Pre-culturing human adipose tissue mesenchymal stem cells under hypoxia increases their adipogenic and osteogenic differentiation potentials.

OBJECTIVES: Hypoxia is an important factor in many aspects of stem-cell biology including their viability, proliferation, differentiation and migration. We evaluated whether low oxygen level (2%) affected human adipose tissue mesenchymal stem-cell (hAT-MSC) phenotype, population growth, viability, apoptosis, necrosis and their adipogenic and osteogenic differentiation potential. MATERIALS AND METHODS: hAT-MSCs from four human donors were cultured in growth medium under either normoxic or hypoxic conditions for 7 days and were then transferred to normoxic conditions to study their differentiation potential. RESULTS: Hypoxia enhanced hAT-MSC expansion and viability, whereas expression of mesenchymal markers such as CD90, CD73 and endothelial progenitor cell marker CD34, remained unchanged. We also found that pre-culturing hAT-MSCs under hypoxia resulted in their enhanced ability to differentiate into adipocytes and osteocytes. CONCLUSIONS: This protocol could be useful for maximizing hAT-MSC potential to differentiate in vitro into the adipogenic and osteogenic lineages, for use in plastic and reconstructive surgery, and in tissue engineering strategies.

In vitro mesenchymal stem cell differentiation after mechanical stimulation.

OBJECTIVES: Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into adipocytic, chondrocytic and osteocytic lineages on suitable stimulation. We have hypothesized that mechanical loading may influence MSC differentiation and alter their phenotype accordingly. MATERIALS AND METHODS: Mouse bone marrow-derived MSC were established in vitro by differential adherence to plastic culture plates and grown in low glucose medium with 10% foetal calf serum and growth factors. Cells grew out and were subcultured up to 20 times. Differentiation protocols were followed for several cell lineages. Clones with trilineage potential were seeded in type I collagen gels and incubated in a tensioning force bioreactor and real-time cell-derived forces were recorded. Gels were fixed and sectioned for light and electron microscopy. RESULTS: Cell monolayers of parent and cloned mouse bone marrow-derived MSC differentiated into adipocytes, osteocytes and chondrocytes, but not into cardiomyocytes, myotubes or neuronal cells. When cast into type I collagen gels and placed in tensioning bioreactors, MSC differentiated into fibroblast-like cells typical of tissue stroma, and upregulated α-smooth muscle actin, but rarely upregulated desmin. Electron microscopy showed collagen and elastin fibre synthesis into the matrix. CONCLUSIONS: These experiments confirmed that MSC cell fate choice depends on minute, cell-derived forces. Applied force could assist in commercial manufacture of cultured bio-engineered prostheses for regenerative medicine as it mimics tissue stresses and constitutes a good model for development of tissue substitutes.

Hypoxia increases Sca-1/CD44 co-expression in murine mesenchymal stem cells and enhances their adipogenic differentiation potential.

Mesenchymal stem cells (MSCs) are usually cultured under normoxic conditions (21% oxygen). However, in vivo, the physiological "niches" for MSCs have a much lower oxygen tension. Because of their plasticity, stem cells are particularly sensitive to their environments, and oxygen tension is one developmentally important stimulus in stem cell biology and plays a role in the intricate balance between cellular proliferation and commitment towards differentiation. Therefore, we investigated here the effect of hypoxia (2% oxygen) on murine adipose tissue (AT) MSC proliferation and adipogenic differentiation. AT cells were obtained from the omental fat and AT-MSCs were selected for their ability to attach to the plastic dishes, and were grown under normoxic and hypoxic conditions. Prior exposure of MSCs to hypoxia led to a significant reduction of ex vivo expansion time, with significantly increased numbers of Sca-1(+) as well as Sca-1(+)/CD44(+)double-positive cells. Under low oxygen culture conditions, the AT-MSC number markedly increased and their adipogenic differentiation potential was reduced. Notably, the hypoxia-mediated inhibition of adipogenic differentiation was reversible: AT-MSCs pre-exposed to hypoxia when switched to normoxic conditions exhibited significantly higher adipogenic differentiation capacity compared to their pre-exposed normoxic-cultured counterparts. Accordingly, the expression of adipocyte-specific genes, peroxisome proliferator activated receptor gamma (Ppargamma), lipoprotein lipase (Lpl) and fatty acid binding protein 4 (Fabp4) were significantly enhanced in hypoxia pre-exposed AT-MSCs. In conclusion, pre-culturing MSCs under hypoxic culture conditions may represent a strategy to enhance MSC production, enrichment and adipogenic differentiation.

Exogenous bone marrow cells do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl2, despite establishment of chimaerism and cell proliferation in bone marrow and spleen.

OBJECTIVE: Various studies have shown that bone marrow stem cells can rescue mice from acute renal tubular damage under a conditioning advantage (irradiation or cisplatin treatment) favouring donor cell engraftment and regeneration; however, it is not known whether bone marrow cells (BMCs) can contribute to repair of acute tubular damage in the absence of a selection pressure for the donor cells. The aim of this study was to examine this possibility. MATERIALS AND METHODS: Ten-week-old female mice were assigned into control non-irradiated animals having only vehicle treatment, HgCl(2)-treated non-irradiated mice, HgCl(2)-treated non-irradiated mice infused with male BMCs 1 day after HgCl(2), and vehicle-treated mice with male BMCs. Tritiated thymidine was given 1 h before animal killing. RESULTS: Donor BMCs could not alleviate non-irradiated mice from acute tubular damage caused by HgCl(2), deduced by no reduction in serum urea nitrogen combined with negligible cell engraftment. However, donor BMCs could home to the bone marrow and spleen and display proliferative activity. This is the first report to show that despite no preparative myeloablation of recipients, engrafted donor BMCs can synthesize DNA in the bone marrow and spleen. CONCLUSIONS: Exogenous BMCs do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl(2), despite establishment of chimerism and cell proliferation in bone marrow and spleen.

Haematopoietic lineage-committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 -induced acute tubular injury.

OBJECTIVE: Our previous studies have demonstrated that endogenous bone arrow cells (BMCs) contribute to renal tubular regeneration after acute tubular injury. The aim of this study was to examine which fraction of BMCs, haematopoietic lineage marrow cells (HLMCs) or mesenchymal stem cells (MSCs), are effective. MATERIALS AND METHODS: Six-week-old female mice were lethally irradiated and were transplanted with female enhanced green fluorescent protein-positive (GFP(+)), plastic on-adherent marrow cells (as a source of HLMCs) plus cloned cultured male GFP(-) MSCs. Four weeks later, they were assigned into two groups: control mice with vehicle treatment and mice treated with HgCl2. Tritiated thymidine was given 1 h before animal killing which occurred at intervals over 2 weeks. Kidney sections were stained for a tubular epithelial marker, cell origin indicated by GFP immunohistochemistry or Y chromosome in situ hybridization; periodic acid-Schiff staining was performed, and samples were subjected to autoradiography. One thousand consecutive renal tubular epithelial cells per mouse, in S phase, were scored as either female (indigenous) GFP+(HLMC-derived) or male (MSC-derived). RESULTS: Haematopoietic lineage marrow cells and MSCs stably engrafted into bone marrow and spleen, but only HLMC-derived cells, not MSCs, were found in the renal tubules and were able to undergo DNA synthesis after acute renal injury. A few MSCs were detected in the renal interstitium, but their importance needs to be further explored. CONCLUSION: Haematopoietic lineage marrow cells, but not cloned cultured MSCs, can play a role not only in normal wear-and-tear turnover of renal tubular cells, but also in repair after tubular injury.

TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons.

TRPA1 is a receptor expressed by sensory neurons, that is activated by low temperature (<17 degrees C) and plant derivatives such as cinnamaldehyde and isoeugenol, to elicit sensations including pain. Using immunohistochemistry, we have, for the first time, localised TRPA1 in human DRG neurons, spinal cord motoneurones and nerve roots, peripheral nerves, intestinal myenteric plexus neurones, and skin basal keratinocytes. TRPA1 co-localised with a subset of hDRG neurons positive for TRPV1, the heat and capsaicin receptor. The number of small/medium TRPA1 positive neurons (< or =50 microm) was increased after hDRG avulsion injury [percentage of cells, median (range): controls 16.5 (7-23); injured 46 (34-55); P<0.005], but the number of large TRPA1 neurons was unchanged [control 19.5 (13-31); injured 21 (11-35)]. Similar TRPA1 changes were observed in cultured hDRG neurons, after exposure to a combination of key neurotrophic factors NGF, GDNF and NT-3 (NTFs) in vitro. We used calcium imaging to examine responses of HEK cells transfected with hTRPA1 cDNA, and of human and rat DRG neurons cultured with or without added NTFs, to cinnamaldehyde (CA) and isoeugenol (IE). Exposure to NTFs in vitro sensitized cultured human sensory neuronal responses to CA; repeated CA exposure produced desensitisation. In rDRG neurons, low (225 microM) CA preincubation enhanced capsaicin responses, while high (450 microM and 2mM) CA caused inhibition which was partially reversed in the presence of 8 bromo cAMP, indicating receptor dephosphorylation. While TRPA1 localisation is more widespread than TRPV1, it represents a promising novel drug target for the treatment of chronic pain and hypersensitivity.

Side populations of gastrointestinal cancers are not enriched in stem cells.

The side population (SP) phenotype, defined as the reserpine-blockable ability to efflux the nucleic acid dye Hoechst 33342, has been claimed to be enriched for stem cells in several human normal tissues, cancers and cell lines, and thus may be useful for the identification and isolation of cancer stem cells. We demonstrated the presence of SP fractions in all of seven tested gastrointestinal cancer cell lines. Four cell lines were selected (HT29, HGT101, Caco2 and HRA19a1.1) for detailed phenotypic and behavioural analysis with respect to stem cell characteristics. Cell surface marker analysis showed that, contrary to non-SP cells, the SPs entirely lack the expression of CD34. This difference, however, disappeared when the cells were cultured, rendering both populations CD34-positive. Expression of other putative stem cell markers (CD133, CD44, Hes-1, beta-catenin, Musashi-1, Oct-4 and CD117) was identical on SP and non-SPs before and after culturing. Sorted SP and non-SP cells were similarly clonogenic in vitro, tumourigenic in vivo, and displayed similar multipotential differentiation potential in vitro and in vivo. Additionally, culturing cytometrically-sorted SP and non-SP cells showed that the populations are interconvertible, each giving rise to the other. Expression of ABCG2 and Mdr-1, two membrane transporter proteins that have been suggested to be responsible for the drug-effluxing capacities of SP cells, including Hoechst 33342, was identical in non-SP and SP cells, indicating that there may be additional factors responsible for the Hoechst effluxing property in gastrointestinal cancer SP cells. Here, we show that the SP and non-SP fractions, albeit phenotypically distinct populations, do not differ with respect to stem cell-like cell number or behaviour. We thus conclude that the concept of the SP phenotype as a universal marker for stem cells does not apply to gastrointestinal cancer cells. These findings stand in contrast to the observations made in many other tissues and harbour important implications for the future search for intestinal cancer stem cell markers.

The cellular origin and proliferative status of regenerating renal parenchyma after mercuric chloride damage and erythropoietin treatment.

OBJECTIVES: In this study, we have sought to establish the cellular origin and proliferative status of the renal parenchyma as it regenerates after damage induced by mercuric chloride, with or without erythropoietin treatments, that might alter the response. MATERIALS AND METHODS: Female mice were irradiated and male whole bone marrow was transplanted into them. Six weeks later recipient mice were assigned to one of four groups: control, mercuric chloride treated, erythropoietin treated and treated with mercuric chloride plus erythropoietin. RESULTS: Tubular injury scores were high 3 days after mercuric chloride and had recovered partially after 14 days, in line with serum urea nitrogen levels. Confocal microscopy confirmed the tubular location of bone marrow-derived cells. A 'four-in-one' analytical technique (identifying cell origin, tubular phenotype, tubular basement membranes and S-phase status) revealed that tubular necrosis increased bone marrow derivation of renal tubular epithelium from a baseline of approximately 1.3% to approximately 4.0%. Erythropoietin increased the haematocrit, but no other effects were detected. CONCLUSION: As 1 in 12 proximal tubular cells in S-phase was derived from bone marrow, we conclude that in the kidney, the presence of bone marrow-derived cells makes a minor but important regenerative contribution after tubular necrosis.

Bone marrow transplantation ameliorates pathology in interleukin-10 knockout colitic mice.

The authors have previously reported the derivation of colonic subepithelial myofibroblasts (SEMFs) in both humans and mice from bone marrow (BM). In the pathogenesis of inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, colonic SEMFs mediate several types of inflammatory response. In the present study, interleukin (IL)-10-/- mice were used as a model of IBD to investigate the involvement of BM-derived cells in the inflamed mucosa. Male whole BM [either C57/BL10 (wild type: WT) or IL-10-/- donor mice] was used to perform bone marrow transplantation (BMT) into both WT and IL-10-/- female mice. Tissue samples were evaluated by immunohistochemistry for alpha-smooth muscle actin expression and by in situ hybridization using a Y-chromosome-specific probe to track the donor-derived colonic SEMFs. The mucosal expression of mRNA for pro-inflammatory cytokines was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, mRNA expression of matrix metalloproteinase (MMP)-7 and osteopontin in the inflamed mucosa was assessed using in situ hybridization. Body weights and histological scores showed that IL-10-/- mice that received WT BM had an improved course of colitis, decreased mucosal pro-inflammatory mRNA expression, and up to 30% of their SEMFs were of BM origin. Conversely, IL-10-/- mice receiving IL-10-/- BM progressed to extensive colitis, and Y probe analysis revealed that up to 45% of colonic SEMFs were of BM origin. WT mice receiving IL-10-/- or WT BM had no signs of colonic inflammation. The expression of MMP-7 and osteopontin was up-regulated in the inflamed mucosa. In conclusion, IL-10-/- mice displayed ameliorated disease activity after WT BMT, whilst colitis was not induced in WT mice by IL-10-/- BMT. The contribution of BM-derived cells to colonic SEMFs was significantly increased in the inflamed mucosa compared with non-inflamed mucosa.

The effect of neurotrophic factors on morphology, TRPV1 expression and capsaicin responses of cultured human DRG sensory neurons.

We have studied the effect of key neurotrophic factors (NTFs) on morphology, levels of the vanilloid receptor-1 (TRPV1) and responses to capsaicin in adult human sensory neurons in vitro. Avulsed dorsal root ganglia (DRG, n = 5) were cultured with or without a combination of nerve growth factor (NGF), glial cell (line)-derived growth factor (GDNF) and neurotrophin3 (NT3) for 5 days. In the absence of NTFs, the diameter of neurons ranged from 20 to 100 microm (mean 42 +/- 4 microm). Adding NTFs caused a significant increase in neuronal sizes, up to 120 microm (mean diameter 62 +/- 5 microm, P < 0.01, t-test), an overall 35% increase of TRPV1-positive neurons (P < 0.003), and notably of large TRPV1-positive neurons > 80 microm (P < 0.05). Responses to capsaicin were significantly enhanced with calcium ratiometry (P < 0.0001). Short duration (1h) exposure of dissociated sensory neurons to NTFs increased numbers of TRPV1-positive neurons, but not of TRPV3, Nav 1.8 and IK1 and the morphological size-distribution remained similar to intact post-mortem DRG neurons. NTFs thus increase size, elevate TRPV1 levels and enhance capsaicin responses in cultured human DRG neurons; these changes may relate to pathophysiology in disease states, and provide an in vitro model to study novel analgesics.

Trefoil factor family-interacting proteins.

Trefoil factor family (TFF) peptides have many in vivo and in vitro effects on restitution, wound healing, apoptosis, cell motility, adhesion and vectorial ion pumping, amongst others. (125)I-TFF peptides bind to cell membranes with classical saturable ability. It would be surprising if there were not TFF-protein interactions that would explain these actions, but to date no convincing TFF-binding partner has been shown which unambiguously takes part in any of these functions. Nevertheless, several TFF-binding proteins exist, including the small intestinal CRP-ductin (muclin), which binds TFF2, and the recently described gastric foveolar proteins TFIZ1 (TFF1-binding) and blottin (TFF2-binding), any of which may yet interact in novel ways to elicit TFF-mediated events. This review describes the expression and, where known, the functions of such proteins.

Circulating mesenchymal stem cells.

Mesenchymal precursor cells (MPCs) are multipotent cells capable of differentiating into various mesenchymal tissues, such as bone, cartilage, fat, tendon and muscle. They are present within both mesenchymal tissues and the bone marrow (BM). If marrow-derived MPCs are to have a role in repair and fibrosis of mesenchymal tissues, transit of these cells through the peripheral blood is to be expected. Although there is evidence for the existence of MPCs within the peripheral blood, results are debated and are not always reproducible. Variations in the methods of cell purification, culture and characterisation may explain the inconsistent results obtained in different studies.

Tomorrow's skeleton staff: mesenchymal stem cells and the repair of bone and cartilage.

Stem cells are regenerating medicine. Advances in stem cell biology, and bone marrow-derived mesenchymal stem cells in particular, are demonstrating that many clinical options once thought to be science fiction may be attainable as fact. The extra- and intra-cellular signalling used by stem cells as they differentiate into lineages appropriate to their destination are becoming understood. Thus, the growth stimuli afforded by LIF, FGF-2 and HGF, as well as the complementary roles of Wnt and Dickkopf-1 in stem cell proliferation are evident. The ability to direct multi-lineage mesenchymal stem sell (MSC) potential towards an osteogenic phenotype by stimulation with Menin and Shh are important, as are the modulatory roles of Notch-1 and PPARgamma. Control of chondrocytic differentiation is effected by interplay of Brachyury, BMP-4 and TGFbeta3. Smads 1, 4 and 5 also play a role in these phenotypic expressions. The ability to culture MSC has led to their use in tissue repair, both as precursor and differentiated cell substitutes, and with successful animal models of bone and cartilage repair using MSC, their clinical use is accelerating. However, MSC also suppress some T-cell functions in transplanted hosts, and could facilitate tumour growth, so a cautious approach is needed.

Recipes for adult stem cell plasticity: fusion cuisine or readymade?

A large body of evidence supports the idea that certain adult stem cells, particularly those of bone marrow origin, can engraft at alternative locations, particularly when the recipient organ is damaged. Under strong and positive selection pressure these cells will clonally expand/differentiate, making an important contribution to tissue replacement. Similarly, bone marrow derived cells can be amplified in vitro and differentiated into many types of tissue. Despite seemingly irrefutable evidence for stem cell plasticity, a veritable chorus of detractors has emerged, some doubting its very existence, motivated perhaps by more than a little self interest. The issues that have led to this situation include the inability to reproduce certain quite startling observations, and extrapolation from the behaviour of embryonic stem cells to suggest that adult bone marrow cells simply fuse with other cells and adopt their phenotype. Although these issues need resolving and, accepting that cell fusion does appear to allow reprogramming of haemopoietic cells in special circumstances, criticising this whole new field because some areas remain unclear is not good science.

Influence of human skin injury on regeneration of sensory neurons.

The regeneration of sensory nerve fibres is regulated by trophic factors released from their target tissue, particularly the basal epidermis, and matrix molecules. Means to modulate this response may be useful for the treatment of neuromas and painful hypertrophic scars and of sensory deficits in skin grafts and flaps. We have developed an in vitro model of sensory neuron regeneration on human skin in order to study the mechanisms of sensory dysfunction in pathological conditions. Adult rat sensory neurons were co-cultured with unfixed cryosections of normal or injured (crushed) human skin for 72 h. Neurons were immunostained for growth-associated protein-43 and the neurite lengths of neuronal cell bodies situated in various skin regions were measured. Two-way analysis of variance was performed. Neurites of sensory cell bodies on epidermis of normal skin were the shortest, with a mean +/- SEM of 75+/-10 micrometer, whereas those of cells on the dermo-epidermal junction were the longest, with a mean +/- SEM of 231+/-18 micrometer. Neurons on the dermo-epidermal junction of injured skin had significantly longer neurites than those on the same region of normal skin (mean +/- SEM = 289+/-21 micrometer). Regeneration of sensory neurons may be influenced by extracellular matrix molecules, matrix-binding growth factors and trophic factors. Altered substrate or trophic factors in injured skin may explain the increase of neurite lengths. This in vitro model may be useful for studying the molecular mechanisms of sensory recovery and the development of neuropathic pain following peripheral nerve injury.

Lung epithelial stem cells.

This review concentrates on recent evidence about lung stem cell origins and plasticity. The range of potential cells which can repopulate the injured lung, classically the basal and mucous secretory cells of the trachea, the Clara cells of the bronchiole, and the type II pneumocyte of the alveolus, has been extended to include the mucus-gland duct cells of the trachea and bronchus. Some evidence suggests that there are variant Clara cells that lack cytochrome P-450 and so are spared toxic activation of xenobiotics, and may aid bronchiolar repopulation after injury, such as with naphthalene. There may even be involvement of the neuroepithelial bodies or cells in this, though the evidence is not yet conclusive. The search for a resident pulmonary multipotent cell for repopulating any lung epithelium has not yet been successful. The picture remains similar to earlier conclusions, in that the local stem or precursor cell is the most likely to contribute to local needs in times of tissue damage. There remains a major challenge for lung cancer treatment, where high-dose chemo- or radio-therapy may be hoped to promote the seeding and repair of lung parenchyma by circulating bone marrow stem cells, as seen in liver models. Patient survival rates do not yet suggest that this occurs to any great extent, but this remains to be shown formally. The effects of prior fibrosis and tumour necrosis are probably confounding factors in this lack of rescue.

Allogeneic skin substitutes applied to burns patients.

Early re-surfacing of burn wounds remains the ideal but is limited by the availability of skin graft donor sites. Cultured grafts overcome these problems and autologous keratinocytes can be grown in culture and placed on a dermal substitute, but this results in delay and requires two operations. We developed an organotypic skin substitute, which achieves cover in one procedure, and have previously found allogeneic cell survival up to 2.5 years after grafting onto clean elective wounds (tattoo removal). Here, we report a short series using the same model applied to burns patients with less than 20% total body surface area affected. The skin substitutes consisted of allogeneic dermal fibroblasts embedded in a collagen gel overlain with allogeneic epidermal keratinocytes, and were grafted to patients with tangentially excised burns. A side-by-side comparison with meshed split-thickness autografts was performed. No grafts became infected. The allogeneic skin substitute showed little effective take at 1 week, and by 2 weeks only small islands of keratinocytes survived. These sites were subsequently covered with meshed split-thickness autograft, which took well. It is concluded that further development of this model is needed to overcome the hostile wound bed seen in burns patients.

Calcium-activated potassium channel SK1- and IK1-like immunoreactivity in injured human sensory neurones and its regulation by neurotrophic factors.

Calcium-activated potassium ion channels SK and IK (small and intermediate conductance, respectively) may be important in the pathophysiology of pain following nerve injury, as SK channels are known to impose a period of reduced excitability after each action potential by afterhyperpolarization. We studied the presence and changes of human SK1 (hSK1)- and hIK1-like immunoreactivity in control and injured human dorsal root ganglia (DRG) and peripheral nerves and their regulation by key neurotrophic factors in cultured rat sensory neurones. Using specific antibodies, hSK-1 and hIK-1-like immunoreactivity was detected in a majority of large and small/medium-sized cell bodies of human DRG. hSK1 immunoreactivity was decreased significantly in cell bodies of avulsed human DRG (n = 8, surgery delay 8 h to 12 months). There was a decrease in hIK1-like immunoreactivity predominantly in large cells acutely (<3 weeks after injury), but also in small/medium cells of chronic cases. Twenty-three injured peripheral nerves were studied (surgery delay 8 h to 12 months); in five of these, hIK1-like immunoreactivity was detected proximally but not distally to injury, whereas neurofilament staining confirmed the presence of nerve fibres in both regions. These five nerves, unlike the others, had all undergone Wallerian degeneration previously and the loss of hIK1-like immunoreactivity may therefore reflect reduced axonal transport of this ion channel across the injury site in regenerated fibres, as well as decreased expression in the cell body. In vitro studies of neonatal rat DRG neurones showed that nerve growth factor (NGF) significantly increased the percentage of hSK1-positive cells, whereas neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) failed to show a significant effect. NT-3 stimulated hIK1 expression, while NGF and GDNF were ineffective. As expected, NGF increased expression of the voltage-gated sodium channel SNS1/PN3 in this system. Decreased retrograde transport of these neurotrophic factors in injured sensory neurones may thus reduce expression of these ion channels and increase excitability. Blockade of IK1-like and other potassium channels by aminopyridines (4-AP and 3,4-DAP) may also explain the paraesthesiae induced by these medications. Selective potassium channel openers are likely to represent novel therapies for pain following nerve injury.

Molecular and cellular analysis of embryonic avian tongue development.

Signalling cascades first described in Drosophila have been found to regulate patterning and outgrowth in a number of structures in higher vertebrates. We sought to determine whether the evolutionarily conserved genes were important during the development of the tongue. In situ hybridisation was used to determine the temporo-spatial expression of a panel of conserved genes. Histological examination and incorporation of BrdU were used to determine the mechanism by which the tongue develops. We show that evolutionarily conserved genes were expressed in distinct dynamic patterns during tongue development. Sonic Hedgehog (Shh) and Patched (Ptc) were found only in the dorsal tongue epithelium. Shh expression was only observed in the suprabasal layers, whereas Ptc was observed in both basal and suprabasal layers. Cell division in the epithelium was concentrated in regions devoid of Shh. Expression of bone morphogenetic protein-7 (BMP) was identical to that of Shh. Shh and Ptc expression were never detected in the mesenchyme. Ectopic expression of Noggin (a potent antagonist of the BMPs) caused severe abnormalities in tongue morphology, including swelling of the mesenchymal component and a thickening of the epithelial layer. Data from this study suggests that the epithelium and mesenchyme express quite different genes during development. However BMP activity acts to inhibit growth in both tissues.

Trefoil factor family (TFF)-domain peptides in the mouse: embryonic gastrointestinal expression and wounding response.

Trefoil factor family (TFF)-domain peptides are mucin-associated molecules that play a role in maintaining gastrointestinal (GI) epithelial integrity. They are expressed in specific patterns in adult mammals, but their embryonic expression has not been clarified. Developmental TFF mRNA in mice was studied by non-isotopic whole mount in situ hybridization. All TFF's (1-3) were seen in the stomach from E13 to E16. TFF1 was gastric at E13, then spread to the small intestine (E15) and caecum on E16. Froin E19 TFF1 expression was gastric. TFF2 was gastric at E13, and absent in lower intestines till E17 when duodenal, small intestinal and caecal expression was seen. Afterwards, TFF2 was confined to the gastric region. TFF3 was in the stomach at E13. On E15 and 16 TFF3 was ubiquitous, except for E15 caecum. From E17, TFF3 was confined to small intestine and the distal gut. WOUNDS: E17 and 18 GI tissues were subjected to incisional wounds in vitro. TFF1 induction was seen only in stomach, after as short as 30 min incubation. TFF2 was only induced at E18 in the stomach. TFF3 was induced within 5 min in the rectum. No change in overall expression patterns were seen after wounding. CONCLUSIONS: TFF expression is developmentally controlled in the GI tract, and appears before mucous cell differentiation in several tissues. Gene regulation is predicted to be under different control(s) in utero compared with post-natal life. The response to incisional wounding of fetal GI tissue shows differences to the adult.