Human corneal limbal organoids maintaining limbal stem cell niche function.

Corneal epithelial stem cells reside in the limbal area between the cornea and conjunctiva. We examined the potential use of limbal organoids as a source of transplantable limbal stem cells. After treating tissue with collagenase, limbal cells were seeded onto Matrigel and cultivated using limbal phenotype maintenance medium. After 1-month, approximately 500 organoids were formed from one donor cornea. Organoids derived from vertical sites (superior and inferior limbus) showed large colony forming efficiency, a higher ratio of slow cycling cells and N-cadherin-expressing epithelial cells compared to horizontal sites. The progenitor markers Keratin (K) 15 and p63 were expressed in epithelial sheets engineered form a single organoid. Organoids transplanted in the limbus of a rabbit limbal deficiency model confirmed the presence of organoid-derived cells extending on to host corneas by immunohistochemistry. Our data show that limbal organoids with a limbal phenotype can be maintained for up to 1 month in vitro which can each give rise to a fully stratified corneal epithelium complete with basal progenitor cells. Limbal organoids were successfully engrafted in vivo to provide epithelial cells in a rabbit limbal deficiency model, suggesting that organoids may be an efficient cell source for clinical use.

Effects of Amniotic Membrane-Derived Fibroblast Supernatant on Corneal Epithelium.

Purpose: To evaluate the effects of human amniotic membrane-derived fibroblast (AMF) cell supernatant (AMF-sup) on corneal epithelium. Methods: The phenotype of AMF cells was analyzed by flow cytometry using cell-surface markers. AMF cells were also induced to form osteoblasts and neural cells, and cell phenotypes were observed by staining and RT-PCR. Cultivated human corneal limbal epithelial sheets generated using AMF-sup were analyzed using immunohistochemistry and colony-forming efficiency, and the wound healing of epithelial defects was observed using a tissue-punch method. The effects of instillation of each supernatant in a rabbit corneal epithelial wound healing model were compared. Results: Mesenchymal stem cell (CD29, CD44, CD73, and CD90) and neural crest (CD49d and CD56) markers were expressed on the AMF cell surface. Following induction of differentiation, isolated AMF cells showed characteristics of osteoblasts and neural cells. Application of AMF-sup resulted in maintenance of the limbal epithelial phenotype and immature state, and significantly promoted wound healing in cultivated human corneal limbal epithelial sheets (P < 0.05) and rabbit corneal epithelium (P < 0.05) compared with the control. Conclusions: These data suggest that AMF cells have multi-differentiation potential, and that AMF-sup is effective in maintaining the limbal epithelial phenotype and promoting corneal epithelial wound healing, which may be of value in ocular surface reconstruction.

[Comparison of conventional tuberculin skin test and QFT-2G, a new method for diagnosis of tuberculosis infection, with the use of the contact score].

BACKGROUND: QuantiFERON TB-Gold (QFT) has recently been developed as a new method for diagnosing tuberculosis (TB) infection. To evaluate the usefulness of QFT, we analyzed the relationship between QFT and the closeness of contact with a source of infection, in comparison with that of the tuberculin skin test (TST). METHODS: Male (n=322) and female (n=340) subjects (4-75 years old) who had contact with an index case received QFT and TST. The diagnostic criterion for TB infection with TST was defined as a test with an erythema diameter of > or = 30 mm. The closeness of contact with an index case was quantified in the "contact score," based on the information obtained with a questionnaire. RESULTS: There was a significant positive correlation between the contact score and QFT-positive rate, while there was no such relationship for TST positivity. The odds ratios for positive QFT rate for the subjects in the 3rd and 4th quartile groups of contact score (taking the QFT-positive rate in the lowest score quartile as unity) were 3.40 (95% confidence interval: 1.07-10.76, p<0.05) and 7.62 (95% confidence interval: 2.60-22.37, p<0.01), respectively. These odds ratios were also significantly greater than unity after adjustment for age, sex, history of BCG vaccination and history of health care-related jobs. There was a wide difference in the QFT-positive rates between the 2nd and 3rd quartiles of contact score (3.5% vs. 11.9%). The borderline value of the contact score between these two quartiles corresponded to 200, which could be a cutoff value for defining a high-risk contact. CONCLUSION: The QFT-positive rates correlated well with closeness of contact, while TST showed a poor correlation. Thus, QFT is considered more useful than TST for diagnosing tuberculosis infection.

Altered lung surfactant system in a Rab38-deficient rat model of Hermansky-Pudlak syndrome.

Several Long-Evans rat substrains carrying the phenotype of oculocutaneous albinism and bleeding diathesis are a rat model of Hermansky-Pudlak syndrome (HPS). The mutation responsible for the phenotype (Ruby) was identified as a point mutation in the initiation codon of Rab38 small GTPase that regulates intracellular vesicle transport. As patients with HPS often develop life-limiting interstitial pneumonia accompanied by abnormal morphology of alveolar type II cells, we investigated lung surfactant system in Long-Evans Cinnamon rats, one strain of the Ruby rats. The lungs showed conspicuous morphology of type II cells containing markedly enlarged lamellar bodies. Surfactant phosphatidylcholine and surfactant protein B were increased in lung tissues and lamellar bodies but not in alveolar lumen. Expression levels of mRNA for surfactant proteins A, B, C, and D were not altered. Isolated type II cells showed aberrant secretory pattern of newly synthesized [(3)H]phosphatidylcholine, i.e., decreased basal secretion and remarkably amplified agonist-induced secretion. [(3)H]phosphatidylcholine synthesis and uptake by type II cells were not altered. Thus Rab38-deficient type II cells appear to carry abnormality in lung surfactant secretion but not in synthesis or uptake. These results suggest that aberrant lung surfactant secretion may be involved in the pathogenesis of interstitial pneumonia in HPS.

A mutation in Rab38 small GTPase causes abnormal lung surfactant homeostasis and aberrant alveolar structure in mice.

The chocolate mutation, which is associated with oculocutaneous albinism in mice, has been attributed to a G146T transversion in the conserved GTP/GDP-interacting domain of Rab38, a small GTPase that regulates intracellular vesicular trafficking. Rab38 displays a unique tissue-specific expression pattern with highest levels present in the lung. The purpose of this study was to characterize the effects of Rab38-G146T on lung phenotype and to investigate the molecular basis of the mutant gene product (Rab38(cht) protein). Chocolate lungs exhibited a uniform enlargement of the distal airspaces with mild alveolar destruction as well as a slight increase in lung compliance. Alveolar type II cells were engorged with lamellar bodies of increased size and number. Hydrophobic surfactant constituents (ie, phosphatidylcholine and surfactant protein B) were increased in lung tissues but decreased in alveolar spaces, consistent with a malfunction in lamellar body secretion and the subsequent cellular accumulation of these organelles. In contrast to wild-type Rab38, native Rab38(cht) proteins were found to be hydrophilic and not bound to intracellular membranes. Unexpectedly, recombinant Rab38(cht) proteins retained GTP-binding activity but failed to undergo prenyl modification that is required for membrane-binding activity. These results suggest that the genetic abnormality of Rab38 affects multiple lysosome-related organelles, resulting in lung disease in addition to oculocutaneous albinism.