Clonal Heterogeneity in the Neuronal and Glial Differentiation of Dental Pulp Stem/Progenitor Cells.

Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs) have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K(+) but not outward Na(+) currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required.

Transglutaminase 6: a protein associated with central nervous system development and motor function.

Transglutaminases (TG) form a family of enzymes that catalyse various post-translational modifications of glutamine residues in proteins and peptides including intra- and intermolecular isopeptide bond formation, esterification and deamidation. We have characterized a novel member of the mammalian TG family, TG6, which is expressed in a human carcinoma cell line with neuronal characteristics and in mouse brain. Besides full-length protein, alternative splicing results in a short variant lacking the second ß-barrel domain in man and a variant with truncated ß-sandwich domain in mouse. Biochemical data show that TG6 is allosterically regulated by Ca(2+) and guanine nucleotides. Molecular modelling indicates that TG6 could have Ca(2+) and GDP-binding sites related to those of TG3 and TG2, respectively. Localization of mRNA and protein in the mouse identified abundant expression of TG6 in the central nervous system. Analysis of its temporal and spatial pattern of induction in mouse development indicates an association with neurogenesis. Neuronal expression of TG6 was confirmed by double-labelling of mouse forebrain cells with cell type-specific markers. Induction of differentiation in mouse Neuro 2a cells with NGF or dibutyryl cAMP is associated with an upregulation of TG6 expression. Familial ataxia has recently been linked to mutations in the TGM6 gene. Autoantibodies to TG6 were identified in immune-mediated ataxia in patients with gluten sensitivity. These findings suggest a critical role for TG6 in cortical and cerebellar neurons.

A multipotent neural crest-derived progenitor cell population is resident within the oral mucosa lamina propria.

Wounds within the oral mucosa, similarly to fetal wounds, exhibit rapid healing with reduced scarring. We hypothesized that a progenitor population resident within the oral mucosal lamina propria (OMLP) contributes to this preferential healing. Progenitor cells (PC) were reliably isolated from the OMLP by differential adhesion to fibronectin. Isolated colonies originating from a single cell demonstrated a rapid initial phase of proliferation, completing in excess of 50 population doublings (PDs) before entering cellular senescence. These data were supported by the expression of active telomerase within both developing colonies and expanded clones as assessed by immunocytochemistry (ICC) and the quantitative telomeric repeat amplification protocol. FACS analysis confirmed expression of the stem cell markers CD44, CD90, CD105, and CD166, but negative expression of CD34 and CD45 ruling out a hematopoietic or fibrocyte origin for these progenitors. A neural crest origin was confirmed by increased colony-forming efficiency (CFE) in the presence of Jagged 1 and the expression of a number of neural crest markers within the developing colonies by ICC and serially passaged clones by Western blotting. The multipotency of this novel PC population was demonstrated by differentiation of the cells down both mesenchymal (chondrogenic, osteoblastic, and adipogenic) and neuronal (neuron and Schwann-like cells) cell lineages. This article reports for the first time, the isolation and characterization of a novel, clonally derived PC population resident within the OMLP. The attributes of this adult stem cell (ASC) population and its accessibility lends itself to future therapeutic applications.

TNFalpha and TGF-beta1 influence IL-18-induced IFNgamma production through regulation of IL-18 receptor and T-bet expression.

Bacterial infections can lead to a state of uncontrolled inflammation and also trigger autoimmune disease. At the centre of this are CD4(+) T cell responses in inflammatory tissues or local lymph nodes which are orchestrated by dendritic cells. IL-18 is a pro-inflammatory cytokine that drives dendritic cell maturation and mediates IFNgamma production. In this study, we demonstrate that in the dendritic precursor-like cell line KG-1, IFNgamma production induced by IL-18 is potentiated (>5-fold) by TNFalpha and completely suppressed by TGF-beta1. IL-18 stimulation rapidly activates different MAPK signalling pathways but only blocking of p38 activation alleviates IFNgamma production. The mechanism through which TNFalpha enhances IL-18 induced IFNgamma production is by promoting IL-18 receptor alpha-chain expression which results in higher levels of p38 activation and induces expression of T-bet, a transcriptional regulator of the IFNG gene. In contrast, TGF-beta1 rapidly suppresses IFNgamma production by limiting IL-18 receptor numbers at the cell surface and preventing induction of T-bet expression. TGF-beta1 experience by cells leads to sustained long-term inactivation of TNFalpha/IL-18-mediated cell activation but not IL-18 induced p38 activation suggesting transcriptional silencing of the T-BET and/or IFNG promoter independent of MAPK signalling. These results demonstrate how IL-18 activity is regulated by pro and anti-inflammatory cytokines and thereby provide insight into the mechanism that controls dendritic cell activity and ultimately leads to resolution of an inflammatory response.

Late stages of embryo progression are a much better predictor of clinical pregnancy than early cleavage in intracytoplasmic sperm injection and in vitro fertilization cycles with blastocyst-stage transfer.

OBJECTIVE: To define and validate metrics of embryo progression and morphology during extended embryo culture and to compare the effects of early cleavage (EC) vs. blastulation stages on clinical pregnancy. DESIGN: Retrospective observational study. SETTING: University-affiliated assisted reproduction center. PATIENT(S): One thousand two hundred ninety-two intracytoplasmic sperm injection and 842 IVF blastocyst-transfer cycles. INTERVENTION(S): The embryo progression index (EPI) was calculated as the area under the curve of total cell number (TCN) over time, by using observed TCN for cleavage-stage embryos and estimated blastocyst TCN according to morphology. The EPI from days 1-3 measured early cleavage, and blastulation was assessed by EPI over extended embryo culture. Blastocyst morphology was converted into numerical blastocyst quality scores (BQSs). Receiver operating characteristic curve analysis was used to evaluate predictors for clinical pregnancy. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Per-cycle mean EPI and mean BQS for all embryos developing into blastocysts, as well as mean BQS of the transferred embryos, were significant predictors of clinical pregnancy in intracytoplasmic sperm injection and IVF cycles. Mean EPI for days 1-3 did not predict outcome. CONCLUSION(S): Early cleavage is a putative marker of embryo quality. Late-stage embryo development is more sensitive and specific in predicting clinical pregnancy than is early cleavage, supporting the use of extended embryo culture for embryo selection. The embryo progression index and BQS may also be used for this purpose.

Precycle administration of GnRH antagonist and microdose HCG decreases clinical pregnancy rates without affecting embryo quality and blastulation.

The outcome of a novel protocol utilizing precycle gonadotrophin-releasing hormone (GnRH) antagonist administration and LH activity support with microdose recombinant human chorionic gonadotrophin (HCG) was compared to GnRH agonist long protocol used in patients undergoing their first ICSI (n=707) or IVF (n=571) cycles, which had resulted in one or two blastocyst transfers. In GnRH antagonist cycles, cetrorelix acetate (3 mg) was administered s.c. 4 days before FSH stimulation and a repeat dose was given when the lead follicular diameter was 13-14 mm. LH support was provided by recombinant HCG (2.5 microg). Embryo progression and blastulation were evaluated using embryo progression indices and blastocyst quality scores. The tested protocol demonstrated reduced implantation and clinical pregnancy rates as compared with GnRH agonist long protocol, although the embryo progression and blastulation parameters and blastocyst quality were comparable among the groups. Logistic regression models further supported the significant negative impact of GnRH antagonist/microdose HCG protocol on clinical pregnancy rates in both ICSI and IVF patients. Assisted reproduction cycles with fresh blastocyst transfers utilizing precycle GnRH antagonist administration and microdose HCG support resulted in lower implantation and clinical pregnancy rates as compared with GnRH agonist cycles, although the embryo progression and blastulation parameters were comparable.

Crosslinking and G-protein functions of transglutaminase 2 contribute differentially to fibroblast wound healing responses.

Tissue transglutaminase (TG2) affects cell-matrix interactions in cell spreading, migration and extracellular matrix (ECM) reorganisation. Using fibroblasts deficient in TG2 or overexpressing normal or crosslinking-deficient enzyme, we show that the extracellular crosslinking activity and intracellular G-protein function in signal transduction contribute differentially to regulation of cell-matrix interactions. TG2-deficient cells displayed normal attachment but delayed spreading on ECM substrata and defects in motility unrelated to crosslinking. Blocking antibodies to TG2 failed to induce similar defects in normal fibroblasts. TG2-deficient fibroblasts had defects in focal adhesion turnover and stress fibre formation, showed changes in focal adhesion kinase (FAK) phosphorylation and failed to activate protein kinase C alpha (PKCalpha). Phospholipase C (PLC) and PKCalpha inhibitors blocked spreading of normal fibroblasts whilst PKC activators induced spreading in TG2-deficient cells. In contrast, ECM remodelling was not only compromised by TG2 deficiency but also by overexpression of dominant negative enzyme and TG inhibitors. TG2 activity increased matrix tension and was required for membrane type 1-MMP (MT1-MMP)-dependent activation of MMP-2. Our results demonstrate that TG2 is involved in the control of dynamic adhesion formation in cell spreading and migration via regulation of phospholipase C activity. By virtue of its crosslinking activity, the enzyme plays a central role in regulating ECM remodelling.

Identification of proteinaceous material in the bone of the dinosaur Iguanodon.

This study has directed attention at the search for bone-related proteins in an extract of demineralized rib bone of the 120 mya Iguanodon. The inner compact bone was demineralized and the GuCl extract resolved into 11 fractions using anion exchange chromatography, which all contained silver-reactive proteins with various amino acid profiles. Two specific fractions, iv and xi, revealed characteristics typical of contemporary phosphoproteins and proteoglycans, respectively. Fraction iv, 43-57 kDa, contained a high ratio of aspartate and serine, although no phosphate was discernable. Fraction xi contained a band of 41-47 kDa and was rich in chondroitin sulphate and hyaluronan. In addition an early eluting fraction was immunoreactive with an antibody against osteocalcin. A cancellous bone fraction from the same bone sample was also analyzed using N-terminal sequencing and revealed potential similarities with cystatin. While we do not claim to have identified the presence of intact proteins, this study has value in demonstrating that extruded extracellular matrix is protected by its capacity to induce mineralization, which subsequently is important in conserving detectable protein products in ancient skeletal tissues.