Human adipose stromal cells expanded in human serum promote engraftment of human peripheral blood hematopoietic stem cells in NOD/SCID mice

Human mesenchymal stem cells (hMSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles, and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle, and neuron. Therefore, hMSC are attractive candidates for cell and gene therapy. The optimal conditions for hMSC expansion require medium supplemented with fetal bovine serum (FBS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FBS proteins. In this study, we cultured human adipose stromal cells (hADSC) and bone marrow stroma cells (HBMSC) in human serum (HS) during their isolation and expansion, and demonstrated that they maintain their proliferative capacity and ability for multilineage differentiation and promote engraftment of peripheral blood-derived CD34(+) cells mobilized from bone marrow in NOD/SCID mice. Our results indicate that hADSC and hBMSC cultured in HS can be used for clinical trials of cell and gene therapies, including promotion of engraftment after allogeneic HSC transplantation.     

Direct comparison of human mesenchymal stem cells derived from adipose tissues and bone marrow in mediating neovascularization in response to vascular ischemia.

BACKGROUND/AIM: Although transplantation of MSC derived from bone marrow or adipose tissues has been shown in proangiogenic action in hindlimb ischemia model of nude mice, little information is available regarding comparison of the angiogenic potency between human adipose stromal cells (hADSC) and bone marrow stromal cells (hBMSC). We compared their therapeutic potential by transplantation of equal numbers of hADSC or hBMSC in a nude mice model of hindlimb ischemia. METHODS AND RESULTS: One day after creating hindlimb ischemia, mice were randomized to receive hADSC transplantation (hADSC group), hBMSC transplantation (hBMSC group), or vehicle transplantation (Control group). Two weeks after transplantation, the laser Doppler perfusion index was significantly higher in the hADSC group and hBMSC group than in the control group. Comparison between hADSC and hBMSC group showed better recovery of blood flow in hADSC group than in hBMSC group. Conditioned media from hADSC (hADSC-CM) showed better in vitro tube formation of hADSC than conditioned media from hBMSC (hBMSC-CM). hADSC showed higher expression of MMP3 and MMP9 than hBMSC. A MMP inhibitor, GM6001, and the transfection of MMP3 or MMP9 siRNA oligonucleotides inhibited in vitro tube formation of hADSC. Transplantation of MMP3 or MMP9 siRNA oligonucleotieds-transfected hADSC showed lower blood flow recovery and higher tissue injury than control oligonucelotide-transfected cells. CONCLUSION: This study showed that hADSC can be an ideal source for therapeutic angiogenesis in ischemic disease in terms of efficacy, accessibility and available tissue amounts.     

Mg2+ in β-TCP/Mg–Zn composite enhances the differentiation of human bone marrow stromal cells into osteoblasts through MAPK-regulated Runx2/Osx

Inducing the osteogenic differentiation from bone marrow stromal cells (BMSCs) might be a potent strategy for treating bone loss and nonunion during fracture and improving fracture healing. Among several signaling pathways involved, mitogen-activated protein kinases (MAPKs) have been reported to play a critical role. Magnesium (Mg)-based alloys, including Mg–Zn alloy, have been used clinically as implants in the musculoskeletal field and could promote BMSC osteogenic differentiation. However, the underlying mechanisms remain unclear. In this study, we produced Mg–Zn alloy consists of Mg and low concentrations of Zn, calcium carbonate, and β-tricalcium phosphate (β-TCP; manifesting process not shown), prepared Mg, Zn, and Mg–Zn extracts, and investigated the specific effects of these extracts on human BMSC (hBMSC) osteogenic differentiation and MAPK signaling. Mg extracts and Mg–Zn extracts could significantly promote the osteogenic differentiation of hBMSCs as manifested as increased alkaline phosphatase levels, enhanced calcium nodules formation, and increased messenger RNA expression and protein levels of osteogenesis markers, including BMPs, Col-I, Runx2, and Osx; in the meantime, Mg culture medium (CM) and Mg–Zn CM both significantly enhanced the activation of MAPK signaling in hBMSCs. By adding ERK1/2 signaling, p38 signaling, or JNK signaling inhibitor to Mg–Zn CM, or conducting p38 MAPK silence in hBMSCs, we revealed that these extracts might promote hBMSC osteogenic differentiation via p38 MAPK signaling and MAPK-regulated Runx2/Osx. In conclusion, Mg²⁺ in β-TCP/Mg–Zn extract promotes the osteogenic differentiation of hBMSCs via MAPK-regulated Runx2/Osx interaction.     

Osteogenic Potency of Nacre on Human Mesenchymal Stem Cells

Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC’s), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC’s led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I–IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC’s.     

Osteogenic oxysterols inhibit the adverse effects of oxidative stress on osteogenic differentiation of marrow stromal cells

The osteoporosis that occurs with aging is associated with reduced number and activity of osteoblastic cells. Aging, menopause, and osteoporosis are correlated with increased oxidative stress and reduced antioxidant defense mechanisms. We previously demonstrated that oxidative stress induced by a variety of compounds such as xanthine/xanthine oxidase (XXO) and minimally oxidized LDL (MM-LDL) inhibit the osteogenic differentiation of osteoprogenitor cells. Oxysterols are a family of products derived from cholesterol oxidation that have important biological activities. Recently, we reported that a specific oxysterol combination consisting of 22(S)- or 22(R)-hydroxycholesterol and 20(S)-hydroxycholesterol has potent osteogenic properties in vitro when applied to osteoprogenitor cells including M2-10B4 (M2) marrow stromal cells. We now demonstrate that this osteogenic combination of oxysterols prevents the adverse effects of oxidative stress on differentiation of M2 cells into mature osteoblastic cells. XXO and MM-LDL inhibited the osteogenic differentiation of M2 cells, demonstrated by the inhibition of markers of osteogenic differentiation: alkaline phosphatase activity, osteocalcin expression and mineralization. Treatment of M2 cells with osteogenic oxysterol combination 22(S)- and 20(S)-hydroxycholesterol both blocked and reversed the inhibition of osteogenic differentiation produced by XXO and MM-LDL in these cells. The protective effect of the oxysterols against oxidative stress was dependent on cyclooxygenase 1 and was associated with the osteogenic property of the oxysterols. These findings further demonstrate the ability of the osteogenic oxysterols to positively regulate osteogenic differentiation of cells, and suggests that the use of these compounds may be a novel strategy to prevent the adverse effects of oxidative stress on osteogenesis.     

Necrotic and apoptotic cells serve as nuclei for calcification on osteoblastic differentiation of human mesenchymal stem cells in vitro

A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase‐3 activation increased in this culture. A pan‐caspase inhibitor (Z‐VAD‐FMK) and anti‐oxidants (Tiron and n ‐acetylcysteine) inhibited osteogenic culture‐induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co‐localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro. Copyright © 2013 John Wiley & Sons, Ltd.     

Salicylideneamino-2-thiophenol enhances osteogenic differentiation through the activation of MAPK pathways in multipotent bone marrow stem cell

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone formation and bone resorption. Therefore, the identification of specific stimulators of bone formation is of therapeutic significance in the treatment of osteoporosis. Salicylideneamino-2-thiophenol (Sal-2) consists of two benzene rings, has been reported to possess antioxidant activity, and is an effective remedy for fever and rheumatic diseases. However, until now the effects of osteoblastic bone formation by Sal-2 were unknown. In this study, we investigated the effects of Sal-2 on osteogenic differentiation of multipotent bone marrow stromal stem cells by alizarin red S staining for osteogenic differentiation, RT-PCR and western blot for alkaline phosphatase (ALP) activity and signaling pathways, FACS analysis and immunofluorescence staining for CD44 and CD51 expression, calcium assays, and immunofluorescence staining for signaling pathways. We found that Sal-2 enhanced the osteogenic differentiation of multipotent bone marrow stromal stem cells. Sal-2 treatment induced the expression and activity of ALP, and enhanced the levels of CD44 and CD51 expression as well as Ca2+ content, in multipotent bone marrow stromal stem cells. Moreover, we found that Sal-2-induced osteogenic differentiation and expression of osteogenesis-related molecules involve the activation of the MAPK and nuclear factor-κB pathways. Our findings provide insight into both the mechanism and effects of Sal-2 on osteogenic differentiation and demonstrate that Sal-2 may be a beneficial adjuvant in stimulating bone formation in osteoporotic diseases.     

Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo

Embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and hepatic stem cells in liver have been known as a useful source that can induce to differentiate into hepatocytes. In this study, we examined whether human adipose tissue-derived stromal cells (hADSC) can differentiate into hepatic lineage in vitro. hADSC, that were induced to differentiate into hepatocyte-like cells by the treatment of HGF and OSM, had morphology similar to hepatocytes. Addition of DMSO enhanced differentiation into hepatocytes. RT-PCR and immunocytochemical analysis showed that hADSC express albumin and alpha-fetoprotein during differentiation. Differentiated hADSC showed LDL uptake and production of urea. Additionally, transplanted hADSC to CCl4-injured SCID mouse model were able to be differentiated into hepatocytes and they expressed albumin in vivo. Mesenchymal stem cells isolated from human adipose tissue are immunocompatible and are easily isolated. Therefore, hADSC may become an alternative source to hepatocyte regeneration or liver cell transplantation.     

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.     

Oxysterol-induced osteogenic differentiation of marrow stromal cells is regulated by Dkk-1 inhibitable and PI3-kinase mediated signaling

Osteoporosis and its complications cause morbidity and mortality in the aging population, and result from increased bone resorption by osteoclasts in parallel with decreased bone formation by osteoblasts. A widely accepted strategy for improving bone health is targeting osteoprogenitor cells in order to stimulate their osteogenic differentiation and bone forming properties through the use of osteoinductive/anabolic factors. We previously reported that specific naturally occurring oxysterols have potent osteoinductive properties, mediated in part through activation of hedgehog signaling in osteoprogenitor cells. In the present report, we further demonstrate the molecular mechanism(s) by which oxysterols induce osteogenesis. In addition to activating the hedgehog signaling pathway, oxysterol-induced osteogenic differentiation is mediated through a Wnt signaling-related, Dkk-1-inhibitable mechanism. Bone marrow stromal cells (MSC) treated with oxysterols demonstrated increased expression of osteogenic differentiation markers, along with selective induced expression of Wnt target genes. These oxysterol effects, which occurred in the absence of beta-catenin accumulation or TCF/Lef activation, were inhibited by the hedgehog pathway inhibitor, cyclopamine, and/or by the Wnt pathway inhibitor, Dkk-1. Furthermore, the inhibitors of PI3-Kinase signaling, LY 294002 and wortmanin, inhibited oxysterol-induced osteogenic differentiation and induction of Wnt signaling target genes. Finally, activators of canonical Wnt signaling, Wnt3a and Wnt1, inhibited spontaneous, oxysterol-, and Shh-induced osteogenic differentiation of bone marrow stromal cells, suggesting the involvement of a non-canonical Wnt pathway in pro-osteogenic differentiation events. Osteogenic oxysterols are, therefore, important small molecule modulators of critical signaling pathways in pluripotent mesenchymal cells that regulate numerous developmental and post-developmental processes.     

A dual role of cholesterol in osteogenic differentiation of bone marrow stromal cells

Osteoblasts, the chief bone-forming cells, are differentiated from mesenchymal stromal/stem cells. Disruption of this differentiation process can cause osteoporosis, a bone disease characterized by low bone mass and deteriorated bone structure. Cholesterol has been implicated in pathogenesis of osteoporosis, and was recently identified as an endogenous activator of Hedgehog (Hh) signaling. However, its pathological and physiological roles in osteoblast differentiation are still poorly understood. Moreover, it is unclear whether these potential roles played by cholesterol are related to its capability to modulate Hh pathway. In this study, we investigated the role of exogenous versus endogenous cholesterol in osteogenesis and Hh pathway activation using ST2 cells, a bone marrow stromal cell line. We found that exogenous cholesterol significantly inhibited alkaline phosphatase (ALP) activity and messenger RNA expression of osteoblast markers genes (Alpl, Sp7, and Ibsp) while modestly activating expression of Gli1 (a readout of Hh signaling) under both basal osteogenic culture condition and Wnt3a treatment. Similarly, exogenous cholesterol suppressed osteogenic response of ST2 cells to sonic Hh (Shh) or purmorphamine (Purmo) treatment, which, however, was accompanied by diminished induction of Gli1, indicating the involvement of a Hh-dependent mechanism. Interestingly, depletion of endogenous cholesterol also reduced Shh-induced ALP activity and Gli1 expression. Likewise, cholesterol depletion inhibited osteogenic response to Purmo, although it did not affect Gli1 induction. Taken together, our findings have demonstrated that cholesterol plays a dual role in osteoblast differentiation likely through both Hh-dependent and -independent mechanisms.     

ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice

Osteogenic differentiation refers to the process of bone formation and remodeling, which is controlled by complex molecular mechanisms. Activin A receptor type I (ACVR1) is reported to be associated with osteogenic differentiation. However, the underlying molecular mechanism remains elusive. Therefore, this study evaluates the function of ACVR1 in osteogenic differentiation through the Wnt signaling pathway. The expression of osteocalcin (Oc) and osterix together with osteogenic differentiation and mineralization was examined in ACVR1-knockout (KO) mouse. Furthermore, the Wnt signaling pathway was inhibited in bone marrow stromal cells (BMSCs) of mice to explore the role of the Wnt signaling pathway in osteogenic differentiation by means of alkaline phosphatase (ALP) activity detection and evaluation of mineralized nodules and calcium content. Subsequently, the effect of ACVR1 on the Wnt signaling pathway was assessed by determining the expression of ACVR1, β-catenin, glycogen synthase kinase 3 β (GSK3β), dickkopf-related protein 1 (DKK1), and frizzled class receptor 1 (FZD1). Both their effects on osteogenic differentiation were further evaluated by determination of Oc, osterix, and Runx2 expression. AVCR1 KO mice exhibited increased Oc and osterix expression and promoted bone resorption and formation. ACVR1-knockout was observed to activate the Wnt signaling pathway with an increase of β-catenin and reductions in GSK3β, DKK1, and FZD1. With the inhibited Wnt signaling pathway expression of Oc, osterix, and Runx2 was decreased, and ALP activity, mineralized nodule, and calcium content in cellular matrix were decreased as well, indicating that inactivation of the Wnt signaling pathway reduced the differentiation of BMSCs into osteoclasts. These findings indicate that ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice.     

Effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement and Oil Red O stain and measurement were employed to assess the effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts. The results showed that except for individual concentrations of zinc ion there was no effect on the proliferation of MSCs and osteoblasts. Zinc ion inhibited the osteogenic differentiation of MSCs at all the concentrations tested. It also inhibited adipogenic differentiation at all concentrations tested except 10(-9)mol/L. Both of the inhibition effects were attenuated with time increasing. Zinc ion depressed adipocytic trans-differentiation of osteoblasts at concentrations of 10(-11) and 10(-10)mol/L, but the effect could be reversed to promote or even be removed when concentration was increased. It suggests that the influence of zinc ion on osteogenic, adipogenic differentiation of MSCs and adipocytic trans-differentiation of osteoblasts depends on zinc ion concentrations and incubation time. The protective effects of zinc ion on bone may be mediated by modulating differentiation of MSCs away from the adipocytes and inhibiting adipocytic trans-differentiation of osteoblasts. This may in turn promote osteoblast formation and reduce secretion of cytokines which may inhibit osteoclast formation and activation. These findings may be valuable for better understanding the mechanism of the effect of zinc ion on bone.     

Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E₂ without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.