Ana María Pino

Abnormal osteogenesis in osteoporotic patients is reflected by altered mesenchymal stem cells dynamics

Bone marrow contains a population of mesenchymal stem cells with the ability to differentiate into cells that form bone, cartilage, adipose, and other connective tissues. Stem cells can be isolated from bone marrow aspirates and expanded in vitro. Presently, most stem cells studies have been performed in cells obtained from “healthy” control subjects. The goal of this study was to compare the functional characteristics of mesenchymal stem cells derived from “healthy” control and osteoporotic postmenopausal women to better understand the mechanisms involved in the pathogenesis of this disease. Osteoporotic and control stem cells have similar morphology and size and express similar cell surface antigens as evidenced by their reactivity with cell specific monoclonal antibodies. Mesenchymal stem cells from osteoporotic women differ from controls in having a lower growth rate than control cells, being refractory to the mitogenic effect of IGF‐1, and exhibiting a deficient ability to differentiate into the osteogenic linage as evidenced by the alkaline phosphatase activity and calcium phosphate deposition. We conclude that in osteoporosis stem cell growth, proliferative response and osteogenic differentiation are significantly affected. Also, the study of mesenchymal stem cells from osteoporotic postmenopausal women may provide a better understanding of the mechanisms involved in the pathogenesis of the osteoporosis. It may also serve to test in vitro in rapid manner novel new therapeutic strategies. J. Cell. Biochem. 75:414–423, 1999.

Involvement of adipogenic potential of human bone marrow mesenchymal stem cells (MSCs) in osteoporosis.

Mesenchymal Stem Cells (MSCs) from bone marrow stroma are capable of differentiating into osteoblasts and adipocytes, among other cell phenotypes. In normal bone marrow osteoblastic and adipocytic cell differentiation occur in favor of bone formation, but this relationship appears disrupted in several bone diseases. In osteoporosis increased bone marrow adipocyte production is counterbalanced by diminished production of osteogenic cells. Since osteoblasts and adipocytes originate from a common MSC precursor cell, quantitative and qualitative stem cell defects may underlie the modified number and function of differentiated cells. This review analyzes experimental evidence which describes differences in the osteogenic/adipogenic potentials of human bone marrow MSCs obtained from control and osteoporotic postmenopausal women. The protective effect exerted by locally generated factors, such as estradiol and leptin, on MSCs differentiation was analyzed, because altered bioavailability of these factors may play a part in osteoporosis triggering. Several properties differ among differentiating MSCs from control and osteoporotic donors. Some of these functional differences may be considered to mirror, at the cell level, the detrimental changes displayed in osteoporosis. Osteoporotic MSCs are characterized by increased adipogenic potential, as shown by increased PPARgamma protein content and diminished inactivation of the transcription factor, as compared to control cells. Leptin exerts a direct protective activity against adipogenesis only in control cells. In contrast, leptin activity in MSCs from osteoporotic women appears hampered, suggesting that inadequate leptin activity contributes to excessive lipid accumulation in bone marrow.

Increased adipogenesis of osteoporotic human‐mesenchymal stem cells (MSCs) characterizes by impaired leptin action

The bone marrow contains mesenchymal stem cells (MSCs) that differentiate to the osteogenic and adipogenic lineages. The fact that the decrease in bone volume of age‐related osteoporosis is accompanied by an increase in marrow adipose tissue implies the importance that the adipogenic process may have in bone loss. We previously observed that MSCs from control and osteoporotic women showed differences in their capacity to differentiate into the osteogenic and adipogenic pathways. In vitro studies indicate that bone marrow stromal cells are responsive to leptin, which increases their proliferation, differentiation to osteoblasts, and the number of mineralized nodules, but inhibits their differentiation to adipocytes. The aim of the present report was to study the direct effect of leptin on control and osteoporotic MSCs analyzing whether the protective effect of leptin against osteoporosis could be expressed by inhibition of adipocyte differentiation. MSCs from control, and osteoporotic donors were subjected to adipogenic conditions, in the absence or in the presence of 62.5 nM leptin. The number of adipocytes, the content of PPARγ protein, and mRNA, and leptin mRNA were measured by flow cytometry, Western blot, and RT‐PCR, respectively. Results indicate that control and osteoporotic MSCs differ in their adipogenic potential as shown by expression of active PPARγ protein. Leptin exerted an antiadipogenic effect only on control MSCs increasing the proportion of inactive phosphorylated PPARγ protein. Finally, results obtained during adipogenesis of osteoporotic cells suggest that this process is abnormal not only because of increased adipocyte number, but because of impaired leptin cells response. J. Cell. Biochem. 103: 1054–1065, 2008.

High affinity leptin receptors are present in human mesenchymal stem cells (MSCs) derived from control and osteoporotic donors

There are disparate observations on central and peripheral effects of leptin, but several studies consistently support its role as a link between fat and bone. Bone marrow stroma contains mesenchymal stem cells (MSCs), which differentiate into osteoblasts and adipocytes, among others. In this study we assessed the expression of leptin receptors protein in MSCs from control and osteoporotic postmenopausal donors and their change during osteogenic and adipogenic differentiation. Also, we assessed the effects of leptin on osteogenic and adipogenic differentiation of these cells. We demonstrated high affinity leptin binding (KD = 0.36 ± 0.02 nM) in both types of cells. Binding was very low under basal, but increased significantly (2–3 times) through osteogenic and adipogenic differentiation. Osteoporotic MSCs showed lower leptin binding capacity than control cells at an early osteogenic and adipogenic differentiation time, which could restrict cell sensitivity to the protective action of leptin. In this regard, we observed that leptin significantly inhibited adipocyte differentiation in control but not in osteoporotic MSCs, while it exerted a low stimulatory effect on calcium deposition (10%–20%) in both types of MSCs cells. In summary, we report the presence of high affinity leptin receptors on control and osteoporotic MSCs, which were modified distinctly by osteogenic and adipogenic stimulation and a direct and distinct effect of leptin on both type of cells.

Phytoplasmas Associated with Grapevine Yellows Disease in Chile

An extensive survey was performed from 2002 to 2006 to detect and identify phytoplasmas associated with Chilean grapevines. Nested polymerase chain reaction assays using phytoplasma universal primer pairs P1/P7 and R16F2n/R2 detected phytoplasmas in 34 out of the 94 samples tested (36%). Restriction fragment length polymorphism (RFLP) analyses, cloning, and sequencing allowed identification of phytoplasmas belonging to ribosomal subgroups 16SrI-B, 16SrI-C, 16SrVII-A, and 16SrXII-A. The 16SrVII-A phytoplasma represents a new finding in grapevine; moreover, variability of the RFLP profile was observed in some of the 16SrXII-A phytoplasmas, indicating possible new ribosomal subgroups. Mixed phytoplasma infections and infections of phytoplasmas together with one or more viruses also occurred.

Synthesis of nanostructured porous silica coatings on titanium and their cell adhesive and osteogenic differentiation properties

Nanostructured porous silica coatings were synthesized on titanium by the combined sol-gel and evaporation-induced self-assembly process. The silica-coating structures were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and nitrogen sorptometry. The effect of the nanoporous surface on apatite formation in simulated body fluid, protein adsorption, osteoblast cell adhesion behavior, and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) is reported. Silica coatings with highly ordered sub-10 nm porosity accelerate early osteoblast adhesive response, a favorable cell response that is attributed to an indirect effect due to the high protein adsorption observed on the large-specific surface area of the nanoporous coating but is also probably due to direct mechanical stimulus from the nanostructured topography. The nanoporous silica coatings, particularly those doped with calcium and phosphate, also promote the osteogenic differentiation of hBMSCs with spontaneous mineral nodule formation in basal conditions. The bioactive surface properties exhibited by the nanostructured porous silica coatings make these materials a promising alternative to improve the osseointegration properties of titanium dental implants and could have future impact on the nanoscale design of implant surfaces.

Changes in cytosolic and nuclear estradiol receptors of normal fallopian tube throughout the menstrual cycle

During the normal menstrual cycle, the changes in estradiol binding to cytosol and nuclear receptors of ampulla, isthmus and fimbria were analyzed. Cytosol estradiol receptor concentration showed little variation throughout the cycle, but were higher at the periovulatory phases. The concentration of nuclear estradiol receptor markedly varied in isthmus and ampulla; being significantly higher at the late proliferative phase than at the secretory phases. A positive linear correlation between nuclear receptor content and plasma estradiol concentration was observed, while a negative one was found with plasma progesterone.

The increased expression of receptor activator of nuclear-κB ligand (RANKL) of multiple myeloma bone marrow stromal cells is inhibited by the bisphosphonate ibandronate

The receptor activator of nuclear factor‐kappaB ligand (RANKL) and interleukin‐1beta are osteoclast activating factors which are abnormally expressed in bone marrow stromal cells and plasma cells of multiple myeloma patients. In this work we analyzed RANKL expression in human bone marrow mesenchymal stromal cells and the effect of the bisphosphonate ibandronate on RANKL expression after IL‐1beta activation of ERK pathway. Mesenchymal stromal cells were obtained from bone marrow iliac aspirates from multiple myeloma patients at stages II/III and non‐osteoporotics control donors; these cells were maintained under long‐term culture conditions. Cells were cultured in the presence or the absence of 5 ng/ml IL‐1beta and/or 5 µM ibandronate, during selected periods. mRNA for RANKL and protein levels were assayed by RT‐PCR and Western blot, respectively. Human bone marrow stromal cell line HS‐5 was used for assessing IL 1beta‐ and ibandronate‐ERK phosphorylation responses. Multiple myeloma mesenchymal stromal cells differentiate from control cells by increased basal RANKL expression. IL‐1beta up regulated RANKL expression showed dependent on activated MEK/ERK pathway. Finally, the bisphosphonate ibandronate, that hindered activation of the MEK/ERK pathway significantly inhibited both basal and IL‐1beta dependent RANKL expression by cells. Results indicate that RANKL expression involves the MEK/ERK pathway in multiple myeloma mesenchymal stromal cells, and that early obstruction of this path, such as that achieved with ibandronate, significantly deters RANKL protein expression. J. Cell. Biochem. 111: 130–137, 2010.

Evidence for a Leydig cell progesterone receptor in the rat

Tritiated promegestone [3H] R 5020 is bound with high affinity by charcoal-treated cytosol prepared from purified Leydig cells. The binding is characterized by high affinity (Kd = 2 x 10(-9) M) and specificity (R 5020 = progesterone greater than testosterone = dehydrotestosterone greater than hydroxyprogesterone greater than cortisol = dexamethasone greater than estradiol) appropriate for progesterone receptors. In vitro, progestin-bound cytosol was quantitatively translocated to nuclei fractions, only if cytosol samples were previously labeled at 25 degrees C. However no translocation of binding activity was observed when previous cytosol labeling was done in the presence of sodium molybdate. Effects of glucocorticoids, androgens and estrogens on the Leydig cell are well documented, the demonstration of a putative progesterone receptor raises the possibility of direct effect of progesterone on the Leydig cell.

Qualitative Aspects of Bone Marrow Adiposity in Osteoporosis

The function of marrow adipocytes and their origin has not been defined although considerable research has centered on their presence in certain conditions, such as osteoporosis. Less work has focused on the qualitative aspects of marrow fat. Bone marrow serum is composed of multiple nutrients that almost certainly relate to functional aspects of the niche. Previous studies using non-invasive techniques have shown that osteoporotic individuals have more marrow fat and that the ratio of saturated: unsaturated fatty acid is high. We recently reported that bone marrow sera from osteoporotic patients with fracture showed a switch toward decreased content of total saturated versus unsaturated fatty acids, compared to patients without fracture highlighting a dynamic relationship between the composition of fatty acids in the bone microenvironment and the metabolic requirements of cells. The relative distribution of fatty acids differed considerably from that in the serum providing further evidence that energy utilization is high and that marrow adipocytes may contribute to this pool. Whether these lipids can affect osteoblast function in a positive or negative manner is still not certain but will require further investigation.