Multilineage Potential of Stable Human Mesenchymal Stem Cell Line Derived from Fetal Marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and β-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders

Effect of self-reported walking difficulty on bone mass and bone resorption marker in Japanese people aged 40?years and over

Background: This study aimed to examine the association of walking difficulty with bone mass or bone turnover among community-dwelling Japanese people aged 40 years and older. Methods: We studied 1097 community-dwelling Japanese people aged 40 years and older (379 men and 718 women) who were invited to participate in periodic health examinations in 2006?2009. Walking difficulty was defined as having difficulty walking 100 m on a level surface (self-administered questionnaire). Calcaneal stiffness index (bone mass) was measured by quantitative ultrasound. Spot urine samples were collected, and urinary N-terminal cross-linking telopeptide of type I collagen (NTx) was measured. Values were corrected for creatinine (Cre) concentration. Results: The prevalence of walking difficulty was significantly higher in women than in men (7.4 vs. 3.4 %, p?=?0.011) and significantly increased with age in men (p for trend?=?0.02) and women (p for trend <0.001). In univariate analysis, men and women with walking difficulty were older (p?<?0.001) and had a lower stiffness index (p?<?0.001), compared with those without walking difficulty. Among women, individuals with walking difficulty had significantly higher urinary NTx/Cre than those without walking difficulty (p?<?0.001); however, this was not so among men (p?=?0.39). Multiple regression analysis adjusted for age, weight, and menopausal status showed a significant association between walking difficulty and lower stiffness index in men (p?=?0.004) and women (p?=?0.005). In women, walking difficulty was significantly associated with higher NTx/Cre (p?=?0.001), but not in men (p?=?0.35). Conclusions: Walking difficulty may contribute to low bone mass in both sexes but might cause high bone turnover in women only

Overexpression of Bone Sialoprotein Leads to an Uncoupling of Bone Formation and Bone Resorption in Mice

The purpose of this study was to determine the effects of bone sialoprotein (BSP) overexpression in bone metabolism in vivo by using a homozygous transgenic mouse line that constitutively overexpresses mouse BSP cDNA driven by the cytomegalovirus (CMV) promoter. CMV-BSP transgenic (TG) mice and wildtype mice were weighed, and their length, BMD, and trabecular bone volume were measured. Serum levels of RANKL, osteocalcin, osteoprotegerin (OPG), TRACP5b, and PTH were determined. Bone histomorphometry, von Kossa staining, RT-PCR analysis, Western blot, MTS assay, in vitro mineralization assay, and TRACP staining were also performed to delineate phenotypes of this transgenic mouse line. Compared with wildtype mice, adult TG mice exhibit mild dwarfism, lower values of BMD, and lower trabecular bone volume. TG mice serum contained increased calcium levels and decreased PTH levels, whereas the levels of phosphorus and magnesium were within normal limits. TG mice serum also exhibited lower levels of osteoblast differentiation markers and higher levels of markers, indicating osteoclastic activity and bone resorption. H&E staining, TRACP staining, and bone histomorphometry showed that adult TG bones were thinner and the number of giant osteoclasts in TG mice was higher, whereas there were no significant alterations in osteoblast numbers between TG mice and WT mice. Furthermore, the vertical length of the hypertrophic zone in TG mice was slightly enlarged. Moreover, ex vivo experiments indicated that overexpression of BSP decreased osteoblast population and increased osteoclastic activity. Partly because of its effects in enhancing osteoclastic activity and decreasing osteoblast population, BSP overexpression leads to an uncoupling of bone formation and resorption, which in turn results in osteopenia and mild dwarfism in mice. These findings are expected to help the development of therapies to metabolic bone diseases characterized by high serum level of BSP