Patent Application: US-201515111883-A

Abstract:
the present invention relates to the field of stem cells . more specifically , the invention provides methods and compositions useful for forming three - dimensional human retinal tissue in vitro . in a specific embodiment , an in vitro method for differentiating hipscs into three - dimensional retinal tissue comprising functional photoreceptors comprises the steps of culturing the hipscs to form aggregates ; transitioning the aggregates into a neural induction medium ; seeding the aggregates on to extracellular matrix coated cell culture substrates ; replacing nim with a chemically - defined differentiation medium ; detaching nr domains ; culturing in suspension ; and adding animal serum or plasma component and retinoic acid .

Description:
it is understood that the present invention is not limited to the particular methods and components , etc ., described herein , as these may vary . it is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only , and is not intended to limit the scope of the present invention . it must be noted that as used herein and in the appended claims , the singular forms “ a ,” “ an ,” and “ the ” include the plural reference unless the context clearly dictates otherwise . thus , for example , a reference to a “ protein ” is a reference to one or more proteins , and includes equivalents thereof known to those skilled in the art and so forth . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . specific methods , devices , and materials are described , although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention . all publications cited herein are hereby incorporated by reference including all journal articles , books , manuals , published patent applications , and issued patents . in addition , the meaning of certain terms and phrases employed in the specification , examples , and appended claims are provided . the definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present invention . retinal development occurs within a very dynamic and complex microenvironment involving highly - coordinated cell - cell interactions through direct contact or diffusible signals 7 , 8 . accordingly , in published studies so far , the differentiation of es or ips cells into retinal cells in vitro typically required an elaborate regime of exogenous factors 5 , 6 , 9 - 14 . here , we succeeded in inducing the differentiation of hipscs into retinal progenitors that self - organized into a 3 - dimensional retinal cup with a simple procedure , involving just a few factors to promote cell survival and photoreceptor maturation ( fig5 ). most importantly , the photoreceptors in our preparations were able to reach the stage of photosensitivity . eye development in the embryo &# 39 ; s neural plate begins with the formation of the eye field ( ef ), a centrally - organized domain consisting of a subpopulation of anterior neuroepithelial cells that have become further specified into retinal progenitors &# 39 ; ( fig5 a ). the ef is characterized by the expression of a group of transcription factors including pax6 , rx , lhx2 , six3 , and six6 , while the surrounding anterior neuroepithelial cells express pax6 and sox1 15 - 17 . in parallel to the native events , our hipsc - derived aggregates , after 8 days of differentiation ( d8 ) in a chemically - defined neural - differentiation medium and attached on matrigel - coated culture dishes , acquired an anterior - neuroepithelial fate expressing pax6 and sox1 ( fig1 a - c ). soon after , retinal progenitor cells expressing lhx2 appeared in the central region of the differentiating aggregates ( fig1 d ). by d12 , well - defined ef - like domains expressing the appropriate transcription factors could be observed ( fig1 e - h ) surrounded by anterior - neuroepithelium - like cells ( fig1 f ). these anterior - neuroepithelium - like cells typically formed rosettes ( fig6 i - 1 ), which although not found in the native situation , are characteristic of these cells in culture 18 . the rt - pcr analyses in fig1 s summarize the temporal sequence of events in culture , showing the gradual loss of the hipscs &# 39 ; pluripotency ( loss of oct4 ), the acquisition of neural fate ( sustained sox2 expression , and appearance of pax6 ), and the progressive differentiation into retinal progenitors . the chronology of expression of the eye - field transcription factors mimicked the in vivo situation , with initial expression of pax6 and six3 , then lhx2 and rx , and eventually six6 15 . thus , without exposure to any “ retinalizing ” exogenous factors , hipscs were still able to differentiate into retinal progenitors that self - organized into ef - like domains surrounded by anterior neuropepithelial - like cells , presenting a cellular organization closely resembling the embryonic anterior neural plate where the ef forms in vivo . the ef in vivo gives rise to the left and right optic vesicles , with their respective retinal progenitors eventually forming the future neural retina ( nr ) and retinal pigment epithelium ( rpe ) ( fig5 a ). cell - fate specification into either nr or rpe is regulated critically by two transcription factors , vsx2 and mitf , which initially are co - expressed in the bipotential progenitor cells but subsequently become restricted to the nr and rpe , respectively 7 , 19 , 20 . again , as in the native situation , the cells within the ef - like domains in our cultures followed the same differentiation sequence ; namely , these cells initially expressed both vsx2 and mitf ( fig1 i ), but subsequently segregated into a central nr - like domain expressing pax6 , lhx2 , rx and vsx2 ( fig1 j - 1 ; fig6 a - d ), and a peripheral rpe - like domain expressing mitf and pax6 ( fig1 l and fig5 e ). between d17 and d25 in culture , these nr and rpe domains transitioned to an optic - cup - like structure , with the nr progressively acquiring a horseshoe - dome shape reminiscent of the inner wall of the optic cup , surrounded by the rpe ( fig1 m - q ; fig5 f and 2 e - h ). similar results were obtained from three different hipsc lines , with the efficiency of nr - domain formation in d20 being 85 . 0 ± 3 . 0 %, 88 . 34 ± 3 . 5 % and 62 . 3 ± 4 . 6 %, respectively ( mean ± sd ) ( fig1 r ). thus , in our cultures , retinal progenitors in the ef domains underwent spontaneous differentiation into nr and rpe efficiently and reproducibly , closely mimicking their in vivo topological organization in the correct temporal sequence . the optic - cup - like shape of the nr domains in our cultures made them easily identifiable and amenable to mechanical detachment one by one , and collection for further culture in suspension ( fig2 a ). the nr domains , collected in d21 - d28 , had a high enrichment of nr progenitors ( 71 . 0 ± 7 . 3 % vsx2 - positive cells vs . 19 . 0 ± 7 . 2 % mitf - positive cells , mean ± sd ; fig2 b ) and , when cultured in suspension , formed 3 - d retinal cups ( fig2 c ) with an efficiency ranging from 50 % to 70 % ( estimated from over 100 nr domains / experiment and 3 independent experiments ), depending on the level of stringency at the time of nr collection . the retinal cup comprised a thick , transparent nr continuous with the adjacent rpe , which appeared bundled at the tip of the retinal cup and became gradually pigmented ( fig7 a ). from the time of nr - domain collection to d35 ( week 5 , or w5 ), the nr presented molecular and histological features resembling the actual features of the human embryonic retina at the same age 21 , including a polarized , pseudostratified epithelium with proliferating cells undergoing interkinetic nuclear migration and expressing the appropriate transcription factors ( fig7 ). during w5 - w7 , the nr cells spontaneously began to differentiate , following the characteristic center - to - periphery wave of neurogenesis and migrating to their corresponding retinal layers ( fig8 ). in order to promote cell survival beyond w7 , the culture medium had to be supplemented with fetal bovine serum ( fbs ), taurine and retinoic acid until w17 , at which time further slight modifications ( less retinoic acid and replacement of the neurobasal supplement b27 by n2 ) were made to induce photoreceptor maturation . these conditions allowed the retinal cups to maintain their shape and steady growth ( longest axis increasing from 0 . 4 ± 0 . 1 mm on d45 to 0 . 7 ± 0 . 1 mm on d90 , mean ± sd ) ( fig9 a - e ) and develop distinguishable layers containing all major retinal cell types , including müller cells ( fig9 f - j ). ganglion cells first appeared in w5 ( fig8 d ) and over time increased in number and migrated toward the emerging ganglion cell layer ( fig2 d - j ), which became well - established by w12 - 13 ( fig2 g ), sometimes including a developing nerve - fiber - like layer ( fig2 k ). photoreceptors ( expressing otx2 ) appeared during w7 and , over the subsequent weeks , populated the developing outer nuclear layer ( where photoreceptor cell bodies are situated in the mature retina ) and expressed recoverin , a well - known phototransduction protein ( fig2 d - f and h - j ). amacrine cells ( expressing ap2α ) and horizontal cells ( expressing prox1 ) also appeared during w7 ( fig2 l ). as time progressed , amacrine and horizontal cells became numerous , and began to segregate to their corresponding layers ( fig2 m - o ). by w21 , the retinal cups presented a well - organized outer nuclear layer , adjoining a developing outer plexiform layer expressing the synaptic - vesicle protein , sv2 ( fig2 p - q ). rod opsin was also detectable in the distal part of photoreceptor cells ( fig2 r ). finally , a developing bipolar cell layer containing postmitotic , vsx2 - expressing bipolar cells appeared after w22 ( fig2 s ). this spatiotemporal pattern of differentiation closely mimics that of the native retina 22 , 23 and was observed in all the retinal cups examined (˜ 60 ) derived from the three different hipsc lines we used ( fig2 t and fig5 k ). in the above culture conditions , although the photoreceptors expressed detectable levels of rod opsin , they did not appear to continue maturing , such as forming outer - segment discs . at the same time , no expression of l / m - and s - cone opsins was apparent . because retinoic acid has been shown to influence photoreceptor differentiation in a time - and concentration - dependent manner 24 , 25 , we reasoned that prolonged exposure to a relatively high retinoic - acid concentration ( 1 μm retinoic acid in w7 - w17 ) might hamper photoreceptor maturation . accordingly , we tried two shorter time windows of retinoic - acid exposure ( w7 - w14 and w10 - w14 , both with 1 μm retinoic acid ; fig1 ). the w10 - w14 condition showed , already at w17 , dispersed cells with higher rod - opsin expression , not just in the distal part of the immature photoreceptors but also in their cell bodies , as in native development 26 ( observed in ˜ 50 % of the retinal cups , n = 8 ; rod - opsin expression remained weak in the remainder ) ( fig3 a - c ). moreover , we began to observe s - opsin expression in some photoreceptors not expressing rod opsin ( fig3 d ). by w21 , 90 % of the retinal cups ( n = 20 ) showed a significant increase in the number of photoreceptors expressing rod opsin , organized in patches throughout the outer nuclear layer or even encompassing the full extent of this layer ( fig3 e ). photoreceptors expressing l / m - or s - opsins were also observed ( fig3 f ). the morphologies of the rods and cones and the localization of their cell bodies , with cones at the outer edge and rods toward the inner edge of the outer nuclear layer , resembled remarkably the native situation 26 ( fig3 g - j ). the photoreceptors showed rounded structures at their distal tip reminiscent of the short nascent outer segments at comparable developmental stages in the human retina 26 ( fig3 g - i arrowheads ). although infrequent , there were also elongated structures resembling more advanced native outer segments in w25 ( fig3 j , arrow ). during w27 - w28 , several ultrastructural features of functional significance appeared in electron microscopy , including an outer limiting membrane , inner segments with numerous mitochondria , basal bodies , and connecting cilia ( fig3 k - m ). although at low frequency , some photoreceptors also showed intracellular membrane discs reminiscent of the outer - segment discs in mature photoreceptors ( fig3 n and fig5 j ). all of these features were very similar to those observed in the human developing retina in vivo 27 , 28 . perhaps most importantly , based on immunocytochemistry with specific antibodies verified in adult human retina ( fig1 j - 1 ), several key proteins involved in rod phototransduction were expressed in the photoreceptors of w25 retinal cups , including the a - subunit of rod transducin ( g t1α ), the α - and β - subunits of the rod cgmp - phosphodiesterase ( pde6 αβ ), the rod cyclic - nucleotide - gated - channel a - subunit ( cnga1 ) and β - subunit ( cngb1 ), and retinal guanylate cyclase 1 ( retgc1 ) ( fig4 a - i ). these proteins increased in expression over time in parallel to rod opsin , as in native human retina 26 ( fig1 a - i ). with perforated - patch recordings in the voltage - clamp mode from the rod - like cells in w25 - w27 rcs , we found 2 out of 13 randomly chosen cells to respond to a light flash ( fig4 j ). this response consisted of the suppression of a standing dark inward current , similar to the situation in native rods ; the speed of the response &# 39 ; s rising phase also resembled that of the native response . the photosensitivities of the two responsive cells were much lower than normal , likely because the rhodopsin level was still low and the downstream phototransduction steps still maturing ; repeated flashes also failed to elicit further responses . possibly for the same reasons , light did not completely suppress the dark inward current ( see also reference 29 for a cgmp - induced current in their preparations ). because of the very low incidence of w27 photoreceptors with visible outer - segment discs in the retinal cups used for recordings , we consider the percentage of responsive cells encountered to be actually very respectable . finally , and with interesting correlation , almost all light - insensitive cells recorded had a much smaller dark inward current ( fig4 k , 1 ). compared to the progressive maturation of photoreceptors , most ganglion cells and amacrine cells , on the other hand , gradually disappeared from the advanced cultures , presumably because they needed additional factors for their long - term survival . in summary , we have developed a simple and highly - efficient strategy for inducing hipscs to differentiate , almost autonomously , into 3 - dimensional retinal tissue in vitro , with spatial and temporal features that replicate the development of the human retina in vivo . the photoreceptors in our system are able to reach an advanced stage of maturation , up to at least the beginning of outer - segment formation and of photosensitivity . to our knowledge , this is the first time that such a developmental step has been achieved in vitro . surprisingly , this degree of photoreceptor maturation does not require physical contact with the rpe , which may have important implications about the intrinsic developmental program in these cells . finally , the success here with human ipscs obviously opens up many exciting possibilities in establishing models for human eye diseases , and hopefully will also take potential therapeutic applications one step closer to reality . without further elaboration , it is believed that one skilled in the art , using the preceding description , can utilize the present invention to the fullest extent . the following examples are illustrative only , and not limiting of the remainder of the disclosure in any way whatsoever . the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds , compositions , articles , devices , and / or methods described and claimed herein are made and evaluated , and are intended to be purely illustrative and are not intended to limit the scope of what the inventors regard as their invention . efforts have been made to ensure accuracy with respect to numbers ( e . g ., amounts , temperature , etc .) but some errors and deviations should be accounted for herein . unless indicated otherwise , parts are parts by weight , temperature is in degrees celsius or is at ambient temperature , and pressure is at or near atmospheric . there are numerous variations and combinations of reaction conditions , e . g ., component concentrations , desired solvents , solvent mixtures , temperatures , pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process . only reasonable and routine experimentation will be required to optimize such process conditions . the following describes one embodiment of the present invention . on day 0 ( d0 ) of differentiation , human ipscs cultured on matrigel - coated plates with mtesr1 medium , were enzymatically detached by dispase treatment and cultured in suspension to induce formation of aggregates . during the following three days , aggregates were gradually transitioned into neural induction medium ( nim ), and seeded onto matrigel - coated dishes on d6 - 7 . on d16 , nim was replaced by a chemically - defined differentiation medium containing dmem / f12 ( 3 : 1 ), 2 % b27 ( without vitamin a , invitrogen ), lx minimum essential media - non essential amino acids ( neaa ), and 1 % antibiotic - antimycotic ( gibco ). on the 4 th week of differentiation ( d22 - 28 ), horseshoe - shaped neural retina ( nr ) domains were manually detached and cultured in suspension , where 3 - dimensional retinal cups gradually formed . on d42 , the medium was supplemented with 10 % fetal bovine serum ( fbs ; gibco ), 100 μm taurine ( sigma ) and 2 mm glutamax ( invitrogen ) to promote cell survival . to induce photoreceptor maturation , all - trans retinoic acid ( ra ; sigma ) was added daily to a final concentration of 1 μm from d63 to d91 - 98 , and decreased to 0 . 5 μm thereafter . three hipsc lines , imr90 - 4 ( wicell ), cb - ipsc6 . 2 and ka . 1 , were used in this study ( table 1 ). all cell lines were obtained with verified normal karyotype and contamination - free . hipscs were maintained on matrigel ( growth - factor - reduced ; bd biosciences ) coated plates with mtesr1 medium ( stemcell technologies ) according to wicell protocols . cells were passaged every 5 - 7 days at approximately 80 % confluence . colonies containing clearly visible differentiated cells were marked and mechanically removed before passaging . the use of human ips cells in this study conforms to the johns hopkins institutional stem cell research oversight ( iscro ) committee . three human ips cell lines of different cell origin and reprogramming method where chosen in order to test the reproducibility of our method across cell lines . the procedure to induce early stages of retinal differentiation was based on a previously described protocol with major modifications 29 , 30 . briefly , on day 0 ( d0 ) of differentiation , hipscs were enzymatically detached by dispase treatment , dissociated into small clumps , and cultured in suspension with mtesr1 medium and 10 μm blebbistatin ( sigma ) to induce aggregate formation . aggregates were gradually transitioned into neural - induction medium ( nim ) containing dulbecco &# 39 ; s modified eagle medium ( dmem )/ f12 ( 1 : 1 ), 1 % n2 supplement ( invitrogen ), lx minimum essential media - non essential amino acids ( neaa ), 2 μg ml − 1 heparin ( sigma ), by replacing the medium with a 3 : 1 ratio of mtesr1 / nim on d1 , 1 : 1 on d2 , and 100 % nim on d3 . on d6 - 7 aggregates were seeded onto matrigel - coated dishes containing nim , and switched to dmem / f12 ( 3 : 1 ) supplemented with 2 % b27 ( without vitamin a , invitrogen ), lx neaa , and 1 % antibiotic - antimycotic ( gibco ) on d16 . thereafter , the medium was changed daily . on the 4 th week of differentiation ( d22 - 28 ), horseshoe - shaped neural retina ( nr ) domains were manually detached with a sharpened tungsten needle under inverted microscope , collected and cultured in suspension at 37 ° c . in a humidified 5 % co 2 incubator in dmem / f12 ( 3 : 1 ) supplemented with 2 % b27 , lx neaa , and 1 % antibiotic - antimycotic where they gradually formed 3 - dimensional retinal cups ( rcs ). thereafter , the medium was changed twice a week . for long - term suspension culture , the medium was supplemented with 10 % fetal bovine serum ( fbs ; gibco ), 100 μm taurine ( sigma ) and 2 mm glutamax ( invitrogen ) beginning on d42 unless otherwise noted . to promote photoreceptor maturation , suspension cultures of rcs were supplemented daily with 1 μm all - trans retinoic acid ( ra ; sigma ) at various time windows : w7 - w17 ; w7 - w14 or w10 - w14 ; subsequently , ra concentration was decreased to 0 . 5 μm . cells growing on adherent conditions were fixed in 4 % paraformaldehyde ( pfa ; sigma ) for 15 min . rcs were fixed in 4 % pfa for 30 min . a human eyeball from a 71 - year old person affected by age - related macular degeneration ( old dominion eye foundation ) was fixed in 4 % pfa for 4 hr . tissue cryopreservation , sectioning , and immunohistochemistry were performed as previously described 31 . antibodies against the following proteins were used at the indicated dilutions : lhx2 ( goat , 1 : 200 , santa cruz , sc - 19344 ), rx ( rabbit , 1 : 500 , abcam , ab86210 ), sox1 ( goat , 1 : 1000 , r & amp ; d , af3369 ), vsx2 ( sheep , 1 : 500 , millipore , ab9016 ), mcm2 ( rabbit , 1 : 1000 , abcam , ab4461 ), otx2 ( rabbit , 1 : 500 , millipore , ab9566 ), recoverin ( rabbit , 1 : 500 , millipore , ab5585 ), caspase 3 ( rabbit , 1 : 500 , cell signaling , asp175 ), hu c / d ( mouse , 1 : 200 , molecular probes , mp21271 ), brn3 ( goat , 1 : 100 , santa cruz , sc - 6026x ), tuj1 ( rabbit , 1 : 2000 , covance , mrb - 435p ), mitf ( mouse , 1 : 50 , neomarkers , ms - 771 - p1 ), prox1 ( rabbit , 1 : 2000 , millipore , ab5475 ), cralbp ( mouse , 1 : 500 , abcam , ab15051 ), phospho - histone h3 ( ph3 , rabbit , 1 : 250 , cell signaling , # 9701l ), rod - opsin ( mouse , 1 : 100 , gift from dr . david hicks ), l / m opsin ( rabbit , 1 : 50 , 000 , gift from dr . jeremy nathans ), s - opsin ( rabbit , 1 : 50 , 000 , gift from dr . jeremy nathans ), phosphodiesterase 6 alpha ( pde6α , rabbit , 1 : 1000 , abcam , ab5659 ) and beta ( pde6β , rabbit , 1 : 2000 , thermo scientific , pa1 - 722 ), hretgcl ( rabbit , 1 : 4000 , gift from dr . alexander m . dizhoor ), g t1α ( rabbit , 1 : 2000 , santa cruz , sc - 389 ), rod cyclic nucleotide gated channel a - subunit ( cnga1 , mouse , 1 : 10 , a gift from dr . robert s . molday ) and β - subunit ( cngb1 , mouse , 1 : 10 , a gift from dr . robert s . molday ). antibodies from the dshb , developed under the auspices of the nichd and maintained by the university of iowa , department of biology , were : pax6 ( mouse , 1 : 50 ), ap2α ( 3b5a , mouse , 1 : 35 ), and sv2 ( mouse , 1 : 1000 ). secondary antibodies used included the corresponding species - specific alexa fluor - 488 , - 546 and - 647 conjugated antibodies ( 1 : 500 , molecular probes ). dapi was used for nuclear counterstaining ( molecular probes ). fluorescence images were acquired with an lsm 510 confocal microscope ( zeiss ). click - it edu imaging kit ( invitrogen , c10337 ) was used according to the manufacturer &# 39 ; s protocol in order to visualize cells undergoing s - phase during the time - window under study . 3 - d rcs were incubated with 50 μg of edu diluted in pbs for 1 hr or 20 hr , then collected and processed for microscopic imaging . an antibody against the dna replication licensing factor mcm2 ( rabbit , 1 : 1000 , abcam , ab4461 ) was used to identify proliferating retinal progenitors , whereas an antibody against phospho - histone h3 ( ph3 , rabbit , 1 : 250 , cell signaling , # 9701l ) was used to identify cells in m phase by immunohistochemistry as described above . total rna isolation was done in triplicate with rnaeasy mini kit ( qiagen ) and followed by dnase i treatment ( qiagen ) to remove potential dna contamination . rna quality was evaluated using a nanodrop1000 spectrophotometer ( thermo scientific ). reverse transcription was performed using the superscript iii rt - pcr kit ( invitrogen ). samples without reverse transcriptase were used as negative controls . pcr was performed with taq dna polymerase ( invitrogen ) on a ptc - 200 thermal cycler ( bio - rad ). cycles ( 30 - 40 depending on primer pair ) were run at 95 ° c . denaturation for 20 s , 60 ° c . annealing for 20 s , and 72 ° c . extension for 30 s . subsequent pcr products were run on 2 % agarose gels . primers used were as follows : oct4 forward 5 ′- cgagcaatttgccaagctcctgaa - 3 ′ ( seq id no : 1 ), reverse 5 ′- tcgggcactgcaggaacaaattc - 3 ′ ( seq id no : 2 ); sox2 forward 5 ′- cccccggcggcaatagca - 3 ′ ( seq id no : 3 ), reverse 5 ′- tcggcgccggggagatacat - 3 ′ ( seq id no : 4 ); pax6 forward 5 ′- cggagtgaatca gctcggtg - 3 ′ ( seq id no : 5 ), reverse 5 ′- ccgcttatactgggctattttgc - 3 ′( seq id no : 6 ); six3 forward 5 ′- cgagcagaagacgcattgcttcaa - 3 ′ ( seq id no : 7 ), reverse 5 ′- cggccttggctatcatacatcaca - 3 ′ ( seq id no : 8 ); lhx2 forward 5 ′- caagatctcggaccgctact - 3 ′ ( seq id no : 9 ), reverse 5 ′- ccgtgg tcagcatcttgtta - 3 ′ ( seq id no : 10 ); rx forward 5 ′- gaatctcgaaatctcagccc - 3 ′ ( seq id no : 11 ), reverse 5 ′- cttcactaatttgctcaggac - 3 ′ ( seq id no : 12 ); six6 forward 5 ′- atttgggacggcgaacagaagaca - 3 ′ ( seq id no : 13 ), reverse 5 ′- atcctggatgggcaactcagatgt - 3 ′ ( seq id no : 14 ); gapdh forward 5 ′- accacagtccatgccatcac - 3 ′ ( seq id no : 15 ), reverse 5 ′- tccaccacc ctgttgctgta - 3 ′ ( seq id no : 16 ). neural retina - domains ( nr ) collected on d22 from two biological replicates were dissociated into single cells with trypsin , fixed in 1 % pfa for 15 minutes , washed with pbs containing 0 . 04 % triton - x - 100 and 2 % donkey serum , and then incubated for 1 hr at rt in primary antibodies at a concentration of 1 μg of antibody per 1 million cells in pbs with 0 . 25 % triton - x - 100 and 2 % donkey serum . cells were then incubated with species - specific alexa fluor - 488 conjugated secondary antibodies for 30 min , washed , and analyzed using a bd accuri c6 flow cytometer ( bd pharmingen ). in all experiments , nonspecific , species - appropriate isotype antibodies were used as controls . data analysis was performed using bd accuri c6 software . cb - ipsc6 . 2 - derived rcs were fixed in a cold , phosphate - buffered , 2 . 5 % glutaraldehyde / 2 % paraformaldehyde mixture , post - fixed in 1 % osmium tetroxide , dehydrated and embedded in epon 812 . semi - thin sections were cut for orientation , and ultrathin sections were cut and stained with uranyl acetate and lead citrate and examined using a transmission electron microscope ( hitachi h7600 ). cb - ipsc6 . 2 - derived rcs were placed in a 1 mm - gap electroporation cuvette with a plasmid solution ( 2 . 3 μg / μl of pcig plasmid expressing nuclear gfp 32 in pbs ) and 4 square pulses of 15 v , 50 - ms duration , and 950 - ms interval were delivered using an ecm 830 electroporation apparatus ( btx , holliston , mass ., usa ). immediately after electroporation , rcs were returned to the cell - culture incubator for 36 hr , at the end of which time - lapse microscopy imaging was performed at 2 - hr intervals for 48 hr using an lsm 710 confocal laser scanning system ( zeiss ) equipped with temperature and co 2 control . in room light , a cb - ipsc6 . 2 - derived rc ( age w25 - w27 ) was embedded in low - melting agarose gel and sliced into 100 - μm - thick slices with a vibratome ( leica vt1000s ). then , in darkness , the eyecup slices were transferred to rc culture medium containing 100 - μm 9 - cis - retinal ( a commercially available analog of 11 - cis - retinal ) and incubated for 1 hr in a light - proof , 95 % o 2 / 5 % co 2 cell - culture incubator at 37 ° c . afterwards , the rc slices , still under light - proof conditions , were transferred and mounted laterally in the recording chamber . all procedures afterwards were performed in infrared or dim - red light . perforated - patch recordings were performed at 35 - 37 ° c . on a zeiss upright microscope equipped with infrared dic optics and imaging . the bath solution ( ames medium equilibrated with 95 % o 2 / 5 % co 2 ) was temperature - controlled and ran at − 5 ml / min through the 1 - ml experimental chamber . all recordings were in the voltage - clamp mode with v hold at − 50 mv , low - pass filtered at 20 hz ( 8 - pole bessel ) and sampled at 500 hz . the pipette solution contained ( in mm ): 110 kcl , 13 nacl , 2 mgcl 2 , 1 cacl 2 , 10 egta , 10 hepes , 0 . 125 amphotericin b , ph 7 . 2 titrated with koh . the cells situated at the outer 1 - 4 layers of cells in the rc slice were chosen for recording because rhodopsin - positive photoreceptors were concentrated in this region . the recorded photoreceptor was stimulated with diffuse white flashes ( 40 - ms duration ) from a mercury arc lamp , attenuated with neutral density filters , with intensity calibrated with a radiometer . aggregates seeded on d7 appeared as colonies under adherent culture conditions . most colonies had clear boundaries before d20 . the percentage of nr domains expressing vsx2 was evaluated by counting the number of vsx2 - positive among dapi - positive colonies on d12 , d16 and d20 . colonies containing & gt ; 5 vsx2 - positive cells were considered nr domains . results represent the average of 3 independent experiments , ˜ 100 colonies per time - point , per cell line , per experiment . to trace the morphological progression of nr and rpe domains , plated aggregates were individually outlined using a microscope objective marker ( nikon ) and imaged every other day from d17 to d25 under an inverted microscope ( nikon ). rcs were imaged every 15 days from d45 until d120 under inverted microscope with 4 × magnification . the length of the longest axis of rcs was measured using image j . results represent the average of 15 - 20 rcs per time point . to approximate the time of generation of the major retinal neuronal cell types , a minimum of 5 rcs were collected each week from w5 to w13 , then every other week until w17 , and once a month thereafter . cell - type - specific markers were used for immunohistochemical identification as described above . 1 . hartong , d . t ., berson , e . l . & amp ; 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