Published: April 21st, 2021
Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.
Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.
Pluripotent stem cells (PSCs) are characterized by their self-renewal and ability to differentiate into all kinds of somatic cells. Thus, organoids derived from PSCs have become an important resource in regenerative medicine research. Retinal degeneration is characterized by the loss of photoreceptors (rods and cones) and retinal pigment epithelium. Retinal cell replacement could be an encouraging treatment for this disease. However, it is not feasible to obtain human retinas for disease research and therapy. Therefore, retinal organoids (ROs) derived from PSCs, which effectively and successfully recapitulate multi-layered native retinal cells, are beneficial for basi....
This study was approved by the institutional Ethics Committee of Beijing Tongren Hospital, Capital Medical University. H9 hESCs were obtained from the WiCell Research Institute and genetically engineered to tdTomato-tagged cell line.
1. Generation of human ROs
The schematic illustration depicts the differentiation protocol to improve precursor cells with COCO (Figure 1). From PSC to ROs, numerous details could cause result variations. It is recommended to record every step and even the catalog number and lot number of every medium to track the entire procedure.
Herein, we provide bright field images for days 6, 12, 18, and 45 (Figure 2). On day 6, the organoids are usually around 600 µ.......
Retinal organoid differentiation is a desirable method for the generation of ample functional retinal cells. The RO is a composite of different retinal cells, such as ganglion cells, bipolar cells, and photoreceptors, generated by pluripotent stems cells toward the neural retina4,5,8,9. Although confluent ROs could be harvested, it is time-consuming, which may require long culturing periods (up.......
We thank members of 502 laboratory for their technical supports and helpful comments regarding the manuscript. This work was partly supported by the Beijing Municipal Natural Science Foundation (Z200014) and National Key R&D Program of China (2017YFA0105300).....
|DAN Domain family of BMP antagonists
|Fetal Bovine Serum (FBS), Qualified for Human Embryonic Stem Cells
|KnockOut Serum Replacement-Multi-Species
|MEM Non-essential Amino Acid Solution (100X)
|Primesurface 96 V-plate
|Cell aggregation in 1.2.7
|Matrigel in 1.1.1
|mTeSR- E8 , PSCs basal medium in 1.1.2
|TrypLE Express in 1.2
|DNase I , in 1.2
|Enzo Life Science
|N-2 Supplement (100X), Liquid, supplemet in medum III
|Trypsin-EDTA (0.25%), phenol red
|organoids dissociation in 2.1.3
|Wnt Antagonist I, IWR-1-endo - Calbiochem
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