Hongshan Liu

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

Cell Therapy of Congenital Corneal Diseases with Umbilical Mesenchymal Stem Cells: Lumican Null Mice

Background Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation. Methodology/Principal Findings Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice. Conclusions/Significance Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.

Collagen V is a dominant regulator of collagen fibrillogenesis: dysfunctional regulation of structure and function in a corneal-stroma-specific Col5a1-null mouse model

Collagen V is a regulatory fibril-forming collagen that forms heterotypic fibrils with collagen I. Deletion of collagen V in the mouse is associated with a lack of fibril assembly in the embryonic mesenchyme, with a resultant lethal phenotype. The current work elucidates the regulatory roles of collagen V during development and growth of tissues. A conditional mouse model with a mutation in Col5a1 was developed using a Cre-loxP approach. Col5a1 was ablated in Col5a1flox/flox mice using a cornea stroma-specific Kera-Cre driver mouse to produce a bitransgenic Col5a1Δst/Δst line that is null for collagen V. This permits analyses of the corneal stroma, a widely used model for studies of collagen V. The collagen-V-knockout stroma demonstrated severe dysfunctional regulation of fibrillogenesis. Fibril diameters were significantly increased, with an abnormal, heterogeneous distribution; fibril structure was abnormal, fibril number was decreased and lamellae were disorganized with decreased stroma thickness. The phenotype was more severe in the anterior versus posterior stroma. Opacity was demonstrated throughout the Col5a1Δst/Δst stroma, with significantly increased haze intensity compared with control mice. These data indicate central regulatory roles for collagen V in fibril and matrix assembly during tissue development, with dysfunctional regulation resulting in a functional loss of transparency.

Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells

TRPV4 is a non-selective cation channel with moderate calcium permeability, which is activated by exposure to hypotonicity. Such a stress induces regulatory volume decrease (RVD) behavior in human corneal epithelial cells (HCEC). We hypothesize that TRPV4 channel mediates RVD in HCEC. Immunohistochemistry revealed centrally and superficially concentrated TRPV4 localization in the corneal tissue. Immunocytochemical and fluorescence activated cell sorter (FACS) analyses identified TRPV4 membrane surface and cytosolic expression. RT-PCR and Western blot analyses identified TRPV4 gene and protein expression in HCEC, respectively. In addition, 4alpha-PDD or a 50% hypotonic medium induced up to threefold transient intracellular Ca2+ ([Ca2+]i) increases. Following TRPV4 siRNA HCEC transfection, its protein expression level declined by 64%, which abrogated these [Ca2+]i transients. Similarly, exposure to either ruthenium red or Ca(2+)-free Ringers solution also eliminated this response. In these transfected cells, RVD declined by 51% whereas in the non-transfected counterpart, ruthenium red and Ca(2+)-free solution inhibited RVD by 54 and 64%, respectively. In contrast, capsazepine, a TRPV1 antagonist, failed to suppress [Ca2+]i transients and RVD. TRPV4 activation contributes to RVD since declines in TRPV4 expression and activity are associated with suppression of this response. In conclusion, there is TRPV4 functional expression in HCEC.

TRPV1 Involvement in Inflammatory Tissue Fibrosis in Mice

We examined whether absence or blocking of transient receptor potential vanilloid subtype 1 (TRPV1) affects the level of inflammation and fibrosis/scarring during healing of injured tissue using an alkali burn model of cornea in mice. A cornea burn was produced with 1 N NaOH instilled into one eye of TRPV1-/- (KO) (n = 88) or TRPV1+/+ (n = 94) mice. Examinations of the corneal surface and eye globe size suggested that the loss of TRPV1 suppressed inflammation and fibrosis/scarring after alkali burn, and this was confirmed by histology, IHC, and gene expression analysis. The loss of TRPV1 inhibited inflammatory cell invasion and myofibroblast generation in association with reduction of expression of proinflammatory and profibrogenic components. Experiments of bone marrow transplantation between either genotype of mice showed that KO corneal tissue resident cells, but not KO bone marrow-derived cells, are responsible for KO-type wound healing with reduced inflammation and fibrosis. The absence of TRPV1 attenuated expression of transforming growth factor β 1 (TGFβ1) and other proinflammatory gene expression in cultured ocular fibroblasts, but did not affect TGFβ1 expression in macrophages. Loss of TRPV1 inhibited myofibroblast transdifferentiation in cultured fibroblasts. Systemic TRPV1 antagonists reproduced the KO type of healing. In conclusion, absence or blocking of TRPV1 suppressed inflammation and fibrosis/scarring during healing of alkali-burned mouse cornea. TRPV1 is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing.

Bone marrow mesenchymal stem cells can differentiate and assume corneal keratocyte phenotype

It remains elusive as to what bone marrow (BM) cell types infiltrate into injured and/or diseased tissues and subsequently differentiate to assume the phenotype of residential cells, for example, neurons, cardiac myocytes, keratocytes, etc., to repair damaged tissue. Here, we examined the possibility of whether BM cell invasion via circulation into uninjured and injured corneas could assume a keratocyte phenotype, using chimeric mice generated by transplantation of enhanced green fluorescent protein (EGFP)+ BM cells into keratocan null (Kera−/−) and lumican null (Lum−/−) mice. EGFP+ BM cells assumed dendritic cell morphology, but failed to synthesize corneal‐specific keratan sulfate proteoglycans, that is KS‐lumican and KS‐keratocan. In contrast, some EGFP+ BM cells introduced by intrastromal transplantation assumed keratocyte phenotypes. Furthermore, BM cells were isolated from Kera‐Cre/ZEG mice, a double transgenic mouse line in which cells expressing keratocan become EGFP+ due to the synthesis of Cre driven by keratocan promoter. Three days after corneal and conjunctival transplantations of such BM cells into Kera−/− mice, green keratocan positive cells were found in the cornea, but not in conjunctiva. It is worthy to note that transplanted BM cells were rejected in 4 weeks. MSC isolated from BM were used to examine if BM mesenchymal stem cells (BM‐MSC) could assume keratocyte phenotype. When BM‐MSC were intrastromal‐transplanted into Kera−/− mice, they survived in the cornea without any immune and inflammatory responses and expressed keratocan in Kera−/− mice. These observations suggest that corneal intrastromal transplantation of BM‐MSC may be an effective treatment regimen for corneal diseases involving dysfunction of keratocytes.

Lumican is required for neutrophil extravasation following corneal injury and wound healing

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum−/− mice and a novel transgenic mouse, Lum−/−,Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum−/− mice and this defect was rescued by the expression of lumican in the corneas of Lum−/−,Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.

Crosstalk between TGF-β and MAPK Signaling during Corneal Wound Healing

PURPOSE The aim of this study was to elucidate the mechanisms governing epithelial cell migration and proliferation during wound healing. METHODS The authors used wound healing of mouse corneal epithelium to examine the role TGF-β signaling plays during the healing process. To achieve this goal, they used transgenic mice in which the TGF-β receptor type II (Tbr2) was conditionally ablated from the corneal epithelium. Epithelium debridement wounds were made, followed by the assessment of cell migration, proliferation, and immunostaining of various signaling pathway components. RESULTS The authors showed that in the absence of TGF-β signaling corneal epithelial wound healing is delayed by 48 hours; this corresponds to a delay in p38MAPK activation. Despite the delayed p38MAPK activation, ATF2, a substrate of p38MAPK, is still phosphorylated, leading to the suppression of cell proliferation at the leading edge of the wound. These data provide evidence that in the absence of TGF-β signaling, the suppression of cell proliferation during the early stages of wound healing is maintained through the JNK activation of ATF2. CONCLUSIONS; Together the data presented here demonstrate the importance of the TGF-β and MAPK signaling pathways in corneal epithelial wound healing.

Aberrant expression of a β-catenin gain-of-function mutant induces hyperplastic transformation in the mouse cornea

β-catenin signaling has been shown to play a fundamental role in embryonic development and tumorigenesis. In this study, we investigated the role of β-catenin (Ctnnb1) in corneal homeostasis and tumorigenesis. Conditional expression of a murine Ctnnb1 gain-of-function mutation alone caused corneal neoplasia and neovascularization, resembling human ocular surface squamous neoplasia (OSSN). These corneas displayed an upregulation of cell proliferative markers (PCNA and p63), while presenting downregulation of both the Pax-6 transcription factor and the corneal differentiation marker cytokeratin 12. In addition, the expression of limbal-type keratin 15 ectopically extended to cornea, but the pattern of conjunctival keratin 4 and epidermal keratin 10 were unchanged. Moreover, epithelial E-cadherin and laminins decreased concomitantly with elevated levels of MMP-7. We also noticed a dramatic upregulation of pro-angiogenic factors (Vegf-A, Vegfr1) and angiopoietins in these corneas. Interestingly, all human OSSN specimens examined revealed nuclear β-catenin immunoreactivity. Taken together, these results argue that β-catenin activation is a crucial step during OSSN pathogenesis. Thus, inhibition of β-catenin might be beneficial for treating this disease.

Wakayama Symposium: Challenges of Future Research in Ocular Surface Cell Biology

During embryonic development, surface ectoderm differentiates to form corneal, conjunctival, and eyelid epidermal epithelia, and glandular epithelium (lacrimal and meibomian glands). Periocular mesenchymal cells of neural crest origin migrate and differentiate, leading to the formation of corneal endothelium and the stromas of the cornea, conjunctiva, eyelids, and trabecular meshwork. The formation of functional ocular surface tissues requires coordinated spatial and temporal expression of transcription factors and signaling molecules of various cytokines and signaling pathways, and the synthesis and remodeling of unique extracellular matrix. Although bidirectional interactions and signaling between mesenchyme and epithelium are considered necessary for embryonic formation of ocular surface tissues and homeostasis in adults, the molecular and cellular mechanisms that regulate such processes remain largely unknown. To investigate possible mechanisms, we have developed mouse models in which the gene functions of ocular surface epithelia and stromas can be altered by Doxycycline induction in spatial and temporal specific manners.