RESUMO
The purpose of this study was to examine the effects of age, gender and population origin on human globe and corneal dimensions and to explore the relationships between the dimensions. Human post-mortem eyes were obtained in Hyderabad (n = 223; range, 0-85 years) and Miami (n = 486; range, 6-103 years). The eyes were freed of extraneous tissues and globe antero-posterior length (GAPL), mean globe diameter (MGD) (average of horizontal and vertical), and corneal horizontal (HCD) and vertical (VCD) diameters were measured using digital calipers. The relationships of age, gender and population origin with globe and corneal dimensions and the relationships between the dimensions were assessed by bivalent and multiple regression analyses. Globe and cornea dimensions increase asymptotically with age until around the late teens but do not change thereafter. Bivariate and multivariate regression analyses of the >20-year-old eyes showed that population was significantly correlated with GAPL, MGD, HCD and VCD. Male globes and corneas were larger than those from females, but the difference did not appear to be statistically significant. All Hyderabad dimensions were significantly larger than those from the Miami. Neither GAPL nor MGD were correlated with the corneal dimensions. GAPL was significantly correlated with MGD as was HCD with VCD.
Assuntos
Envelhecimento/fisiologia , Biometria/métodos , Córnea/anatomia & histologia , Olho/anatomia & histologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Comprimento Axial do Olho/anatomia & histologia , Criança , Pré-Escolar , Córnea/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Doadores de TecidosRESUMO
Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to -40 °C and 3 °C/min to -120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.
Assuntos
Córnea/crescimento & desenvolvimento , Transplante de Córnea/métodos , Criopreservação/normas , Endotélio Corneano/ultraestrutura , Temperatura Baixa , Córnea/patologia , Córnea/ultraestrutura , Transplante de Córnea/efeitos adversos , Dimetil Sulfóxido/farmacologia , Endotélio Corneano/citologia , Endotélio Corneano/efeitos dos fármacos , Humanos , Microscopia Eletrônica de Varredura , Espanha , Bancos de TecidosRESUMO
Human oral mucosa stem cells (hOMSCs) arise from the neural crest, they can self-renew, proliferate, and differentiate to several cell lines and could represent a good source for application in tissue engineering. Because of their anatomical location, hOMSCs are easy to isolate, have multilineage differentiation capacity and express embryonic stem cells markers such as-Sox2, Oct3/4 and Nanog. We have used SHEM (supplemented hormonal epithelial medium) media and cultured hOMSCs over human amniotic membrane and determined the cell's capacity to differentiate to an epithelial-like phenotype and to express corneal specific epithelial markers-CK3, CK12, CK19, Pan-cadherin and E-cadherin. Our results showed that hOMSCs possess the capacity to attach to the amniotic membrane and express CK3, CK19, Pan-Cadherin and E-Cadherin without induction with SHEM media and expressed CK12 or changed the expression pattern of E-Cadherin to a punctual-like feature when treated with SHEM media. The results observed in this study show that hOMSCs possess the potential to differentiate toward epithelial cells. In conclusion, our results revealed that hOMSCs readily express markers for corneal determination and could provide the ophthalmology field with a therapeutic alternative for tissue engineering to achieve corneal replacement when compared with other techniques. Nevertheless, further studies are needed to develop a predictable therapeutic alternative for cornea replacement.
Assuntos
Diferenciação Celular/genética , Epitélio Corneano/crescimento & desenvolvimento , Células-Tronco Mesenquimais/citologia , Mucosa Bucal/crescimento & desenvolvimento , Âmnio/crescimento & desenvolvimento , Células Cultivadas , Córnea/citologia , Córnea/crescimento & desenvolvimento , Córnea/metabolismo , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Mucosa Bucal/citologia , Engenharia Tecidual/tendênciasRESUMO
Diseases and injuries that compromise the ocular surface cause considerable patient distress and have long term consequences for their quality of life. Treatment modalities that can address the delicate balance of tissue regeneration, inflammation and maintenance of corneal transparency are therefore needed. We have recently formulated two novel eye drops from placental tissues: cord blood platelet lysate (CBED) and amniotic membrane extract eye drops (AMED), which can be used to treat severe ocular disorders. Here we characterise these two preparations by measuring: (a) growth factors (GF) and cytokines composition, (b) promotion of human corneal epithelial cell (HCEC) growth and (c) effects on immune cells in a lymphocyte culture assay. Finally, their bioavailability was assayed in an ex vivo porcine corneal model. We show that both preparations contain GF and cytokines that were able to promote the in vitro growth of HCEC and support repair in an in vitro scratch test. When assessed in a lymphocyte culture, both favoured immune suppression reducing the cellular expression of NKG2D and CD107a as well as the production of interferon gamma (IFN-γ) in natural killer, NKT and T cells. Regarding bioavailability, CBED active molecules were found mainly in the pre-corneal fraction with some penetration into the corneal fraction, in an ex vivo model. In summary, both placental-derived allogeneic preparations, CBED and AMED, display regenerative and immunomodulatory capabilities. These results will help define mechanisms of action and the best indications and doses of each product for use in a particular patient and support the development of off-the-shelf therapies for ocular surface pathologies in which wound healing defects and inflammatory events are contributing factors.
Assuntos
Córnea/efeitos dos fármacos , Doenças da Córnea/tratamento farmacológico , Soluções Oftálmicas/farmacologia , Regeneração/efeitos dos fármacos , Âmnio/química , Animais , Plaquetas/imunologia , Proliferação de Células/efeitos dos fármacos , Córnea/crescimento & desenvolvimento , Doenças da Córnea/patologia , Células Epiteliais/efeitos dos fármacos , Feminino , Sangue Fetal/química , Sangue Fetal/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Soluções Oftálmicas/química , Placenta/química , Placenta/imunologia , Gravidez , Qualidade de Vida , Suínos , CicatrizaçãoRESUMO
It is well known that human crystalline lens shape, dimensions and optical properties change throughout life and influence whole eye refraction. However, it is not clear if lens properties are associated with other ocular parameters. The purpose of the present study was to investigate the relationship of corneal and external globe dimensions with adult lens diameter (LD), lens thickness (LT) and lens power (LP) in order to determine if external factors influence lens properties. Postmortem human eyes (n = 66, age = 20-78 years) were obtained from the Ramayamma International Eye Bank, Hyderabad, India. Globe antero-posterior length (GAPL) and mean (average of horizontal and vertical) diameters of cornea (MCD) and globe (MGD) were measured using digital calipers. Eyes were dissected to produce ocular structures that contain the lens maintained in its accommodating framework, including intact zonules, ciliary body and sections of sclera. Specimens were mounted in a mechanical lens stretching system. LD, LT and LP were measured using high magnification retro-illumination photography, slit illumination photography and Scheiner principle-based optical system respectively in the unstretched (accommodated) state. Relationships between external globe and corneal dimensions and LD, LT or LP were assessed by multiple regression analysis. Age (0.012 ± 0.003 mm/year; p<0.001) and GAPL (0.185 ± 0.045 mm/mm; p<0.001) were significant (p<0.0001) predictors of LD. After adjusting for age-related increases, LD appears to be positively correlated with GAPL. Age (0.010 ± 0.004 mm/year; p = 0.009) and GAPL (-0.143 ± 0.060 mm/mm; p = 0.02) were significant (p = 0.001) predictors of LT. After adjusting for the age-related increase, LT appears to be negatively correlated with GAPL. Only age was a significant predictor of LP (-0.26 ± 0.04 D/year; p<0.001). The results suggest that, apart from aging, lens diameter and thickness are dependent on the anteroposterior length of the eye globe. Lens power is not influenced by globe dimensions.
Assuntos
Acomodação Ocular/fisiologia , Envelhecimento/fisiologia , Biometria/métodos , Córnea/anatomia & histologia , Olho/anatomia & histologia , Cristalino/anatomia & histologia , Refração Ocular/fisiologia , Adulto , Idoso , Córnea/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Adulto JovemRESUMO
A proper skin barrier function requires constant formation of stratum corneum, i.e. the outermost layer of epidermis composed of terminally differentiated keratinocytes. The complex process of converting proliferative basal keratinocytes into corneocytes relies on programmed changes in the activity of many well-established genes. Much remains however to be investigated about this process, e.g. in conjunction with epidermal barrier defects due to genetic errors as in ichthyosis. To this end, we re-analyzed two sets of microarray-data comparing altered gene expression in differentiated vs. proliferating keratinocytes and in the skin of patients with autosomal recessive congenital ichthyosis (ARCI) vs. healthy controls, respectively. We thus identified 24 genes to be upregulated in both sets of array and not previously associated with keratinocyte differentiation. For 10 of these genes (AKR1B10, BLNK, ENDOU, GCNT4, GLTP, RHCG, SLC15A1, TMEM45B, TMEM86A and VSNL1), qPCR analysis confirmed the array results and subsequent immunostainings of normal epidermis showed superficial expression of several of the proteins. Furthermore, induction of keratinocyte differentiation using phorbol esters (PMA) resulted in increased expression of eight of the genes, whereas siRNA silencing of PPARδ, a transcription factor supporting differentiation, had the opposite effect. In summary, our results identify ten new candidate genes seemingly involved in human epidermal keratinocyte differentiation and possibly important for epidermal repair in a genetic skin disease characterized by barrier failure.
Assuntos
Diferenciação Celular/genética , Córnea/metabolismo , Ictiose/genética , PPAR delta/genética , Pele/crescimento & desenvolvimento , Proliferação de Células/genética , Córnea/crescimento & desenvolvimento , Epiderme/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Ictiose/patologia , Queratinócitos/metabolismo , Proteínas de Membrana/genética , Organogênese/genética , PPAR delta/antagonistas & inibidores , Ésteres de Forbol/farmacologia , RNA Interferente Pequeno/genéticaRESUMO
The tissue response to injury is a complex process. The cornea is an excellent model for studying wound repair processes because of its simple anatomy, easy accessibility, and normal avascular state. Here, we describe two corneal repair models in mice: an epithelial/mechanical injury model and a stromal/chemical injury model. The two models induce different repair responses, and consequently enable the study of independent repair processes. Here, we describe how these two wound models may be used to study basic cellular and molecular mechanisms of corneal repair.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Córnea/crescimento & desenvolvimento , Epitélio Corneano/crescimento & desenvolvimento , Cicatrização , Animais , Córnea/patologia , Lesões da Córnea/patologia , Lesões da Córnea/terapia , Epitélio Corneano/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Plasmacytoid dendritic cells (pDCs) are crucial for corneal homeostasis through secretion of various anti-angiogenic molecules and growth factors. Due to its avascular nature, only a limited number of adoptively transferred cells home to the cornea, when administered systemically. In addition, local adoptive transfer of cells poses several challenges and the clinical application of commonly used techniques is limited. Herein, we detail a novel approach for local adoptive transfer of pDCs to the cornea for the treatment of corneal wounds. This approach utilizes a commonly used fibrin sealant as a means of transferring previously isolated cells locally on the cornea. The technique is simple, reproducible, and is accompanied with successful transfer and integration of a substantial number of the cells to the cornea. Application of this approach to transfer pDCs promotes corneal wound healing. Furthermore, this technique can be applied for adoptive transfer of any cell of interest to the cornea.
Assuntos
Transferência Adotiva/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Dendríticas/transplante , Imunoterapia Adotiva/métodos , Cicatrização , Animais , Córnea/crescimento & desenvolvimento , Córnea/patologia , Lesões da Córnea/patologia , Lesões da Córnea/terapia , Epitélio Corneano/crescimento & desenvolvimento , Epitélio Corneano/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The corneal ultrastructure of the pre- and post-metamorphic stages of the neotenic axolotl Ambystoma mexicanum is examined using light microscopy and both scanning and transmission electron microscopy to reveal whether there are any morphological changes associated with a switch in lifestyle. Although the complement of corneal layers remains the same, there are significant quantitative changes in corneal, epithelial and stromal thickness, epithelial and endothelial cell size and density, and the thickness of Bowman's layer and Desçemet's membrane. Microholes in the epithelium and vertical sutures within the stroma are predominant features in the pre-metamorphic stage but are rarely seen in the post-metamorphic stage. There are also significant quantitative centro-peripheral differences in the thickness of the whole cornea, primarily due to differences in the thickness of the stroma in both metamorphic stages. These changes may reflect the physiological demands on the cornea as it switches from a purely aquatic to an amphibious lifestyle, which includes venturing onto land.
Assuntos
Córnea/ultraestrutura , Metamorfose Biológica/fisiologia , Ambystoma mexicanum , Animais , Córnea/crescimento & desenvolvimento , Substância Própria/ultraestrutura , Endotélio Corneano/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos AnimaisRESUMO
Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.
Assuntos
Epitélio Corneano/crescimento & desenvolvimento , Transportador 1 de Aminoácido Excitatório/genética , Proteínas de Homeodomínio/genética , Células-Tronco/citologia , Fatores de Transcrição/genética , Animais , Divisão Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Túnica Conjuntiva/crescimento & desenvolvimento , Córnea/crescimento & desenvolvimento , Homeostase/genética , Humanos , Limbo da Córnea/crescimento & desenvolvimento , Camundongos , Camundongos TransgênicosRESUMO
Here, we evaluated the effects of PEDF (pigment epithelium-derived factor) and PEDF peptides on cone-photoreceptor cell damage in a mouse model of focal LED-induced phototoxicity (LIP) in vivo. Swiss mice were dark-adapted overnight, anesthetized, and their left eyes were exposed to a blue LED placed over the cornea. Immediately after, intravitreal injection of PEDF, PEDF-peptide fragments 17-mer, 17-mer[H105A] or 17-mer[R99A] (all at 10 pmol) were administered into the left eye of each animal. BDNF (92 pmol) and bFGF (27 pmol) injections were positive controls, and vehicle negative control. After 7 days, LIP resulted in a consistent circular lesion located in the supratemporal quadrant and the number of S-cones were counted within an area centered on the lesion. Retinas treated with effectors had significantly greater S-cone numbers (PEDF (60%), 17-mer (56%), 17-mer [H105A] (57%), BDNF (64%) or bFGF (60%)) relative to their corresponding vehicle groups (≈42%). The 17-mer[R99A] with no PEDF receptor binding and no neurotrophic activity, PEDF combined with a molar excess of the PEDF receptor blocker P1 peptide, or with a PEDF-R enzymatic inhibitor had undetectable effects in S-cone survival. The findings demonstrated that the cone survival effects were mediated via interactions between the 17-mer region of the PEDF molecule and its PEDF-R receptor.
Assuntos
Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/farmacologia , Peptídeos/farmacologia , Retina/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Serpinas/farmacologia , Animais , Córnea/efeitos dos fármacos , Córnea/crescimento & desenvolvimento , Córnea/metabolismo , Dermatite Fototóxica , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Humanos , Camundongos , Fatores de Crescimento Neural/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos/genética , Fotoperíodo , Receptores de Neuropeptídeos/genética , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Serpinas/metabolismoRESUMO
MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses. RNA-seq combined with quantitative proteomics based on multiplexed isobaric tandem mass tag labeling was performed in LECs transfected with miR-146a mimic vs. mimic control. Western blot and immunostaining were used to confirm the expression of some targeted genes/proteins. A total of 251 differentially expressed mRNAs and 163 proteins were identified. We found that miR-146a regulates the expression of multiple genes in different pathways, such as the Notch system. In LECs and organ-cultured corneas, miR-146a increased Notch-1 expression possibly by downregulating its inhibitor Numb, but decreased Notch-2. Integrated transcriptome and proteome analyses revealed the regulatory role of miR-146a in several other processes, including anchoring junctions, TNF-α, Hedgehog signaling, adherens junctions, TGF-ß, mTORC2, and epidermal growth factor receptor (EGFR) signaling, which mediate wound healing, inflammation, and stem cell maintenance and differentiation. Our results provide insights into the regulatory network of miR-146a and its role in fine-tuning of Notch-1 and Notch-2 expressions in limbal epithelium, which could be a balancing factor in stem cell maintenance and differentiation.
Assuntos
MicroRNAs/genética , Proteoma/genética , Receptores Notch/genética , Transcriptoma/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Córnea/crescimento & desenvolvimento , Córnea/metabolismo , Células Epiteliais/metabolismo , Epitélio/crescimento & desenvolvimento , Receptores ErbB/genética , Extremidades/crescimento & desenvolvimento , Regulação da Expressão Gênica/genética , Proteínas Hedgehog/genética , Humanos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética , Cicatrização/genéticaRESUMO
Keratins are the forming units of intermediate filaments (IF) that provide mechanical support, and formation of desmosomes between cells and hemi desmosomes with basement membranes for epithelium integrity. Keratin IF are polymers of obligate heterodimer consisting one type I keratin and one type II keratin molecules. There are 54 functional keratin genes in human genome, which are classified into three major groups, i.e., epithelial keratins, hair follicle cell-specific epithelial keratins and hair keratins. Their expression is cell type-specific and developmentally regulated. Corneal epithelium expresses a subgroup of keratins similar to those of epidermal epithelium. Limbal basal stem cells express K5/K14, and K8/K18 and K8/K19 IF suggesting that there probably are two populations of limbal stem cells (LSCs). In human, LSCs at limbal basal layer can directly stratify and differentiate to limbal suprabasal cells that express K3/K12 IF, or centripetally migrate then differentiate to corneal basal transient amplifying cells (TAC) that co-express both K3/K12 and K5/K14 prior to moving upward and assuming suprabasal cells phenotype of only K3/K12 expression that signifies corneal type epithelium differentiation. In rodent, the differentiated cornea epithelial cells express K5/K12 in lieu of K3/K12, because K3 allele exists as a pseudogene and does not encode a functional K3 protein. The basal corneal cells of new-born mice originate from surface ectoderm during embryonic development slowly commit to differentiation of becoming TAC co-expressing K5/K12 and K5/K14 IF. However, the centripetal migration may still occur at a slower rate in young mice, which is accelerated during wound healing. In this review, we will discuss and compare the cornea-specific keratins expression patterns between corneal and epidermal epithelial cells during mouse development, and between human and mouse during development and homeostasis in adult, and pathology caused by a mutation of keratins.
Assuntos
Córnea/metabolismo , Queratinas/biossíntese , Animais , Diferenciação Celular , Células Cultivadas , Córnea/crescimento & desenvolvimento , Humanos , Limbo da Córnea/crescimento & desenvolvimento , Limbo da Córnea/metabolismo , Células-Tronco/citologiaRESUMO
PURPOSE: To quantitatively describe the structural corneal changes from infancy to early adulthood using ultrasound biomicroscopy. METHODS: In this prospective study, 168 ultrasound biomicroscopy images were obtained from 24 healthy eyes of 24 patients who consented and enrolled in the Pediatric Anterior Segment Imaging Innovation Study. Their ages ranged from birth to 26 years. An established ultrasound biomicroscopy imaging protocol including seven views of one eye per patient were obtained and measured using ImageJ software (National Institutes of Health). Twelve corneal structural parameters were measured. Means were compared between younger and older groups. RESULTS: Among the 12 measured structures, 5 demonstrated statistically significant differences (P < .05) between patients younger than 1 year and patients older than 1 year. The mean values for corneal cross-sectional width and length, central corneal thickness, and radii of curvature (anterior and posterior) were significantly different in patients younger than 1 year. Curvature and limbus-to-limbus dimensions changed more dramatically than thickness and tissue density. When comparing the youngest to oldest subgroups, anterior curvature flattened (6.14 to 7.55 radius), posterior curvature flattened (5.53 to 6.72 radius), angle-to-angle distance increased (8.93 to 11.40 mm), and endothelial cross-sectional distance increased (10.63 to 13.61 mm). CONCLUSIONS: Pediatric corneal structures change with age. The most significant changes occur in the first months of life, with additional changes later in childhood. This study further demonstrates the importance of age in pediatric corneal imaging analysis. [J Pediatr Ophthalmol Strabismus. 2020;57(4):238-245.].
Assuntos
Córnea/diagnóstico por imagem , Córnea/crescimento & desenvolvimento , Microscopia Acústica , Adolescente , Adulto , Biometria , Criança , Pré-Escolar , Paquimetria Corneana , Estudos Transversais , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Prospectivos , Adulto JovemRESUMO
The corneal endothelium forms a leaky barrier between the corneal stroma and the aqueous humor of the anterior chamber. This cell monolayer maintains the corneal stroma in a state of relative dehydration, a process called deturgescence, which is required in order to obtain corneal stromal transparency. Endothelial dysfunctions lead to visual impairment that ultimately can only be treated surgically via the corneal transplantation of a functional endothelium. Shortages of corneas suitable for transplantation has motivated research toward new alternatives involving in vitro corneal endothelial cell (CEC) expansion.This chapter describes current methods that allow isolate and culture CECs. In brief, Descemet membrane is peeled out of the cornea and digested in order to obtain CECs. Cells are then seeded and cultured.
Assuntos
Técnicas de Cultura de Células/métodos , Córnea/crescimento & desenvolvimento , Células Endoteliais/citologia , Endotélio Corneano/crescimento & desenvolvimento , Animais , Transplante de Córnea , Endotélio Corneano/citologia , HumanosRESUMO
The cultivation of corneal-limbal cells in vitro represents an excellent means to generate models to study cornea function and disease processes. These in vitro expanded cornea-limbal epithelial cell cultures are rich in stem cells for cornea, and hence can be used as a cell therapy for cornea-limbal deficiency. This chapter details the primary culture of these cornea-limbal cells, which can be used as model for further studies of the cornea surface.
Assuntos
Técnicas de Cultura de Células/métodos , Córnea/crescimento & desenvolvimento , Epitélio Corneano/citologia , Limbo da Córnea/citologia , Âmnio/crescimento & desenvolvimento , Epitélio Corneano/crescimento & desenvolvimento , Humanos , Limbo da Córnea/crescimento & desenvolvimentoRESUMO
The cornea is the outermost transparent and refractive barrier surface of the eye necessary for vision. Development of the cornea involves the coordinated production of extracellular matrix, epithelial differentiation, and endothelial cell expansion to produce a highly transparent tissue. Here we describe the production of multilayered three-dimensional organoids from human-induced pluripotent stem cells. These organoids have the potential for multiple downstream applications which are currently unattainable using traditional in vitro techniques.
Assuntos
Técnicas de Cultura de Células/métodos , Epitélio Corneano/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Diferenciação Celular/genética , Proliferação de Células/genética , Córnea/citologia , Córnea/crescimento & desenvolvimento , Epitélio Corneano/crescimento & desenvolvimento , Matriz Extracelular/genética , Humanos , Organoides/crescimento & desenvolvimentoRESUMO
CRISPR/Cas9 gene editing holds the promise of sequence-specific alteration of the genome to achieve therapeutic benefit in the treated tissue. Cas9 is an RNA-guided nuclease in which the sequence of the RNA can be altered to match the desired target. However, care must be taken in target choice and RNA guide design to ensure both maximum on-target and minimum off-target activity. The cornea is an ideal tissue for gene therapy due to its small surface area, accessibility, immune privilege, avascularity, and ease of visualization. Herein, we describe the design, testing, and delivery of Cas9 and guide RNAs to target genes expressed in the cornea.
Assuntos
Sistemas CRISPR-Cas/genética , Substância Própria/citologia , Edição de Genes/métodos , Regeneração/genética , Córnea/citologia , Córnea/crescimento & desenvolvimento , Substância Própria/crescimento & desenvolvimento , Terapia Genética/métodos , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Gene delivery approaches using adeno-associated virus (AAV) vectors are currently the preferred method for human gene therapy applications and have demonstrated success in clinical trials for a diverse set of diseases including retinal blindness. To date, no clinical trials using AAV gene therapy in the anterior eye have been initiated; however, corneal gene delivery appears to be an attractive approach for treating both corneal and ocular surface diseases. Multiple preclinical studies by our lab and others have demonstrated efficient AAV vector-mediated gene delivery to the cornea for immunomodulation, anti-vascularization, and enzyme supplementation. Interestingly, the route of AAV vector administration and nuances such as administered volume influence vector tropism and transduction efficiency. In this chapter, a detailed protocol for AAV vector production and specific approaches for AAV-mediated gene transfer to the cornea via subconjunctival and intrastromal injections are described.
Assuntos
Córnea/crescimento & desenvolvimento , Oftalmopatias/genética , Terapia Genética/métodos , Transdução Genética/métodos , Animais , Córnea/patologia , Dependovirus/genética , Oftalmopatias/terapia , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Retina/crescimento & desenvolvimento , Retina/patologia , Transgenes/genéticaRESUMO
Tissue engineering is a flourishing field of regenerative medicine that allows the reconstruction of various tissues of our body, including the cornea. In addition to addressing the growing need for organ transplants, such tissue-engineered substitutes may also serve as good in vitro models for fundamental and preclinical studies. Recent progress in the field of corneal tissue engineering has led to the development of new technologies allowing the reconstruction of a human bi-lamellar cornea. One unique feature of this model is the complete absence of exogenous material. Indeed, these human corneal equivalents are exclusively composed of untransformed human corneal fibroblasts (hCFs) entangled in their own extracellular matrix, as well as untransformed human corneal epithelial cells (hCECs), both of which isolated from donor corneas. The reconstructed human bi-lamellar cornea thereby exhibits a well-organized stroma as well as a well-differentiated epithelium. This chapter describes the methods used for the isolation and culture of hCFs, the production and assembly of hCFs stromal sheets, the seeding of hCECs, and the maturation of the tissue-engineered cornea.