The anabolic effect of inorganic polyphosphate on chondrocytes is mediated by calcium signalling.

Inorganic polyphosphates (polyP) are polymers composed of phosphate residues linked by energy-rich phosphoanhydride bonds. As polyP can bind calcium, the hypothesis of this study is that polyP enters chondrocytes and exerts its anabolic effect by calcium influx through calcium channels. PolyP treatment of cartilage tissue formed in 3D culture by bovine chondrocytes showed an increase in proteoglycan accumulation but only when calcium was also present at a concentration of 1.5 mM. This anabolic effect could be prevented by treatment with either ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid or the calcium channel inhibitors gadolinium and nifedipine. Calcium and polyP cotreatment of chondrocytes in monolayer culture resulted in calcium oscillations that were polyP chain length specific and were inhibited by gadolinium and nifedipine. The calcium influx resulted in increased gene expression of sox9, collagen type II, and aggrecan which was prevented by treatment with either calphostin, an inhibitor of protein kinase C, and W7, an inhibitor of calmodulin; suggesting activation of the protein kinase C-calmodulin pathway. Tracing studies using  4',6-diamidino-2-phenylindole, Mitotracker Red, and/or Fura-AM staining showed that polyP was detected in the nucleus, mitochondria, and intracellular vacuoles suggesting that polyP may also enter the cell. PolyP colocalizes with calcium in mitochondria. This study demonstrates that polyP requires the influx of calcium to regulate chondrocyte matrix production, likely via activating calcium signaling. These findings identify the mechanism regulating the anabolic effect of polyP in chondrocytes which will help in its clinical translation into a therapeutic agent for cartilage repair.

An expanded population of CD34+ cells from frozen banked umbilical cord blood demonstrate tissue repair mechanisms of mesenchymal stromal cells and circulating angiogenic cells in an ischemic hind limb model.

Peripheral vascular disease affects ~20 % of the population over 50 years of age and is a complication of type 2 diabetes. Cell therapy studies revealed that cells from older or diabetic donors have a reduced capacity to induce tissue repair compared to healthy and younger cells. This fact greatly impedes the use of autologous cells for treatment. Umbilical cord blood CD34+ cells are a source of angiogenic cells but unlike bone marrow CD34+ angiogenic cells, achieving clinically significant cell numbers has been difficult without in vitro expansion. We report here that culturing CD34+/CD45+ blood cells from frozen umbilical cord blood units in a medium supplemented with FGF4, SCF and FLT3-ligand produced a population of cells that remain CD34+/CD45+ but have an increased capacity for tissue healing. The cultured CD34+ cells were compared directly to non-cultured CD34+ cells in a mouse model of ischemia. Cultured CD34+ cells demonstrated strong paracrine signaling as well as the capacity to differentiate into endothelial cells, smooth muscle and striated muscle. We observed an improvement in blood flow and a significant reduction in foot necrosis. A second study was completed to assess the safety of the cells. No adverse effects were associated with the injection of the cultured cells. Our method described here for culturing umbilical cord blood cells resulted in cells with a strong paracrine effect that induces substantial tissue repair in a murine model of hind limb ischemia and evidence of engraftment and differentiation of the cultured cells into new vasculature and muscle.

Identification of genes expressed by immune cells of the colon that are regulated by colorectal cancer-associated variants.

A locus on human chromosome 11q23 tagged by marker rs3802842 was associated with colorectal cancer (CRC) in a genome-wide association study; this finding has been replicated in case-control studies worldwide. In order to identify biologic factors at this locus that are related to the etiopathology of CRC, we used microarray-based target selection methods, coupled to next-generation sequencing, to study 103 kb at the 11q23 locus. We genotyped 369 putative variants from 1,030 patients with CRC (cases) and 1,061 individuals without CRC (controls) from the Ontario Familial Colorectal Cancer Registry. Two previously uncharacterized genes, COLCA1 and COLCA2, were found to be co-regulated genes that are transcribed from opposite strands. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. In colon tissues, COLCA1 co-localizes with crystalloid granules of eosinophils and granular organelles of mast cells, neutrophils, macrophages, dendritic cells and differentiated myeloid-derived cell lines. COLCA2 is present in the cytoplasm of normal epithelial, immune and other cell lineages, as well as tumor cells. Tissue microarray analysis demonstrates the association of rs3802842 with lymphocyte density in the lamina propria (p = 0.014) and levels of COLCA1 in the lamina propria (p = 0.00016) and COLCA2 (tumor cells, p = 0.0041 and lamina propria, p = 6 × 10(-5)). In conclusion, genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer. Histologic analyses indicate the involvement of immune pathways.

The effect of umbilical cord blood cells on outcomes after experimental traumatic spinal cord injury.

STUDY DESIGN: A cytokine expression profile of umbilical cord blood (UCB) derived multipotential stem cells (MPSC) was produced. We then transplanted MPSCs into a rat model of spinal cord injury (SCI) and assessed neurologic function as well as spinal cord histology. OBJECTIVE: To determine if MPSCs transplanted into a rat model of acute SCI would lead to a beneficial neurologic effect. SUMMARY OF BACKGROUND DATA: Conditioned medium from UCB contains factors that could promote healing of endogenous neural tissues. Previously, our laboratory has demonstrated that UCB hematopoietic cells can develop into MPSCs capable of differentiating into multiple cell types including oligodendrocyte-like cells. METHODS: We cultured MPSCs from UCB cells using fibroblast growth factor 4, stem cell factor and fms-like tyrosine kinase receptor-3 ligand supplemented serum-free medium. Using a cytokine antibody array, we produced a cytokines expression profile of MPSCs. We then transplanted MPSCs into an immunosuppressed rat model of SCI and assessed neurologic function weekly for 6 weeks by the Basso, Beattie, and Bresnahan locomotor test. The spinal cords were examined histologically and lesion areas quantified. RESULTS: We detected elevated levels of cytokines and growth factors with known neuroprotective, angiogenic, and anti-inflammatory effects in the MPSC conditioned media. The SCI rats treated with MPSCs showed a significant improvement in Basso, Beattie, and Bresnahan scores after 6 weeks compared with the group that received vehicle only. Immunohistochemistry revealed transplanted human cells were present in the injured spinal cord after 1 week, but were no longer present by 6 weeks. There was a trend for the lesion size in treated rats to be smaller than that of the control group. CONCLUSION: We conclude that UCB MPSCs improve neurologic function of rats with acute SCI, possibly by the release of factors that reduce secondary injury.

Control of vertebrate skeletal mineralization by polyphosphates.

BACKGROUND: Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization. PRINCIPAL FINDINGS/METHODOLOGY: The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation. CONCLUSIONS/SIGNIFICANCE: We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.

Neural progenitors, neurons and oligodendrocytes from human umbilical cord blood cells in a serum-free, feeder-free cell culture.

We have previously demonstrated that lineage negative cells (Lin(neg)) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone, muscle, endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin(neg) UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin, neurofilament, A2B5 and Sox2) are expressed in Lin(neg) cells cultured in FGF4, SCF, Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase, GalC, Olig2 and the late-stage marker MOSP are observed, as is the Schwann cell marker PMP22. In summary, Lin(neg) UCB cells, when appropriately cultured, are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation.

Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation.

We report on a subset of cells that co-purify with CD45-positive/Lineage minus (CD45(pos)/Lin(minus)) hematopoietic cells that are capable of in vitro differentiation into multi-potential cells including cells with neuroectoderm properties. Although these cells are CD45 positive and have properties similar to CD45-negative mesenchymal progenitor cells (MPC) derived from bone marrow (BM), they are neither hematopoietic cells nor mesenchymal cells. These CD45(pos)/Lin(minus) cells can be expanded in vitro, express the stem cell genes Oct-4 and Nanog and can be induced to differentiate into endothelial cells, osteoblasts, muscle cells and neural cells at frequencies similar to those reported for bone marrow mesenchymal cells. Long-term culture of these cells followed by transplantation into NOD/SCID mice resulted in positive bone marrow stromal cell engraftment but not hematopoietic engraftment, suggesting that despite their CD45-positive status these cells do not have the same properties as hematopoietic stem cells. Clonal cell analysis determined that the culture period caused a broadening in the differentiation potential of the starting population.

Effect of a statin on an in vitro model of endometriosis.

OBJECTIVE: To determine the inhibitory effect of a statin on angiogenesis in a three-dimensional (3-D) culture of human endometrial fragments in vitro. Angiogenesis has been proposed as an important mechanism in the pathogenesis of endometriosis, and statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) have been shown to have anti-inflammatory and anti-angiogenic activity. DESIGN: Experimental in vitro study of human endometrial biopsies and 3-D culture in fibrin matrix. SETTING: Research laboratory at a university-affiliated infertility center. PATIENT(S): Forty-six normal ovulating women undergoing infertility treatment. INTERVENTION(S): Endometrial samples obtained from the fundus of the uterine cavity were minced, and the fragments were placed in a three-dimensional fibrin matrix culture system. MAIN OUTCOME MEASURE(S): Presence or absence of proliferation of stromal cells and invasion of the fibrin matrix, presence or absence of vessel sprouting, and immunohistochemical characterization of cellular components. RESULT(S): During the 1st week of culture, invasion of stromal cells into the fibrin matrix occurred in the control group and in some wells outgrowths were observed. After 2 weeks, endometrial glands were observed in the outgrowths at a distance from the main tissue and were growing in conjunction with new vessel formation until the end of culture period. A concentration-dependent effect of lovostatin was seen on cell growth and angiogenesis in the experimental groups. In the presence of 5 and 10 microM of statin, angiogenesis was abolished, and cell proliferation was inhibited. In the presence of 1 microM of lovastatin, angiogenesis was reduced, but cell proliferation was not affected. CONCLUSION(S): The statins were shown to be effective in inhibiting the mechanisms of cell proliferation and angiogenesis in an experimental model for the development of endometriosis-like tissue.

Nicotine-induced Disturbances of Meiotic Maturation in Cultured Mouse Oocytes: Alterations of Spindle Integrity and Chromosome Alignment.

We investigated whether nicotine exposure in vitro of mouse oocytes affects spindle and chromosome function during meiotic maturation (M-I and M-II). Oocytes in germinal vesicle (GV) stage were cultured in nicotine for 8 h or for 16 h, to assess effects in M-I and in metaphase II (M-II). The latter culture setting used the three protocols: 8 h nicotine then 8 h medium (8N + 8M); 16 h nicotine (16N); 8 h medium then 8 h nicotine (8M + 8N). Non-toxic concentrations of nicotine at 1.0, 2.5, 5.0 and 10.0 mmol/L were used. Spindle-chromosome configurations were analyzed with wide-field optical sectioning microscopy. In 8 h cultures, nicotine exposure resulted in dose-related increased proportions of M-I oocytes with defective spindle-chromosome configurations. A dose-related delayed entry into anaphase I was also detected. In 16 h cultures, nicotine exposure for the first 8 h (8N + 8M), or for 16 h (16N), resulted in dose- and time-related increased proportions of oocytes arrested in M-I (10 mmol/L; 8 h: 53.2%, controls 9.6%; 16 h: 87.6%, controls 8.5%). Defects in M-I spindles and chromosomes caused M-I arrest leading to dose-related decreased proportions of oocytes that reached metaphase-II (10 mmol/L 8 h: 46.8%, controls 90.4%;16 h: 12.4%, controls 91.5%). A delayed anaphase-I affected the normal timing of M-II, leading to abnormal oocytes with dispersed chromosomes, or with double spindles and no polar body. Nicotine exposure during the second 8 h (8M + 8N) resulted in dose-related, increased proportions of M-II oocytes with defective spindles and chromosomes (10 mmol/L: 42.9%, controls 2.0%). Nicotine has no adverse effects on GV break down, but induces spindle and chromosome defects compromising oocyte meiotic maturation and development.

Three-dimensional in vitro culture of endometrial explants mimics the early stages of endometriosis.

OBJECTIVE: To reproduce the earliest phases of endometriosis using a new in vitro model in which cells from a cultured endometrial fragment can proliferate, invade, reconstitute new endometrial-like tissue, and generate blood vessels. DESIGN: Experimental in vitro study. SETTING: A hospital-based academic research institute. PATIENT(S): Five normal ovulating women undergoing surgery for various benign gynecological indications. INTERVENTION(S): Endometrial samples obtained from the fundus of the uterine cavity were placed in a three-dimensional fibrin matrix culture system. MAIN OUTCOME MEASURE(S): Degree of proliferation of stromal cells and invasion of the fibrin matrix, gland, and stroma formation, vessel sprouting, and immunohistochemical characterization of various cellular components. RESULT(S): During the first week of culture, an endometrial cell outgrowth was observed from the original fragments in 120 of 144 wells (83.3%). Subsequently, cell outgrowths could be quantified in 132 (91.6%), 129 (89.5%), and 127 (88.1%) of the wells after 15, 60, and 90 days, respectively. An invasion of the matrix by the human endometrial cells led to the formation of tubular structures that coalesced into tissue, architecturally resembling endometrium and in which the glands were immunohistochemically positive for cytokeratin. New capillaries, immunohistochemically positive for CD31 and vimentin, sprouted from the endometrial outgrowths at the beginning of the fifth week of culture. CONCLUSION(S): These data show that cells from endometrial explants can proliferate and invade a fibrin matrix in vitro generating new glands, stroma, and vessels consistent with endometriosis. The three-dimensional fibrin matrix used in the present study provides an opportunity to observe the earliest biological events of endometriosis in a quantifiable way.