概要: Death of photoreceptors and/or Retinal Pigment Epithelium (RPE) cells is a common cause of age related and inherited retinal dystrophies, thus their replenishment from renewable stem cell sources is a well sought therapeutic goal. Human pluripotent stem cells provide a useful cell source in view of their limitless self-renewal capacity and potential to differentiate into all key retinal cell types either in isolation or as part of three dimensional retinal organoids. Photoreceptor precursors have been isolated from differentiating human pluripotent stem cells either through application of cell surface markers or fluorescent reporter approaches and shown to share a transcriptional profile akin to foetal photoreceptors. In this study we investigated the transcriptional profile of CRX+ photoreceptor precursors derived from human embryonic stem cells (hESC) using single cell RNA sequencing and their engraftment capacity in an animal model of retinitis pigmentosa (C3H/rd1). Single cell RNA seq analysis revealed the presence of dominant cell cluster which displayed the hallmarks of early cone photoreceptor expression. When transplanted subretinally into the C3H/rd1 mice, the Crx positive cells settled next to the inner nuclear layer of host retina, matured into cone photoreceptors and made connections with the inner neurones of the host retina. Cellular transfer between the host retina and donor photoreceptors was investigated and shown to be minimal. Together our data provide valuable molecular insights into the transcriptional profile of human pluripotent stem cells derived CRX+ photoreceptor precursors and indicate their usefulness as a source of transplantable cone photoreceptors.
项目整体设计: CRX-GFP human ESC line was differentiated to retinal organoids. At day 90 CRX+ and CRX- cells were purified by flow activated cell sorting and subjected to single cell RNA-seq. RNA-seq of bulk CRX+ and CRX- from the same experiment was carried out in parallel.
human ESC line (CRX-GFP) was expanded in mTeSR_1 (StemCell Technologies) at 37C and 5% CO2 on 6 well plates pre-coated with Low Growth Factor Matrigel (Corning) as described in our previous publication (Collin et al. 2015, , Collin et al.Stem Cells. 2016 Feb;34:311-21) ). Differentiation to reti-l organoids was performed as described in Mellough et al. 2015 (Stem Cells. 2015 Aug;33(8):2416-30) with minor modifications which included addition of 10M Y27632 dihydrochloride for the first 48 hours of differentiation.
处理方案:
Reti-l organoids at day 90 of differentiation were disassociated to single cells using the embryoid body disassociation kit (Miltenyi Biotech) following the manufacturer__ instructions. CRX-GFP+ cells were sorted using a FACS-ARIA flow cytometric activated cell sorter (BD Biosciences). Single cells were loaded onto the C1 Single-Cell Auto Prep fluidic circuit (Fluidigm). Cell capture efficiency was assessed using the Zeiss Axiovert imaging system and empty sites, or sites with more than one cell were excluded from further a-lysis. Cell lysis, reverse transcription, cDNA amplification were performed using the SMARTer PCR cDNA synthesis kit (Clontech).
提取方案:
Full length cDNA libraries were prepared using the Illumina Nextera XT DNA library preparation kit (Illumina). ExteRNAl RNA Control Consortium (ERCC) RNA spike-in Mix_ (Ambion) was added to each cell sample prior to sequencing. Libraries were multiplexed as 24-48 libraries per lane and 75 bp single end reads were sequenced on the Ilumi- HiSeq 2500.
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测序信息
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poly(A)+ RNA
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PAIRED
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ILLUMINA
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Illumina NextSeq 500
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Unspecific
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文章
CRX Expression in Pluripotent Stem Cell-Derived Photoreceptors Marks a Transplantable Subpopulation of Early Cones.
