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Microarray Characterization of Human Embryonic Stem Cell–Derived Retinal Cultures

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Microarray Characterization of Human Embryonic Stem Cell–Derived Retinal Cultures Retinal Cell Biology Microarray Characterization of Human Embryonic Stem Cell–Derived Retinal Cultures Deepak A. Lamba1,2 and Thomas A. Reh2,3 PURPOSE. A number of protocols have been published to induce photoreceptor cells to cause significant visual impairment retinal determination from human embryonic stem cells (hESC) and blindness. The mammalian retina does not have an and induced pluripotent stem cells (iPSC). Although all these innate capacity to regenerate the dying cells (see Ref. 1 for studies have shown some degree of expression of markers of review); visual prosthetics or cell replacement strategies are retinal cells, fewer than 30 markers are typically used to char- therefore being pursued in an effort to restore some vision acterize the ESC-derived retinal cells. Hence, it is not known to the most severely affected patients. Human embryonic whether they express all the genes present in normal develop- stem cells (hESCs), because of their property of pluripo- ing retinal cells. To assess the efficiency of their retinal deter- tency, may have a significant role in cell replacement ther- mination protocol at the transcriptome level and to understand apies in the eye. However, this same property requires that the changes in human retinal gene expression patterns during the cells be directed in their differentiation to retinal cell development, the authors conducted a microarray-based anal- fates before use in therapies to minimize the risks of devel- ysis comparing human retina to hESC-derived retinal cells. oping teratomas. Several groups, including our own, have METHODS. The authors extracted total RNA from 60-day, 80-day, previously shown that a combination of signaling molecules and 96-day human fetal retina and hESC-derived retinal cells at can direct ESCs to retinal cell fates (see Discussion). The 3 weeks and 9 weeks after induction. RNA was subjected to retinal determination (RD) protocol we developed uses a analysis using a commercial microarray. Data were normalized combination of a BMP inhibitor (e.g., Noggin), a Wnt Inhib- using Affymetrix Power Tools and analyzed using commercial itor (DKK-1), and the growth factor IGF-1 (RD factors). With microarray software. the RD protocol we can specifically induce retinal determi- nation in hESCs,2 hiPSCs,3 and mouse ESCs (La Torre et al., RESULTS. On K-median clustering analysis, the authors found unpublished observations, 2010) with very high efficiency. that overall there was a very high correlation between genes We also found that photoreceptors from these hESC-derived expressed in human fetal retina and those in ESC-derived reti- retinal progenitors have the capacity to convey some light nal cultures. The cultures were at similar developmental ages responsiveness in a mouse model of Leber congenital amau- to the corresponding fetal retinal ages. They found only 1% of rosis4 showing in principle that cell-based replacement ther- the genes on the array to be expressed at a higher level in apy might be applicable to some forms of inherited retinal ESC-derived retinal cells than in fetal retina, and most of these degeneration. were expressed in the retinal pigment epithelium and ciliary Before these hESC-derived retinal cells can be used ther- epithelium. apeutically, they must be highly characterized. The presence CONCLUSIONS. In sum, gene array profiling provides an effec- of undifferentiated ESCs in a transplant may result in tera- tive method for characterization of the efficiency of directed toma formation in the recipient eye, whereas the presence differentiation of hESCs to retinal cells. (Invest Ophthalmol of other ESC-derived tissues, such as muscle or bone, would Vis Sci. 2011;52:4897–4906) DOI:10.1167/iovs.10-6504 likely interfere with normal visual function. In our earlier studies, we used quantitative PCR (QPCR) to assess the levels of markers of the various lineages. However, these ereditary and age-related photoreceptor degenerative studies provided only a limited view of the status of differ- Hdiseases can lead to the death of sufficient numbers of entiation of the population of cells treated with the RD factors. Here, we instead used cDNA microarrays to get a more global view of the efficiency of our RD protocol. We From the Departments of 1Ophthalmology and 3Biological Struc- performed microarray analysis on hESC-derived cells from ture and the 2Institute of Stem Cell and Regenerative Medicine, Uni- two different time points (3 weeks and 9 weeks) and on versity of Washington, Seattle, Washington. human fetal retinas from three different ages (60, 80, and 96 Supported by National Institutes of Health Grants 1 PO1 days after conception). This allowed us to compare the GM081619-01 and TA-CBT-0608-0464-UWA-WG (TAR). Human fetal efficiency of our protocol and to stage our cells with relation tissue was obtained through the Laboratory of Developmental Biology project supported by National Institutes of Health Award Number to human retinal development in vivo. Our results indicate 5R24HD000836 from the Eunice Kennedy Shriver National Institute of that most cells in the cultures treated with the RD protocol Child Health and Human Development. The content does not neces- develop as retinal progenitors and ultimately differenti- sarily represent the official views of the Eunice Kennedy Shriver Na- ate into retinal cells. Markers for nonocular tissues were tional Institute of Child Health and Human Development of the Na- absent or expressed at very low levels, though we did find tional Institutes of Health. evidence for the presence of pigmented epithelial cells and Submitted for publication August 31, 2010; revised December 1, ciliary epithelial cells in the cultures. This is the first study to 2010; accepted January 11, 2011. Disclosure: D.A. Lamba, None; T.A. Reh, None assess the purity of hESC-derived retinal cells at the tran- Corresponding author: Thomas A. Reh, Institute of Stem Cell and scriptome level, and it provides a baseline for comparison Regenerative Medicine, University of Washington, P.O. Box 357420, for future studies using other directed differentiation proto- Seattle, WA 98195; [email protected]. cols. Investigative Ophthalmology & Visual Science, June 2011, Vol. 52, No. 7 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. 4897 Downloaded from jov.arvojournals.org on 09/27/2021 4898 Lamba et al. IOVS, June 2011, Vol. 52, No. 7 METHODS mately 150 clumps/mL (100 cells/clump) in a six-well, ultra-low attach- ment plate (VWR, West Chester, PA). These EBs were cultured for 3 Cell Culture and Retinal Induction days in the presence of mouse Noggin (R&D Systems, Minneapolis, The H-1 (WA01) hESC line was obtained from the WiCell Research MN), human recombinant DKK-1 (R&D Systems), and human recom- Institute. The cells were cultured and passaged on a feeder layer made binant insulin-like growth factor-1 (IGF-1; R&D Systems). On the fourth of irradiated mouse embryonic fibroblasts in accordance with the day, EBs were plated onto poly-D-lysine-extracellular matrix (Matrigel; University of Washington Institutional Animal Care and Use Commit- Collaborative Research, Inc., Bedford, MA)–coated plates and cultured tee–approved protocols, as previously described.2 Retinal induction in the presence of DMEM/F12, B-27 supplement, N-2 supplement was performed as previously described. Briefly, embryoid bodies (EBs) (Invitrogen), mouse Noggin, human recombinant DKK-1, human re- were formed by treating undifferentiated hES cell colonies with type IV combinant IGF-1, and human recombinant basic fibroblast growth collagenase (Invitrogen, Carlsbad, CA) and resuspending approxi- factor (R&D Systems). The media were changed every 2 to 3 days for FIGURE 1. hESC-derived retinal cells at 3 weeks (A–C) and 9 weeks (D–L). At 3 weeks, RD cells expressed Sox2 (A, red), Pax6 (B, C, green), and HuC/D (C, red). (D, E) Floating retinal aggregates at 9 weeks with rosettes and RPE. On immunostaining, the centers of the rosettes express Sox2 (F, G, red), Sox9 (F, H, green), and Pax6 (F, I, white; F, merge). The rosettes also express Nrl (J, red) and Recoverin (K, red). (L) HuC/D (green) expressing ganglion and amacrine cells within and around Sox9-expressing neural progenitors (red) in rosettes. Downloaded from jov.arvojournals.org on 09/27/2021 IOVS, June 2011, Vol. 52, No. 7 ESC-Derived Retinal Cells 4899 up to 3 weeks. Thereafter, the cells were cultured in the media without Images were captured using a confocal microscope (Olympus, Tokyo, any growth factors. For further aggregate culture, rosette-like regions Japan). Image analysis was performed using confocal imaging software from the plate were manually transferred to low-attachment plates and (Volocity; Improvision; PerkinElmer, Waltham, MA) and graphics edit- allowed to self-aggregate and grow. Half the media were changed every ing software (Photoshop; Adobe, Mountain View, CA). 2 days. Microarray and QPCR Analysis Immunocytochemistry and Immunohistochemistry For microarray analysis, human eyes from fetal ages 60, 80, 96, and hESC-derived retinal aggregates were fixed with 4% paraformaldehyde, 145 days were obtained from the University of Washington’s Labo- embedded in OCT, and cryosectioned. The cells were analyzed with ratory of Developmental Biology without any identifiers. Fresh tis- the following antibodies: rabbit anti-recoverin (gift from Jim Hurley, sue was received within 2 to 3 hours postmortem, and the retina University of Washington), mouse anti-Hu C/D (Molecular Probes), was dissected of all extraocular tissue and retinal pigment epithe- mouse anti-Pax6 (DHSB), goat anti-Sox2, rabbit anti-Nrl (gift from lium (RPE) in cold HBSS. For RPE microarray, RPEs from the 145 Anand Swaroop), and rabbit anti-Sox9 (Abcam, Cambridge, MA). Sec- fetal day eye was used. The retinas were lysed in reagent (Trizol; ondary antibody staining was performed using the corresponding Al- Invitrogen). Similarly, 3-week hESC-derived retinal cells and retinal exa Fluor-488, Alexa Fluor -568, and Cy5 fluorescent-tagged antibodies aggregates at 9 weeks after retinal induction were lysed in reagent (Molecular Probes, Eugene, OR).
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