Shuanhu Zhou

Age‐related intrinsic changes in human bone‐marrow‐derived mesenchymal stem cells and their differentiation to osteoblasts

In vivo and in vitro studies indicate that a subpopulation of human marrow‐derived stromal cells (MSCs, also known as mesenchymal stem cells) has potential to differentiate into multiple cell types, including osteoblasts. In this study, we tested the hypothesis that there are intrinsic effects of age in human MSCs (17–90 years). We tested the effect of age on senescence‐associated β‐galactosidase, proliferation, apoptosis, p53 pathway genes, and osteoblast differentiation in confluent monolayers by alkaline phosphatase activity and osteoblast gene expression analysis. There were fourfold more human bone MSCs (hMSCs) positive for senescence‐associated β‐galactosidase in samples from older than younger subjects (P < 0.001; n = 17). Doubling time of hMSCs was 1.7‐fold longer in cells from the older than the younger subjects, and was positively correlated with age (P = 0.002; n = 19). Novel age‐related changes were identified. With age, more cells were apoptotic (P = 0.016; n = 10). Further, there were age‐related increases in expression of p53 and its pathway genes, p21 and BAX. Consistent with other experiments, there was a significant age‐related decrease in generation of osteoblasts both in the STRO‐1+ cells (P = 0.047; n = 8) and in adherent MSCs (P < 0.001; n = 10). In sum, there is an age‐dependent decrease in proliferation and osteoblast differentiation, and an increase in senescence‐associated β‐galactosidase‐positive cells and apoptosis in hMSCs. Up‐regulation of the p53 pathway with age may have a critical role in mediating the reduction in both proliferation and osteoblastogenesis of hMSCs. These findings support the view that there are intrinsic alterations in human MSCs with aging that may contribute to the process of skeletal aging in humans.

Engineered human mesenchymal stem cells: a novel platform for skeletal cell mediated gene therapy

Human mesenchymal stem cells (hMSCs) are pluripotent cells that can differentiate to various mesenchymal cell types. Recently, a method to isolate hMSCs from bone marrow and expand them in culture was described. Here we report on the use of hMSCs as a platform for gene therapy aimed at bone lesions.

Cooperation Between TGF-β and Wnt Pathways During Chondrocyte and Adipocyte Differentiation of Human Marrow Stromal Cells†

Human marrow stromal cells have the potential to differentiate to chondrocytes or adipocytes. We show interactions between TGF‐β and Wnt signaling pathways during stimulation of chondrogenesis and inhibition of adipogenesis. Combining these signals may be useful in marrow stromal cell therapies.

Estrogen modulates estrogen receptor alpha and beta expression, osteogenic activity, and apoptosis in mesenchymal stem cells (MSCs) of osteoporotic mice.

In the mouse, ovariectomy (OVX) leads to significant reductions in cancellous bone volume while estrogen (17β‐estradiol, E2) replacement not only prevents bone loss but can increase bone formation. As the E2‐dependent increase in bone formation would require the proliferation and differentiation of osteoblast precursors, we hypothesized that E2 regulates mesenchymal stem cells (MSCs) activity in mouse bone marrow. We therefore investigated proliferation, differentiation, apoptosis, and estrogen receptor (ER) α and β expression of primary culture MSCs isolated from OVX and sham‐operated mice. MSCs, treated in vitro with 10−7 M E2, displayed a significant increase in ERα mRNA and protein expression as well as alkaline phosphatase (ALP) activity and proliferation rate. In contrast, E2 treatment resulted in a decrease in ERβ mRNA and protein expression as well as apoptosis in both OVX and sham mice. E2 up‐regulated the mRNA expression of osteogenic genes for ALP, collagen I, TGF‐β1, BMP‐2, and cbfa1 in MSCs. In a comparison of the relative mRNA expression and protein levels for two ER isoforms, ERα was the predominant form expressed in MSCs obtained from both OVX and sham‐operated mice. Cumulatively, these results indicate that estrogen in vitro directly augments the proliferation and differentiation, ERα expression, osteogenic gene expression and, inhibits apoptosis and ERβ expression in MSCs obtained from OVX and sham‐operated mice. Co‐expression of ERα, but not ERβ, and osteogenic differentiation markers might indicate that ERα function as an activator and ERβ function as a repressor in the osteogenic differentiation in MSCs. These results suggest that mouse MSCs are anabolic targets of estrogen action, via ERα activation. J. Cell. Biochem. Suppl. 36: 144–155, 2001.

Systemically administered rhBMP‐2 promotes MSC activity and reverses bone and cartilage loss in osteopenic mice

Osteoporosis is a disease manifested in drastic bone loss resulting in osteopenia and high risk for fractures. This disease is generally divided into two subtypes. The first, post‐menopausal (type I) osteoporosis, is primarily related to estrogen deficiency. The second, senile (type II) osteoporosis, is mostly related to aging. Decreased bone formation, as well as increased bone resorption and turnover, are thought to play roles in the pathophysiology of both types of osteoporosis. In this study, we demonstrate in murine models for both type I (estrogen deficiency) and type II (senile) osteopenia/osteoporosis that reduced bone formation is related to a decrease in adult mesenchymal stem cell (AMSC) number, osteogenic activity, and proliferation. Decreased proliferation is coupled with increased apoptosis in AMSC cultures obtained from osteopenic mice. Recombinant human bone morphogenetic protein (rhBMP‐2) is a highly osteoinductive protein, promoting osteogenic differentiation of AMSCs. Systemic intra‐peritoneal (i.p.) injections of rhBMP‐2 into osteopenic mice were able to reverse this phenotype in the bones of these animals. Moreover, this change in bone mass was coupled to an increase in AMSCs numbers, osteogenic activity, and proliferation as well as a decrease in apoptosis. Bone formation activity was increased as well. However, the magnitude of this response to rhBMP‐2 varied among different stains of mice. In old osteopenic BALB/c male mice (type II osteoporosis model), rhBMP‐2 systemic treatment also restored both articular and epiphyseal cartilage width to the levels seen in young mice. In summary, our study shows that AMSCs are a good target for systemically active anabolic compounds like rhBMP‐2. J. Cell. Biochem. 86: 461–474, 2002.

The Melatonin MT1 Receptor Axis Modulates Mutant Huntingtin-Mediated Toxicity

Melatonin mediates neuroprotection in several experimental models of neurodegeneration. It is not yet known, however, whether melatonin provides neuroprotection in genetic models of Huntingtons disease (HD). We report that melatonin delays disease onset and mortality in a transgenic mouse model of HD. Moreover, mutant huntingtin (htt)-mediated toxicity in cells, mice, and humans is associated with loss of the type 1 melatonin receptor (MT1). We observe high levels of MT1 receptor in mitochondria from the brains of wild-type mice but much less in brains from HD mice. Moreover, we demonstrate that melatonin inhibits mutant htt-induced caspase activation and preserves MT1 receptor expression. This observation is critical, because melatonin-mediated protection is dependent on the presence and activation of the MT1 receptor. In summary, we delineate a pathologic process whereby mutant htt-induced loss of the mitochondrial MT1 receptor enhances neuronal vulnerability and potentially accelerates the neurodegenerative process.

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow‐derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF‐β) and Wnt/β‐catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF‐β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF‐β activation of β‐catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF‐β1 requires TGF‐β type I receptor ALK‐5, Smad3, phosphoinositide 3‐kinases (PI3K), and protein kinase A (PKA) to stabilize β‐catenin, and needs ALK‐5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β‐catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF‐β1 on bone sialoprotein (BSP) expression, suggested that TGF‐β1 cooperated with β‐catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF‐β1 activates β‐catenin signaling pathway via ALK‐5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF‐β and β‐catenin signaling supports the view that β‐catenin signaling is a mediator of TGF‐βs effects on osteoblast differentiation of hMSCs. J. Cell. Biochem. 112: 1651–1660, 2011.

Vitamin D metabolism and action in human bone marrow stromal cells.

Vitamin D metabolites are important effectors of bone and mineral homeostasis. Extrarenal conversion of 25-hydroxyvitamin D (25OHD) to the biologically active form of vitamin D, 1 alpha,25-dihydroxyvitamin D [1,25(OH)(2)D] is catalyzed in several cell types by the 1 alpha-hydroxylase (CYP27B1), but little is known about the expression or regulation of CYP27B1 in human bones. We examined whether human bone marrow stromal cells (hMSCs, also known as mesenchymal stem cells) participate in vitamin D metabolism and whether vitamin D hydroxylases in hMSCs are influenced by the vitamin D status of the individual from whom the hMSCs were obtained. We also investigated the effects of vitamin D metabolites on osteoblast differentiation and the role of IGF-I in the regulation of CYP27B1. In a series of 27 subjects, vitamin D hydroxylases in hMSCs were expressed at different levels and were correlated with serum 25OHD, 1,25(OH)(2)D, and PTH. In vitro treatment with 25OHD up-regulated CYP27B1 and IGF-I in hMSCs; IGF-I also up-regulated CY27B1 expression and stimulated osteoblast differentiation. When hydroxylation of 25OHD was blocked by ketoconazole, a cytochrome P450 inhibitor, 25OHD was no longer able to induce CYP27B1 expression. In summary, these findings show that human bone marrow stromal cells have the molecular machinery both to metabolize and respond to vitamin D. We propose that circulating 25OHD, by virtue of its local conversion to 1,25(OH)(2)D catalyzed by basal CYP27B1 in hMSCs, amplifies vitamin D signaling through IGF-I up-regulation, which in turn induces CYP27B1 in a feed-forward mechanism to potentiate osteoblast differentiation initiated by IGF-I.

Effects of 25-Hydroxyvitamin D3 on Proliferation and Osteoblast Differentiation of Human Marrow Stromal Cells Require CYP27B1/1α-Hydroxylase

1,25‐Dihydroxyvitamin D3 [1,25(OH)2D3] has many noncalcemic actions that rest on inhibition of proliferation and promotion of differentiation in malignant and normal cell types. 1,25(OH)2D3 stimulates osteoblast differentiation of human marrow stromal cells (hMSCs), but little is known about the effects of 25‐hydroxyvitamin D3 [25(OH)D3] on these cells. Recent evidence shows that hMSCs participate in vitamin D metabolism and can activate 25(OH)D3 by CYP27B1/1α‐hydroxylase. These studies test the hypothesis that antiproliferative and prodifferentiation effects of 25(OH)D3 in hMSCs depend on CYP27B1. We studied hMSCs that constitutively express high (hMSCshi‐1α) or low (hMSCslo‐1α) levels of CYP27B1 with equivalent expression of CYP24A1 and vitamin D receptor. In hMSCshi‐1α, 25(OH)D3 reduced proliferation, downregulated proliferating cell nuclear antigen (PCNA), upregulated p21Waf1/Cip1, and decreased cyclin D1. Unlike 1,25(OH)2D3, the antiapoptotic effects of 25(OH)D3 on Bax and Bcl‐2 were blocked by the P450 inhibitor ketoconazole. The antiproliferative effects of 25(OH)D3 in hMSCshi‐1α and of 1,25(OH)2D3 in both samples of hMSCs were explained by cell cycle arrest, not by increased apoptosis. Stimulation of osteoblast differentiation in hMSCshi‐1α by 25(OH)D3 was prevented by ketoconazole and upon transfection with CYP27B1 siRNA. These data indicate that CYP27B1 is required for 25(OH)D3s action in hMSCs. Three lines of evidence indicate that CYP27B1 is required for the antiproliferative and prodifferentiation effects of 25(OH)D3 on hMSCs: Those effects were not seen (1) in hMSCs with low constitutive expression of CYP27B1, (2) in hMSCs treated with ketoconazole, and (3) in hMSCs in which CYP27B1 expression was silenced. Osteoblast differentiation and skeletal homeostasis may be regulated by autocrine/paracrine actions of 25(OH)D3 in hMSCs.

Recombinant TGF‐β1 stimulates bone marrow osteoprogenitor cell activity and bone matrix synthesis in osteopenic, old male mice

We have previously hypothesized that the osteopenic changes seen in the skeletons of old male BALB/c mice are due to reductions in the availability and/or synthesis of bone TGF‐β which results in fewer, less osteogenic marrow osteoprogenitor cells (CFU‐f; OPCs) and lower levels of bone formation. Among other things, this hypothesis would predict that introducing exogenous TGF‐β into old mice (growth factor replacement) should stimulate marrow CFU‐f and increase bone formation. In the present study, we have tested this prediction and, indirectly the hypothesis, by injecting human recombinant TGF‐β1, i.p., into both young adult (4 month) and old mice (24 month). The effects of the growth factor on the skeleton were then assessed by measurements of trabecular bone volume, bone formation, fracture healing, and the number, proliferative, apoptotic, and alkaline phosphatase activity of marrow CFU‐f/OPCs. Our data show that the introduction of 0.5 or 5.0 ug/day of TGF‐β1 into old mice for 20 days 1) increases trabecular bone volume, bone formation and the mineral apposition rate, 2) augments fracture healing, 3) increases the number and size of CFU‐f colonies, and 4) increases proliferation and diminishes apoptosis of CFU‐f in primary bone marrow cultures. Importantly, these stimulatory effects of injected growth factor are apparently age‐specific, i.e., they are either not seen in young animals or, if seen, are found at much lower levels. While these observations do not exclude other possible mechanisms for the osteopenia of old mice, they provide further support for the hypothesis that, with age, diminished TGF‐β synthesis or availability results in a reduction in the marrow osteoprogenitor pool and bone formation. The findings also demonstrate that the latter changes can be reversed, at least transiently, by introducing exogenous TGF‐β1. J. Cell. Biochem. 73:379–389, 1999.