Illumina HiSeq 4000 paired end sequencing; GSM3162655: Rebecca-Oligo_HMMKJ_L7_AAGAGGCA-ACTCTAGG; Homo sapiens; RNA-Seq

Rebecca-Oligo_HMMKJ_L7_AAGAGGCA-ACTCTAGG

We report a method for deriving oligodendrocyte lineage cells from human pluripotent stem cells (hPSCs) in three-dimensional (3D) culture called human oligodendrocyte spheroids (hOLS). To characterize oligodendrocyte-lineage cells in hOLS, we isolated O4+ cells by immunopanning and performed deep single cell RNA sequencing. We sequenced 295 cells and compared their profiles to unsorted cells isolated from primary human fetal cortex, primary human adult cortex, and hCS. Clustering of all cells using the t-distributed stochastic neighbor embedding (tSNE) approach revealed a distinct populations of SOX10+ oligodendrocytes, within which the O4+ cells derived from hOLS clustered most closely to oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes from the primary human adult cortical tissue. Additionally, subpopulations of OPCs, newly formed oligodendrocytes, and myelinating oligodendrocytes derived were observed in the hOLS-derived cluster. To further assess the state of oligodendrocyte-lineage cells in hOLS, we performed a Monocle analysis which revealed a spectrum of oligodendrocyte-lineage stages in hOLS ranging from dividing cells that closely resembled primary OPCs to mature cells that closely resembled primary oligodendrocytes. Overall design: Examination of gene expression in single oligodendrocyte-lineage cells derived from human pluripotent stem cells in three-dimensional culture

Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures

Submitted on 01-JAN-2019

We report a method for deriving oligodendrocyte lineage cells from human pluripotent stem cells (hPSCs) in three-dimensional (3D) culture called human oligodendrocyte spheroids (hOLS). To characterize oligodendrocyte-lineage cells in hOLS, we isolated O4+ cells by immunopanning and performed deep single cell RNA sequencing. We sequenced 295 cells and compared their profiles to unsorted cells isolated from primary human fetal cortex, primary human adult cortex, and hCS. Clustering of all cells using theÂ t-distributed stochastic neighbor embedding (tSNE) approach revealed a distinct populations of SOX10+ oligodendrocytes, within which the O4+ cells derived from hOLS clustered most closely to oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes from the primary human adult cortical tissue. Additionally, subpopulations of OPCs, newly formed oligodendrocytes, and myelinating oligodendrocytes derived were observed in the hOLS-derived cluster. To further assess the state of oligodendrocyte-lineage cells in hOLS, we performed a Monocle analysis which revealed a spectrum of oligodendrocyte-lineage stages in hOLS ranging from dividing cells that closely resembled primary OPCs to mature cells that closely resembled primary oligodendrocytes. Overall design: Examination of gene expression in single oligodendrocyte-lineage cells derived from human pluripotent stem cells in three-dimensional culture

Single cells were sorted into lysis buffer according to a published Smartseq2  protocol (Picelli, S. et al. 2014). ERCC RNA Spike-In Mix was added at 1: 2.4X10^7  dilution in the buffer. Sorted cells in 96-well plates were used for library preparation following the  Smartseq2 protocol (Picelli, S. et al. 2014) with modifications. Briefly, plates were  incubated at 72˚C, 3 min for annealing with Oligo-dT 30 VN. Reverse transcription  mixture was added 6ul per well, and RT was performed at 42˚C for 90 min, followed  by 70˚C, 5 min. To amplify the whole transcriptome, 15ul of DNA amplification  mixture was added and cDNA was amplified for 21 cycles. Amplified cDNA was then  purified using PCRClean DX beads. cDNA quality and quantity were assessed using a  Fragment Analyzer (AATI, High Sensitivity NGS Fragment Analysis Kit:1 bp - 6000  bp), and samples with concentrations below 0.08ng/ul or abnormal peak patterns  were filtered out. A total of 295 cDNA samples (retaining 88.9% of initially sorted)  were diluted down to 0.15ng/ul (only if above it) and consolidated into a 384-well  plate using a Mosquito X1 (TTP Labtech). Library preparation was performed using  Nextera XT DNA Sample Prep Kit (Illumina, Cat: FC-131-1096) and Nextera XT 384  dual index primers, with the help of a Mosquito HTS machine for liquid transfer.  Tagmentation was performed in 1.2ul at 55˚C for 10 min and amplification was  performed in 4ul system, 10 cycles. In the end, all samples were pooled (0.5ul each)  together into a 1.5ml Eppendorf tube, and the mixed libraries were purified twice  with PCRClean DX beads. The final sample was reconstituted in 50ul EB buffer, and  the concentration and peaks were measured by Qubit and Bioanalyzer before  Illumina sequencing (Hiseq). Single cell RNA-seq

Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures

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