fibre bioreactors

The transplantation of human bone marrow stromal cells (BMSCs) is a novel immunotherapeutic approach that is currently being explored in many clinical settings. Evidence suggests that the efficacy of cell transplantation is directly associated with soluble factors released by human BMSCs. In order to harness these secreted factors, we integrated BMSCs into large-scale hollow-fibre bioreactor devices in which the cells, separated by a semipermeable polyethersulphone (PES) membrane, can directly and continuously release therapeutic factors into the blood stream. BMSCs were found to be rapidly adherent and exhibited long-term viability on PES fibres. The cells also preserved their immunophenotype under physiological fluid flow rates in the bioreactor, and exhibited no signs of differentiation during device operation, but still retained the capacity to differentiate into osteoblastic lineages. BMSC devices released growth factors and cytokines at comparable levels on a per-cell basis to conventional cell culture platforms. Finally, we utilized a potency assay to demonstrate the therapeutic potential of the collected secreted factors from the BMSC devices. In summary, we have shown that culturing BMSCs in a large-scale hollow-fibre bioreactor is feasible without deleterious effects on phenotype, thus providing a platform for collecting and delivering the paracrine secretions of these cells.

Safety of Bovine Sera for Production of Mesenchymal Stem Cells for Therapeutic Use

Human Gene TherapyVol. 22, No. 6 Letter to the EditorSafety of Bovine Sera for Production of Mesenchymal Stem Cells for Therapeutic UseJens Reinhardt, Albert Stühler, and Johannes BlümelJens ReinhardtAdvanced Therapy Medicinal Products, Tissue Preparations, Division of Medicinal Biotechnology, Paul-Ehrlich-Institut, D-63225 Langen, Germany.Search for more papers by this author, Albert StühlerViral Safety, Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany.Search for more papers by this author, and Johannes BlümelViral Safety, Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany.Search for more papers by this authorPublished Online:8 Jun 2011https://doi.org/10.1089/hum.2011.2501AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Safety of Bovine Sera for Production of Mesenchymal Stem Cells for Therapeutic Use." , 22(6), p. 775FiguresReferencesRelatedDetailsCited byGaps in the knowledge of human platelet lysate as a cell culture supplement for cell therapy: a joint publication from the AABB and the International Society for Cell & Gene Therapy14 August 2019 | Transfusion, Vol. 59, No. 11Gaps in the knowledge of human platelet lysate as a cell culture supplement for cell therapy: a joint publication from the AABB and the International Society for Cell & Gene TherapyCytotherapy, Vol. 21, No. 9Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry2 March 2018 | Scientific Reports, Vol. 8, No. 1Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applicationsCytotherapy, Vol. 20, No. 9Growth Factor Content in Human Sera Affects the Isolation of Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow17 October 2016 | Frontiers in Cell and Developmental Biology, Vol. 4Bioprocessing of human mesenchymal stem/stromal cells for therapeutic use: Current technologies and challengesBiochemical Engineering Journal, Vol. 108Mesenchymal Stem Cells Derived from Dental Pulp: A ReviewStem Cells International, Vol. 2016Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against StrokeStem Cells International, Vol. 2016Results from a horizon scan on risks associated with transplantation of human organs, tissues and cells: from donor to patient1 May 2014 | Cell and Tissue Banking, Vol. 16, No. 1Standardization of Good Manufacturing Practice–compliant production of bone marrow–derived human mesenchymal stromal cells for immunotherapeutic applicationsCytotherapy, Vol. 17, No. 2Cell Manufacturing for Clinical Applications18 August 2014 | Stem Cells, Vol. 32, No. 9Manufacturing and banking of mesenchymal stem cells23 January 2013 | Expert Opinion on Biological Therapy, Vol. 13, No. 5GMP Requirements and Quality Management of Cellular products19 August 2013MSCs: Clinical Applications and European Regulatory Aspects4 December 2012Large-scale production of human mesenchymal stem cells for clinical applications16 April 2012 | Biotechnology and Applied Biochemistry, Vol. 59, No. 2Safety of Mesenchymal Stem Cells for Clinical ApplicationStem Cells International, Vol. 2012Ex Vivo Expansion of Human Mesenchymal Stem Cells in Defined Serum-Free MediaStem Cells International, Vol. 2012 Volume 22Issue 6Jun 2011 InformationCopyright 2011, Mary Ann Liebert, Inc.To cite this article:Jens Reinhardt, Albert Stühler, and Johannes Blümel.Safety of Bovine Sera for Production of Mesenchymal Stem Cells for Therapeutic Use.Human Gene Therapy.Jun 2011.775-775.http://doi.org/10.1089/hum.2011.2501Published in Volume: 22 Issue 6: June 8, 2011PDF download

Kif3a deficiency reverses the skeletal abnormalities in Pkd1 deficient mice by restoring the balance between osteogenesis and adipogenesis.

Pkd1 localizes to primary cilia in osteoblasts and osteocytes. Targeted deletion of Pkd1 in osteoblasts results in osteopenia and abnormalities in Runx2-mediated osteoblast development. Kif3a, an intraflagellar transport protein required for cilia function, is also expressed in osteoblasts. To assess the relationship between Pkd1 and primary cilia function on bone development, we crossed heterozygous Pkd1- and Kif3a-deficient mice to create compound Pkd1 and Kif3a-deficient mice. Pkd1 haploinsufficiency (Pkd1 +/Δ) resulted in osteopenia, characterized by decreased bone mineral density, trabecular bone volume, and cortical thickness. In addition, deficiency of Pkd1 resulted in impaired osteoblastic differentiation and enhanced adipogenesis in both primary osteoblasts and/or bone marrow stromal cell cultures. These changes were associated with decreased Runx2 expression, increased PPARγ expression, and impaired hedgehog signaling as evidenced by decreased Gli2 expression in bone and osteoblast cultures. In contrast, heterozygous Kif3a +/Δ mice display no abnormalities in skeletal development or osteoblast function, but exhibited decreased adipogenic markers in bone and impaired adipogenesis in vitro in association with decreased PPARγ expression and upregulation of Gli2. Superimposed Kif3a deficiency onto Pkd1 +/Δ mice paradoxically corrected the effects of Pkd1 deficiency on bone mass, osteoblastic differentiation, and adipogenesis. In addition, Runx2, PPARγ and Gli2 expression in bone and osteoblasts were normalized in compound double Pkd1 +/Δ and Kif3a +/Δ heterozygous mice. The administration of sonic hedgehog, overexpression of Gli2, and the PC1 C-tail construct all increased Gli2 and Runx2-II expression, but decreased PPARγ2 gene expression in C3H10T1/2 cells. Our findings suggest a role for Pkd1 and Kif3a to counterbalance the regulation of osteogenesis and adipogenesis through differential regulation of Runx2 and PPARγ by Gli2.

Non-Hematopoietic Stromal Cells Sense Toll-Like Receptor 4 Agonists and Consequently Enhance Myelopoiesis.

AbstractAbstract 2583Hematopoiesis is tightly regulated by growth factors acting on stem and progenitor cells (HSPCs) in the bone marrow. During systemic infections cytokines are elevated in serum, myelopoiesis is enhanced, and myeloid CFUs and granulocytes increase in circulation. However, the underlying mechanisms of this “emergency” myelopoietic response have not been defined. Sensing of conserved pathogen-associated products by specialized pattern-recognition receptors such as Toll-like receptors (TLRs) is crucial for rapid responses to infection. Based on the well-known regulatory function of the BM microenvironment, we hypothesized that bone marrow stromal cells (BMSCs) express TLRs and possess all functional properties required to sense microbes and drive emergency myelopoiesis. Human BMSCs expressed Tlr1, Tlr5, and Tlr6 at similar levels and Tlr3 and Tlr4 mRNA at about 2-log higher compared to dendrititc cells (DCs). Stimulation of BMSCs with the TLR4 agonist LPS led to de novo expression of G-csf and Gm-csf, and increased M-csf, Il-6, and Il-11 expression. In line with this, LPS induced production of G-CSF and GM-CSF protein and significantly enhanced the secretion of M-CSF, IL-6, and IL-11. Using LPS-stimulated BMSC culture supernatant in myeloid CFU assays led to a 2.5-fold higher myeloid CFU activity compared to un-stimulated BMSC supernatant. This effect was partly mimicked by adding G-, M-, and GM-CSF to the methylcellulose cultures. Importantly, direct LPS stimulation of CB CD34+ cells had no effect. Furthermore, co-culture of BMSCs and CB CD34+ cells together with LPS for 12 days led to approximate 2-fold higher recovery of immuno-phenotypically primitive CD34+ cells, and retained up to 8-fold more CD34+ cells in divisions 0–3 as compared to LPS-free co-cultures as measured by CFSE dilution. When subjected to cytokine-supplemented myeloid CFU assays or transplanted into newborn RAG2-/- γc-/- mice to evaluate lymphoid differentiation, recovered CD34+ cells from LPS-stimulated BMSC cultures gave rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus proving maintenance of primitive hematopoietic progenitors. To elucidate the in vivo relevance of the findings and to clarify the contribution of stromal vs. hematopoietic cell expressed TLR4, we generated chimeras with TLR4-/- hematopoiesis in a wild-type (WT) background (hematopoietic-TLR4-/-) and WT hematopoiesis in a TLR4-/- background (non-hematopoietic-TLR4-/-). Chimeric, WT, and TLR4-/- mice were injected with LPS and hallmarks of myelopoietic responses such as G-CSF expression, myeloid cell mobilization from the BM, and increased myeloid cell production in the BM was evaluated. Significant G-csf mRNA induction could be observed in the BM of WT and hematopoietic-TLR4-/- mice. To a much lesser, non-significant extent, this effect could be observed also in non-hematopoietic-TLR4-/- mice, while no transcripts were detectable in TLR4-/- mice. Accordingly, serum G-CSF levels significantly increased 10-fold in WT and hematopoietic-TLR4-/- mice after LPS injection, but no increase was detectable in non-hematopoietic-TLR4-/- and TLR4-/- mice. LPS injection also resulted in a significant decrease in BM cellularity accompanied by an increase of spleen cell numbers only in WT and hematopoietic-TLR4-/- mice. Furthermore, Gr-1highCD11blow/+ mature myeloid cells were significantly reduced whereas Gr-1lowCD11blow/+ immature promyelocytes and myelocytes significantly increased (2.5-fold) in the BM of WT and hematopoietic-TLR4-/- mice. In contrast, similar changes in cellular composition could not be observed in TLR4-/- and non-hematopoietic-TLR4-/- mice, while a small, but still significant 1.25-fold increase in immature Gr-1lowCD11blow/+ cells was detectable in non-hematopoietic-TLR4-/- mice. Finally, inflammation-induced Sca-1 upregulation on HSPCs and increasing frequencies of GMPs were only observed in WT and hematopoietic-TLR4-/- mice. Collectively, our in vitro data demonstrate that human BMSCs are able to sense pathogens and stimulate emergency myelopoiesis but also prevent loss of HSPCs by enhancing their maintenance. Importantly, in vivo signaling via non-hematopoietic cell-expressed TLR4 is sufficient and is the main mechanism regulating both the release of mature myeloid cells from and the enhanced myeloid cell production in the bone marrow during systemic challenges. Disclosures:No relevant conflicts of interest to declare.

Comparison of beneficial effects of undifferentiated cultured bone marrow stromal cells and omental adipose‐derived nucleated cell fractions on sciatic nerve regeneration

Adipose tissue is a good source for isolation of cells with stem-cell-like properties. The effects of undifferentiated cultured bone marrow stromal cells (BMSCs) and omental adipose-derived nucleated cells (OADNCs) on peripheral nerve regeneration were compared in a rat nerve regeneration model. A 10-mm sciatic nerve defect was bridged using a vein graft. In one group, the vein was filled with BMSCs and in the other group with OADNCs. Functional study, morphometric indices, and immunohistochemistry indicated there was no significant difference (P > 0.05) between groups in recovery of regenerated axons at 4, 8, and 12 weeks after surgery. OADNCs enhanced regeneration similar to undifferentiated BMSCs. These observations suggest OADNCs represent an effective and cost-saving cell population due to the shortened time interval from tissue collection to cell injection as well as procedural simplicity. This approach is clinically translatable toward new methods for enhanced peripheral nerve repair without the limitations of BMSC.

Bone marrow stromal cells as an inducer for cardiomyocyte differentiation from mouse embryonic stem cells

Bone marrow stromal cells (BMSCs) secrete soluble factors and display varied cell-biological functions. To confirm the ability and efficiency of BMSCs to induce embryonic stem cells (ESCs) into cardiomyocytes, mouse embryoid bodies (EBs) were co-cultured with rat BMSCs. After about 10 days, areas of rhythmically contracting cells in more solid aggregates became evident with bundle-like structures formed along borders between EB outgrowth and BMSC layer. ESC-derived cardiomyocytes exhibited sarcomeric striations when stained with troponin I (Trop I), organized in separated bundles. Besides, the staining for connexin 43 was detected in cell-cell junctions, which demonstrated that ESC-derived cardiomyocytes were coupled by gap junction in culture. The related genes of cardiomyocytes were found in these beating and no-beating EBs co-cultured with BMSCs. In addition, an improved efficiency of cardiomyocyte differentiation from ESC-BMSC co-culture was found in the serum-free medium: 5-fold up-regulation in the number of beating area compared with the serum medium. Effective cardiac differentiation was also recognized in transfer filter assay and in condition medium obtained from BMSC culture. A clear increase in the expression of cardiac genes and TropI protein confirmed further cardiac differentiation by BMP4 and Retinoic Acid (RA) treatment. These results demonstrate that BMSCs can induce cardiomyocyte differentiation from ESCs through soluble factors and enhance it with BMP4 or RA treatment. Serum-free ESC-BMSC co-culture represents a defined in vitro model for identifying the cardiomyocyte-inducing activity from BMSCs and, in addition, a straightforward experimental system for assessing clinical applications.

Characterization of conditioned medium of cultured bone marrow stromal cells

It has been recognized that bone marrow stromal cell (BMSC) transplantation has beneficial effects on spinal cord injury in animal models and therapeutic trials. It is hypothesized that BMSCs provide microenvironments suitable for axonal regeneration and secrete some trophic factors to rescue affected cells from degeneration. However, the molecular and cellular mechanisms of the trophic factors involved remain unclear. In the present study, we examined the effects of trophic factors secreted by rat BMSCs using bioassays involving cultured hippocampal neurons. The conditioned medium (CM) as well as non-contact co-culture of BMSCs promoted neurite outgrowth and suppressed TUNEL-positive cells compared to serum-free D-MEM. Protein analyses of the CM by antibody-based protein array analysis and ELISA revealed that the CM contained insulin-like growth factor (IGF)-1, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-β1. DNA microarray analysis revealed that neurons highly expressed receptors of IGF-1 and TGF-β1. However, their expression indices remained unchanged even after the CM treatment. The individual trophic factors mentioned above or their combinations were less effective at promoting neuronal survival and neurite outgrowth than the CM. The present study showed that BMSCs secreted various kinds of molecules into the culture medium including trophic factors to promote neuronal survival and neurite outgrowth. The main trophic factors responsible remain to be elucidated.

Disruption of the Fgf2 gene activates the adipogenic and suppresses the osteogenic program in mesenchymal marrow stromal stem cells

Here we determine the Fibroblast Growth Factor-2 (FGF2) dependency of the time course of changes in bone mass in female mice. This study extends our earlier reports that knockout of the FGF2 gene ( Fgf2) caused low turnover bone loss in Fgf2 − / − male mice by examining bone loss with age in Fgf2 −/− female mice, and by assessing whether reduced bone formation is associated with differentiation of bone marrow stromal cells (BMSCs) towards the adipocyte lineage. Bone mineral density (BMD) was similar in 3-month-old female Fgf2 +/+ and Fgf2 −/− mice but was significantly reduced as early as 5 months of age in Fgf2 −/− mice. In vivo studies showed that there was a greater accumulation of marrow fat in long bones of 14 and 20 month old Fgf2 −/− mice compared with Fgf2 +/+ littermates. To study the effect of disruption of FGF2 on osteoblastogenesis and adipogenesis, BMSCs from both genotypes were cultured in osteogenic or adipogenic media. Reduced alkaline phosphatase positive (ALP), mineralized colonies and a marked increase in adipocytes were observed in Fgf2 −/− BMSC cultures. These cultures also showed an increase in the mRNA of the adipogenic transcription factor PPARγ2 as well as the downstream target genes aP2 and adiponectin. Treatment with exogenous FGF2 blocked adipocyte formation and increased ALP colony formation and ALP activity in BMSC cultures of both genotypes. These results support an important role for endogenous FGF2 in osteoblast (OB) lineage determination. Alteration in FGF2 signaling may contribute to impaired OB bone formation capacity and to increased bone marrow fat accumulation both of which are characteristics of aged bone.

Impaired osteoblast function in GPRC6A null mice

GPRC6A is a widely expressed orphan G protein–coupled receptor that senses extracellular amino acids, osteocalcin, and divalent cations in vitro. GPRC6A null (GPRC6A−/−) mice exhibit multiple metabolic abnormalities including osteopenia. To investigate whether the osseous abnormalities are a direct function of GPRC6A in osteoblasts, we examined the function of primary osteoblasts and bone marrow stromal cell cultures (BMSCs) in GPRC6A−/− mice. We confirmed that GPRC6A−/− mice exhibited a decrease in bone mineral density (BMD) associated with reduced expression of osteocalcin, ALP, osteoprotegerin, and Runx2-II transcripts in bone. Osteoblasts and BMSCs derived from GPRC6A−/− mice exhibited an attenuated response to extracellular calcium-stimulated extracellular signal-related kinase (ERK) activation, diminished alkaline phosphatase (ALP) expression, and impaired mineralization ex vivo. In addition, siRNA-mediated knockdown of GPRC6A in MC3T3 osteoblasts also resulted in a reduction in extracellular calcium-stimulated ERK activity. To explore the potential relevance of GPRC6A function in humans, we looked for an association between GPRC6A gene polymorphisms and BMD in a sample of 1000 unrelated American Caucasians. We found that GPRC6A gene polymorphisms were significantly associated with human spine BMD. These data indicate that GRPC6A directly participates in the regulation of osteoblast-mediated bone mineralization and may mediate the anabolic effects of extracellular amino acids, osteocalcin, and divalent cations in bone. © 2010 American Society for Bone and Mineral Research.

Multilineage differentiation of dental follicle cells and the roles of Runx2 over‐expression in enhancing osteoblast/cementoblast‐related gene expression in dental follicle cells

Dental follicle cells (DFCs) provide the origin of periodontal tissues, and Runx2 is essential for bone formation and tooth development. In this study, pluripotency of DFCs was evaluated and effects of Runx2 on them were investigated. The DFCs were induced to differentiate towards osteoblasts, adipocytes or chondrocytes, and alizarin red staining, oil red O staining or alcian blue staining was performed to reveal the differentiated states. Bone marrow stromal cells (BMSCs) and primary mouse fibroblasts served as controls. DFCs were also infected with recombinant retroviruses encoding either full-length Runx2 or mutant Runx2 without the VWRPY motif. Western blot analysis, real-time real time RT-PCR and in vitro mineralization assay were performed to evaluate the effects of full-length Runx2 or mutant Runx2 on osteogenic/cementogenic differentiation of the cells. The above-mentioned staining methods demonstrated that DFCs were successfully induced to differentiate towards osteoblasts, adipocytes or chondrocytes respectively, confirming the existence of pluripotent mesenchymal stem cells in dental follicle tissues. However, staining intensity in DFC cultures was weaker than in BMSC cultures. Real-time PCR analysis indicated that mutant Runx2 induced a more pronounced increase in expression levels of OC, OPN, Col I and CP23 than full-length Runx2. Mineralization assay also showed that mutant Runx2 increased mineralization nodule formation more prominently than full-length Runx2. Multipotent DFCs can be induced to differentiate towards osteoblasts, adipocytes or chondrocytes in vitro. Runx2 over-expression up-regulated expression levels of osteoblast/cementoblast-related genes and in vitro enhanced osteogenic differentiation of DFCs. In addition, mutant Runx2-induced changes in DFCs were more prominent than those induced by full-length Runx2.