Publications

BACKGROUND: Corneal stromal stem cells (CSSC) reduce corneal inflammation, prevent fibrotic scarring, and regenerate transparent stromal tissue in injured corneas. These effects rely on factors produced by CSSC to block the fibrotic gene expression. This study investigated the mechanism of the scar-free regeneration effect.

METHODS: Primary human CSSC (hCSSC) from donor corneal rims were cultivated to passage 3 and co-cultured with mouse macrophage RAW264.7 cells induced to M1 pro-inflammatory phenotype by treatment with interferon-γ and lipopolysaccharides, or to M2 anti-inflammatory phenotype by interleukin-4, in a Transwell system. The time-course expression of human transforming growth factor β3 (hTGFβ3) and hTGFβ1 were examined by immunofluorescence and qPCR. TGFβ3 knockdown for > 70% in hCSSC [hCSSC-TGFβ3(si)] was achieved by small interfering RNA transfection. Naïve CSSC and hCSSC-TGFβ3(si) were transplanted in a fibrin gel to mouse corneas, respectively, after wounding by stromal ablation. Corneal clarity and the expression of mouse inflammatory and fibrosis genes were examined.

RESULTS: hTGFβ3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition. Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGFβ3 at days 1 and 3 post-injury, along with the reduced expression of mouse inflammatory genes (CD80, C-X-C motif chemokine ligand 5, lipocalin 2, plasminogen activator urokinase receptor, pro-platelet basic protein, and secreted phosphoprotein 1). By day 14, hCSSC treatment significantly reduced the expression of fibrotic and scar tissue genes (fibronectin, hyaluronan synthase 2, Secreted protein acidic and cysteine rich, tenascin C, collagen 3a1 and α-smooth muscle actin), and the injured corneas remained clear. However, hCSSC-TGFβ3(si) lost these anti-inflammatory and anti-scarring functions, and the wounded corneas showed intense scarring.

CONCLUSION: This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFβ3, inducing a scar-free tissue response.

Stem cell-based therapy to restore the function of abnormal trabecular meshwork (TM) and decrease intraocular pressure (IOP) provides a novel approach to treat open-angle glaucoma. However, molecular mechanism for stem cells homing and anchoring to the TM remains unclear. This study aimed to discover the function of integrins in homing and integration of exogenous TM stem cells (TMSCs) to the TM. Integrin expression in TMSCs and fibroblasts was evaluated by quantitative real-time PCR (qPCR), flow cytometry, immunofluorescent staining, and western blotting. Expression of integrin ligand fibronectin was detected in cultured TM cells and murine TM tissue by immunostaining. Cell affinity to TM cells or fibronectin matrix was examined to compare TMSCs with TMSCs functionally blocked with an α5β1 integrin antibody. TMSCs and TMSCs with α5β1 integrin-blocking were intracamerally injected into wild-type mice. Wholemounts and cryosections were analyzed to discover cell distribution and integration at 3 days and 1 month. IOP was measured to detect possible changes. We discovered that human TMSCs expressed a higher level of α5β1 integrin than fibroblasts, but similar levels of αvβ3 and αvβ5 integrin. Upregulation of fibronectin was found in both TM cells treated with dexamethasone for 14 days and murine TM tissues damaged by laser photocoagulation. TMSCs were able to attach to the TM cells and fibronectin matrix in vitro. When the surface α5β1 integrin was blocked, the attached cell numbers were significantly reduced. Both TMSCs and TMSCs incubated with an α5β1 integrin-blocking antibody could home to the mouse TM after injection. TMSCs blocked with the α5β1 integrin-blocking antibody were not retained in the TM tissue at 1 month. The injected cells did not affect mouse IOP. In conclusion, highly expressed α5β1 integrin participates in maintaining TMSCs anchored and integrated to the TM, which would be crucial for stem cell-based therapy for glaucoma.

Stem cells from the eye hold a great potential for vision restoration and can also be used for regeneration in other tissues. In this study, we characterized the stem cell properties of Trabecular meshwork stem cells (TMSCs) after long-term cryopreservation (∼8 years). TMSCs derived from four donors were examined for their viability and proliferation, as well as stem cell marker expression. Spheroid formation, colony formation, and multipotency were investigated. We observed that TMSCs were fully viable with variable proliferation ability. They expressed the stem cell markers CD90, CD166, CD105, CD73, OCT4, SSEA4, Notch1, KLF4, ABCG2, Nestin, and HNK1 detected by flow cytometry, quantitative polymerase chain reaction, or immunofluorescent staining. They could form spheroids and colonies after thawing. All TMSCs were able to differentiate into osteocytes, neural cells, and trabecular meshwork (TM) cells, but not adipocytes. Differentiated TM cells responded to dexamethasone treatment with increased expression of myocilin and angiopoietin-like 7 (ANGPTL7). In a nutshell, our study demonstrated that TMSCs retain their stem cell properties after long-term cryopreservation and hence can be an effective cell therapy source for various clinical applications.

Human Schlemm's canal (HSC) cells are critical for understanding outflow physiology and glaucoma etiology. However, primary donor cells frequently used in research are difficult to isolate. HSC cells exhibit both vascular and lymphatic markers. Human adipose-derived stem cells (ADSCs) represent a potential source of HSC due to their capacity to differentiate into both vascular and lymphatic endothelial cells, via VEGF-A and VEGF-C. Shear stress plays a critical role in maintaining HSC integrity, function, and PROX1 expression. Additionally, the human trabecular meshwork (HTM) microenvironment could provide cues for HSC-like differentiation. We hypothesize that subjecting ADSCs to VEGF-A or VEGF-C, shear stress, and co-culture with HTM cells could provide biological, mechanical, and cellular cues necessary for HSC-like differentiation. To test this hypothesis, effects of VEGF-A, VEGF-C, and shear stress on ADSC differentiation were examined and compared to primary HSC cells in terms of cell morphology, and HSC marker expression using qPCR, immunoblotting, and immunocytochemistry analysis. Furthermore, the effect of co-culture with HTM cells on porous scaffolds on ADSC differentiation was studied. Treatment with VEGF-C under shear stress is effective in differentiating ADSCs into PROX1-expressing HSC-like cells. Co-culture with HTM cells on porous scaffolds leads to HTM/ADSC-derived HSC-like constructs that regulate through-flow and respond as expected to dexamethasone. STATEMENT OF SIGNIFICANCE: We successfully generated human Schlemm's canal (HSC) like cells from adipocyte-derived stem cells induced by biochemical and biomechanical cues as well as bioengineered human trabecular meshwork (HTM) on micropatterned, porous SU8 scaffolds. These stem cell-derived HSC-like cells co-cultured with HTM cells on SU8 scaffolds can regulate through-flow, and in particular, are responsive to steroid treatment as expected. These findings show that ADSC-derived HSC-like cells have the potential to recreate the ocular outflow pathway for in vitro glaucoma drug screening. To the best of our knowledge, it is the very first time to demonstrate derivation of Schlemm's canal-like cells from stem cells. It provides an important alternative source to primary Schlemm's canal cells that are very difficult to be isolated and cultured from human donors.

Glaucoma is a leading cause of irreversible blindness worldwide. Reducing intraocular pressure is currently the only effective treatment. Elevated intraocular pressure is associated with increased resistance of the outflow pathway, mainly the trabecular meshwork (TM). Despite great progress in the field, the development of novel and effective treatment for glaucoma is still challenging. In this study, we reported that human induced pluripotent stem cells (iPSCs) can be cultured as colonies and monolayer cells expressing OCT4, alkaline phosphatase, SSEA4 and SSEA1. After induction to neural crest cells (NCCs) positive to NGFR and HNK1, the iPSCs can differentiate into TM cells. The induced iPSC-TM cells expressed TM cell marker CHI3L1, were responsive to dexamethasone treatment with increased expression of myocilin, ANGPTL7, and formed CLANs, comparable to primary TM cells. To the best of our knowledge, this is the first study that induces iPSCs to TM cells through a middle neural crest stage, which ensures a stable NCC pool and ensures the high output of the same TM cells. This system can be used to develop personalized treatments using patient-derived iPSCs, explore high throughput screening of new drugs focusing on TM response for controlling intraocular pressure, and investigate stem cell-based therapy for TM regeneration.

The trabecular meshwork (TM) is an ocular tissue that maintains intraocular pressure (IOP) within a physiologic range. Glaucoma patients have reduced TM cellularity and, frequently, elevated IOP. To establish a stem cell-based approach to restoring TM function and normalizing IOP, human adipose-derived stem cells (ADSCs) were induced to differentiate to TM cells in vitro. These ADSC-TM cells displayed a TM cell-like genotypic profile, became phagocytic, and responded to dexamethasone stimulation, characteristic of TM cells. After transplantation into naive mouse eyes, ADSCs and ADSC-TM cells integrated into the TM tissue, expressed TM cell markers, and maintained normal IOP, outflow facility, and extracellular matrix. Cell migration and affinity results indicated that the chemokine pair CXCR4/SDF1 may play an important role in ADSC-TM cell homing. Our study demonstrates the possibility of applying autologous or allogeneic ADSCs and ADSC-TM cells as a potential treatment to restore TM structure and function in glaucoma.

Corneal opacities affect vision for millions of individuals worldwide. Fibrotic scar tissues accumulate in reaction to inflammatory responses and remain permanently in corneal stroma, and conventionally correctable only by donor corneal transplantation. Numerous studies have explored innovative approaches to reverse corneal scarring through non-surgical means; however, existing mouse models limit these studies, due to the lack of visibility of scar tissue in mouse corneas with steep curvature. Here, we reported that corneal scarring was modelled using a transgenic mouse line, Tg(Col3a1-EGFP)DJ124Gsat, in which enhanced green fluorescence protein (EGFP) reporter expression was driven by the promoter of collagen 3a1 (COL3a1), a stromal fibrosis gene. Similar to wildtype, Col3a1-EGFP transgenic corneas developed opacities after wounding by alkali burn and mechanical ablation, respectively, as examined under stereomicroscopy and Spectral Domain optical coherent tomography. The time course induction of EGFP was aligned with Col3a1 upregulation and matched with the elevated expression of other fibrosis genes (α-smooth muscle actin, fibronectin and tenascin C). Measured by flow cytometry and enzyme-linked immunosorbent assay, increased number of EGFP expressing cells and fluorescent intensities were correlated to corneal thickening and scar volume. After treatment with human corneal stromal stem cells or their exosomes, EGFP expression was downregulated together with the reduction of scar volume and fibrosis gene expression. These results have demonstrated that the transgenic mouse line, Tg(Col3a1-EGFP)DJ124Gsat, can be a valuable tool for the detection of corneal fibrosis and scarring in vivo, and will be useful in monitoring the changes of corneal fibrosis over time.

Age-related human trabecular meshwork (HTM) cell loss is suggested to affect its ability to regulate aqueous humor outflow in the eye. In addition, disease-related HTM cell loss is suggested to lead to elevated intraocular pressure in glaucoma. Induced pluripotent stem cell (iPSC)-derived trabecular meshwork (TM) cells are promising autologous cell sources that can be used to restore the declining TM cell population and function. Previously, an in vitro HTM model is bioengineered for understanding HTM cell biology and screening of pharmacological or biological agents that affect trabecular outflow facility. In this study, it is demonstrated that human iPSC-derived TM cells cultured on SU-8 scaffolds exhibit HTM-like cell morphology, extracellular matrix deposition, and drug responsiveness to dexamethasone treatment. These findings suggest that iPSC-derived TM cells behave like primary HTM cells and can thus serve as reproducible and scalable cell sources when using this in vitro system for glaucoma drug screening and further understanding of outflow pathway physiology, leading to personalized medicine.

BACKGROUND: Corneal stromal stem cells (CSSC) reduce corneal inflammation, prevent fibrotic scarring, and regenerate transparent stromal tissue in injured corneas. These effects rely on factors produced by CSSC to block the fibrotic gene expression. This study investigated the mechanism of the scar-free regeneration effect.

METHODS: Primary human CSSC (hCSSC) from donor corneal rims were cultivated to passage 3 and co-cultured with mouse macrophage RAW264.7 cells induced to M1 pro-inflammatory phenotype by treatment with interferon-γ and lipopolysaccharides, or to M2 anti-inflammatory phenotype by interleukin-4, in a Transwell system. The time-course expression of human transforming growth factor β3 (hTGFβ3) and hTGFβ1 were examined by immunofluorescence and qPCR. TGFβ3 knockdown for > 70% in hCSSC [hCSSC-TGFβ3(si)] was achieved by small interfering RNA transfection. Naïve CSSC and hCSSC-TGFβ3(si) were transplanted in a fibrin gel to mouse corneas, respectively, after wounding by stromal ablation. Corneal clarity and the expression of mouse inflammatory and fibrosis genes were examined.

RESULTS: hTGFβ3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition. Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGFβ3 at days 1 and 3 post-injury, along with the reduced expression of mouse inflammatory genes (CD80, C-X-C motif chemokine ligand 5, lipocalin 2, plasminogen activator urokinase receptor, pro-platelet basic protein, and secreted phosphoprotein 1). By day 14, hCSSC treatment significantly reduced the expression of fibrotic and scar tissue genes (fibronectin, hyaluronan synthase 2, Secreted protein acidic and cysteine rich, tenascin C, collagen 3a1 and α-smooth muscle actin), and the injured corneas remained clear. However, hCSSC-TGFβ3(si) lost these anti-inflammatory and anti-scarring functions, and the wounded corneas showed intense scarring.

CONCLUSION: This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFβ3, inducing a scar-free tissue response.

Stem cell-based therapy to restore the function of abnormal trabecular meshwork (TM) and decrease intraocular pressure (IOP) provides a novel approach to treat open-angle glaucoma. However, molecular mechanism for stem cells homing and anchoring to the TM remains unclear. This study aimed to discover the function of integrins in homing and integration of exogenous TM stem cells (TMSCs) to the TM. Integrin expression in TMSCs and fibroblasts was evaluated by quantitative real-time PCR (qPCR), flow cytometry, immunofluorescent staining, and western blotting. Expression of integrin ligand fibronectin was detected in cultured TM cells and murine TM tissue by immunostaining. Cell affinity to TM cells or fibronectin matrix was examined to compare TMSCs with TMSCs functionally blocked with an α5β1 integrin antibody. TMSCs and TMSCs with α5β1 integrin-blocking were intracamerally injected into wild-type mice. Wholemounts and cryosections were analyzed to discover cell distribution and integration at 3 days and 1 month. IOP was measured to detect possible changes. We discovered that human TMSCs expressed a higher level of α5β1 integrin than fibroblasts, but similar levels of αvβ3 and αvβ5 integrin. Upregulation of fibronectin was found in both TM cells treated with dexamethasone for 14 days and murine TM tissues damaged by laser photocoagulation. TMSCs were able to attach to the TM cells and fibronectin matrix in vitro. When the surface α5β1 integrin was blocked, the attached cell numbers were significantly reduced. Both TMSCs and TMSCs incubated with an α5β1 integrin-blocking antibody could home to the mouse TM after injection. TMSCs blocked with the α5β1 integrin-blocking antibody were not retained in the TM tissue at 1 month. The injected cells did not affect mouse IOP. In conclusion, highly expressed α5β1 integrin participates in maintaining TMSCs anchored and integrated to the TM, which would be crucial for stem cell-based therapy for glaucoma.