In the past year, beside using genetically modified mouse lines to examine how growth factors function in regulating tissue morphogenesis during embryonic development and maintenance of tissue functions in normal and disease in adults, we have also examined the efficacy of using bone marrow stem cells and umbilical cord mesenchymal stem cells in treating corneal diseases. Our results demonstrated that both bone marrow cells and umbilical cord mesenchymal stem cells could become corneal stromal cells, when they were transplanted to the stroma of keratocan and lumican knock out mice. The results suggest that transplantation of somatic stem cells from adults is a feasible regiment to cure certain genetic and/or acquired corneal diseases.

Herein we present evidence that Cre/LoxP recombination happens during gametogenesis in several driver mouse lines that express tissue-specific Cre outside the gonads. PCR and recombination analysis demonstrate recombination of floxed allele occurs during transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in testis. Thereby, target floxed allele(s) may be ubiquitously ablated in experimental animals intended for tissue-specific gene deletion. Thus, gametogenesis-associated recombination should always be examined in tissue-specific gene-ablation studies (Manuscript submitted).

2. Keratocan and Lumican Regulates Neutrophil Infiltration and Corneal Clarity in LPS (Lipopolysaccharides)-induced Keratitis through Direct CXC Chemokine Interaction Keratocan and lumican are keratansulfate proteoglycans (KSPG), which have a critical role in maintaining corneal clarity by regulating collagen fibril diameter and interfibrillar spacing in the corneal stroma. LPS-induced keratitis in wildtype mice developed increased corneal thickness and haze due to infiltration of neutrophils to the corneal stroma. Corneal thickness increases caused by LPS were significantly lower in Kera^-/- and Lum^-/- mice than wild type mice. Further, LPS injected Lum^-/- mice had elevated corneal haze levels compared with that of Kera^-/- and wild type mice. Interestingly, neutrophil infiltration was inhibited in Kera^-/- and Lum^-/- mice at 6 and 24 hours post-stimulation, with Lum^-/- corneas having the fewest number of neutrophils. Reconstituting keratocan and lumican in corneas of Kera^-/- and Lum^-/- mice with adeno-keratocan and adeno-lumican viral vectors restore the tissue accessibility to neutrophils in LPS induced inflammatory response. Moreover, we demonstrate that lumican and keratocan can bind the CXC chemokine KC during a corneal inflammatory response suggesting a novel role of corneal proteoglycans in chemokine gradient formation. These data support a significant role for both of these proteoglycans in a corneal inflammatory response with respect to corneal edema (Manuscript submitted).

Bone marrow stem cells (BMSC) are multipotent and capable of differentiating to various cell types, e.g., myocardial cells, hepatocytes, neurons, epidermal and gastrointestinal epithelial cells. We examined whether BMSC could differentiate to keratocan expressing cells: a characteristic of corneal keratocytes. Bone marrow cells obtained from green fluorescent, Kera-Cre/ZEG, Kera-Cre/DTRred bitransgenic mice were intrastromally injected into corneas of Kera^-/- knockout, and Kera^-/-/DTRred, and Kera-Cre/DTRred bitransgenic mice mice. Immunostaining with goat anti-keratocan antibodies confirmed that freshly isolated BMC were keratocan negative. Green BMC injected into corneal stroma began to change shape and assumed dendritic and polygonal morphology at one week of intrastromal injection. Keratocan expression was detected in stroma and around green BMC in the recipient corneas. EGFP negative BMC isolated from Kera-Cre/ZEG mice were intrastromally injected to corneas of Kera^-/-, Kera^-/-/DTRred and Kera-Cre/DTRred mice. In 6 days, injected BMC became EGFP positive and displayed a dendritic and polygonal morphology, suggesting the modification of ZEG transgene by Cre recombinase driven by keratocan promoter in Kera-Cre/ZEG BMC injected. Image analysis with confocal microscope showed the segregation of red fluorescence from the green, indicating that the donor BMC cells underwent differentiation and exhibited keratocyte phenotype instead of fusion with host keratocytes. Our results indicate that corneal stroma provides an environment for BMSC differentiation to assume a phenotype of keratocan expression, characteristics of keratocytes.

To examine the possibility of clonal allelic activation of Krt12 gene of limbal stem cells undergoing corneal type epithelium differentiation, we determined the expression pattern of Cre in corneal epithelium of Krt12-Cre knock-in mice that would express Cre recombinase by corneal epithelial cells when the Krt12^Cre allele was transcribed. Excision of LoxP elements modified genomic DNA then leads to the expression of second reporter genes, e.g., EGFP (Enhanced Green Fluorescence Protein) and AP (Alkaline Phosphatase) in corneas of bitransgenic Krt12^Cre/w/ROZA-EGFP, Krt12^Cre/w/ZEG and Krt12^Cre/w/ZAP mouse lines. Our results indicated that clonal limbal stem cells randomly activate one single or both Krt12 alleles in the process of terminal differentiation, and provides evidence that clonal allelic activation may account for mild phenotype in epithelium of heterozygous dominant negative mutation (Manuscript in preparation).

We have prepared mouse lines in which genes of interest can be specifically ablated in corneal epithelium and stroma. However, the cryptic Cre activities during gametogenesis severely compromise our ability to obtain experiemnetal mice of correct genotypes. To overcome the pitfalls of cryptic Cre recombinase expression during gametogenesis, we develop a strategy of breeding tri-transgenic Krt12^rtTA/rtTA/tet-O-Cre/cJun^f/f and Krt12^rtTA/rtTA/tet-O-Cre/Tbr2^f/f (type II receptor of TGF-β) mice. The ablation of cJun^f/f and Tbr2^f/f are achieved by feeding experimental mice with doxycycline for two weeks. Preliminary results indicated that ablation of cJun and Tbr2 caused delayed healing of epithelium debridement. Understanding of signaling pathways involving cJun and Tbr2 will allow us to develop treatment regiments of diseases that exhibit altered signaling of cJun and TGF-β.

The formation of ocular surface tissues, e.g., cornea, conjunctiva, eyelids, lacrimal glands, and meibomian glands, was not well understood. Dry eye syndromes severely compromise the functions of ocular tissues and represent as one of the major causes of office visits in eye care. There is a great need for a better understanding on how the ocular tissue morphogenesis takes place during embryonic development and the maintenance of tissue functions in adult. We will determine the cell lineages of ocular surface tissues, e.g., corneal endothelium, keratocytes, and stromal cells in iris, ciliary body, eyelids with bitransgenic Kera-Cre/ZEG and Wnt1-Cre/ZEG mice. The distribution of EGFP positive cells will be determined in ocular surface tissues 100-150 µm thick vibratome slices with ZEISS LSM confocal microscope. Thereby, the ocular surface cells derived from neural crest cells at different stages of embryonic development can be determined.

3. Stem Cell Therapy

To expand the possible use of different types of somatic stem cells in treating genetic and/or acquired corneal diseases, we will use umbilical cord stem cells to further confirm that the umbilical cord mesenchymal cells can become corneal keratocytes when transplanted to keratocan and lumican knock out mice.

1. Keynote speaker in the Asian ARVO in Singapore, March, 2007

2. Invited Speaker in Department of Ophthalmology, Wakayama medical University, Japan, June, 2007

3. Presented fifteen papers in 2007 annual ARVO meeting, Fort Lauderdfale Fl