Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells.

Type 1 diabetes is caused by the destruction of pancreatic beta-cells by T cells of the immune system. Islet transplantation is a promising therapy for diabetes mellitus. Bone marrow stem cells (BMSC) have the capacity to differentiate into various cell lineages including endocrine cells of the pancreas. To investigate the conditions that allow BMSC to differentiate into insulin-producing cells, a novel in vitro method was developed by using the histone deacetylase inhibitor, trichostatin A (TSA). BMSC, cultured in presence of TSA, differentiated into islet-like clusters under appropriate culture conditions. These islet-like clusters were similar to the cells of the islets of the pancreas. The islet-like clusters showed endocrine gene expression typical for pancreatic beta-cell development and function, such as insulin (I and II), glucagon, somatostatin, GLUT-2, pancreatic duodenal homeobox-1 (PDX-1), and Pax 4. Immunocytochemistry confirmed islet-like clusters contained pancreatic hormones. The colocalization of insulin and C-peptide was also observed. Enzyme-linked immunosorbent assay analysis demonstrated that insulin secretion was regulated by glucose. Western blot analysis demonstrated the presence of stored insulin. Electron microscopy of the islet-like cells revealed an ultrastructure similar to that of pancreatic beta-cells, which contain insulin granules within secretory vesicles. These findings suggest that histone-deacetylating agents could allow the differentiation of BMSC into insulin-producing beta-cells.

hPTH-fragments (53-84) and (28-48) antagonize the stimulation of calcium release and repression of alkaline phosphatase activity by hPTH-(1-34) in vitro.

Different C-terminal fragments of parathyroid hormone (PTH)-(1-84) in blood participate in the regulation of calcium homeostasis by PTH-(1-84), and an antagonizing effect for the large carboxyl-terminal parathyroid hormone (C-PTH)-fragment (7-84) on calcium release has been described in vivo and in vitro. In this study the smaller C-PTH-fragment (53-84) and mid-regional PTH fragment (28-48), which represent discrete areas of activity in the PTH-(7-84) molecule, were assayed for their effects on calcium release and alkaline phosphatase (ALP) activity in a chick bone organ culture system. Neither PTH-(28-48) nor PTH-(53-84) had any effect on calcium release into the medium and both fragments stimulated ALP activity in the bone tissue, suggesting that the cAMP/PKA signalling pathway was not affected by these fragments. However they suppressed the calcium release induced by PTH-(1-34) and attenuated the down regulation of ALP activity caused by PTH-(1-34), suggesting that the effect on the cAMP/PKA signalling pathway may be indirectly. In conclusion, the study shows that the PTH-fragments (53-84) and (28-48) antagonize the PTH-(1-34) induced effects on calcium release and inhibition of ALP activity in a chick bone organ culture system.

Effect of donor characteristics, technique of harvesting and in vitro processing on culturing of human marrow stroma cells for tissue engineered growth of bone.

The aim of this study was to assess the effect of donor characteristics and the technique of harvesting and in vitro processing on the efficacy of culturing of human mesenchymal stem cells (hMSCs) for tissue engineered growth of bone. Cultures of hMSCs were derived from iliac crest bone marrow aspirates (21 donors, age 11-76) and from cancellous bone grafting material (32 donors, age 13-84). Age had no significant effect on the ability to isolate and culture hMSCs, although the failure rate was 55.6% in donors beyond the age of 60, while it varied between 14.3% and 22.2% in donors under 60 years of age. Male and female donors had comparable failure rates (27.3% and 28.6%, respectively). Culturing of hMSCs was successful in 90.4% of marrow aspirates from 21 donors and in 62.5% of cancellous bone specimens from 35 donors. This difference was statistically significant (P=0.023). Regression analysis confirmed that at simultaneous testing of the three variables, only the source of cells significantly affected the result (P=0.043). Morphological evaluation of the unfractionated primary population showed a change in cell shape of the adherent cells from a triangular into thin spindle-shaped elongated form, which remains until confluence. When the cultures were exposed to osteoinductive medium, various morphotypes expressing different levels of alkaline phosphatase and secreting different amounts of mineral were evident. Morphology of marrow stroma cells (MSCs) from marrow aspirates was not different from MSCs derived from cancellous bone specimens. Expression of osteogenic markers in MSCs as shown by PCR as well, did not differ between the two sources. It is concluded that marrow aspirates and cancellous bone specimens produce comparable populations of MSCs. However, bone marrow aspirates from donors under the age of 60 years rather than cancellous bone chips are favourable for isolation and expansion of hMSCs for tissue engineered growth of bone.

Vascular endothelial growth factor (VEGF-A) expression in human mesenchymal stem cells: autocrine and paracrine role on osteoblastic and endothelial differentiation.

Angiogenesis is essential in bone fracture healing for restoring blood flow to the fracture site. Vascular endothelial growth factor (VEGF) and its receptor have been implicated in this process. Despite the importance of angiogenesis for the healing processes of damaged bones, the role of VEGF signaling in modulation of osteogenic differentiation in human mesenchymal stem cells has not been investigated in great detail. We examined the expression of VEGF-A and VEGFR-1 in human adult mesenchymal stem cells derived from trabecular bone (hTBCs). VEGF-A was found to be secreted in a differentiation dependent manner during osteogenesis. Transcripts for VEGF-A were also seen to be elevated during osteogenesis. In addition, transcripts for VEGF-A and the corresponding receptor VEGFR-1 were upregulated under hypoxic conditions in undifferentiated hTBCs. To investigate the signaling of VEGF-A on osteogenesis recombinant hTBCs were generated. High expression of VEGF-A stimulated mineralization, whereas high expression of sFLT-1, an antagonist to VEGF-A, reduced mineralization suggesting that VEGF-A acts as autocrine factor for osteoblast differentiation. In addition, VEGF-A secreted by hTBCs promotes sprouting of endothelial cells (HUVE) demonstrating a paracrine role in blood vessel formation. In summary, an in vitro analysis of transgene effects on cellular behavior can be used to predict an effective ex vivo gene therapy.

Properties of human trabecular bone cells from elderly women: implications for cell-based bone engraftment.

Tissue-engineered bone regeneration offers new therapeutic options in the treatment of patients with fractures. Due to the changes in the hormone levels, elderly women are most affected by bone fractures and thus constitute one of the future target groups of cell-based bone therapy. For designing cell-based therapy approaches, a better understanding of individual-dependent variations in bone-derived cells of the host is required. In this study, a simple, high-yield procedure is described for the collection of cells from bone tissue of a high number of elderly women. The cultured cells display stem cell characteristics indicated by the presence of a CD13+, CD44+, CD90+, CD147+, CD14-, CD34-, CD45- and CD144- cell populations and by a stable undifferentiated phenotype as well as by the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic lineage even after 27 cell doublings. A high variability in the number of cell-forming units (CFUs) within a donor population of 34 samples, in the morphology within 50 donors, in the expression of alkaline phosphatase within 15 samples and in the responsiveness to BMP-2 was evident, but no age-related correlation could be found. In conclusion, the data indicate that individual variations in cell number, cell morphology and in the osteogenic potential of progenitor cells of the patient may be relevant for a successful treatment of bone fractures in the elderly by cell-based therapy approaches.

The T-box transcription factor Brachyury mediates cartilage development in mesenchymal stem cell line C3H10T1/2.

The BMP2-dependent onset of osteo/chondrogenic differentiation in the acknowledged pluripotent murine mesenchymal stem cell line (C3H10T1/2) is accompanied by the immediate upregulation of Fibroblast Growth Factor Receptor 3 (FGFR3) and a delayed response by FGFR2. Forced expression of FGFR3 in C3H10T1/2 is sufficient for chondrogenic differentiation, indicating an important role for FGF-signaling during the manifestation of the chondrogenic lineage in this cell line. Screening for transcription factors exhibiting a chondrogenic capacity in C3H10T1/2 identified that the T-box containing transcription factor Brachyury is upregulated by FGFR3-mediated signaling. Forced expression of Brachyury in C3H10T1/2 was sufficient for differentiation into the chondrogenic lineage in vitro and in vivo after transplantation into muscle. A dominant-negative variant of Brachyury, consisting of its DNA-binding domain (T-box), interferes with BMP2-mediated cartilage formation. These studies indicate that BMP-initiated FGF-signaling induces a novel type of transcription factor for the onset of chondrogenesis in a mesenchymal stem cell line. A potential role for this T-box factor in skeletogenesis is further delineated from its expression profile in various skeletal elements such as intervertebral disks and the limb bud at late stages (18.5 d.p.c.) of murine embryonic development.

Lipopolysaccharide and porin of Roseobacter denitrificans, confirming its phylogenetic relationship to the alpha-3 subgroup of Proteobacteria.

Roseobacter denitrificans has rough (R)-type lipopolysaccharide, containing 2-keto-3-deoxyoctonate but no hepatoses. Its lipid A has a glucosamine-containing, phosphorylated backbone. It contains the rare 3-oxotetradecanoic (3-oxomyristic) acid as the only amide-bound fatty acid and ester-bound 3-hydroxydecanoic acid, this pattern being characteristic for the alpha-3 subgroup of Proteobacteria. Treatment of the major outer-membrane protein (porin, apparent molecular mass 88 kDa) of Roseobacter denitrificans with EDTA (2 mM, 30 degrees C, 20 min) resulted in the dissociation of the oligomers into monomers (apparent molecular mass 35 kDa). EDTA-sensitive dissociation has so far been observed only within the alpha-3 subgroup of Proteobacteria. The 12 N-terminal amino acids of the monomers exhibit sequence homology with the porins of Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas blastica. Renaming of Roseobacter denitrificans as Rhodobacter denitrificans is suggested.