Winston W. Kao

From Hair to Cornea: Toward the Therapeutic Use of Hair Follicle-Derived Stem Cells in the Treatment of Limbal Stem Cell Deficiency

Limbal stem cell deficiency (LSCD) leads to severe ocular surface abnormalities that can result in the loss of vision. The most successful therapy currently being used is transplantation of limbal epithelial cell sheets cultivated from a limbal biopsy obtained from the patients healthy, contralateral eye or cadaveric tissue. In this study, we investigated the therapeutic potential of murine vibrissae hair follicle bulge‐derived stem cells (HFSCs) as an autologous stem cell (SC) source for ocular surface reconstruction in patients bilaterally affected by LSCD. This study is an expansion of our previously published work showing transdifferentiation of HFSCs into cells of a corneal epithelial phenotype in an in vitro system. In this study, we used a transgenic mouse model, K12rtTA/rtTA/tetO‐cre/ROSAmTmG, which allows for HFSCs to change color, from red to green, once differentiation to corneal epithelial cells occurs and Krt12, the corneal epithelial‐specific differentiation marker, is expressed. HFSCs were isolated from transgenic mice, amplified by clonal expansion on a 3T3 feeder layer, and transplanted on a fibrin carrier to the eye of LSCD wild‐type mice (n = 31). The HFSC transplant was able to reconstruct the ocular surface in 80% of the transplanted animals; differentiating into cells with a corneal epithelial phenotype, expressing Krt12, and repopulating the corneal SC pool while suppressing vascularization and conjunctival ingrowth. These data highlight the therapeutic properties of using HFSC to treat LSCD in a mouse model while demonstrating a strong translational potential and points to the niche as a key factor for determining stem cell differentiation. STEM CELLS 2011;29:57–66

Age-related changes in the meibomian gland.

The purpose of this study was to characterize the age-related changes of the mouse meibomian gland. Eyelids from adult C57Bl/6 mice at 2, 6, 12 and 24 months of age were stained with specific antibodies against peroxisome proliferator activated receptor gamma (PPARgamma) to identify differentiating meibocytes, Oil Red O (ORO) to identify lipid, Ki67 nuclear antigen to identify cycling cells, B-lymphocyte-induced maturation protein-1 (Blimp1) to identify potential stem cells and CD45 to identify immune cells. Meibomian glands from younger mice (2 and 6 months) showed cytoplasmic and perinuclear staining with anti-PPARgamma antibodies with abundant ORO staining of small, intracellular lipid droplets. Meibomian glands from older mice (12 and 24 months) showed only nuclear PPARgamma localization with less ORO staining and significantly reduced acinar tissue (p < 0.04). Acini of older mice also showed significantly reduced (p < 0.004) numbers of Ki67 stained nuclei. While Blimp1 appeared to diffusely stain the superficial ductal epithelium, isolated cells were occasionally stained within the meibomian gland duct and acini of older mice that also stained with CD45 antibodies, suggesting the presence of infiltrating plasmacytoid cells. These findings suggest that there is altered PPARgamma receptor signaling in older mice that may underlie changes in cell cycle entry/proliferation, lipid synthesis and gland atrophy during aging. These results are consistent with the hypothesis that mouse meibomian glands undergo age-related changes similar to those identified in humans and may be used as a model for age-related meibomian gland dysfunction.

Therapeutic effects of adenoviral gene transfer of bone morphogenic protein-7 on a corneal alkali injury model in mice

An alkali burn in the cornea is a common serious clinical problem often leading to permanent visual impairment. Since transforming growth factor-β (TGF-β) is involved in the response to corneal injury, we evaluated the therapeutic effects of adenoviral gene transfer of mouse bone morphogenic proten-7 (BMP-7), which has antagonistic effects on TGF-β in tissue fibrosis. Burned cornea did not express endogenous BMP-7 mRNA and protein. Resurfacing of the burned cornea by invading conjunctival epithelium was accelerated by adenoviral introduction of BMP-7. Exogenous BMP-7 expression also suppressed myofibroblast generation, appearance of monocytes/macrophages and expression of MCP-1, TGF-βs, and collagen I α2 chain in the affected stroma. Ectopic BMP-7 did not suppress stromal neovascularization throughout the interval studied and also did not reduce VEGF mRNA expression at Day 10. Ectopic BMP-7 in burned corneal tissue resulted in activation of Smad1/5/8 signaling and partial suppression of the phospho-Smad2 signal. These data suggest that overexpression of BMP-7 is an effective strategy for treatment of ocular alkali burns.

Lumican Binds ALK5 to Promote Epithelium Wound Healing

Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.

Impaired cornea wound healing in a tenascin C-deficient mouse model.

We investigated the effects of loss of tenascin C on the healing of the stroma using incision-injured mice corneas. Tenascin C was upregulated in the stroma following incision injury to the cornea. Wild-type (WT) and tenascin C-null (knockout (KO)) mice on a C57BL/6 background were used. Cell culture experiments were also conducted to determine the effects of the lack of tenascin C on fibrogenic gene expression in ocular fibroblasts. Histology, immunohistochemistry and real-time reverse transcription PCR were employed to evaluate the healing process in the stroma. The difference in the incidence of wound closure was statistically analyzed in hematoxylin and eosin-stained samples between WT and KO mice in addition to qualitative observation. Healing of incision injury in corneal stroma was delayed, with less appearance of myofibroblasts, less invasion of macrophages and reduction in expression of collagen Iα1, fibronectin and transforming growth factor β1 (TGFβ1) in KO mice compared with WT mice. In vitro experiments showed that the loss of tenascin C counteracted TGFβ1 acceleration of mRNA expression of TGFβ1, and of collagen Iα1 and of myofibroblast conversion in ocular fibroblasts. These results indicate that tenascin C modulates wound healing-related fibrogenic gene expression in ocular fibroblasts and is required for primary healing of the corneal stroma.

Keratocan is Expressed by Osteoblasts and Can Modulate Osteogenic Differentiation

Keratocan is an extracellular matrix protein that belongs to the small leucine-rich proteoglycan family that also includes lumican, biglycan, decorin, mimecan, and fibromodulin. Members of this family are known to play a role in regulating cellular processes such as proliferation and modulation of osteoprogenitor lineage differentiation. The aims of this study were to evaluate the expression pattern of the keratocan within the osteoprogenitor lineage and to assess its role in regulating osteoblast maturation and function. Results from gene expression analyses of cells at different maturation stages within the osteoblast lineage indicate that keratocan is differentially expressed by osteoblasts and shows little or no expression by osteocytes. During primary osteoblast cultures, high keratocan mRNA expression was observed on day 14, whereas lower expression was detected at days 7 and 21. To assess the effects of keratocan on osteoprogenitor cell differentiation, we evaluated primary calvarial cell cultures from keratocan-deficient mice. The mineralization of calvarial osteoblast cultures derived from keratocan null (Kera–/–) mice was lower than in wild-type osteoblast cultures. Furthermore, analysis of RNA derived from Kera−/− calvarial cell cultures showed a reduction in the mature osteoblast differentiation markers, that is, bone sialoprotein and osteocalcin. In addition, we have evaluated the bone formation in keratocan-deficient mice. Histomorphometric analysis indicated that homozygous knockout mice have significantly decreased rates of bone formation and mineral apposition. Taken together, our results demonstrate the expression of keratocan by osteoblast lineage cells and its ability to modulate osteoblast function.

Lumican Exhibits Anti-Angiogenic Activity in a Context Specific Manner

A series of overexpression studies have shown that lumican suppresses angiogenesis in tumors produced from pancreatic adenocarcinoma, fibrosarcoma, and melanoma tumor cells. Despite lumican’s anti-angiogenic activity, a clear correlation of differential expression of lumican in various cancers and cancer malignancy has failed to emerge. Therefore, we hypothesized that either 1.) endogenously expressed lumican is not anti-angiogenic or alternatively that 2.) lumican exhibits angiostatic activity only in limited microenvironments. Previously, lumican was shown to suppress tumor growth and angiogenesis in subcutaneously injected PanO2 pancreatic adenocarcinoma cells. Therefore, to determine if endogenously expressed lumican is anti-angiogenic we subcutaneously injected PanO2 cells into wild-type and lumican knockout mice and compared tumor growth and vascular densities of the resulting tumors. We found that tumors grown in lumican knockout animals were larger and contained significantly elevated vascular densities compared to those grown in wild-type mice. Interestingly however lumican knockout animals did not exhibit enhanced angiogenesis in aortic ring assays, matrigel plugs, or healing wound biopsies raising the possibility that lumican suppresses angiogenesis only in tumor microenvironments. To test this possibility, we sought a tumor model wherein lumican did not exhibit anti-angiogenic activity. Utilizing the 4T1 breast cancer model, we found that lumican suppressed 4T1 tumor growth and lung metastasis, but not angiogenesis. In conclusion, these results show that the angiostatic activity of lumican is dependent on currently undefined microenvironmental cues and therefore helps to understand why differential expression of lumican does not consistently correlate with human tumor malignancy.

Role of EGF receptor signaling on morphogenesis of eyelid and meibomian glands

The epidermal growth factor receptor (EGFR) signaling has a pivotal role in the regulation of morphogenesis during development and maintenance of homeostasis in adult eyelid and its adnexa. Studies have demonstrated that during eyelid morphogenesis the EGFR signaling pathway is responsible for keratinocyte and mesenchymal cell proliferation and migration at the eyelid tip. For meibomian gland morphogenesis, EGFR signaling activation stimulates meibomian gland epithelial cell proliferation. EGFR signaling pathway functions through multiple downstream signals such as ERK, Rho/ROCK and integrin and is regulated by a variety of upstream signals including Adam17, GPR48 and FGFR signaling. Herein we review the literature that describe the role of EGFR and its related signaling pathways in eyelid and meibomian gland morphogenesis.

A Mouse Model of Schnyder Corneal Dystrophy with the N100S Point Mutation

Schnyder corneal dystrophy (SCD) is a rare autosomal dominant disease in humans, characterized by abnormal deposition of cholesterol and phospholipids in cornea caused by mutations in the UbiA prenyltransferase domain containing 1 (UBIAD1) gene. In this study, we generated a mouse line carrying Ubiad1 N100S point mutation using the CRISPR/Cas9 technique to investigate the pathogenesis of SCD. In vivo confocal microscopy revealed hyper-reflective dot-like deposits in the anterior cornea in heterozygotes and homozygotes. No significant change was found in corneal epithelial barrier function or wound healing. Electron microscopy revealed abnormal mitochondrial morphology in corneal epithelial, stromal, and endothelial cells. Mitochondrial DNA copy number assay showed 1.27 ± 0.07 fold change in homozygotes versus 0.98 ± 0.05 variation in wild type mice (P < 0.05). Lipidomic analysis indicated abnormal metabolism of glycerophosphoglycerols, a lipid class found in mitochondria. Four (34:1, 34:2, 36:2, and 44:8) of the 11 glycerophosphoglycerols species identified by mass spectrometry showed a significant increase in homozygous corneas compared with heterozygous and wild-type mouse corneas. Unexpectedly, we did not find a difference in the corneal cholesterol level between different genotypes by filipin staining or lipidomic analysis. The Ubiad1N100S mouse provides a promising animal model of SCD revealing that mitochondrial dysfunction is a prominent component of the disease. The different phenotype in human and mouse may due to difference in cholesterol metabolism between species.