Cultured mammalian limbal stem cells, methods for generating the same, and uses thereof

A technology of corneal limbal stem cells and corneal epithelial cells, applied in the field of cultured mammalian limbal stem cells, their production and use, which can solve the problems of limited supply of limbal stem cells, inability to repair the surface of the eye, and improve vision

Inactive Publication Date: 2017-08-18
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alternatively, the graft may produce a distinct corneal epithelium but lack sufficient limbal stem cells resulting in an abnormal epithelial surface and poor healing, resulting in an inability to repair the ocular surface and improve vision
Therefore, these methods intended to supply limbal stem cells to eyes with limbal stem cell deficiency have severe limitations, which may be attributed to the limited number of self-regenerating undifferentiated limbal stem cells present in grafts or grafts. limited supply

Method used

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  • Cultured mammalian limbal stem cells, methods for generating the same, and uses thereof
  • Cultured mammalian limbal stem cells, methods for generating the same, and uses thereof
  • Cultured mammalian limbal stem cells, methods for generating the same, and uses thereof

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Experimental program
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Embodiment 1

[0285] Materials and methods

[0286] Human Pathology Samples

[0287] Corneal epithelial squamous metaplasia and all other tissues were obtained as deidentified surgical specimens, fixed in 5% formalin, embedded in paraffin, sectioned and stained for immunofluorescence studies.

[0288] Isolation and Culture of Limbal Stem Cells and Skin Epidermal Stem Cells

[0289]Post mortem human eyeballs were obtained from an eye bank, the marginal region was removed and washed in cold PBS with 100 IU penicillin and 100 μg / ml streptomycin, and cut into small pieces. Cell clusters were obtained by digestion with 0.2% collagenase IV for 2 hours at 37°C and single cells were obtained by further digestion with 0.25% trypsin-EDTA for 15 min at 37°C. Primary cells were seeded on plastic plates coated with 2% growth factor-reduced Matrigel (354230, BD Biosciences). Limbal stem cells from GFP-labeled rats and rabbits were isolated and cultured using the same method as human LSCs.

[0290] Hu...

Embodiment 2

[0357] Materials and methods

[0358] animal

[0359] ROSA mT / mG Mice were previously described (PMID: 17868096;28) and maintained homozygous. P0-3.9-GFPCre mice expressing the EGFP-Cre recombinase fusion protein under the control of the Pax6P0 enhancer were maintained on the FVB background and PCR genotyped as described (29).

[0360] Pedigree Tracing

[0361] By making homozygous GFP reporter mice (ROSA mT / mG ) were crossed with lens-specific Cre transgenic mice (P0-3.9-GFPCre) for lineage tracing experiments, in which Cre expression was under the control of the mouse Pax6 ectodermal enhancer. Eyes were dissected at postnatal (P) day 1 and P60 and fixed overnight in 4% formaldehyde. Tissues were then incubated in 10% sucrose and embedded in optimal cutting temperature medium for cryosectioning. Frozen sections were washed in PBS and imaged on a Zeiss Axio Imager fluorescence microscope.

[0362] Isolation and Culture of Human LSCs and Skin Epidermal Stem Cells (SESCs)...

Embodiment 3

[0423] Method for preparing donor corneal repair material

[0424] 1. Materials

[0425] Limbal Stem Cell Medium or Limbal Stem Cell Maintenance Medium: DMEM / Nutrient Mixture F-12 (DMEM:F-12 with a volume:volume ratio of 3:1) supplemented with: 10% Fetal Bovine Serum , 0.4μg / ml hydrocortisone, 10 -10 μM cholera toxin, 5μg / ml transferrin, 2×10 -9 M 3,3',5-triiodo-L-thyronine, 5 μg / ml insulin, 10 ng / ml epidermal growth factor (EGF), 100 U / ml penicillin, and 100 μg / ml streptomycin. After the 4th cell passage, a ROCK inhibitor was added to the limbal stem cell medium to maintain the LSCs in a proliferative state, such as by adding 1 μM Y-27632. The above components DMEM, F12 medium and fetal bovine serum were purchased from (Life Technologies), the remaining components were purchased from Sigma, USA.

[0426]Limbal stem cell differentiation medium: CnT-30 or equivalent (CellnTec Advanced Cell Systems AG, Bern, Switzerland). Other limbal stem cell differentiation media that ...

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Abstract

The invention provides an isolated limbal stem or progenitor cell (LSC) population or LSC-like population comprising a chemically synthesized, recombinant or isolated nucleic acid encoding PAX6 mtegrated into a chromosome, or alternatively, not integrated remaining as an extrachromosomal genetic material, wherein the isolated LSC population is substantially free of non-LSC cells or wherein the LSC-like population is substantially free of non- LSC-like cells, or wherein the isolated LSC or LSC-like population is substantially free of non-LSC and non-LSC-like cells and uses thereof.

Description

[0001] This application claims the benefit of US Provisional Application No. 62 / 018,396, filed June 27, 2014, which is hereby incorporated by reference in its entirety. [0002] Various publications are cited throughout this application. The entire disclosures of these publications are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. technical field [0003] The field of the invention relates to methods and compositions for the treatment of ophthalmic conditions, diseases and injuries. In particular, the field of the invention relates to methods, kits and compositions for treating conditions, diseases, defects and injuries of the cornea and ocular surfaces. The present disclosure relates to preparations of cultured mammalian limbal stem cells derived from limbal tissue. In preferred embodiments, the limbal stem cell line is self-renewing and has the ability to differentiate into corneal epith...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N2510/00C12N2513/00C12N2533/90C12N5/0621A61P17/00A61P17/02A61P27/02A61P27/10A61P31/00A61P35/00A61P43/00A61K35/30C12N2501/727C12N2501/11C12N2501/235C12N2501/33A61K35/36C12N5/0625C12N2501/998C12N2506/03C12N2509/00C12Q1/6883C12Q2600/106
Inventor K·张H·欧阳R·候H·蔡
Owner RGT UNIV OF CALIFORNIA
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