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Spatial and temporal development of Müller glial cells in hiPSC-derived retinal organoids facilitating the cell enrichment and transcriptome analysis

ABSTRACT: Müller glial cells (MGs) play important roles in human retina during physiological and pathological conditions. However, there are still many obstacles to obtain large numbers of human MGs in vitro so far, which hinder the further study of MGs. Human induced pluripotent stem cells (hiPSCs) have capacity to differentiate into almost all body cells, even develop complex, organized tissues, including retinal organoids (ROs) with all cell subtypes, providing many opportunities in study of retinal development and disorders. This study explored the development of MGs within hiPSC-derived ROs and the approach to isolate and expand these MGs. In ROs, MG precursors expressing SOX9 and Ki67 appeared since differentiation day 60 (D60), while SOX9+ CRALBP+ GS+ and Ki67- mature MGs developed from D150. MGs isolated from ROs aged older than 120 days could be expanded and exhibited a spindle-like morphology under adherent culture conditions. These expanded cells expressed MG specific markers SOX9, vimentin, nestin, CRALBP and GS, and responded to L-glutamate stimuli revealed by whole-cell patch-clamp recordings. They could be passaged several times, yielding large numbers of cells in a short period. In addition, they did not transdifferentiate into other types of retinal cells after subretinal transplantation in NOD/SCID mice. This study firstly clarified the timecourse of human MG development in ROs, and established a simple approach to expand and enrich these cells from ROs, paving the way for downstream investigation and application of human MGs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE188698 | GEO | 2022/06/08

REPOSITORIES: GEO

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Project description:Native host plant insect resistance in the maize inbred line Mp708 was developed by traditional plant breeding. Resistant Mp708 thwarts feeding by fall armyworm (Spodoptera frugiperda [J.E. Smith]; Lepidoptera: Noctuidae), numerous other lepidopteran pests, and the coleopteran western corn rootworm. This broad resistance makes it an excellent model for studying native host plant resistance mechanisms. In response to caterpillar feeding, Mp708 rapidly mobilizes Mir1-CP, a unique cysteine protease that appears to translocate from roots to the maize midwhorl where it accumulates. This accumulation correlates with a significant reduction in caterpillar growth resulting from diminished food utilization. In addition, the peritrophic membrane (PM) that surrounds the food bolus in the mudgut (MG) is severely damaged in caterpillars fed on sweet corn callus transformed to express the gene encoding Mir1-CP or on midwhorl tissue from resistant Mp708 maize. Functions of the PM include assisting digestion and protecting the epithelium of the caterpillar MG from physical and chemical damage. Consequently, the reduced growth of caterpillars that feed on Mp708 is probably due to the action of Mir1-CP on PM physiology. In fact, previous in vitro studies indicated that Mir1-CP was capable of permeabilizing the PM. The present study used both targeted (qRT-PCR) and global (mRNA-seq) transcriptome analyses to explore the effect of eating Mir1-CP expressing Mp708 maize on abundance of transcripts in the MG of fall armyworm larvae in comparison to MGs from larvae fed on susceptible Tx601 maize that does not express Mir1-CP. Expression of genes encoding proteins involved in PM production is upregulated in MGs from fall armyworm fed on Mp708. Also, several digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on Tx601. Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function.

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Spatial and temporal development of Müller glial cells in hiPSC-derived retinal organoids facilitating the cell enrichment and transcriptome analysis

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