Stem cell transplantation rescued a primary open-angle glaucoma mouse model.

Glaucoma is a leading cause of irreversible blindness. In this study, we investigated if transplanted stem cells are able to rescue a glaucoma mouse model with transgenic myocilin Y437H mutation and explored the possible mechanisms. Human trabecular meshwork stem cells (TMSCs) were intracamerally transplanted which reduced mouse intraocular pressure, increased outflow facility, protected the retinal ganglion cells and preserved their function. TMSC transplantation also significantly increased the TM cellularity, promoted myocilin secretion from TM cells into the aqueous humor to reduce endoplasmic reticulum stress, repaired the TM tissue with extracellular matrix modulation and ultrastructural restoration. Co-culturing TMSCs with myocilin mutant TM cells in vitro promoted TMSCs differentiating into phagocytic functional TM cells. RNA sequencing revealed that TMSCs had upregulated genes related to TM regeneration and neuroprotection. Our results uncovered therapeutic potential of TMSCs for curing glaucoma and elucidated possible mechanisms by which TMSCs achieve the treatment effect.

Two-step induction of trabecular meshwork cells from induced pluripotent stem cells for glaucoma.

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.

Adipose-derived stem cells integrate into trabecular meshwork with glaucoma treatment potential.

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.

Fidelity of long-term cryopreserved adipose-derived stem cells for differentiation into cells of ocular and other lineages.

Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPARγ2. ADSCs could differentiate into neural cells, stained positive to ß-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.

Endoplasmic Reticulum Stress Response of Trabecular Meshwork Stem Cells and Trabecular Meshwork Cells and Protective Effects of Activated PERK Pathway.

Purpose: This study aimed to investigate the differential responses of trabecular meshwork stem cells (TMSCs) and trabecular meshwork (TM) cells to endoplasmic reticulum (ER) stress inducers. Methods: Human TM cells and TMSCs were exposed to tunicamycin, brefeldin A, or thapsigargin. Cell apoptosis was evaluated by flow cytometry. ER stress markers were detected by quantitative PCR, Western blotting, and immunostaining. Morphologic changes were evaluated by transmission electron microscopy. Cells were treated with the PERK inhibitor GSK2606414 or the elF2α dephosphorylation inhibitor Salubrinal together with tunicamycin to evaluate their effects on ER stress. Results: Both TMSCs and TM cells underwent apoptosis after 48- and 72-hour treatment with ER stress inducers. ER stress triggered the unfolded protein response (UPR) with increased expression of GRP78, sXBP1, and CHOP, which was significantly lower in TMSCs than TM cells. Swollen ER and mitochondria were detected in both TMSCs and TM cells. Neither GSK2606414 nor salubrinal alone activated UPR. GSK2606414 significantly reduced cell survival rates after tunicamycin treatment, and salubrinal increased cell survival rates. The increased expression of GRP78, sXBP1, CHOP, and GADD34 peaked at 6 or 12 hours and lasted longer in TM cells than TMSCs. Salubrinal treatment dramatically increased OCT4 and CHI3L1 expression in TMSCs. Conclusions: In response to ER stress inducers, TMSCs activated a lower level of UPR and lasted shorter than TM cells. Inhibition of elF2α dephosphorylation had a protective mechanism against cell death. Stem cells combined with salubrinal may be a more effective way for TM regeneration in glaucoma.

Stemness and Regenerative Potential of Corneal Stromal Stem Cells and Their Secretome After Long-Term Storage: Implications for Ocular Regeneration.

Purpose: To assess the stemness and regenerative potential of cryopreserved corneal stromal stem cells (cryo-CSSCs) after long-term storage. We also used the secretome from these cells to observe the effect on wound-healing capacity of corneal fibroblasts and on the expression of fibrotic markers during wound healing. Methods: CSSCs were obtained from three donors and stored in liquid nitrogen for approximately 10 years. Post thaw, cryo-CSSCs were characterized for stemness using phenotypic and genotypic markers along with colony-forming efficiency and three-dimensional spheroid formation. Multilineage differentiation was observed by differentiation into osteocytes, adipocytes, neural cells, and keratocytes. Secretome was harvested by culturing cryo-CSSCs in log phase. Wound-healing capacity was observed by live-cell time-lapse microscopy. Statistical analysis was done using 1-way ANOVA and Tukey posttest. Results: CSSCs displayed good viability post thaw and showed >90% expression of stem cell markers CD90, CD73, CD105, STRO1, and CD166. cryo-CSSCs also expressed stem cell genes OCT4, KLF4, and ABCG2, and could also form colonies and three-dimensional spheroids. Multipotency assessment showed that all three cryo-CSSCs could differentiate into osteocytes, adipocytes, neural cells, as shown by ß-III tubulin and neurofilament antibody staining and corneal keratocytes as observed by staining for Kera C, J19, and collagen V antibodies. The secretome derived from these three populations could promote the wound healing of corneal fibroblasts and reduce the expression of fibrotic markers SPARC and fibronectin. Conclusions: CSSCs maintained their stemness and multipotency after long-term storage, and secretome derived from these cells can be of paramount importance for corneal regeneration and prevention of fibrosis.

A laser-induced mouse model with long-term intraocular pressure elevation.

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.

Bioengineering organized, multilamellar human corneal stromal tissue by growth factor supplementation on highly aligned synthetic substrates.

Recapitulating the microstructure of the native human corneal stromal tissue is believed to be a key feature in successfully engineering the corneal tissue. The stratified multilayered collagen fibril lamellae with orthogonal orientation determine the robust biomechanical properties of this tissue, and the uniform collagen fibril size and interfibrillar spacing are critical to its optical transparency. The objective of this investigation was to develop a highly organized collagen-fibril construct secreted by human corneal stromal stem cells (hCSSCs) to mimic the human corneal stromal tissue. In culture on a highly aligned fibrous substrate made from poly(ester urethane) urea, the fibroblast growth factor-2 (FGF-2, 10 ng/mL) and transforming growth factor-beta 3 (TGF-ß3, 0.1 ng/mL) impacted the organization and abundance of the secreted collagen fibril matrix. hCSSCs differentiated into keratocytes with significant upregulation of the typical gene markers, including KERA, B3GnT7, and CHST6. FGF-2 treatment stimulated hCSSCs to secrete collagen fibrils strongly aligned in a single direction, whereas TGF-ß3 induced collagenous layers with orthogonal fibril orientation. The combination of FGF-2 and TGF-ß3 induced multilayered lamellae with orthogonally oriented collagen fibrils, in a pattern mimicking the human corneal stromal tissue. The constructs were 60-70 µm thick and had an increased content of cornea-specific extracellular matrix components, including keratan sulfate, lumican, and keratocan. The approach of combining substrate cues with growth factor augmentation offers a new means to engineer well-organized, collagen-based constructs with an appropriate nanoscale structure for corneal repair and regeneration.

Stem cells from trabecular meshwork home to TM tissue in vivo.

PURPOSE: To investigate the potential of human trabecular meshwork stem cells (TMSCs) for homing to mouse TM tissue and survival in vivo. METHODS: Human TMSCs and fibroblasts were labeled with fluorescent membrane dye DiO and injected into normal mouse anterior chamber. Stem cell and TM cell markers were identified by immunofluorescent staining of cryosections or tissue whole mounts. Apoptosis was determined by TUNEL assay. Replicating and inflammatory cells were detected by bromodeoxyuridine (BrdU) incorporation and anti-CD45 staining, respectively. Quantitative RT-PCR detected gene expression of injected cells after isolation by fluorescence activated cell sorting. Intraocular pressure was measured using a TonoLab rebound tonometer. RESULTS: Expanded cultures of DiO-labeled TMSCs expressed stem cell markers preferentially in DiO positive cells, demonstrating a slow-cycling, label-retaining stem cell phenotype. DiO-labeled TMSCs injected into the anterior chamber of normal mice localized primarily in TM, remaining in the tissue at least 4 months. Within 1 week, TM-associated TMSCs began expressing TM marker protein CHI3L1. Fibroblasts injected in mouse anterior chamber showed distributed localization in corneal endothelium, lens epithelium, and TM and did not express CHI3L1. Little apoptosis was detected in injected TM tissue and intraocular pressure was not elevated during the experiment. Dividing cells or CD45-staining cells were not detected after TMSC-injection. CONCLUSIONS: Stem cells isolated from human TM and expanded in vitro exhibit the ability to home to the TM and differentiate into TM cells in vivo. Such cells present a potential for development of a novel cell-based therapy for glaucoma.