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Nicotinamide promotes formation of retinal organoids from human pluripotent stem cells via enhanced neural cell fate commitment

ABSTRACT: Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) recapitulate key features of human retinogenesis and thus provide a promising platform to study the mechanisms of retinal development and disease in a human context and to evaluate therapies. Although multiple differentiation protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs thus limiting their utility in research. To improve the efficiency and robustness of RO generation, we set out to identify factors that promote neural retina differentiation from hPSCs. We report here that nicotinamide (NAM) treatment at the early stage of differentiation significantly improved RO yield across 8 hPSC lines from different donors. Importantly, NAM treatment enabled efficient production of ROs from cell lines that would otherwise fail to generate meaningful number of ROs. Further analyses revealed that NAM treatment promotes neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field commitment and RO generation. This effect is mediated, at least in part, through inhibition of BMP signaling. Thus, our modified protocol with a simple NAM treatment improves the yield of ROs in all lines tested, and importantly greatly benefits to cell lines that previously proved to be intractable in retinal differentiation. Our data should facilitate the broader use of human ROs for disease modeling applications requiring the use of multiple cell lines or a larger scale of cell source such as therapy screening.

ORGANISM(S): Homo sapiens

PROVIDER: GSE164884 | GEO | 2022/06/14

REPOSITORIES: GEO

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Project description:Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However the effective use of hPSCs for cell therapy has lagged far behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons derived from human PSCs generally display poor in vivo performance. There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor plate-based strategy for the derivation of human DA neurons that efficiently engraft, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor plate precursors are derived from hPSCs in days following exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signaling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive in vitro molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of hPSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in PD animal models in three host species. Long-term engraftment in 6-OHDA-lesioned mouse and rats demonstrates robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into Parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models tested indicate considerable promise for the development of cell based therapies in PD. Differentiated hESC with three conditions (LSB, LSB/S/F8, LSB/S/F8/CHIR) were subjected to RNA extraction in specific timepoint (day 0, 1, 3, 5, 7, 11, 13, 25) and hybridization on Illumina microarrays. Each sample has 3 or 4 biological repeats. Based on previous study* of dual SMAD inhibition neural induction, we developed new midbrain dopamine neuron protocol. It depends on time specific treatment of below factors (LSB/S/F8/CHIR): L (LDN193189 (BMP inhibitor) , day 0-11), SB (SB431542 (TGF-b signal inhibitor), day 0-5), S (SHH + Purmorphamine (Smo agonist), day 1-7), F8 (FGF8, day 1-7) and CHIR (CHIR99021 (GSK3b inhibitor), day 3-13) LSB and LSB/S/F8 are limited control conditions of dual SMAD only (LSB) or traditional patterning with Sonic and FGF (LSB/S/F8) *Chambers,S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275-280 (2009).

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Nicotinamide promotes formation of retinal organoids from human pluripotent stem cells via enhanced neural cell fate commitment

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