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Systems and methods for producing retinal progenitors

A technology of retinal progenitor cells and stem cells, applied in the field of systems and methods for generating retinal progenitor cells, capable of solving problems such as not showing effectiveness

Active Publication Date: 2020-09-25
NAT UNIV OF SINGAPORE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, neither has been shown to be effective in restoring vision

Method used

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  • Systems and methods for producing retinal progenitors
  • Systems and methods for producing retinal progenitors
  • Systems and methods for producing retinal progenitors

Examples

Experimental program
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Effect test

Embodiment Embodiment 1

[0111] Cell line development of HEKs overexpressing LN-523 and LN-323

[0112] exist Figure 1A A flowchart describing the steps for producing recombinant laminin-523 and laminin-323 is provided in . Briefly, human recombinant laminin expression plasmids encoding laminin α-3, β-2 and γ-3 chains and containing three different antibiotic resistance genes were generated and sequentially transfected into HEK cells. To generate LN-523, expression plasmids encoding laminin α-5, β-2 and γ-3 chains were sequentially transfected into HEK cells. Stable clonal cell lines overexpressing the desired laminin chains were established. Third, identify potential positive clones using qPCR and Western blotting. Fourth, switch the cell culture medium to serum-free medium for positive laminin clones. Fifth, the cell culture medium containing the secreted laminin is gel filtered. Finally, western blotting and mass spectrometry were performed to confirm the identity of the laminin chains.

[0...

Embodiment 2

[0144] Two independent single-cell RNA-sequencing datasets were generated using day 20 and day 30 retinal progenitor cells derived from H1 embryonic stem cells. H1 cells were cultured with the laminin protocol of Example 1 for 20 and 30 days. In each case, RNA was isolated using a 10× Genomics kit and sequenced using the Illumina Hi-Seq3000 sequencing platform. Reads were mapped to the human genome (Ensembl version 90) and quantified using Cell Ranger 2.1.1 10x Genomics software. A custom reference transcriptome generated by genotype (protein coding, lincRNA, and antisense) filtering of the Ensembl transcriptome was provided to the Cell Ranger software. Cell Ranger was run with the expected cell number parameter (expected cells) set to 3000.

[0145] The estimated cell numbers for the Cell Ranger at day 20 and day 30 were 1,790 and 3,881, respectively. The total reads on days 20 and 30 were 631,803,330 and 687,721,993, respectively. The percentage of reads mapped to the hu...

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Abstract

Retinal progenitors and mature photoreceptor cells can be produced by differentiating human embryonic stem cells on a surface having a laminin matrix thereon made of two laminins. One laminin is laminin-521, and the other laminin is either laminin-323 or laminin-523. Stem cells plated on this substrate can be differentiated using various cell culture mediums to obtain the retinal progenitors and mature photoreceptor cells.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to PCT Application No. PCT / SG2017 / 050520, filed October 17, 2017, which is incorporated by reference in its entirety. Background technique [0003] The present disclosure relates to methods for generating retinal progenitor cells (such as photoreceptor progenitor cells or ganglion progenitor cells) or retinal photoreceptor cells from human pluripotent stem cells (retinal photoreceptor cell) systems and methods. Compositions and substrates for such systems and methods are also disclosed. Transplantation of the resulting retinal progenitor cells or retinal photoreceptor cells is also disclosed and encompassed herein. These methods, compositions and systems comprising the same are useful in the treatment of eye diseases such as macular degeneration (MD) or retinitis pigmentosa (RP). [0004] Worldwide, the leading causes of blindness are age-related macular degeneration (AMD) and retinit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A61K35/30A61P27/02
CPCC12N5/062C12N2500/98C12N2501/13C12N2501/15C12N2501/385C12N2501/415C12N2506/02C12N2533/52C12N2501/999C12N2506/45C12P21/02
Inventor 卡尔·特里格瓦松蔡慧冈艾达·莫雷诺莫拉尔
Owner NAT UNIV OF SINGAPORE
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