Metabolism of Acetaminophen by Enteric Epithelial Cells Mitigates Hepatocellular Toxicity In Vitro.

The gut-liver axis is defined by dietary and environmental communication between the gut, microbiome and the liver with its redox and immune systems, the overactivation of which can lead to hepatic injury. We used media preconditioning to mimic some aspects of the enterohepatic circulation by treating the human Caco-2 intestinal epithelial cell line with 5, 10 and 20 mM paracetamol (N-acetyl-para-aminophenol; APAP) for 24 h, after which cell culture supernatants were transferred to differentiated human hepatic HepaRG cells for a further 24 h. Cell viability was assessed by mitochondrial function and ATP production, while membrane integrity was monitored by cellular-based impedance. Metabolism by Caco-2 cells was determined by liquid chromatography with tandem mass spectrometry. Caco-2 cell viability was not affected by APAP, while cell membrane integrity and tight junctions were maintained and became tighter with increasing APAP concentrations, suggesting a reduction in the permeability of the intestinal epithelium. During 24 h incubation, Caco-2 cells metabolised 64-68% of APAP, leaving 32-36% of intact starting compound to be transferred to HepaRG cells. When cultured with Caco-2-preconditioned medium, HepaRG cells also showed no loss of cell viability or membrane integrity, completely in contrast to direct treatment with APAP, which resulted in a rapid loss of cell viability and membrane integrity and, ultimately, cell death. Thus, the pre-metabolism of APAP could mitigate previously observed hepatotoxicity to hepatic tight junctions caused by direct exposure to APAP. These observations could have important implications for the direct exposure of hepatic parenchyma to APAP, administered via the intravenous route.

Socioeconomic deprivation is associated with reduced efficacy of an insulin adjustment education program for people with type 1 diabetes.

BACKGROUND: The Dose Adjustment for Normal Eating (DAFNE) course teaches insulin dose adjustment to match dietary carbohydrates and improve glycaemic control in participants with type 1 diabetes mellitus (T1DM). We investigated the association between socioeconomic deprivation and reduction in HbA1c as a marker of sustained glycaemic control, after attending DAFNE education. METHODS: This retrospective observational study identified adults with T1DM who attended DAFNE training in NHS Lothian, South East Scotland. We extracted age, sex, postcode-based Scottish Index of Multiple Deprivation (SIMD) quintiles and annual HbA1c measurements available four years before and after course attendance. We calculated mean HbA1c before (baseline) and after attendance at DAFNE, across four annual measurements. Change in mean HbA1c (mmol/mol) was categorised into three groups: decrease (≥ - 2.5), no change (<±2.5), increase (≥ + 2.5). We used multivariable ordinal logistic regression, with baseline mean HbA1c as a covariate, to investigate the association of SIMD quintile with reduction in mean HbA1c. RESULTS: 335 participants were included. Age and sex distribution were similar across SIMD quintiles (Mean age = 45, range 21-91, 59% women). Lower SIMD quintiles (greater deprivation) had higher baseline mean HbA1c (SIMD 1: 76.0, SIMD 5: 69.0). Higher SIMD quintiles (lower deprivation) were associated with lower odds of no change/increase in mean HbA1c (SIMD 5, odds ratio = 0.25, 95% confidence interval 0.10, 0.58, p = 0.001, multivariable analysis). CONCLUSION: Socioeconomic deprivation was associated with higher baseline mean HbA1c and lower reduction in HbA1c following DAFNE education. Future research could explore causes and how best to support participants from deprived areas. PREVIOUS SUBMISSIONS: This work has not been previously submitted to a journal. This work was presented as a poster at The ABCD Conference 2021 and the abstract (of no more than 300 words) from the meeting has been published: Innes CWD, Henshall DE, Wilson B, Poon M, Morley SD, Ritchie SA. Socioeconomic deprivation is associated with reduced efficacy of an insulin adjustment education programme for people with type 1 diabetes. Br J Diabetes. 2021; 21: 293-296.

Oxygen Plasma Substrate and Specific Nanopattern Promote Early Differentiation of HepaRG Progenitors.

Fully differentiated HepaRG™ cells are the hepatic cell line of choice for in vitro study in toxicology and drug trials. They are derived from a hepatoblast-like progenitor (HepaRG-P) that differentiates into a coculture of hepatocyte-like and cholangiocyte-like cells. This process that requires 2 weeks of proliferation followed by 2 weeks of differentiation using dimethyl sulfoxide (DMSO) can be time consuming and costly. Identifying a method to accelerate HepaRG-Ps toward a mature lineage would save both time and money. The ability to do this in the absence of DMSO would remove the possibility of confounding toxicology results caused by DMSO induction of CYP pathways. It has been shown that tissue culture substrates play an important role in the development and maturity of a cell line, and this is particularly important for progenitor cells, which retain some form of plasticity. Oxygen plasma treatment is used extensively to modify cell culture substrates. There is also evidence that patterned rather than planar surfaces have a positive effect on proliferation and differentiation. In this study, we compared the effect of standard tissue culture plastic (TCP), oxygen plasma coated (OPC), and nanopatterned substrates (NPS) on early differentiation and function of HepaRG-P cells. Since NPS were OPC we initially compared the effect of TCP and OPC to enable comparison between all three culture surfaces using OPC as control to asses if patterning further enhanced early differentiation and functionality. The results show that HepaRG-P's grown on OPC substrate exhibited earlier differentiation, proliferation, and function compared with TCP. Culturing HepaRG-P's on OPC with the addition of NPS did not confer any additional advantage. In conclusion, OPC surface appeared to enhance hepatic differentiation and functionality and could replace traditional methods of differentiating HepaRG-P cells into fully differentiated and functional HepaRGs earlier than standard methods. Impact statement We show significantly earlier differentiation and function of HepaRG progenitor cells when grown in dimethyl sulfoxide-free medium on oxygen plasma substrates versus standard tissue culture plastic. Further investigation showed that nanopatterning of oxygen plasma substrates did not confer any additional advantage over smooth oxygen plasma, although one pattern (DSQ120) showed comparable early differentiation and function.

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling.

Gene expression analysis by quantitative real-time polymerase chain reaction (RT-qPCR) is routinely used in biomedical studies. The reproducibility and reliability of the data fundamentally depends on experimental design and data interpretation. Despite the wide application of this assay, there is significant variation in the validation process of gene expression data from research laboratories. Since the validity of results depends on appropriate normalisation, it is crucial to select appropriate reference gene(s), where transcription of the selected gene is unaffected by experimental setting. In this study we have applied geNorm technology to investigate the transcription of 12 'housekeeping' genes for use in the normalisation of RT-qPCR data acquired using a widely accepted HepaRG hepatic cell line in studies examining models of pre-clinical drug testing. geNorm data identified a number of genes unaffected by specific drug treatments and showed that different genes remained invariant in response to different drug treatments, whereas the transcription of 'classical' reference genes such as GAPDH (glyceralde- hyde-3-phosphate dehydrogenase) was altered by drug treatment. Comparing data normalised using the reference genes identified by geNorm with normalisation using classical housekeeping genes demonstrated substantial differences in the final results. In light of cell therapy application, RT-qPCR analyses has to be carefully evaluated to accurately interpret data obtained from dynamic cellular models undergoing sequential stages of phenotypic change.

Application of Impedance-Based Techniques in Hepatology Research.

There are a variety of end-point assays and techniques available to monitor hepatic cell cultures and study toxicity within in vitro models. These commonly focus on one aspect of cell metabolism and are often destructive to cells. Impedance-based cellular assays (IBCAs) assess biological functions of cell populations in real-time by measuring electrical impedance, which is the resistance to alternating current caused by the dielectric properties of proliferating of cells. While the uses of IBCA have been widely reported for a number of tissues, specific uses in the study of hepatic cell cultures have not been reported to date. IBCA monitors cellular behaviour throughout experimentation non-invasively without labelling or damage to cell cultures. The data extrapolated from IBCA can be correlated to biological events happening within the cell and therefore may inform drug toxicity studies or other applications within hepatic research. Because tight junctions comprise the blood/biliary barrier in hepatocytes, there are major consequences when these junctions are disrupted, as many pathologies centre around the bile canaliculi and flow of bile out of the liver. The application of IBCA in hepatology provides a unique opportunity to assess cellular polarity and patency of tight junctions, vital to maintaining normal hepatic function. Here, we describe how IBCAs have been applied to measuring the effect of viral infection, drug toxicity /IC50, cholangiopathies, cancer metastasis and monitoring of the gut-liver axis. We also highlight key areas of research where IBCAs could be used in future applications within the field of hepatology.

Computer simulation of neutral drift among limbal epithelial stem cells of mosaic mice.

The use of mice that are mosaic for reporter gene expression underlies many lineage-tracing studies in stem cell biology. For example, using mosaic LacZ reporter mice, it was shown that limbal epithelial stem cells (LESCs) around the periphery of the cornea maintain radial sectors of the corneal epithelium and that radial stripe numbers declined with age. Originally, the corneal results were interpreted as progressive, age-related loss or irreversible inactivation of some LESC clones. In this study we used computer simulations to show that these results could also be explained by stochastic replacement of LESCs by neighbouring LESCs, leading to neutral drift of LESC populations. This was shown to reduce the number of coherent clones of LESCs and hence would coarsen the mosaic pattern in the corneal epithelium without reducing the absolute number of LESCs. Simulations also showed that corrected stripe numbers declined more slowly when LESCs were grouped non-randomly and that mosaicism was rarely lost unless simulated LESC numbers were unrealistically low. Possible reasons why age-related changes differ between mosaic corneal epithelia and other systems, such as adrenal cortices and intestinal crypts, are discussed.

Acetaminophen cytotoxicity is ameliorated in a human liver organotypic co-culture model.

Organotypic liver culture models for hepatotoxicity studies that mimic in vivo hepatic functionality could help facilitate improved strategies for early safety risk assessment during drug development. Interspecies differences in drug sensitivity and mechanistic profiles, low predictive capacity, and limitations of conventional monocultures of human hepatocytes, with high attrition rates remain major challenges. Herein, we show stable, cell-type specific phenotype/cellular polarity with differentiated functionality in human hepatocyte-like C3A cells (enhanced CYP3A4 activity/albumin synthesis) when in co-culture with human vascular endothelial cells (HUVECs), thus demonstrating biocompatibility and relevance for evaluating drug metabolism and toxicity. In agreement with in vivo studies, acetaminophen (APAP) toxicity was most profound in HUVEC mono-cultures; whilst in C3A:HUVEC co-culture, cells were less susceptible to the toxic effects of APAP, including parameters of oxidative stress and ATP depletion, altered redox homeostasis, and impaired respiration. This resistance to APAP is also observed in a primary human hepatocyte (PHH) based co-culture model, suggesting bidirectional communication/stabilization between different cell types. This simple and easy-to-implement human co-culture model may represent a sustainable and physiologically-relevant alternative cell system to PHHs, complementary to animal testing, for initial hepatotoxicity screening or mechanistic studies of candidate compounds differentially targeting hepatocytes and endothelial cells.

Fibrinogen production is enhanced in an in-vitro model of non-alcoholic fatty liver disease: an isolated risk factor for cardiovascular events?

BACKGROUND: Cardiovascular disease (CVD) remains the major cause of excess mortality in patients with non-alcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the individual contribution of NAFLD to CVD risk factors in the absence of pathogenic influences from other comorbidities often found in NAFLD patients, by using an established in-vitro model of hepatic steatosis. METHODS: Histopathological events in non-alcoholic fatty liver disease were recapitulated by focused metabolic nutrient overload of hepatoblastoma C3A cells, using oleate-treated-cells and untreated controls for comparison. Microarray and proteomic data from cell culture experiments were integrated into a custom-built systems biology database and proteogenomics analysis performed. Candidate genes with significant dysregulation and concomitant changes in protein abundance were identified and STRING association and enrichment analysis performed to identify putative pathogenic pathways. RESULTS: The search strategy yielded 3 candidate genes that were specifically and significantly up-regulated in nutrient-overloaded cells compared to untreated controls: fibrinogen alpha chain (2.2 fold), fibrinogen beta chain (2.3 fold) and fibrinogen gamma chain (2.1 fold) (all rank products pfp <0.05). Fibrinogen alpha and gamma chain also demonstrated significant concomitant increases in protein abundance (3.8-fold and 2.0-fold, respectively, p <0.05). CONCLUSIONS: In-vitro modelling of NAFLD and reactive oxygen species formation in nutrient overloaded C3A cells, in the absence of pathogenic influences from other comorbidities, suggests that NAFLD is an isolated determinant of CVD. Nutrient overload-induced up-regulation of all three fibrinogen component subunits of the coagulation cascade provides a possible mechanism to explain the excess CVD mortality observed in NAFLD patients.

Cell proliferation, movement and differentiation during maintenance of the adult mouse adrenal cortex.

Appropriate maintenance and regeneration of adult endocrine organs is important in both normal physiology and disease. We investigated cell proliferation, movement and differentiation in the adult mouse adrenal cortex, using different 5-bromo-2'-deoxyuridine (BrdU) labelling regimens and immunostaining for phenotypic steroidogenic cell markers. Pulse-labelling showed that cell division was largely confined to the outer cortex, with most cells moving inwards towards the medulla at around 13-20 µm per day, though a distinct labelled cell population remained in the outer 10% of the cortex. Pulse-chase-labelling coupled with phenotypic immunostaining showed that, unlike cells in the inner cortex, most BrdU-positive outer cortical cells did not express steroidogenic markers, while co-staining for BrdU and Ki67 revealed that some outer cortical BrdU-positive cells were induced to proliferate following acute adrenocorticotropic hormone (ACTH) treatment. Extended pulse-chase-labelling identified cells in the outer cortex which retained BrdU label for up to 18-23 weeks. Together, these observations are consistent with the location of both slow-cycling stem/progenitor and transiently amplifying cell populations in the outer cortex. Understanding the relationships between these distinct adrenocortical cell populations will be crucial to clarify mechanisms underpinning adrenocortical maintenance and long-term adaptation to pathophysiological states.

Increased corneal epithelial turnover contributes to abnormal homeostasis in the Pax6(+/-) mouse model of aniridia.

We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6(+/-) mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6(+/-) mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6(+/-) mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6(+/-) and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6(+/-) heterozygotes but BrdU-LRCs were also present in Pax6(+/-) corneas. It seems likely that Pax6(+/-) LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6(+/-) cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6(+/-) corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6(+/-) than WT mice. This implies that epithelial cell loss is higher in Pax6(+/-) mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6(+/-) mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.

Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice.

We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn't fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.

Stem cells and corneal epithelial maintenance: insights from the mouse and other animal models.

Maintenance of the corneal epithelium is essential for vision and is a dynamic process incorporating constant cell production, movement and loss. Although cell-based therapies involving the transplantation of putative stem cells are well advanced for the treatment of human corneal defects, the scientific understanding of these interventions is poor. No definitive marker that discriminates stem cells that maintain the corneal epithelium from the surrounding tissue has been discovered and the identity of these elusive cells is, therefore, hotly debated. The key elements of corneal epithelial maintenance have long been recognised but it is still not known how this dynamic balance is co-ordinated during normal homeostasis to ensure the corneal epithelium is maintained at a uniform thickness. Most indirect experimental evidence supports the limbal epithelial stem cell (LESC) hypothesis, which proposes that the adult corneal epithelium is maintained by stem cells located in the limbus at the corneal periphery. However, this has been challenged recently by the corneal epithelial stem cell (CESC) hypothesis, which proposes that during normal homeostasis the mouse corneal epithelium is maintained by stem cells located throughout the basal corneal epithelium with LESCs only contributing during wound healing. In this chapter we review experimental studies, mostly based on animal work, that provide insights into how stem cells maintain the normal corneal epithelium and consider the merits of the alternative LESC and CESC hypotheses. Finally, we highlight some recent research on other stem cell systems and consider how this could influence future research directions for identifying the stem cells that maintain the corneal epithelium.

Normal X-inactivation mosaicism in corneas of heterozygous FlnaDilp2/+ female mice--a model of human filamin A (FLNA) diseases.

BACKGROUND: Some abnormalities of mouse corneal epithelial maintenance can be identified by the atypical mosaic patterns they produce in X-chromosome inactivation mosaics and chimeras. Human FLNA/+ females, heterozygous for X-linked, filamin A gene (FLNA) mutations, display a range of disorders and X-inactivation mosaicism is sometimes quantitatively unbalanced. FlnaDilp2/+ mice, heterozygous for an X-linked filamin A (Flna) nonsense mutation have variable eye, skeletal and other abnormalities, but X-inactivation mosaicism has not been investigated. The aim of this study was to determine whether X-inactivation mosaicism in the corneal epithelia of FlnaDilp2/+ mice was affected in any way that might predict abnormal corneal epithelial maintenance. RESULTS: X-chromosome inactivation mosaicism was studied in the corneal epithelium and a control tissue (liver) of FlnaDilp2/+ and wild-type (WT) female X-inactivation mosaics, hemizygous for the X-linked, LacZ reporter H253 transgene, using ß-galactosidase histochemical staining. The corneal epithelia of FlnaDilp2/+ and WT X-inactivation mosaics showed similar radial, striped patterns, implying epithelial cell movement was not disrupted in FlnaDilp2/+ corneas. Corrected stripe numbers declined with age overall (but not significantly for either genotype individually), consistent with previous reports suggesting an age-related reduction in stem cell function. Corrected stripe numbers were not reduced in FlnaDilp2/+ compared with WT X-inactivation mosaics and mosaicism was not significantly more unbalanced in the corneal epithelia or livers of FlnaDilp2/+ than wild-type Flna+/+ X-inactivation mosaics. CONCLUSIONS: Mosaic analysis identified no major effect of the mouse FlnaDilp2 mutation on corneal epithelial maintenance or the balance of X-inactivation mosaicism in the corneal epithelium or liver.

Transition from organogenesis to stem cell maintenance in the mouse adrenal cortex.

Mice showing mosaic expression of an appropriate marker gene that is activated during development provide simple tools for investigating cell lineages. We used the mosaic ß-galactosidase staining patterns in adrenal cortices of 21OH/ LacZ transgenic mice to study both organogenesis and maintenance of the adult tissue. Randomly orientated mosaic patterns present in embryonic day 14.5 (E14.5) adrenals changed progressively during the perinatal period from discrete spots, via patches and radial arrays, to radial stripes, which first emerged between postnatal days 0 and 7 (P0 and P7). The mosaic radial stripe pattern was fully established by P21 and remained unchanged throughout the adult period (8-52 weeks). The mouse adrenal gland grew continuously between E14.5 and P21, including the period during which stripes emerge. Ki67-positive, proliferative cells in the adrenal cortex were mainly localized to the outer cell layers between E18.5 and P3. By P10, cell proliferation had increased, and the proliferative region had expanded but was still mainly confined to the outer cortex. Correlation of changes in mosaic patterns in 21OH/LacZ adrenal cortices with the locations of adrenocortical cell proliferation suggest that the radial stripes arise by edge-biased growth during the perinatal period, even if they are maintained by stem cells in adults. The stability of the adult stripe pattern suggests that stem cell function is unchanged between 8 and 52 weeks.

Effects of aberrant Pax6 gene dosage on mouse corneal pathophysiology and corneal epithelial homeostasis.

BACKGROUND: Altered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6⁺/⁻ heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6(+/Sey-Neu) (Pax6⁺/⁻) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77(Tg/-) transgenics, which over-express Pax6 and model human PAX6 duplication. METHODOLOGY/PRINCIPAL FINDINGS: We used electron microscopy to investigate ocular defects in Pax6⁺/⁻ heterozygotes (low Pax6 levels) and PAX77(Tg/-) transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZ(Tg/-)) mice to investigate corneal epithelial maintenance by LESC clones in Pax6⁺/⁻ and PAX77(Tg/-) mosaic mice. PAX77(Tg/-) mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6⁺/⁻ mosaics were corrected by introducing the PAX77 transgene (in Pax6⁺/⁻, PAX77(Tg/-) mosaics). Pax6(Leca4/+), XLacZ(Tg/-) mosaic mice (heterozygous for the Pax6(Leca4) missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZ(Tg/-) mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6⁺/⁻ and PAX77(Tg/-) mosaic corneas, suggesting Pax6 under- and over-expression both affect LESC clones. CONCLUSIONS/SIGNIFICANCE: Pax6⁺/⁻ and PAX77(Tg/-) genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK.

Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium.

BACKGROUND: The mouse corneal epithelium is a continuously renewing 5-6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age. RESULTS: The initial pattern of corneal epithelial patches in XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks. CONCLUSION: Striping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.

Identification of a human ortholog of the mouse Dcpp gene locus, encoding a novel member of the CSP-1/Dcpp salivary protein family.

Salivary fluid, the collective product of numerous major and minor salivary glands, contains a range of secretory proteins that play key defensive, digestive, and gustatory roles in the oral cavity. To understand the distinct protein "signature" contributed by individual salivary glands to salivary secretions, we studied a family of proteins shown by in vitro mRNA translation to be abundantly expressed in mouse sublingual glands. Molecular cloning, Southern blotting, and restriction fragment length polymorphism analyses showed these to represent one known and two novel members of the common salivary protein (CSP-1)/Demilune cell and parotid protein (Dcpp) salivary protein family, the genes for which are closely linked in the T-complex region of mouse chromosome 17. Bioinformatic analysis identified a putative human CSP-1/Dcpp ortholog, HRPE773, expressed predominantly in human salivary tissue, that shows 31% amino acid identity and 45% amino acid similarity to the mouse Dcpp query sequence. The corresponding human gene displays a similar structure to the mouse Dcpp genes and is located on human chromosome 16 in a region known to be syntenic with the T-complex region of mouse chromosome 17. The predicted mouse and human proteins both display classical NH(2)-terminal signal sequences, putative jacalin-related lectin domains, and potential N-linked glycosylation sites, suggesting secretion via sublingual saliva into the oral cavity where they may display antimicrobial activity or provide a defensive coating to enamel. Identification of a human CSP-1/Dcpp ortholog therefore provides a key tool for investigation of salivary protein function in human oral health and disease.

Mosaic patch patterns in chimeric and transgenic mice suggest that directional growth in the adrenal cortex begins in the perinatal period.

Staining for beta-galactosidase (beta-gal) reporter activity in adrenal glands from adult, fetal and neonatal 21-OH/LacZ transgenic mice revealed mosaic patch patterns that were qualitatively similar to those seen in LacZ <--> wild-type mouse chimeras, at similar developmental stages. This suggests that, as in chimeras, the transgenic patch pattern may reflect cell lineage relationships. Consequently, 21-OH/LacZ transgenic mice could be useful as a simpler alternative to chimeras for the analysis of clonal growth and cell mixing during adrenocortical organogenesis. Embryonic day 16.5 (E16.5) adrenal cortices of 21-OH/LacZ transgenic mice displayed a punctate patch pattern, but by E18.5 "stripes" appeared to be emerging and were clearly visible by the day of birth (P0) and three days later (P3), consistent with the adult mosaic striped pattern. This suggests that adrenocortical organogenesis involves an initial period of randomly oriented clonal growth, followed by directional growth which begins in the perinatal period.