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. 2011 Apr 29;6(4):e18992.
doi: 10.1371/journal.pone.0018992.

Transplantation of Adult Mouse iPS Cell-Derived Photoreceptor Precursors Restores Retinal Structure and Function in Degenerative Mice

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Free PMC article

Transplantation of Adult Mouse iPS Cell-Derived Photoreceptor Precursors Restores Retinal Structure and Function in Degenerative Mice

Budd A Tucker et al. PLoS One. .
Free PMC article

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Abstract

This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs), could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characterization of adult mouse dsRed-iPS cells.
A–L: Microscopic/Immunocytochemical analysis comparing ES and dsRed-iPS cell morphology (A, G) and expression of the pluripotency markers Oct4 (B, H), c-MYC (C, I), Sox2 (D, J), Nanog (E, K) and Klf4 (F, L). M: Focused microarray analysis performed on RNA isolated from both ES and dsRed-iPS cells for the expression of genes known to be associated with pluripotency. N–S: Histological analysis of dsRed-iPS cell generated teratomas (N) for production of cells/tissues specific to ectodermal (O&P) mesodermal (Q&R) and endodermal (S) germ layers. Scale Bar = 100 µm.
Figure 2
Figure 2. Differentiation of dsRed-iPS cells into retinal photoreceptors using defined culture conditions.
A: Schematic diagram illustrating the differentiation paradigm utilized in this study. B: Focused microarray analysis of undifferentiated (D0) and differentiated (D33) dsRed-iPS cells for pluripotency (Sox2, Klf4, c-MYC, Nanog and Eras), retinal progenitor (Chx10 and Lhx2) and retinal photoreceptor (CRX, recoverin, rhodopsin, blue-opsin, red/green-opsin and ROM-1) gene expression. C: RT-PCR analysis of undifferentiated (D0) and differentiated (D33) dsRed-iPS cells for expression of the retinal specification/photoreceptor genes Pax6, RX, NRL, cone-opsin, rhodopsin, recoverin and CRX (GAPDH was including as an internal control). D: Western blot analysis of D0 undifferentiated, D5 embryoid bodies and D33 differentiated dsRed-iPS cells for expression of the retinal photoreceptor markers CRX, recoverin and rhodopsin. E–G: Immunocytochemical analysis of photoreceptor marker (CRX, recoverin and rhodopsin) expression. H: Average number of cells expressing the photoreceptor markers CRX, recoverin and rhodopsin at D33 post-differentiation. I–J: Intracellular Ca2+ levels as a function of time (I) and peak (J) intracellular Ca2+ concentrations for D0 undifferentiated and D33 differentiated iPS cells in response to 1 nM glutamate stimulation. Scale bar = 25µm. *p<0.05, **p<0.001. ***p<0.0001.
Figure 3
Figure 3. Transplantation of SSEA1- dsRed-iPS derived photoreceptor precursor cells induces retinal outer nuclear layer repopulation.
A: Histological staining of a teratoma containing Rho-/- eye at 21 days post-injection of a heterogeneous population of SSEA1-containing D33 differentiated cells. B: Percentage of animals developing teratomas after receiving either heterogeneous undepleted SSEA1-containing (n = 5), SSEA1-enriched (n = 5), one round of SSEA1-depleted (n = 10) or two round of SSEA1-depleted cell transplants at 21 days post injection. C: Number of animals to and time taken for the development of teratomas in animals receiving either SSEA1-enriched or SSEA1-depleted cell transplants over a 16 week post-op period. D–L: Immunocytochemical analysis performed on rho-/- retinal degenerative eyes 21 days after receiving subretinal injections of SSEA1-depleted cells targeted against expression of the photoreceptor markers recoverin (E), rhodopsin (F), ROM-1 (G&H) and opsin (I), the synapse markers synaptophysin (J) and bassoon (K&L), the bipolar cell marker PKCα (K&L) and the iPS cell marker dsRed (D–J). TP = transplant site. Scale bar = 50 µm.
Figure 4
Figure 4. Transplantation of SSEA1-dsRed-iPS derived photoreceptor precursor cells induces increased electroretinal function as determined by ERG and light induced c-Fos expression.
A–C: Representative ERG (A, 5 db flash under scotopic conditions), average peak b-wave amplitudes for rho-/- mice 21 days after receiving subretinal SSEA1-depleted cell injections (B, n = 6 of 10 animals that received transplants, only animals with recovery in ERG response above baseline were chosen for this analysis, the 4 animals that were not chosen were found to have poor cellular integration due to extensive cell death post- transplantation), and correlation between peak b-wave amplitude and iPS cell derived photoreceptor layer repopulation (C, n = 10). D–E: Immunocytochemical analysis targeted against the immediate early gene c-Fos and the iPSC marker dsRed performed on rho-/- transplant and contralateral control mouse eyes at 21 days post-subretinal injection of SSEA1-negative dsRed-iPS derived photoreceptor precursor cells. F: Number of cells per microscopic section expressing c-Fos. A significant increase in the number of cells within the retinal inner nuclear layer expressing c-Fos was detected in Rho-/- eyes that had received subretinal injections of SSEA1-negative dsRed-iPSC derived photoreceptor precursor cells as compared to contralateral control eyes following light exposure. Scale bar = 50 µm.

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