Chemical group-dependent plasma polymerisation preferentially directs adipose stem cell differentiation towards osteogenic or chondrogenic lineages.

Human adipose derived stem cells (ADSCs) are being explored for the repair of craniofacial defects due to their multi-differentiation potential and ease of isolation and expansion. Crucial to using ADSCs for craniofacial repair is the availability of materials with appropriate biomechanical properties that can support their differentiation into bone and cartilage. We tested the hypothesis that different modifications of chemical groups on the surface of a nanocomposite polymer could increase human ADSC adhesion and selectively enhance their osteogenic and chondrogenic differentiation. We show that the COOH modification significantly promoted initial cell adhesion and proliferation over 14days compared to NH2 surfaces. Expression of focal adhesion kinase and vinculin was enhanced after plasma surface polymerisation at 24h. The COOH modification significantly enhanced chondrogenic differentiation as indicated by up-regulation of aggrecan and collagen II transcripts. In contrast, NH2 group functionalised scaffolds promoted osteogenic differentiation with significantly enhanced expression of collagen I, alkaline phosphatase and osteocalcin both at the gene and protein level. Finally, chorioallantoic membrane grafting demonstrated that both NH2 and COOH functionalised scaffolds seeded with ADSCs were biocompatible and supported vessel ingrowth apparently to a greater degree than unmodified scaffolds. In summary, our study shows the ability to direct ADSC chondrogenic and osteogenic differentiation by deposition of different chemical groups through plasma surface polymerisation. Hence this approach could be used to selectively enhance bone or cartilage formation before implantation in vivo to repair skeletal defects. STATEMENT OF SIGNIFICANCE: Human adipose derived stem cells (hADSCs) are an exciting stem cell source for regenerative medicine due to their plentiful supply and ease of isolation. However, the optimal environmental cues to direct stem cells towards certain lineages change have to has not been identified. We have shown that by modifying the surface of the scaffold with specific chemical groups using plasma surface polymerisation techniques we can control ADSCs differentiation. This study shows that ADSCs can be differentiated towards osteogenic and chondrogenic lineages on amine (NH2) and carboxyl (COOH) modified scaffolds respectively. Plasma polymerisation can be easily applied to other biomaterial surfaces to direct stem cell differentiation for the regeneration of bone and cartilage.

Calcification response of a key phytoplankton family to millennial-scale environmental change.

Coccolithophores are single-celled photosynthesizing marine algae, responsible for half of the calcification in the surface ocean, and exert a strong influence on the distribution of carbon among global reservoirs, and thus Earth's climate. Calcification in the surface ocean decreases the buffering capacity of seawater for CO2, whilst photosynthetic carbon fixation has the opposite effect. Experiments in culture have suggested that coccolithophore calcification decreases under high CO2 concentrations ([CO2(aq)]) constituting a negative feedback. However, the extent to which these results are representative of natural populations, and of the response over more than a few hundred generations is unclear. Here we describe and apply a novel rationale for size-normalizing the mass of the calcite plates produced by the most abundant family of coccolithophores, the Noëlaerhabdaceae. On average, ancient populations subjected to coupled gradual increases in [CO2(aq)] and temperature over a few million generations in a natural environment become relatively more highly calcified, implying a positive climatic feedback. We hypothesize that this is the result of selection manifest in natural populations over millennial timescales, so has necessarily eluded laboratory experiments.

MHC-class-II are expressed in a subpopulation of human neural stem cells in vitro in an IFNγ-independent fashion and during development.

Expression of major histocompatibility antigens class-2 (MHC-II) under non-inflammatory conditions is not usually associated with the nervous system. Comparative analysis of immunogenicity of human embryonic/fetal brain-derived neural stem cells (hNSCs) and human mesenchymal stem cells with neurogenic potential from umbilical cord (UC-MSCs) and paediatric adipose tissue (ADSCs), while highlighting differences in their immunogenicity, led us to discover subsets of neural cells co-expressing the neural marker SOX2 and MHC-II antigen in vivo during human CNS development. MHC-II proteins in hNSCs are functional, and differently regulated upon differentiation along different lineages. Mimicking an inflammatory response using the inflammatory cytokine IFNγ induced MHC-II up-regulation in both astrocytes and hNSCs, but not in UC-MSCs and ADSCs, either undifferentiated or differentiated, though IFNγ receptor expression was comparable. Together, hypoimmunogenicity of both UC-MSCs and ADSCs supports their suitability for allogeneic therapy, while significant immunogenicity of hNSCs and their progeny may at least in part underlie negative effects reported in some patients following embryonic neural cell grafts. Crucially, we show for the first time that MHC-II expression in developing human brains is not restricted to microglia as previously suggested, but is present in discrete subsets of neural progenitors and appears to be regulated independently of inflammatory stimuli.

Nanotechnology for Stimulating Osteoprogenitor Differentiation.

BACKGROUND: Bone is the second most transplanted tissue and due to its complex structure, metabolic demands and various functions, current reconstructive options such as foreign body implants and autologous tissue transfer are limited in their ability to restore defects. Most tissue engineering approaches target osteoinduction of osteoprogenitor cells by modifying the extracellular environment, using scaffolds or targeting intracellular signaling mechanisms or commonly a combination of all of these. Whilst there is no consensus as to what is the optimal cell type or approach, nanotechnology has been proposed as a powerful tool to manipulate the biomolecular and physical environment to direct osteoprogenitor cells to induce bone formation. METHODS: Review of the published literature was undertaken to provide an overview of the use of nanotechnology to control osteoprogenitor differentiation and discuss the most recent developments, limitations and future directions. RESULTS: Nanotechnology can be used to stimulate osteoprogenitor differentiation in a variety of way. We have principally classified research into nanotechnology for bone tissue engineering as generating biomimetic scaffolds, a vector to deliver genes or growth factors to cells or to alter the biophysical environment. A number of studies have shown promising results with regards to directing ostroprogenitor cell differentiation although limitations include a lack of in vivo data and incomplete characterization of engineered bone. CONCLUSION: There is increasing evidence that nanotechnology can be used to direct the fate of osteoprogenitor and promote bone formation. Further analysis of the functional properties and long term survival in animal models is required to assess the maturity and clinical potential of this.

A matter of identity - Phenotype and differentiation potential of human somatic stem cells.

Human somatic stem cells with neural differentiation potential can be valuable for developing cell-based therapies, including treatment of birth-related defects, while avoiding issues associated with cell reprogramming. Precisely defining the "identity" and differentiation potential of somatic stem cells from different sources, has proven difficult, given differences in sets of specific markers, protocols used and lack of side-by-side characterization of these cells in different studies. Therefore, we set to compare expression of mesenchymal and neural markers in human umbilical cord-derived mesenchymal stem cells (UC-MSCs), pediatric adipose-derived stem cells (p-ADSCs) in parallel with human neural stem cells (NSCs). We show that UC-MSCs at a basal level express mesenchymal and so-called "neural" markers, similar to that we previously reported for the p-ADSCs. All somatic stem cell populations studied, independently from tissue and patient of origin, displayed a remarkably similar expression of surface markers, with the main difference being the restricted expression of CD133 and CD34 to NSCs. Expression of certain surface and neural markers was affected by the expansion medium used. As predicted, UC-MSCs and p-ADSCs demonstrated tri-mesenchymal lineage differentiation potential, though p-ADSCs display superior chondrogenic differentiation capability. UC-MSCs and p-ADSCs responded also to neurogenic induction by up-regulating neuronal markers, but crucially they appeared morphologically immature when compared with differentiated NSCs. This highlights the need for further investigation into the use of these cells for neural therapies. Crucially, this study demonstrates the lack of simple means to distinguish between different cell types and the effect of culture conditions on their phenotype, and indicates that a more extensive set of markers should be used for somatic stem cell characterization, especially when developing therapeutic approaches.

Evolution of ocean temperature and ice volume through the mid-Pleistocene climate transition.

Earth's climate underwent a fundamental change between 1250 and 700 thousand years ago, the mid-Pleistocene transition (MPT), when the dominant periodicity of climate cycles changed from 41 thousand to 100 thousand years in the absence of substantial change in orbital forcing. Over this time, an increase occurred in the amplitude of change of deep-ocean foraminiferal oxygen isotopic ratios, traditionally interpreted as defining the main rhythm of ice ages although containing large effects of changes in deep-ocean temperature. We have separated the effects of decreasing temperature and increasing global ice volume on oxygen isotope ratios. Our results suggest that the MPT was initiated by an abrupt increase in Antarctic ice volume 900 thousand years ago. We see no evidence of a pattern of gradual cooling, but near-freezing temperatures occur at every glacial maximum.

Delayed osteoprogenitor differentiation in cleft-palate models.

Failure of palatal shelf fusion results in cleft palate (CP) and may lead to malformation of palatal bones and undergrowth of the maxilla. It is not known whether defects in bone formation may contribute to this phenotype. We tested the hypothesis that impaired fusion of developing palatal shelves affects palatal bone development using palate organotypic cultures. Using two different approaches, we show that induction of cleft results in increased expression of pre-osteoblast and early osteoblast markers, Twist1, Snai1 and Runx2, and decreased expression of more mature markers of bone differentiation, collagen-1 and osteopontin, indicating delayed osteoblast differentiation in CPs. This, together with the increase in immature osteoblasts and proliferation observed in non-fused palatal shelves, suggests that palatal osteoblast differentiation is at least partly modulated by shelf fusion. Delayed osteoblast differentiation may therefore contribute to defects in gross morphology and function of the maxilla in CP patients.

Successful neural regeneration in amniotes: the developing chick spinal cord.

Although early after birth the central nervous system is more plastic than in the adult, it already displays limited regenerative capability. This becomes severely impaired at specific stages of embryonic development; however, the precise cellular and molecular basis of this loss is not fully understood. The chick embryo provides an ideal model for direct comparisons of regenerating and non-regenerating spinal cord within the same species because of its accessibility in ovo, the extensive knowledge of chick neural development and the molecular tools now available. Regenerative ability in the chick is lost at around E13, a relatively advanced stage of spinal cord development. This is most likely due to a complex series of events: there is evidence to suggest that developmentally regulated changes in the early response to injury, expression of inhibitory molecules and neurogenesis may contribute to loss of regenerative capacity in the chick spinal cord.

Potential use of craniosynostotic osteoprogenitors and bioactive scaffolds for bone engineering.

The cranial bone has a very limited regenerative capability. Patients with craniosynostosis (the premature fusion of cranial sutures, leading to skull abnormalities) often require extensive craniofacial reconstruction and repeated surgery. The possibility of grafting autologous osteoprogenitor cells seeded on bioabsorbable matrices is of great potential for inducing regeneration of craniofacial structure and protecting the brain from external insult. To this purpose we have studied the behaviour of normal and craniosynostotic mouse osteoblast cell lines, and of human primary osteoprogenitors from craniosynostotic patients. We have monitored their ability to grow and differentiate on plastic and on a scaffold composed of bioactive glass and bioabsorbable polymer by live fluorescent labelling and expression of bone differentiation markers. Cells from syndromic patients display a behaviour very similar to that observed in the stable mouse cell line we generated by introducing the human FGFR2-C278F, a mutation found in certain craniosynostosis, into MC3T3 osteblastic cells, indicating that the mutated cell line is a valuable model for studying the cellular response of human craniosynostotic osteoblasts. Both normal and mutated calvarial osteoprogenitors can attach to the bioactive scaffold, although mutated cells display adhesion defects when cultured on plastic. Furthermore, analysis of bone differentiation markers in human osteoblasts shows that the composite mesh, unlike PLGA(80) plates, supports bone differentiation. The ability of the mesh to support homing and differentiation in both normal and mutant osteoprogenitors is important, in view of further developing autologous biohybrids to repair cranial bone deficits also in craniosynostotic patients undergoing extensive reconstructive surgery.

Changes in response to spinal cord injury with development: vascularization, hemorrhage and apoptosis.

Chick embryos are capable of functional spinal cord regeneration following crush injury until embryonic day 13. Developmental changes occurring thereafter result in failure to regenerate. Secondary injury mechanisms can result in apoptotic cell death and make a major contribution to cell loss after trauma. We report here that around embryonic day 13 there is a dramatic increase in blood vessel numbers in the spinal cord, and that the extent of hemorrhage in response to injury increases with developmental age. This is paralleled by increased apoptosis and subsequent cavitation in spinal cords injured at embryonic day 15 as compared with embryonic day 11. Following spinal cord injury at embryonic day 15, apoptotic cell death is extensive and spreads to the same extent as the hemorrhage. When hemorrhage is reduced by treatment with the hemostatic drug desmopressin the extent of apoptosis and cavity formation in spinal cords injured at embryonic day 15 decreases. Furthermore, manipulations of embryonic day 11 spinal cords that increase hemorrhage also increase apoptosis and result in cavitation in contrast to the effective repair typical of this stage. Altogether these results suggest that cavity formation occurring at developmental stages non-permissive for regeneration is largely due to changes in the extent of apoptosis that are related to vascularization and hemorrhage.

Gene expression during palate fusion in vivo and in vitro.

UNLABELLED: Failure of secondary palate fusion during embryogenesis is a cause of cleft palate. Disappearance of the medial epithelial seam (MES) is required to allow merging of the mesenchyme from both palatal shelves. This involves complex changes of the medial edge epithelial (MEE) cells and surrounding structures that are controlled by several genes whose spatio-temporal expression is tightly regulated. We have carried out morphological analyses and used a semi-quantitative RT-PCR technique to evaluate whether morphological changes and modulation in the expression of putative key genes, such as twist, snail, and E-cadherin, during the fusion process in palate organ culture parallel those observed in vivo, and show that this is indeed the case. We also show, using the organotypic model of palate fusion, that the down-regulation of the transcription factor snail that occurs with the progression of palate development is not dependent on fusion of the palatal shelves. ABBREVIATIONS: dsg1, desmoglein1; EMT, epithelial-mesenchymal transition; MEE, medial edge epithelium; MES, medial epithelial seam; RT-PCR, reverse-transcriptase polymerase chain-reaction.

Expression of FGF2 in the limb blastema of two Salamandridae correlates with their regenerative capability.

Limb regenerative potential in urodeles seems to vary among different species. We observed that Triturus vulgaris meridionalis regenerate their limbs significantly faster than T. carnifex, where a long gap between the time of amputation and blastema formation occurs, and tried to identify cellular and molecular events that may underlie these differences in regenerative capability. Whereas wound healing is comparable in the two species, formation of an apical epidermal cap (AEC), which is required for blastema outgrowth, is delayed for approximately three weeks in T. carnifex. Furthermore, fewer nerve fibres are present distally early after amputation, consistent with the late onset of blastemal cell proliferation observed in T. carnifex. We investigated whether different expression of putative blastema mitogens, such as FGF1 and FGF2, in these species may underlie differences in the progression of regeneration. We found that whereas FGF1 is detected in the epidermis throughout the regenerative process, FGF2 onset of expression in the wound epidermis of both species coincides with AEC formation and initiation of blastemal cell proliferation, which is delayed in T. carnifex, and declines thereafter. In vitro studies showed that FGF2 activates MCM3, a factor essential for DNA replication licensing activity, and can be produced by blastemal cells themselves, indicating an autocrine action. These results suggest that FGF2 plays a key role in the initiation of blastema growth.

Differential regulation of fibroblast growth factor receptors in the regenerating amphibian spinal cord in vivo.

Unlike mammals, adult urodele amphibians can regenerate their spinal cord and associated ganglia, but the molecular mechanisms controlling regeneration are not fully understood. We have recently shown that expression of FGF2, a member of the fibroblast growth factor family, is induced in the progenitor cells of the regenerating spinal cord and appears to play a role in their proliferation and possibly in their differentiation. In order to investigate which receptor(s) may mediate FGF2 signaling and their role in regeneration, we have studied expression of the four fibroblast growth factor receptors, FGFR1, FGFR2, FGFR3 and FGFR4, and of the spliced variants, sFGFR and KGFR, in the regenerating spinal cord of the adult urodele, Pleurodeles waltl, following tail amputation. We show that all FGFRs are expressed in normal and regenerating spinal cord, with the exception of the spliced variants that are expressed only in non-neural tissues of the tail. FGFR1 and 4 show the more interesting spatio-temporal patterns of expression. They are not detectable in the ependymal cells of normal cords, from which neural progenitors for regeneration are believed to originate, though they are expressed in some mature neurons. During regeneration, significant up-regulation of FGFR1 precedes that of FGFR4 in the ependymal tube from which the new cord will form. FGFR4 is highly expressed in these cells at later stages of regeneration, when neuronal differentiation is becoming apparent, and like FGFR1 is also expressed in some newborn neurons. In addition to the known form of FGFR1, the antibody against this receptor reacts also with a non-phosphorylated protein that appears to be present only during regeneration, and might represent a yet undescribed variant of the receptor. Altogether this study shows that fibroblast growth factor signaling is finely modulated during tail and spinal cord regeneration, and points to FGFR1 and FGFR4 as key players in this process, suggesting that FGFR1 is primarily associated with proliferation of progenitor cells and FGFR4 with early stages of neuronal differentiation.

FGF2 promotes skeletogenic differentiation of cranial neural crest cells.

The cranial neural crest gives rise to most of the skeletal tissues of the skull. Matrix-mediated tissue interactions have been implicated in the skeletogenic differentiation of crest cells, but little is known of the role that growth factors might play in this process. The discovery that mutations in fibroblast growth factor receptors (FGFRs) cause the major craniosynostosis syndromes implicates FGF-mediated signalling in the skeletogenic differentiation of the cranial neural crest. We now show that, in vitro, mesencephalic neural crest cells respond to exogenous FGF2 in a dose-dependent manner, with 0.1 and 1 ng/ml causing enhanced proliferation, and 10 ng/ml inducing cartilage differentiation. In longer-term cultures, both endochondral and membrane bone are formed. FGFR1, FGFR2 and FGFR3 are all detectable by immunohistochemistry in the mesencephalic region, with particularly intense expression at the apices of the neural folds from which the neural crest arises. FGFRs are also expressed by subpopulations of neural crest cells in culture. Collectively, these findings suggest that FGFs are involved in the skeletogenic differentiation of the cranial neural crest.

FGF-2 Up-regulation and proliferation of neural progenitors in the regenerating amphibian spinal cord in vivo.

Regeneration of the spinal cord occurs spontaneously in adult urodele amphibians. The key cells in this regenerative process appear to be the ependymal cells that following injury migrate and proliferate to form the ependymal tube from which the spinal cord regenerates. Very little is known about the signal(s) that initiates and maintains the proliferative response of these cells. Fibroblast growth factor 2 (FGF-2) has been shown to play a role in maintaining neural progenitor cell cycling in vitro and may be important for neuronal survival and axonal growth after injury. We have investigated its role in regeneration of the spinal cord in vivo following tail amputation in the adult salamander, Pleurodeles waltl. We show that only the low-molecular-weight form of FGF-2 is found in Pleurodeles and that in the normal cord it is expressed in a subset of neurons, but is hardly detectable in ependymal cells. Tail amputation results in induction of FGF-2 in the ependymal cells of the regenerating structure, and later in regeneration FGF-2 is up-regulated in some newborn neurons. FGF-2 pattern of expression in the ependymal tube parallels that of proliferation. Furthermore, exogenous FGF-2 significantly increases ependymal cell proliferation in vivo. Overall our results strongly support the view that one important role of FGF-2 during spinal cord regeneration in Pleurodeles is to induce proliferation of neural progenitor cells.

Quality control of 99Mo/99Tcm generators: results of a survey of the Radiopharmacy Working Group of the Italian Association of Nuclear Medicine (AIMN).

A multicentre survey of the quality control of 99Tcm generators has been completed: 245 generators from seven different commercial sources were tested over a period of 2 years. The results indicate that the mean pH of the eluates was 5.8 +/- 0.6; the aluminium contents were typically < 10 ppm; the radiochemical purity was 99.8 +/- 0.4% and the median 99Mo content was 3.8 x 10(-4) percent. The elution profiles gave a volume of 1.9 ml to obtain 50% of the total eluted activity and of 4.9 ml to obtain 95%. Other radionuclide impurities and heavy metal breakthrough were evaluated by graphite furnace absorption spectrometry and inductively coupled plasma mass spectrometry. National guidelines for the standardization of radiopharmacy procedures are currently being compiled.

RA regulation of keratin expression and myogenesis suggests different ways of regenerating muscle in adult amphibian limbs.

Formation of a regeneration blastema following limb amputation is believed to occur through a process of dedifferentiation. It has been suggested, however, that the cells contributed to the blastema by the stump muscle are satellite-like cells, rather than cells originated by dedifferentiation. We have previously shown that simple epithelial keratins 8 and 18 are expressed in the mesenchymal progenitor cells of the regenerating amphibian limb and in cultured cells with myogenic potential, and that their expression appears to be causally related to changes in proliferation and differentiation. We show here that retinoic acid (RA) affects the expression of these keratins differently in myogenic cells originated from normal limb and limb blastema. Furthermore, we find that the effects of RA on proliferation, myogenic differentiation and adhesion of these cells also differ. In fact, whereas RA does not affect keratin expression, proliferation or myogenic differentiation in blastemal cells, it does decrease keratin levels and thymidine incorporation and increase myogenesis in cells from normal limb. Conversely, RA increases cell adhesion only in blastemal cells. Significantly, these effects of RA on cultured cells are consistent with those observed in vivo. Overall the results presented here suggest that in the urodele limb there are two distinct cell populations with myogenic potential, one originating from dedifferentiation and one equivalent to the satellite cells of the mammalian muscle, which are likely to be primarily involved in blastema formation and muscle repair, respectively.

Hedgehog family member is expressed throughout regenerating and developing limbs.

Members of the hedgehog family have been shown to play a key role in many developmental processes, including limb patterning and chondrogenesis. We have therefore investigated whether members of this family are also expressed during regeneration of the adult urodele limb and are regulated by retinoic acid (RA), since this derivative induces proximodistal duplications in regenerating limbs, and has been shown to regulate sonic hedgehog (shh) in the developing limbs of birds and mammals. We report here that a newt homologue of Xenopus banded hedgehog, called N-bhh, is uniformly expressed by mesenchymal blastemal cells from the initial stages of regeneration and is up-regulated by RA. In addition, we show that N-bhh is uniformly expressed in the early limb bud of the newt embryo. Since bhh has not been detected in developing limbs of higher vertebrates, its expression in developing and regenerating newt limbs may be related to the regenerative capability of urodeles.

Stability and plasticity of neural crest patterning and branchial arch Hox code after extensive cephalic crest rotation.

The extent to which the spatial organisation of craniofacial development is due to intrinsic properties of the neural crest is at present unclear. There is some experimental evidence supporting the concept of a prepattern established within crest while contiguous with the neural plate. In experiments in which the neural tube and premigratory crest are relocated within the branchial region, crest cells retain patterns of gene expression appropriate for their position of origin after migration into the branchial arches, resulting in skeletal abnormalities. But in apparent conflict with these findings, when crest is rerouted by late deletion of adjacent crest, infilling crest alters its pattern of gene expression to match its new location, and a normal facial skeleton results. In order to reconcile these findings thus identify processes of relevance to the course of normal development, we have performed a series of neural tube and crest rotations producing a more extensive reorganisation of cephalic crest than has been previously described. Lineage analysis using DiI labelling of crest derived from the rotated hindbrain reveals that crest does not migrate into the branchial arch it would have colonised in normal development, rather it simply populates the nearest available branchial arches. We also find that crest adjacent to the grafted region contributes to a greater number of branchial arches than it would in normal development, resulting in branchial arches containing mixed cell populations not occurring in normal development. We find that after exchange of first and third arch crest by rotation of r1-7, crest alters its expression of hoxa-2 and hoxa-3 to match its new location within the embryo resulting in the reestablishment of the normal branchial arch Hox code. A facial skeleton in which all the normal components are present, with some additional ectopic first arch structures, is formed in this situation. In contrast, when second and third arch crest are exchanged by rotation of r3 to 7, ectopic Hox gene expression is stable, resulting in the persistence of an abnormal branchial arch Hox code and extensive defects in the hyoid skeleton. We suggest that the intrinsic properties of crest have an effect on the spatial organisation of structures derived from the branchial arches, but that exposure to increasingly novel environments within the branchial region or "community effects" within mixed populations of cells can result in alterations to crest Hox code and morphogenetic fate. In both classes of operation we find that there is a tight link between the resulting branchial arch Hox code and a particular skeletal morphology.