Amélie Slembrouck

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

Significance Human induced pluripotent stem cells (hiPSCs) could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases. The production of retinal cells from hiPSCs for personalized therapeutic approaches must comply with certain criteria, such as safety, efficiency, reproducibility, and low production cost. Here, we report a simple and scalable retinal differentiation process for the generation of retinal pigmented epithelial cells and neural retinal tissues containing retinal progenitor cells. These progenitors can be differentiated into all retinal cell types, including retinal ganglion cells and precursors of photoreceptors, which could find important applications in regenerative medicine. This method also provides an accessible in vitro model to investigate mechanisms involved in human retinogenesis and retinal diseases. Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.

Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all horizontal cell subtypes in the chick retina.

During development, progenitor cells of the retina give rise to six principal classes of neurons and the Müller glial cells found within the adult retina. The pancreas transcription factor 1 subunit a (Ptf1a) encodes a basic-helix-loop-helix transcription factor necessary for the specification of horizontal cells and the majority of amacrine cell subtypes in the mouse retina. The Ptf1a-regulated genes and the regulation of Ptf1a activity by transcription cofactors during retinogenesis have been poorly investigated. Using a retrovirus-mediated gene transfer approach, we reported that Ptf1a was sufficient to promote the fates of amacrine and horizontal cells from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina. Both GABAergic H1 and non-GABAergic H3 horizontal cells were induced following the forced expression of Ptf1a. We describe Ptf1a as a strong, negative regulator of Atoh7 expression. Furthermore, the Rbpj-interacting domains of Ptf1a protein were required for its effects on cell fate specification. Together, these data provide a novel insight into the molecular basis of Ptf1a activity on early cell specification in the chick retina.

Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno‐Free and Feeder‐Free Conditions

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two‐step xeno‐free/feeder‐free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self‐forming neuroretinal‐like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation‐compatible CD73+ photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC‐derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73+ photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP‐compliant retinal cell manufacturing protocol allowing large‐scale production and banking of hiPSC‐derived retinal cells and tissues. Stem Cells 2017;35:1176–1188

Involvement of Bcl-2-Associated Transcription Factor 1 in the Differentiation of Early-Born Retinal Cells

Retinal progenitor proliferation and differentiation are tightly controlled by extrinsic cues and distinctive combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we have characterized Bcl-2-associated transcription factor (Bclaf1) during rodent retinogenesis. Bclaf1 expression is restricted to early-born cell types, such as ganglion, amacrine, and horizontal cells. Analysis of developing retinas in Bclaf1-deficient mice revealed a reduction in the numbers of retinal ganglion cells, amacrine cells and horizontal cells and an increase in the numbers of cone photoreceptor precursors. Silencing of Bclaf1expression by in vitro electroporation of shRNA in embryonic retina confirmed that Bclaf1 serves to promote amacrine and horizontal cell differentiation. Misexpression of Bclaf1 in late retinal progenitors was not sufficient to directly induce the generation of amacrine and horizontal cells. Domain deletion analysis indicated that the N-terminal domain of Bclaf1 containing an arginine-serine-rich and a bZip domain is required for its effects on retinal cell differentiation. In addition, analysis revealed that Bclaf1 function occurs independently of its interaction with endogenous Bcl-2-related proteins. Altogether, our data demonstrates that Bclaf1expression in postmitotic early-born cells facilitates the differentiation of early retinal precursors into retinal ganglion cells, amacrine cells, and horizontal cells rather than into cone photoreceptors.

Generation of an induced pluripotent stem cell (iPSC) line from a patient with autosomal dominant retinitis pigmentosa due to a mutation in the NR2E3 gene

A human iPSC line was generated from fibroblasts of a patient affected with autosomal dominant Retinitis Pigmentosa (RP) carrying the mutation p.Gly56Arg in the NR2E3 gene. The transgene-free iPSCs were generated with the human OSKM transcription factors using the Sendai-virus reprogramming system. iPSCs contained the expected c.166G>A substitution in exon 2 of NR2E3, expressed the expected pluripotency markers, displayed in vivo differentiation potential to the three germ layers and had normal karyotype. This cellular model will provide a powerful tool to study the pathogenesis of NR2E3-associated RP. Resource table.

The protein tyrosine phosphatase interacting protein 51 (PTPIP51) is required for the differentiation of photoreceptors

Proliferation and differentiation of retinal progenitor cells (RPCs) are tightly controlled by extrinsic cues and distinct combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we investigated the role of the protein tyrosine phosphatase interacting protein 51 (PTPIP51) in the differentiation of RPCs. The expression pattern of PTPIP51 was analyzed by immunostaining during post-natal retinal development in the rat. Ex vivo electroporation has been used to silence or misexpress PTPIP51 in post-natal retinal explants, and the retinal phenotype was investigated after 3-7days in vitro (div). PTPIP51 expression in the retina started postnatally and was maintained throughout adulthood, especially in retinal ganglion cells and in the inner segment of photoreceptor cells. Silencing of Ptpip51 expression in postnatal retina failed to modify the commitment of late RPCs in the different lineages but severely impaired the final differentiation of photoreceptors, observed by a decrease in the fraction of Rhodopsin-positive cells after 7div. By contrast, misexpression of PTPIP51 in early or late RPCs failed to modify the differentiation of the RPCs. Our data demonstrate that PTPIP51 is implicated in the differentiation process of immature photoreceptors. Because PTPIP51 is specifically localized in the inner segment, PTPIP51 may contribute to the complex stage of maturation of the apical segment of these cells.

Establishment of an induced pluripotent stem (iPS) cell line from dermal fibroblasts of an asymptomatic patient with dominant PRPF31 mutation

A human iPS cell line was generated from fibroblasts of a phenotypically unaffected patient from a family with PRPF31-associated retinitis pigmentosa (RP). The transgene-free iPS cells were generated with the human OSKM transcription factors using the Sendai-virus reprogramming system. iPS cells contained the expected c.709-734dup substitution in exon 8 of PRPF31, expressed the expected pluripotency markers, displayed in vivo differentiation potential to the three germ layers and had normal karyotype. This cellular model will provide a powerful tool to study the unusual pattern of inheritance of PRPF31-associated RP.