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

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. Overall design: hiPSCs (N>1×10^5 cells per experiment, 2 independent experiments), different stage ROs (N=3~5 ROs per experiment, 2 independent experiments), different passage MGs from D150-ROs (n>1X10^6 cells per experiment, 3 independent experiments) were collected and sent to Novogene Biotechnology Co. (Beijing, China) and Gene Denovo Biotechnology Co. (Guangzhou, China) for RNA extraction, library construction, sequencing and data analyses. Total RNA was extracted using Trizol reagent kit (Invitrogen) according to the manufacturer's protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Oligo (dT) beads were used to isolate the poly mRNA from the total RNA. The enriched mRNA was fragmented and reverse transcribed into cDNA using random primers. After synthesis of the second strand, the cDNA was purified, end-repaired and ligated to Illumina sequencing adapters. The ligation products were size selected, amplified, and sequenced. Raw reads were filtered and the clean reads were obtained. Afterwards, an index of the reference genome was built, and paired-end clean reads were mapped to the reference genome using HISAT2. The mapped reads of each sample were assembled by using StringTie. Gene expression levels were quantified, and for each transcription region, a fragment per kilobase of transcript per million mapped reads (FPKM) value was calculated to quantify its expression abundance and variations. Differential expression analysis was performed by DESeq2 software between two different groups. The genes/transcripts with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change = 2 was considered differentially expressed genes/transcripts. Co-analyses of these data were performed by Gene Denovo Biotechnology Co. (Guangzhou, China) using a quarter median correction approach to reduce the error from different batches of data.