Methods of Obtaining Retinal Progenitor Cells, Retinal Pigment Epithelial Cells, and Neural Retinal Cells
A neural and cellular technology, applied in the field of obtaining retinal progenitor cells, retinal pigment epithelial cells and neural retinal cells, can solve the problem of low efficiency
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Embodiment 1
[0062] Example 1: 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, INSERM U676, Paris, France) were cultured in Duchenne's modified Eagle's culture with high glucose Glutamax II in a / 95% air incubator DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 1X MEM non-essential amino acids (Life Technologies). This medium is called "fibroblast medium". Established human iPS cells were maintained in mitomycin-C-inactivated mouse embryogenesis in ReproStem (ReproCell) medium with 10 ng / ml human recombinant fibroblast growth factor 2 (FGF2) (Preprotech). Fibroblasts (MEFs) on feeder layer (Zenith). Cells ...
Embodiment 2
[0104] Example 2: Differentiation of Retinal Progenitor Cells to Late-born Retinal Cell Types
[0105] Persistent maintenance of isolated NR-like structures in floating cultures, as demonstrated by qRT-PCR, allowed RPCs to further differentiate into late retinal cell types (Figs. 9A-9E). Indeed, after first expressing the premature retinal markers of mature RGC cells (BRN3A and BRN3B), amacrine cells (calretinin and GAD2) and horizontal cells (LIM) (Fig. 9A and Fig. 9B), it was observed that To the emergence of markers of late retinal cell types, corresponding to cone (R / GOPSIN, BLUE OPSIN and CONE ARRESTIN) and rod photoreceptors (RHODOPSIN and RECOVERIN) (Fig. 9C and Fig. 9D), bipolar cells (PKCα) and Müller glial cells (GLAST1) (Fig. 9E). Between day 21 (Fig. 9F) and day 42 (Fig. 9G), most NR-like structures lost their sheet-like appearance and developed OTX2-containing + 、CRX + and RECOVERIN +Internal rosettes of cells, corresponding to differentiated photoreceptors ...
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 increased from day 14 (Fig. 3J) to day 28 (Fig. 3L, Figure 5 B) Gradually increase between. 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 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). Treatment with DAPT for one week between days 21 and 28 increased the production of photoreceptor precursors, as CRX on day 28 + and RECOVRIN + The nu...
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