Modulating osteogenesis of mesenchymal stem cells by modifying growth factor availability

Growth factors control the proliferation and differentiation of osteoprogenitor cells. This study explores the effects of modulating growth factors (VEGF, IGF-1, FGF-2 and BMP-2) on osteogenesis of mesenchymal stem cells (MSCs) in vitro. Constant and profiled delivery protocols, in accordance with protein expression in vitro, were applied to deliver or neutralize growth factors. Cell number, alkaline phosphatase (ALP-2) and osteocalcin (OC) expression, and mineralization were measured as outcome variables. Profiled addition of VEGF increased MSC proliferation. Constant and profiled application of FGF-2 and neutralization of IGF-1 and BMP-2 decreased ALP-2 levels. Profiled addition of BMP-2 vastly increased OC release from MSCs, but constant addition of IGF-1, constant and profiled neutralization of IGF-1 and FGF-2 reduced OC levels. Constant addition of IGF-1 and FGF-2, as well as profiled loading of FGF-2 decreased mineralization of MSCs. This study indicated that endogenous IGF-1 and FGF-2 are essential to osteogenesis; excess IGF-1 and FGF-2 were inhibitory to bone formation. Selective, temporally specific addition of growth factors, such as BMP-2 and VEGF appears to be an important strategy to enhance osteogenesis.     

TNFR1 signaling resistance associated with female stem cell cytokine production is independent of TNFR2-mediated pathways

End-organ ischemia is a common source of patient morbidity and mortality. Stem cell therapy represents a novel treatment modality for ischemic diseases and may aid injured tissues through the release of beneficial paracrine mediators. Female bone marrow mesenchymal stem cells (MSCs) have demonstrated a relative resistance to detrimental TNF receptor 1 (TNFR1) signaling and are thought to be superior to male stem cells in limiting inflammation. However, it is not known whether sex differences exist in TNF receptor 2 (TNFR2)-ablated MSCs. Therefore, we hypothesized that 1) sex differences would be observed in wild-type (WT) and TNFR2-ablated MSC cytokine signaling, and 2) the production of IL-6, VEGF, and IGF-1 in males, but not females, would be mediated through TNFR2. MSCs were harvested from male and female WT and TNFR2 knockout (TNFR2KO) mice and were subsequently exposed to TNF (50 ng/ml) or LPS (100 ng/ml). After 24 h, supernatants were collected and measured for cytokines. TNF and LPS stimulated WT stem cells to produce cytokines, but sex differences were only seen in IL-6 and IGF-1 after TNF stimulation. Ablation of TNFR2 increased VEGF and IGF-1 production in males compared with wild-type, but no difference was observed in females. Female MSCs from TNFR2KOs produced significantly lower levels of VEGF and IGF-1 compared with male TNFR2KOs. The absence of TNFR2 signaling appears to play a greater role in male MSC cytokine production. As a result, male, but not female stem cell cytokine production may be mediated through TNFR2 signaling cascades.     

FGF-2对人骨髓间充质干细胞增殖和向成骨细胞分化的影响

探讨体外培养条件下,成纤维细胞生长因子-2（FGF-2）和地塞米松（Dex）对第7代人骨髓间充质干细胞（MSCs）增殖和向成骨细胞分化的作用以及两者联合使用的效应。MSCs经含FGF-2或／和Dex的培养液作用后,于不同时间采用MTT法测定细胞增殖情况;对硝基苯磷酸（pNPP）法测定碱性磷酸酶（ALP）活性;ELISA法测定骨钙蛋白（OC）含量;茜素红S染色法对沉积的钙盐进行染色。发现：（1）FGF-2组细胞的生长速度为对照组的1．31倍,Dex／FGF-2组细胞的生长速度为FGF-2组的1．12倍。（2）Dex组的ALP活性、OC含量和细胞外基质钙盐沉积分别为对照组的17．0倍、2．12倍和10．56倍,并能形成成熟的羟基磷灰石（HA）结晶和骨结节;FGF-2组的ALP活性比对照组降低了76．7％,虽然OC含量、钙盐沉积增加,但不能形成成熟的HA结晶和骨结节;FGF-2对Dex诱导的ALP活性增加和HA结晶形成有拮抗作用。由此证明：（1）FGF-2可促进MSCs的增殖,Dex对MSCs的增殖无明显作用;Dex能增强FGF-2对MSCs的促增殖效应。（2）Dex可使MSCs分化为成熟的成骨细胞,是一个有效的成骨细胞分化诱导剂;FGF-2可使MSCs分化为未成熟的成骨细胞;FGF-2拮抗Dex诱导MSCs分化为成熟的成骨细胞。     

Interactions between important regulatory proteins and human alphaB crystallin

Protein pin arrays assessed interactions between alphaB crystallin and 12 regulatory proteins, including EGF, FGF-2, IGF-1, NGF-beta, TGF-beta, VEGF, insulin, beta-catenin, caspase-3, caspase-8, Bcl-2, and Bcl-xL, which are important in cellular differentiation, proliferation, signaling, cytoskeletal assembly, and apoptosis. FGF-2, NGF-beta, VEGF, insulin, and beta-catenin had strong interactions with human alphaB crystallin peptides, and the alphaB crystallin interactive sequences for these proteins were identified. The seven remaining proteins (EGF, IGF-1, TGF-beta, caspase-3, caspase-8, BCl-2, and Bcl-xL) did not interact with alphaB crystallin. The alphaB crystallin sequences that interacted with FGF-2, NGF-beta, VEGF, insulin, and beta-catenin overlapped with sequences that selectively interact with partially unfolded proteins, suggesting a common function for alphaB crystallin in chaperone activity and the regulation of cell growth and differentiation. Chaperone assays conducted with full-length alphaB crystallin and synthetic alphaB crystallin peptides confirmed the ability of alphaB crystallin to protect against the aggregation of FGF-2 and VEGF, suggesting that alphaB crystallin protects these proteins against unfolding and aggregation under conditions of stress. This is the first report in which sequences involved in interactions with regulatory proteins, including FGF-2, NGF-beta, VEGF, insulin, and beta-catenin, were identified in a small heat shock protein.     

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.     

FGF-2 increases colony formation, PTH receptor, and IGF-1 mRNA in mouse marrow stromal cells.

FGF-2 stimulates bone formation in vitro and in vivo in rats. However, there are limited studies in mice and no data on the mechanism(s) by which FGF-2 induces bone formation. We assessed whether short-term FGF-2 treatment of marrow stromal cells from young mice would increase alkaline phosphatase-positive (ALP), mineralized colony formation and expression of genes important in osteoblast maturation. Short-term treatment with FGF-2 (0.01-1.0 nM) for the first 3 days of a 14- or 21-day culture period increased the number of ALP mineralized colonies in bone marrow stromal cells. FGF-2 (0.1 nM) increased the mRNAs for type 1 collagen: osteocalcin, runt domain/core binding factor, PTH/PTHR receptor, and insulin-like growth factor 1 (IGF-1) at 14 and 21 days. We conclude that short-term FGF-2 treatment enhances osteoblast maturation in vitro. Furthermore, the anabolic effect of FGF-2 may be attributed in part to regulation of IGF-1 in osteoblasts.     

Mesenchymal stem cell-conditioned media: A novel alternative of stem cell therapy for quality wound healing

Mesenchymal stem cells-conditioned media (MSCs-CM) contains several growth factors and cytokines, thus may be used as a better alternative to stem cell therapy, which needs to be elucidated. The present study was conducted to evaluate the therapeutic potential of caprine, canine, and guinea pig bone marrow-derived MSCs-CM in excision wound healing in a guinea pig model. MSCs were obtained from bone marrow, expanded ex vivo and characterized as per ISCT criteria. CM was collected assayed by western blot to ascertain the presence of important secretory biomolecules. Quantitative estimation by enzyme-linked immunosorbent assay was done for a vascular epidermal growth factor (VEGF) and interleukin-6 (IL-6) in caprine MSCs-CM and optimum time for collection of CM was decided as 72 hr. CM from all the species was lyophilized by freeze-drying method. Full-thickness (2 × 2 cm²) excision skin wounds were created in guinea pigs (six animals in each group) and respective lyophilized CM mixed with laminin gel was applied topically at weekly interval. On Day 28, histopathological examinations of healed skin were done by hemotoxylin and eosin staining. MSCs were found to secrete important growth factors and cytokines (i.e., VEGF, transforming growth factor-β1, fibroblast growth factor-2, insulin-like growth factor-1, stem cell factor, and IL-6) as demonstrated by immunohistochemistry and western blot assay. It was found that allogenic and xenogenic application of CM significantly improved quality wound healing with minimal scar formation. Thus, MSCs-CM can be used allogenically as well as xenogenically for quality wound healing.     

Viability of human mesenchymal stem cells seeded on crosslinked entropy-elastic gelatin-based hydrogels

Biomimetic polymer network systems with tailorable properties based on biopolymers represent a class of degradable hydrogels that provides sequences for protein adsorption and cell adhesion. Such materials show potential for in vitro MSC proliferation as well as in vivo applications and were obtained by crosslinking different concentrations of gelatin using varying amounts of ethyl lysine diisocyanate in the presence of a surfactant in pH 7.4 PBS solution. Material extracts, which were tested for cytotoxic effects using L929 mouse fibroblasts, were non-toxic. The hydrogels were seeded with human bone marrow-derived MSCs and supported viable MSCs for the incubation time of 9 d. Preadsorption of fibronectin on materials improved this biofunctionality.     

Immunophenotypic characteristics of multipotent mesenchymal stromal cells that affect the efficacy of their use in the prevention of acute graft vs host disease

BACKGROUNDMultipotent mesenchymal stromal cells (MSCs) are widely used in the clinic due to their unique properties, namely, their ability to differentiate in all mesenchymal directions and their immunomodulatory activity. Healthy donor MSCs were used to prevent the development of acute graft vs host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). The administration of MSCs to patients was not always effective. The MSCs obtained from different donors have individual characteristics. The differences between MSC samples may affect their clinical efficacy.AIMTo study the differences between effective and ineffective MSCs.METHODSMSCs derived from the bone marrow of a hematopoietic stem cells donor were injected intravenously into allo-BMT recipients for GVHD prophylaxis at the moment of blood cell reconstitution. Aliquots of 52 MSC samples that were administered to patients were examined, and the same cells were cultured in the presence of peripheral blood mononuclear cells (PBMCs) from a third-party donor or treated with the pro-inflammatory cytokines IL-1β, IFN and TNF. Flow cytometry revealed the immunophenotype of the nontreated MSCs, the MSCs cocultured with PBMCs for 4 d and the MSCs exposed to cytokines. The proportions of CD25-, CD146-, CD69-, HLA-DR- and PD-1-positive CD4+ and CD8+ cells and the distribution of various effector and memory cell subpopulations in the PBMCs cocultured with the MSCs were also determined.RESULTSDifferences in the immunophenotypes of effective and ineffective MSCs were observed. In the effective samples, the mean fluorescence intensity (MFI) of HLA-ABC, HLA-DR, CD105, and CD146 was significantly higher. After MSCs were treated with IFN or cocultured with PBMCs, the HLA-ABC, HLA-DR, CD90 and CD54 MFI showed a stronger increase in the effective MSCs, which indicated an increase in the immunomodulatory activity of these cells. When PBMCs were cocultured with effective MSCs, the proportions of CD4+ and CD8+central memory cells significantly decreased, and the proportion of CD8+CD146+ lymphocytes increased more than in the subpopulations of lymphocytes cocultured with MSC samples that were ineffective in the prevention of GVHD; in addition, the proportion of CD8+effector memory lymphocytes decreased in the PBMCs cocultured with the effective MSC samples but increased in the PBMCs cocultured with the ineffective MSC samples. The proportion of CD4+CD146+ lymphocytes increased only when cocultured with the inefficient samples.CONCLUSIONFor the first time, differences were observed between MSC samples that were effective for GVHD prophylaxis and those that were ineffective. Thus, it was shown that the immunomodulatory activity of MSCs depends on the individual characteristics of the MSC population.     

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation

Background aimsMesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α–pretreated human bone marrow–derived MSCs on resting or activated T cells.MethodsMSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed.ResultsUnprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions.ConclusionsUnprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo.     

The Insulin-like Growth Factor-1 Binding Protein Acid-labile Subunit Alters Mesenchymal Stromal Cell Fate

Age-related osteoporosis is accompanied by an increase in marrow adiposity and a reduction in serum insulin-like growth factor-1 (IGF-1) and the binding proteins that stabilize IGF-1. To determine the relationship between these proteins and bone marrow adiposity, we evaluated the adipogenic potential of marrow-derived mesenchymal stromal cells (MSCs) from mice with decreased serum IGF-1 due to knockdown of IGF-1 production by the liver or knock-out of the binding proteins. We employed 10–16-week-old, liver-specific IGF-1-deficient, IGFBP-3 knock-out (BP3KO) and acid-labile subunit knock-out (ALSKO) mice. We found that expression of the late adipocyte differentiation marker peroxisome proliferator-activated receptor γ was increased in marrow isolated from ALSKO mice. When induced with adipogenic media, MSC cultures from ALSKO mice revealed a significantly greater number of differentiated adipocytes compared with controls. MSCs from ALSKO mice also exhibited decreased alkaline-phosphatase positive colony size in cultures that were stimulated with osteoblast differentiation media. These osteoblast-like cells from ALSKO mice failed to induce osteoclastogenesis of control cells in co-culture assays, indicating that impairment of IGF-1 complex formation with ALS in bone marrow alters cell fate, leading to increased adipogenesis.     

Human mesenchymal stem cells stimulated by TNF-alpha, LPS, or hypoxia produce growth factors by an NF kappa B- but not JNK-dependent mechanism

Understanding the mechanisms by which adult stem cells produce growth factors may represent an important way to optimize their beneficial paracrine and autocrine effects. Components of the wound milieu may stimulate growth factor production to promote stem cell-mediated repair. We hypothesized that tumor necrosis factor-alpha (TNF-alpha), endotoxin (LPS), or hypoxia may activate human mesenchymal stem cells (MSCs) to increase release of vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF-1), or hepatocyte growth factor (HGF) and that nuclear factor-kappa B (NF kappa B), c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) mediates growth factor production from human MSCs. To study this, human MSCs were harvested, passaged, divided into four groups (100,000 cells, triplicates) and treated as follows: 1) with vehicle; 2) with stimulant alone [24 h LPS (200 ng/ml), 24 h TNF-alpha (50 ng/ml), or 24 h hypoxia (1% O2)]; 3) with inhibitor alone [NF kappa B (PDTC, 1 mM), JNK (TI-JIP, 10 microM), or ERK (ERK Inhibitor II, 25 microM)]; and 4) with stimulant and the various inhibitors. After 24 h incubation, MSC activation was determined by measuring supernatants for VEGF, FGF2, IGF-1, or HGF (ELISA). TNF-alpha, LPS, and hypoxia significantly increased human MSC VEGF, FGF2, HGF, and IGF-1 production versus controls. Stem cells exposed to injury demonstrated increased activation of NF kappa B, ERK, and JNK. VEGF, FGF2, and HGF expression was significantly reduced by NF kappa B inhibition (50% decrease) but not ERK or JNK inhibition. Moreover, ERK, JNK, and NF kappa B inhibitor alone did not activate MSC VEGF expression over controls. Various stressors activate human MSCs to increase VEGF, FGF2, HGF, and IGF-1 expression, which depends on an NFkB mechanism.     

Involvement of FGF and BMP family proteins and VEGF in early human kidney development

The spatial and temporal pattern of the appearance of the fibroblast growth factor proteins (FGF-8 and FGF-10), the bone morphogenetic proteins (BMP-2/4 subfamily and BMP-7) and the vascular endothelial growth factor protein (VEGF) was investigated in the human mesonephros and metanephros of the 5-9 week-old conceptuses. In the mesonephros, both FGF's and BMP's were found in all structures and their expression slightly decreased in the early fetal period. VEGF positivity appeared in all mesonephric structures, and increased in the fetal period coincidently with formation of the mesonephric blood vessel network. In the metanephros, FGF-8 first appeared only in the metanephric mesenchyme, but from the 7th week on, its reactivity increased and spread to other metanephric structures. FGF-10 positive cells appeared in all metanephric structures already in the 5th week, and slightly intensified with progression of development. Cell survival and nephrogenesis in the permanent kidney might be associated with the appearance of both growth factors. Both BMP-2/4 and BMP-7 displayed a similar pattern of reactivity in all metanephric structures, and their reactivity intensified with advancing development. Alterations in their pattern of appearance might lead to the formation of small and dysplastic kidneys. Already in the earliest developmental stages, VEGF protein appeared in all metanephric structures. At later stages, VEGF showed more intense reaction in the collecting system than in the differentiating nephrons and interstitium. Due to VEGF involvement in vasculogenesis and angiogenesis, abnormal VEGF appearance might lead to impaired formation of the blood vessel network in the human permanent kidney.     

Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells

CD14-positive monocytes obtained from human peripheral blood were cultured with GM-CSF and IL-4. During the early culture phase immature dendritic cells (DCs) developed which not only expressed CD1a, HLA-DR and CD86, but also expressed the endothelial cell markers von Willebrand factor (vWF), VE-cadherin and VEGF receptors Flt-1 and Flt-4. Further maturation of DCs was achieved by prolonged cultivation with TNFalpha. These cells showed typical DC morphology and like professional antigen-presenting cells (APCs) expressed CD83 and high levels of HLA-DR and CD86. However, if immature DCs were grown with VEGF, bFGF and IGF-1 on fibronectin/vitronectin-coated culture dishes, a marked change in morphology into caudated or oval cells occurred. In the presence of these angiogenic growth factors the cultured cells developed into endothelial-like cells (ELCs), characterized by increased expression of vWF, KDR and Flt-4 and a disappearance of CD1a and CD83. Addition of IL-4 and Oncostatin M also increased VE-cadherin expression, and the loosely adherent cells formed clusters, cobblestones and network-like structures. vWF- expressing ELCs mainly originated from CD1a-positive cells, and VEGF was responsible for the decrease in the expression of the DC markers CD1a and CD83. In mixed leukocyte cultures, mature DCs were more potent APCs than ELCs. Moreover, Ac-LDL uptake, and the formation of tubular structures on a plasma matrix was restricted to ELCs. These results suggest that in the presence of specific cytokines immature DCs have the potential to differentiate along different lineages, i.e. into a cell type resembling ELCs.     

Vitamin A differentially regulates RANKL and OPG expression in human osteoblasts

Mouse marrow, which contains osteoblast and osteoclast precursors, was grown in the presence of calcitriol and/or basic fibroblast growth factor (FGF-2). RAW 264.7 cells were differentiated into osteoclast-like cells in the presence of receptor activator of NF-kappaB-Ligand (RANK-L) and/or FGF-2. FGF-2 alone supported osteoclastogenesis in mouse marrow cultures, but not by RAW 264.7 cells alone. Although FGF-2 supported low levels of osteoclastogenesis in mouse marrow cultures, it strongly inhibited the high levels of osteoclastogenesis triggered by calcitriol. Adding excess recombinant-RANK-L to the cultures did not relieve this inhibition. After mouse marrow osteoclasts were differentiated, FGF-2 dose-dependently inhibited bone resorptive activity. FGF-2 increased the tendency of RAW 264.7 osteoclast-like cells to fuse into very large giant cells and induced reorganizations of the actin cytoskeleton in mature, RANK-L-induced RAW 264.7 osteoclast-like cells. These results suggest that FGF-2 has both direct and indirect effects on osteoclast formation and bone resorption.     

The distribution of the growth factors FGF-2 and VEGF, and their receptors, in growing red deer antler

The cellular distributions of the growth factors FGF-2 and VEGF, and their receptors FGFR1, FGFR2 and FGFR3, and VEGFR-2 respectively, were visualized by immunohistochemistry and light microscopy in sections of growing red deer antler. Both of these signalling systems were widely expressed in the integument and osteocartilaginous compartments. FGF-2 was found in the same cells as all three FGFRs, indicating that FGF signalling may be principally autocrine. The patterns of labelling for VEGF and its receptor were similar to those seen for FGF-2 and FGFR-3, in both compartments. Our data are consistent with the findings of others in suggesting that FGF-2 induces expression of VEGF, to stimulate and maintain high rates of neovascularisation and angiogenesis, thereby providing nutrients to both velvet and bone as they rapidly grow and develop. The presence of FGF and VEGF and their receptors in epithelial cells suggests that these signalling systems play a role in skin development, raising the possibility that one or both may be involved in the close coupling of the coordinated growth of the integument and osteocartilage of antler, a process which is poorly understood at present.     

IGF-1 induced vascular endothelial growth factor secretion in head and neck squamous cell carcinoma

Elevated vascular endothelial growth factor (VEGF) levels correlate with increased progression and poor prognosis of head and neck squamous cell carcinomas (HNSCC). VEGF expression is regulated by hypoxia and cytokines, including insulin-like growth factor-1 (IGF-1). In this report, we examined IGF-1 signaling and VEGF expression in SCC-9 cells. IGF-1 and the chemical hypoxia agent, cobalt chloride, each stimulated VEGF secretion and VEGF promoter activation. Cobalt chloride increased Hif-1alpha protein levels and HIF-1 dependent activation of the enolase promoter. IGF-1 increased these parameters only in the presence of cobalt chloride. IGF-1 stimulated PI-3K/Akt and Erk/MAPK pathways in SCC-9 cells, each contributing to Hif-1alpha expression and VEGF secretion. SCC-9 cells express the VEGF receptors Flk-1 and neuropilin-1, with VEGF treatment increasing VEGF promoter activity and VEGF secretion that was attenuated by the Flk-1 tyrosine kinase inhibitor, ZM 323881. These results demonstrate the presence of an IGF-1 regulated VEGF autocrine loop in HNSCC.     

Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells

Insulin-like growth factor 1 (IGF-1), the most abundant growth factor in the bone matrix, maintains bone mass in adulthood. We now report that IGF-1 released from the bone matrix during bone remodeling stimulates osteoblastic differentiation of recruited mesenchymal stem cells (MSCs) by activation of mammalian target of rapamycin (mTOR), thus maintaining proper bone microarchitecture and mass. Mice with knockout of the IGF-1 receptor (Igf1r) in their pre-osteoblastic cells showed lower bone mass and mineral deposition rates than wild-type mice. Further, MSCs from Igf1rflox/flox mice with Igf1r deleted by a Cre adenovirus in vitro, although recruited to the bone surface after implantation, were unable to differentiate into osteoblasts. We also found that the concentrations of IGF-1 in the bone matrix and marrow of aged rats were lower than in those of young rats and directly correlated with the age-related decrease in bone mass. Likewise, in age-related osteoporosis in humans, we found that bone marrow IGF-1 concentrations were 40% lower in individuals with osteoporosis than in individuals without osteoporosis. Notably, injection of IGF-1 plus IGF binding protein 3 (IGFBP3), but not injection of IGF-1 alone, increased the concentration of IGF-1 in the bone matrix and stimulated new bone formation in aged rats. Together, these results provide mechanistic insight into how IGF-1 maintains adult bone mass, while also providing a further rationale for its therapeutic targeting to treat age-related osteoporosis.