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Methods for obtaining retinal progenitors, retinal pigmented epithelial cells and neural retinal cells

A technique for retinal progenitor cells and retinal cells, applied in the field of obtaining retinal progenitor cells, retinal pigment epithelial cells and neural retinal cells, can solve the problem of low efficiency

Active Publication Date: 2016-04-13
SORBONNE UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different approaches previously developed, despite real advances, still suffer from drawbacks often associated with differentiation of pluripotent stem cells into highly specialized cell types
These protocols for directed differentiation of hES or hiPS cells to photoreceptors require several steps, add several molecules, and are rather inefficient

Method used

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  • Methods for obtaining retinal progenitors, retinal pigmented epithelial cells and neural retinal cells
  • Methods for obtaining retinal progenitors, retinal pigmented epithelial cells and neural retinal cells
  • Methods for obtaining retinal progenitors, retinal pigmented epithelial cells and neural retinal cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Transplantation of human non-integrated induced pluripotent stem cells to retinal neurons and retinal pigment epithelial cells Reliable and efficient differentiation of cells

[0063] 1.1 Experimental procedure

[0064] Culture of human fibroblasts and iPS cells

[0065] At 37°C, standard 5% CO 2 Mature human dermal fibroblasts (AHDF) from an 8-year-old boy (gifted by Dr. Rustin, INSERMU676, Paris, France) were cultured in Duchenne's modified Eagle's medium with high glucose Glutamax II ( DMEM) (Life Technologies) supplemented with 10% FBS (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 1XMEM non-essential amino acids (Life Technologies). This medium is referred to as "fibroblast medium". Established human iPS cells were maintained in mitomycin-C inactivated mouse embryonic cells in ReproStem (ReproCell) medium with 10 ng / ml human recombinant fibroblast growth factor 2 (FGF2) (Preprotech) Fibroblast (MEF) trophoblast (Zenith). Cells we...

Embodiment 2

[0104] Example 2: Differentiation of retinal progenitor cells into late-born retinal cell types

[0105] Sustained maintenance of isolated NR-like structures in floating cultures, as demonstrated by qRT-PCR, allowed further differentiation of RPCs into late-born retinal cell types (Figures 9A-9E). Indeed, after first expressing premature retinal markers of mature RGC cells (BRN3A and BRN3B), amacrine cells (calretinin and GAD2), and horizontal cells (LIM) (Figure 9A and Figure 9B), it was observed that Emergence of markers of retinal cell types to late life, corresponding to cones (R / GOPSIN, BLUEOPSIN, and CONEARRESTIN) and rod photoreceptors (RHODOPSIN and RECOVERIN) (Figures 9C and 9D), bipolar cells (PKCα) ) and Müller glia (GLAST1) (Fig. 9E). Between day 21 (Fig. 9F) and day 42 (Fig. 9G), most of the NR-like structures lost their sheet-like appearance and developed OTX2-containing + , CRX + and RECOVERIN + The inner rosettes of cells, corresponding to differentiated ...

Embodiment 3

[0106] Example 3: Acceleration of Photoreceptor Precursor Generation by Notch Inhibition

[0107] Immunohistochemical analysis using antibodies against CRX and RECOVERIN demonstrated that the number of photoreceptor precursors varied from day 14 (Figure 3J) to day 28 (Figure 3L, Figure 5 B) gradually increased. Between days 21 and 35, NR-like structures lost their lamellar structure and developed internal rosettes containing CRX and RECOVRIN positive cells ( Figure 5 B). Interestingly, on day 21, continuous addition of the Notch inhibitor DAPT for 7 days was sufficient to significantly increase the number of CRX-positive cells and RECOVRIN-positive cells ( Figure 5 B). Subsequent treatment with DAPT also resulted in a large increase in the number of cells expressing CRX and RECOVERIN from day 28 to day 35 ( Figure 5 B). One week of treatment with DAPT increased photoreceptor precursor production between days 21 and 28, as CRX at day 28 increased the production of photor...

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Abstract

The present invention pertains to a method for in vitro obtaining human retinal progenitors, comprising the steps of (i) placing an adherent culture of human pluripotent stem cells in a pro-neural medium; and (ii) maintaining this culture in said pro-neural medium until the appearance of pigmented cells and / or of neuroepithelial-like structures. Advantageously, additional steps can be perfonned to obtain RPE cells and / or precursors of the neural retina.

Description

Background technique [0001] Impaired or complete loss of function of photoreceptor or supporting retinal pigment epithelium (RPE) cells is a major cause of irreversible blindness in retinal diseases such as hereditary retinal degeneration and age-related macular degeneration (AMD). Retinal ganglion cell (RGC) death in glaucoma also leads to irreversible loss of vision. Rescue of the degenerated retina is a major challenge, and cell replacement is one of the most promising approaches (Pearson et al., 2012; Barber et al., 2013). The use of human pluripotent stem cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells opens new avenues for human retinal degenerative diseases. Human ES (hES) and iPS (hiPS) cells can be used as an unlimited source of retinal cells (photoreceptors, RPE and RGC) for tissue transplantation, where human ES (hES) and iPS (hiPS) cells have unlimited potential in culture. The ability to expand while maintaining their pluripotent state ...

Claims

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Application Information

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IPC IPC(8): C12N5/0793C12N5/071C12N5/079
CPCC12N5/062C12N2501/115C12N2501/42C12N2502/1323C12N2506/03C12N2533/54C12N5/0621C12N5/0623C12N2527/00
Inventor 萨夏·赖奇曼奥利维尔·古瑞奥约瑟-阿兰·萨赫尔
Owner SORBONNE UNIV
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