Publications

Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200-300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.

Recapitulation of human corneal stromal tissue is believed to be among the most challenging steps in engineering human corneal tissue because of the difficulty in reproducing its highly-ordered hierarchical ultrastructure, which imparts its robust biomechanical properties and optical transparency. In this study, we compared the feasibility of utilizing human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on a highly-aligned fibrous substrate made from poly(ester urethane) urea. In the serum-free keratocyte differentiation medium supplemented with FGF-2 (10 ng/mL) and TGF-β3 (0.1 ng/mL), hCSSCs successfully differentiated into keratocytes and secreted multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 60-70 μm thick and abundant in cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. Under the identical conditions, hCFs tended to differentiate into myofibroblasts and deposited a less-organized collagen-fibrillar construct in a pattern with similarities to corneal scar tissue due to a lack of cornea-specific ECM components. These observations demonstrated that hCSSCs showed a much greater potential, under proper substrate and growth factor guidance, to facilitate the generation of a biological human cornea equivalent. Unlike hCSSCs, hCFs were less responsive to these environmental cues and under identical culture conditions generated an ECM that poorly mimicked the native, functional tissue structure and composition.

PURPOSE: To develop and characterize a mouse model with intraocular pressure (IOP) elevation after laser photocoagulation on the trabecular meshwork (TM), which may serve as a model to investigate the potential of stem cell-based therapies for glaucoma.

METHODS: IOP was measured in 281 adult C57BL/6 mice to determine normal IOP range. IOP elevation was induced unilaterally in 50 adult mice, by targeting the TM through the limbus with a 532-nm diode laser. IOP was measured up to 24 weeks post-treatment. The optic nerve damage was detected by electroretinography and assessed by semiautomatic counting of optic nerve axons. Effects of laser treatment on the TM were evaluated by histology, immunofluorescence staining, optical coherence tomography (OCT) and transmission electron microscopy (TEM).

RESULTS: The average IOP of C57BL/6 mice was 14.5 ± 2.6 mmHg (Mean ± SD). After laser treatment, IOP averaged above 20 mmHg throughout the follow-up period of 24 weeks. At 24 weeks, 57% of treated eyes had elevated IOP with the mean IOP of 22.5 ± 2.5 mmHg (Mean ± SED). The difference of average axon count (59.0%) between laser treated and untreated eyes was statistically significant. Photopic negative response (PhNR) by electroretinography was significantly decreased. CD45+ inflammatory cells invaded the TM within 1 week. The expression of SPARC was increased in the TM from 1 to 12 weeks. Histology showed the anterior chamber angle open after laser treatment. OCT indicated that most of the eyes with laser treatment had no synechia in the anterior chamber angles. TEM demonstrated disorganized and compacted extracellular matrix in the TM.

CONCLUSIONS: An experimental murine ocular hypertension model with an open angle and optic nerve axon loss was produced with laser photocoagulation, which could be used to investigate stem cell-based therapies for restoration of the outflow pathway integrity for ocular hypertension or glaucoma.

Conventional allograft therapy for corneal scarring is widespread and successful, but donor tissue is not universally available, and some grafts fail owing to rejection and complications such as endothelial failure. We investigated direct treatment of corneal scarring using autologous stem cells, a therapy that, if successful, could reduce the need for corneal grafts. Mesenchymal cells were expanded from small superficial, clinically replicable limbal biopsies of human cadaveric corneo-scleral rims. Limbal biopsy-derived stromal cells (LBSCs) expanded rapidly in media containing human serum, were highly clonogenic, and could generate spheres expressing stem cell genes (ABCG2, Nestin, NGFR, Oct4, PAX6, and Sox2). Human LBSCs differentiated into keratocytes expressing characteristic marker genes (ALDH3A1, AQP1, KERA, and PTGDS) and organized a thick lamellar stroma-like tissue containing aligned collagen and keratan sulfate proteoglycans when cultured on aligned nanofiber substrata. When engrafted into mouse corneal wounds, LBSCs prevented formation of light-scattering scar tissue containing fibrotic matrix components. The presence of LBSCs induced regeneration of ablated stroma with tissue exhibiting lamellar structure and collagen organization indistinguishable from that of native tissue. Because the limbus can be easily biopsied from either eye of an affected individual and LBSCs capable of corneal stromal remodeling can be expanded under xeno-free autologous conditions, these cells present a potential for autologous stem cell-based treatment of corneal stromal blindness.

Emulating corneal stromal tissue is believed to be the most challenging step in bioengineering an artificial human cornea because of the difficulty in reproducing its highly ordered microstructure, the key to the robust biomechanical properties and optical transparency of this tissue. We conducted a comparative study to assess the feasibility of human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on groove-patterned silk substrates. In serum-free keratocyte differentiation medium, hCSSCs successfully differentiated into keratocytes secreting multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 90-100 μm thick, containing abundant cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. In contrast, hCFs tended to differentiate into myofibroblasts that deposited less organized collagen in a pattern resembling that of corneal scar tissue. RGD surface coupling coupling was an essential factor in enhancing cell attachment, orientation, proliferation, differentiation and ECM deposition on the silk substratum. These results demonstrated that an approach of combining hCSSCs with an RGD surface-coupled patterned silk film offers a powerful tool to develop highly ordered collagen fibril-based constructs for corneal regeneration and corneal stromal tissue repair.

Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200-300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.

Recapitulation of human corneal stromal tissue is believed to be among the most challenging steps in engineering human corneal tissue because of the difficulty in reproducing its highly-ordered hierarchical ultrastructure, which imparts its robust biomechanical properties and optical transparency. In this study, we compared the feasibility of utilizing human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on a highly-aligned fibrous substrate made from poly(ester urethane) urea. In the serum-free keratocyte differentiation medium supplemented with FGF-2 (10 ng/mL) and TGF-β3 (0.1 ng/mL), hCSSCs successfully differentiated into keratocytes and secreted multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 60-70 μm thick and abundant in cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. Under the identical conditions, hCFs tended to differentiate into myofibroblasts and deposited a less-organized collagen-fibrillar construct in a pattern with similarities to corneal scar tissue due to a lack of cornea-specific ECM components. These observations demonstrated that hCSSCs showed a much greater potential, under proper substrate and growth factor guidance, to facilitate the generation of a biological human cornea equivalent. Unlike hCSSCs, hCFs were less responsive to these environmental cues and under identical culture conditions generated an ECM that poorly mimicked the native, functional tissue structure and composition.

PURPOSE: To develop and characterize a mouse model with intraocular pressure (IOP) elevation after laser photocoagulation on the trabecular meshwork (TM), which may serve as a model to investigate the potential of stem cell-based therapies for glaucoma.

METHODS: IOP was measured in 281 adult C57BL/6 mice to determine normal IOP range. IOP elevation was induced unilaterally in 50 adult mice, by targeting the TM through the limbus with a 532-nm diode laser. IOP was measured up to 24 weeks post-treatment. The optic nerve damage was detected by electroretinography and assessed by semiautomatic counting of optic nerve axons. Effects of laser treatment on the TM were evaluated by histology, immunofluorescence staining, optical coherence tomography (OCT) and transmission electron microscopy (TEM).

RESULTS: The average IOP of C57BL/6 mice was 14.5 ± 2.6 mmHg (Mean ± SD). After laser treatment, IOP averaged above 20 mmHg throughout the follow-up period of 24 weeks. At 24 weeks, 57% of treated eyes had elevated IOP with the mean IOP of 22.5 ± 2.5 mmHg (Mean ± SED). The difference of average axon count (59.0%) between laser treated and untreated eyes was statistically significant. Photopic negative response (PhNR) by electroretinography was significantly decreased. CD45+ inflammatory cells invaded the TM within 1 week. The expression of SPARC was increased in the TM from 1 to 12 weeks. Histology showed the anterior chamber angle open after laser treatment. OCT indicated that most of the eyes with laser treatment had no synechia in the anterior chamber angles. TEM demonstrated disorganized and compacted extracellular matrix in the TM.

CONCLUSIONS: An experimental murine ocular hypertension model with an open angle and optic nerve axon loss was produced with laser photocoagulation, which could be used to investigate stem cell-based therapies for restoration of the outflow pathway integrity for ocular hypertension or glaucoma.

Conventional allograft therapy for corneal scarring is widespread and successful, but donor tissue is not universally available, and some grafts fail owing to rejection and complications such as endothelial failure. We investigated direct treatment of corneal scarring using autologous stem cells, a therapy that, if successful, could reduce the need for corneal grafts. Mesenchymal cells were expanded from small superficial, clinically replicable limbal biopsies of human cadaveric corneo-scleral rims. Limbal biopsy-derived stromal cells (LBSCs) expanded rapidly in media containing human serum, were highly clonogenic, and could generate spheres expressing stem cell genes (ABCG2, Nestin, NGFR, Oct4, PAX6, and Sox2). Human LBSCs differentiated into keratocytes expressing characteristic marker genes (ALDH3A1, AQP1, KERA, and PTGDS) and organized a thick lamellar stroma-like tissue containing aligned collagen and keratan sulfate proteoglycans when cultured on aligned nanofiber substrata. When engrafted into mouse corneal wounds, LBSCs prevented formation of light-scattering scar tissue containing fibrotic matrix components. The presence of LBSCs induced regeneration of ablated stroma with tissue exhibiting lamellar structure and collagen organization indistinguishable from that of native tissue. Because the limbus can be easily biopsied from either eye of an affected individual and LBSCs capable of corneal stromal remodeling can be expanded under xeno-free autologous conditions, these cells present a potential for autologous stem cell-based treatment of corneal stromal blindness.

Emulating corneal stromal tissue is believed to be the most challenging step in bioengineering an artificial human cornea because of the difficulty in reproducing its highly ordered microstructure, the key to the robust biomechanical properties and optical transparency of this tissue. We conducted a comparative study to assess the feasibility of human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on groove-patterned silk substrates. In serum-free keratocyte differentiation medium, hCSSCs successfully differentiated into keratocytes secreting multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 90-100 μm thick, containing abundant cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. In contrast, hCFs tended to differentiate into myofibroblasts that deposited less organized collagen in a pattern resembling that of corneal scar tissue. RGD surface coupling coupling was an essential factor in enhancing cell attachment, orientation, proliferation, differentiation and ECM deposition on the silk substratum. These results demonstrated that an approach of combining hCSSCs with an RGD surface-coupled patterned silk film offers a powerful tool to develop highly ordered collagen fibril-based constructs for corneal regeneration and corneal stromal tissue repair.