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Webinar: hear from our 3 panelists discussing regulatory, technology, and scientific perspectives to accelerate your compound through the clinical development pipeline with predictive retina models

A liquid nitrogen bank containing suspension of stem cells

Disease Modelling

Assays

  • Cell viability assay
  • Key markers analysis
  • Quantitative immunofluorescence
  • Gene expression
  • Photoreceptor degeneration
  • Phagocytosis of photoreceptor outer segment (RPE)
  • Transmission (TEM) and Scanning (SEM) electron microscopy

Models

  • Human retinal organoids
  • Retinal pigment epithelium (RPE)
  • Derived from healthy donor, patients or or CRISPR/Cas9 gene-edited iPSCs

Timeline

  • Rapid
  • 3-6 months

In vitro retinal disease modelling for retinal therapy

Newcells offers a fast and reliable in vitro safety and efficacy service for the evaluation of novel compounds for retinal therapy using complex human retinal organoid or RPE models developed in house. Both models are iPSC derived allowing gene-edited lines to be engineered using CRISPR/Cas9 for direct comparison between WT and mutant phenotypes after differentiation. Targeted mutations can model retinopathies, more specifically monogenic inherited retinopathies such as retinitis pigmentosa (RP), Stargardt disease, Usher’s syndrome and Leber congenital amaurosis.

Service outputs

  • Cell viability assay
  • Key markers analysis
  • Qualitative immunofluorescence
  • Gene expression
  • Photoreceptor degeneration
  • Phagocytosis of photoreceptor outer segment (RPE)
  • Transmission (TEM) and Scanning (SEM) electron microscopy

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A microscope image of retinal organoids
Cone photoreceptor cells labelled with anti-Opsin (Red/Green) antibody.

CRISPR-Cas9

WT & mutant

High throughput

Newcells Retinal Models

Accelerate your lead compound selection by understanding their mode of action in functional retinal tissue

1.

Recapitulate the architecture and function of the human retina

2.

Retinal disease modelling with genetic engineering techniques

3.

Accelerate drug discovery

Example 1: Phenotypic analysis of Retinitis Pigmentosa mutation Close Open

Following the generation of retinal organoids from RP Type 11 patients with a mutation in pre-mRNA processing factor 31 (PRPF31), the mechanism of retinal dysfunction associated with the disease has been elucidated. Large-scale transcriptome analyses identified cell type-specific and patient-specific mis-splicing of PRPF31 target genes affected by PRPF31 mutations, providing unprecedented molecular characterisation of splicing-factor RP clinical phenotypes. The cellular defects unravelled in this work included dysfunctional RPE, disrupted cilia morphology in photoreceptors, progressive cellular degeneration, and cellular stress, as shown by phenotypic rescue. This work was the first demonstration of the cellular phenotypes associated with RP using patient derived organoids or RPEs.

Rp11 patients
Transmission electron microscopy images showing shorter cilia in patient-derived photoreceptors, with abnormal bulbous morphology (red star). Scale bar (500 nm)
Mean Cillium Length
Quantification of cilia length and frequency in photoreceptors showing significant reduction in RP11 patients compared to the controls

This work and expertise was established in Newcells Biotech co-founder’s lab Prof. Lako, Professor of Stem Cell Sciences, Biosciences Institute, Faculty of Medical Sciences,
Newcastle University

Buskin A, et al., Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa. Nat Commun. 2018 Oct 12;9(1):4234. doi: 10.1038/s41467-018-06448-y.

Accelerate retinal therapy development Close Open

iPSC technology allows the engineering of patient-specific retinal organoids and RPE, offering the chance to investigate disease mechanisms and evaluate novel therapeutics. As our models are derived from human iPSCs, patient-specific gene-edited lines can be engineered allowing direct comparison between WT and mutants.

The retinal organoids are obtained through a carefully controlled differentiation process recapitulating the timeline of embryonic retinogenesis. At day 150, the organoids comprise all key cell types and are functional, allowing the testing of new compounds by simply adding them to the plate. The cell structure integrity and the gene expression profiles of key markers for the main cell types, such as photoreceptors, is assessed. We also perform qualitative imaging and microscopy to provide the full picture of the drug profile.

RPE and retinal organoids can be generated from the parental iPSC line allowing complementary analyses of the retinal tissue and epithelium.

One of the main advantages of our models for retinal disease modelling is their human origin, allowing us to generate robust and predictive data for transitional and clinical studies.

Example 2: Retinal disease modelling in vitro: AMD Close Open

Age related Macular degeneration (AMD) disease mechanisms are largely unknown; thus patient-derived retinal organoids and RPE are very valuable tools for disease modelling. Complement and ARMS/HTRA genes are known to increase the risk of AMD, but the role of the other genes and how they increase the risk of being affected by AMD is not yet fully understood. Newcells have generated iPSC-derived RPE from individuals with low-risk and high-risk CFH (Y402H) complement polymorphisms to model AMD. Compared to low-risk individuals, the high-risk iPSC-derived RPE cells show characteristics typical of AMD including cellular, structural, and functional deficiencies associated with inflammation, cellular stress, accumulation of lipid droplets and deposits that mimic AMD. These studies can be carried out in both our retinal organoids and companion RPE.

AMD
iPSC-derived retinal pigment epithelium (RPE) modelling AMD. iPSC-derived RPE generated from high-risk Y402H AMD donors (F180 and F181) show ultrastructural changes when compared to low-risk Y402H donors (F018 and F116). (A): Microvilli length is decreased in high-risk donor RPE. (B): Mitochondrial area was increased in high-risk donor RPE. (C): Mitochondrial number was decreased in high-risk donor RPE. (D): The number of vacuole structures was greatly increased in high-risk donor RPE. (E): Examples of low-risk iPSC-RPE cells: left hand side, F018; right hand side, F116; (F): Examples of high-risk iPSC-RPE cells: left hand side, F180; right hand side, F181; red asterisk indicates vacuoles.
Service overview Close Open

Our service provides insights into the molecular basis of inherited retinopathies

We can also test the efficacy of novel treatments in vitro to accelerate lead development or model phenotypic rescue. Finally, we can also access and compare the safety of new drugs or viral vectors on both healthy and patient-specific derived retinal tissues.

Our service is fully customised as we generate the patient-specific lines on your behalf, or we work with your mutated iPSC lines if they are available. We will then differentiate them into organoids and RPE. For incoming lines, we first require a feasibility study.

Our differentiation protocol spans 150 to 210 days to mimic embryonic retinogenesis. Our project timelines however, are short as our streamlined manufacturing process releases a regular supply of tissue. The robust data generated by our scientific experts will guide you in confidence for key decision-making steps during drug development.

An example of disease modelling packages includes a set of assays to assess cell viability, photoreceptor or RPE functionality and degeneration, key marker expression and localisation. We can also assess the safety of new chemical drugs or new viral vectors such as AAV.

Assay design
Models

Retinal organoids with photoreceptors (cone and rod), retinal ganglion cells, horizontal cells and amacrine cells (derived from healthy (WT) or gene-edited patient-specific iPSCs).

Retinal pigment epithelium (RPE) cells (2D cobblestones monolayer) (derived from healthy (WT) or gene-edited patient-specific iPSCs.

CRISPR/Cas9 Gene-editing service

Assay format

96-well plates (retinal organoids)

24-well plates (RPE)

Species

Human

Assay readout
  • Cell viability assay (ATP depletion assay, LDH release and microscopy)
  • Qualitative immunofluorescence with cell specific markers & cell death markers
  • Gene expression profile of key marker gene by RT-qPCR
  • Microscopy: EM, 2D-TEM and SEM
  • Cytokine secretion assay (with RPE only)
  • Trans-epithelial electric resistance (TEER) (with RPE only)
Time points and replicates
  • Retinal organoids available at D60, D120, D150, D150 & D210 of differentiation
  • Data points are usually performed in triplicates or quadruples with a minimum of 10 organoids

Models to choose from for this service

Retinal organoids

The retinal organoids are iPSC-derived and recapitulate the complex structure of the human retina with laminar cell organisation mimicking embryonic development. They contain the outer photoreceptor segment of the retina that responds to light.

Find out more
A microscope image of retinal organoids
Cone photoreceptor cells labelled with anti-Opsin (Red/Green) antibody.

Retinal pigment epithelium (RPE)

A functional monolayer in vitro model of retinal pigment epithelial cells generated from human iPSCs recapitulating phagocytosis of photoreceptor outer segments. The RPE cells are pigmented and displays typical cobblestone morphology.

Find out more
Image of RPE model
RPE cells displaying cobblestone morphology. Cells were immunolabeled with tight-junction ZO-1 marker (shown in green) and co-stained with nuclei marker, Hoechst (shown in blue).

CONTACT US

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