Stem Cell Reports. 2014 May 22;2(6):853-65. doi: 10.1016/j.stemcr.2014.04.010.

Derivation of Traceable and Transplantable Photoreceptors from Mouse Embryonic Stem Cells

Sarah Decembrini and Yvan Arsenijevic

Unit of Gene Therapy & Stem Cell Biology, Jules-Gonin Eye Hospital, University of Lausanne, Switzerland.

 

Abstract

Retinal degenerative diseases resulting in the loss of photoreceptors affect millions of people worldwide. Up to now, although gene therapy applied at the very early stage of the disease has been shown to slow down the degeneration process, it has been proved to be less effective with advanced retinal diseases, in which a remarkable photoreceptor loss irreversibly modifies the retina architecture. Therefore alternative therapies to treat late-stage diseases have been developed. Stem cell-based therapy is currently pursued as a potential approach in the treatment of advanced retinal degenerative diseases, since photoreceptor cells collected directly from new-born mice and transplanted into adult wild-type retinas restore photosensitivity as well as some visual function in mouse models of retinal degeneration (Pearson et al., 2013). As in prospective, the human fetal retina is not a suitable source of photoreceptors, other sources of cells have been considered. Among them, pluripotent stem cells (ESCs and iPSCs) have emerged as renewable source for the production of retinal cell types including photoreceptors.Our study provides a proof-of-principle that ESC-derived rod photoreceptors can integrate adult mouse retinas after transplantation. In order to produce an unlimited number of transplantable rods, a photoreceptor-specific embryonic stem cell (ESC) line has been derived from the Crx-GFP mouse strain originating from Cepko’s laboratory (Samson et al., 2010). The isolation of the Crx-GFP ESC line together with the development of a fine-tuned 3D culture system provides a unique tool to follow mammalian retinogenesis, to produce a large number of photoreceptors recapitulating the in vivo retinogenesis and to trace the transplantation of in vitro-derived photoreceptor cells. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. Moreover, integrated rods show a directional light-mediated translocation of phototransduction pathway proteins indicating that the cells respond to light stimuli. The colocalization of the presynaptic ribbon synapses with the postsynaptic endogenous rod bipolar dendrite terminals suggests a correct integration into the recipient retina.

Taken together, the results described in our manuscript provide a comprehensive study showing unambiguously that postmitotic photoreceptors generated from ESC-derived retina tissues can morphologically integrate adult recipient retinas.

PMID: 24936471

 

Supplement

During the last 10 years, the transplantation of rod photoreceptors derived from mouse retinas has been extensively investigated as an alternative therapy to treat advanced retinal diseases with extensive loss of photoreceptor cells. The grounds on which this therapy is built are essentially two: the inefficacy of the gene therapy to treat late-stage diseases by gene replacement and the impossibility for the mammalian retinas to regenerate lost photoreceptors spontaneously. Because the developing retina is not a suitable and renewable source of photoreceptors, many groups focused their research on pluripotent stem cells in view of their unlimited potential to supply the desired cell phenotype. In order to follow photoreceptor development and to trace cells after transplantation, we decided to derive a photoreceptor-specific ESC line from a transgenic mouse expressing GFP under the control of the endogenous photoreceptor-specific promoter Crx (Samson et al., 2009). To generate the ESC lines, blastocysts were isolated from a Crx-GFP-positive female 3.5 days postcoitum. Importantly, similarly to the Crx protein, the GFP is expressed in photoreceptors throughout the retinogenesis. This line was isolated mostly because previous studies on photoreceptor transplantation are based on viral vectors to label ESCs or photoreceptor progenitors prior to transplantation. As known this procedure may promote the generation of false-positive results by enabling the transduction of recipient-retina photoreceptors and can cause genotoxicity.

In parallel, we fine-tuned a 3D culture system, recently published in 2011 by Sasai’s group (Eiraku et al., 2011) allowing the self-generation of mouse optic cups in a dish (Figure 1). The choice of the 3D versus 2D culture system was due mostly to the high and reliable yield of photoreceptor cells achieved with the 3D culture. These cells have been extensively characterized and express all the markers and features borne by integration-competent photoreceptors. The 3D culture system established in this study is based on a multi-step protocol able to recapitulate the key morphogenetic phases that bridge mouse embryonic development to the retinogenesis. The first step involves the formation of aggregates by seeding 3×105 pluripotent cells in a low-binding 96-well plate. Then, a combination of matrix proteins and exogenous factors such as those contained in Matrigel or more simply by adding Entactin/Laminin and Nodal, steers toward the neuroepithelium specification from which one or more optic vesicles first evaginate and then rapidly invaginate to form the optic cups. This last tissue will form the retina. The generation of retinal cells and photoreceptor maturation is headed by the use of medium supplements like B27 and N2 along with the use of hyperoxic conditions (40% O2) applied within a specific time window corresponding to the photoreceptor differentiation. The oxygen supply and conditions established in this paper induce a better environment to sustain the photoreceptor survival in vitro and probably mimic the high oxygen level existing at the choroid proximity.

 

 

DS fig1DS fig2

Fig 1. Schematic of the 3D culture system leading the retina-like structure formation.

A) Crx-GFP mESC line on MEF and (B) on gelatine. (C) ESC dissociation in trypsin-EDTA. (D) Cell seeding in specific medium conditions to form aggregates in 96-low binding plates. (E) Induction of a neuroectodermal ring in the aggregate border (in grey) followed by optic vesicle evaginations (F) optic vesicles in grey and mother aggregate in blue. (G-H) Optic vesicle invagination to form the optic cups. (I) Picture of a well-structured retina bearing lens. White harrow and dashed line indicate the lens. Photoreceptors are GFP-positive.

 

All the ameliorations performed in this work allowed a reproducible and robust production of retina-like tissues containing multilayered photoreceptor rows. From eight up to ten million Crx-GFP photoreceptor cells were routinely generated and simply harvested from a 96-multiwell plate.

The competence of ESC-derived photoreceptors to morphologically integrate adult NOD/SCID retinas after transplantation was tested by grafting Crx-GFP flow-sorted cells derived from retina-like structures into adult recipient mice.

As the ESC line derived in this paper allows the tracing of photoreceptor development, we evaluated the most appropriate developmental stage for transplantation, by grafting photoreceptors collected from cultures at three differentiation time points (days 22, 25, and 30). The analysis of the transplant was performed 3 weeks after the injection. A different integration competence was observed between photoreceptors collected at days 22, 25, and 30 of culture. Photoreceptors at day 25 showed the highest integration competence (around 799 ± 73 cells per retina). Integrated photoreceptors were well orientated within the ONL of the recipient retina and showed the typical morphology of mature rods bearing external segments and ribbon synapses (Figure 2). Moreover, integrated rods showed a certain degree of functionality displaying a directional light-mediated translocation of phototransduction pathway proteins from the cell body towards the outer segment or vice versa. The colocalization of the presynaptic ribbon synapses with the postsynaptic endogenous rod bipolar dendrite terminals suggests signal transmission between the cells, but this remains to be demonstrated functionally. Day 25 ESC-derived photoreceptors resemble rods collected directly from PN4 retinas known to be the best cell source for transplantation (MacLaren et al., 2006; Pearson et al., 2013)

 

DS fig3

Fig 2. Adult NOD-SCID mouse retina transplanted with ESC-derived photoreceptors after 25 days of culture.

A) Section of a ESC-derived retina after 25 days of culture. (B) Adult recipient retinas showing integrated photoreceptors (in green) bearing external segments and spherule synapses (white arrows). Dapi staining in blue. GFP-positive cells in green. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. A) Scale bar, 15 um (B) 30um.

 

We conclude that a 3D protocol coupled with ESCs provides a favourable situation to recapitulate the different steps of retinogenesis allowing the generation of retinal cells closely resembling natural ones, thus opening a variety of application fields.

Perspectives: the ability and efficacy of the 3D culture system to generate transplantation-competent photoreceptors now need to be validated in a functional test paradigm to evaluate whether these cells can adequately mediate light signals and restore a visual function. In the future, the transplantation of human pluripotent stem cell-derived photoreceptors will provide the proof-of-principle of the cell replacement therapy feasibility in human. In prospect, the development of other breakthrough techniques such as the iPSCs and CRISPR combined with the use of an appropriate 3D culture system will serve regenerative medicine by encouraging cell grafting, disease modelling, and drug screening.

The importance of this study is two-fold

Firstly, the present work shows that the 3D culture protocol established to generate whole retinas allows the recapitulation of mammal retinogenesis in vitro and therefore offers the possibility to follow photoreceptor development in a dish. Furthermore, the possibility carried by the Crx-GFP ESC line to trace photoreceptors offers the opportunity to characterize cells at different stages of their development bringing new prospects for cell biology investigations

Secondly, the Crx-GFP ESC line along with the 3D protocol enables the tracing of transplanted cells. By these means, morphologically integrated GFP-positive photoreceptors revealed a correct maturation after transplantation into adult retinas.

 

References

  1. Pearson RA, Barber AC, Rizzi M, Hippert C, Xue T, West EL, et al. Restoration of vision after transplantation of photoreceptors. Nature 2012;485:99-103.
  2. Samson M, Emerson MM, Cepko CL. Robust marking of photoreceptor cells and pinealocytes with several reporters under control of the Crx gene. Developmental dynamics : an official publication of the American Association of Anatomists 2009;238:3218-25.
  3. Eiraku M, Takata N, Ishibashi H, Kawada M, Sakakura E, Okuda S, et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 2011;472:51-6.
  4. MacLaren RE, Pearson RA, MacNeil A, Douglas RH, Salt TE, Akimoto M, et al. Retinal repair by transplantation of photoreceptor precursors. Nature 2006;444:203-7.

 

Acknowledgements: This study was supported by Novartis Stiftung, Velux foundation, Provisu Foundation and Fondation Asile des Aveugles (Lausanne).

 

Contacts

Sarah Decembrini, PhD
Unit Gene Therapy & Stem Cell Biology Université de Lausanne Hôpital Jules-Gonin 15, av. de France 1004 Lausanne Tel .+4121 626 8515 Fax: +4121 626 88 88

Prof. Yvan Arsenijevic, PhD
Head of the Unit of Gene Therapy & Stem Cell Biology, Service d’Ophtalmologie, Université de Lausanne, Hôpital Jules-Gonin 15, av. de France, 1004 Lausanne. Tel .+4121 626 82 60; Fax: +4121 626 88 88; Email: yvan.arsenijevic@fa2.ch

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