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Ratnesh K Singh, Ramya Krishna Mallela, Pamela Cornuet, Igor O Nasonkin; Derivation of retinal cells and retinal organoids from pluripotent stem cells for CRISPR-Cas9 engineering and retinal repair. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3591.
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© ARVO (1962-2015); The Authors (2016-present)
Repairing retina by cell replacement is challenging. We hypothesize that engineering stem cell-derived retinal cells to express and secrete trophic factors (TFs) will sustain the function of degenerating neurons after retinal grafting and improve the survival of the transplanted cells, promote specific retinal fate acquisition and synaptic integration.
Human embryonic stem cells (hESC, H1) was grown on matrigel in mTeSR1 medium, differentiated to retinal cells (0->3 months) and tested by quantitative RT-PCR for the expression of major retinal markers.
We noted sharp upregulation (control: hESCs, data and standard deviation, 4wk) of key genes Rx [20.0 +/-3.4], Lhx2 [7.7 +/-0.1], Pax6 [16.6+/-2.1], Six6 [35.2 +/- 11.8]), Foxg1 [43.3 +/-3.8], Otx2 [7.5 +/-0.1], MITF [100,4 +/-0.0], retinal progenitor genes Sox1 (13.9 +/-1.1], DCX [26.0 =/-0.1], Ascl1 [134.2+/-12.5], NeuroD1 [377.3+/-5.5]. Lgr5 surged at 2 wk [307+/-6.0] and remained high at 3 (280 +/- 2.7] and 4 [276+/- 9.5] wk. We noted the gradual upregulation of photoreceptor genes Nrl, Nr2e3 [5.6 +/-0.5], Pde6b [4.9 +/- 0.3], Opn1sw [31.4+/-2.2], Opn1mw 8.9+/-0.3], Rcvrn [7.2+/-0.1], Chx2, Crx]), horizontal cells (Prox1, Calbindin D28 [26.2 +/- 0.6 sd]), amacrines (Calretinin [1.5 +/-0.0 sd], Pax6), retinal ganglion cells (Brn3a, 3b, 3c [1.7 -2.3], Isl-1 [14.8 +/-1.9], Pax6). A number of key retinal markers were further upregulated by 3 mo: Chx10 [314,0 +/-12.3], Crx [13.4+/-0.5], Nrl [4.9+/-0.0], Dlx2 [46+/-3.6], Brn3a [4.0+/-1.0], Brn3b [18.3+/-0.1], Math5 [15.0+/-0.1], Pax6 [19.5+/-0.2], Ascl1 and NeuroD1 markers remained high [144,2+/-6.3 and 338.0+/-6.6]. A large number of retina-like organoids appeared on the plates (4-12 wk). We are currently testing these organoids for the ability to undergo maturation in vivo after subretinal transplantation, and in vitro in long term differentiation and for the ability to steadily secrete TFs after CRISPR-Cas9 engineering.
Our strategy resulted in very efficient derivation of retinal cells and allows us to proceed with genetic modification of cells (CRISPR-Cas9) to overexpress TF and test our cells in degenerating retinas of mice.
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