概要: Human pluripotent stem cells (hPSCs) provide both powerful models for studying cellular differentiations, and unlimited sources of cells for regenerative medicine. However, a comprehensive single cell level differentiation roadmap for hPSCs has not been achieved yet. Here, we used high throughput single-cell RNA-sequencing (scRNA-seq) method, based on optimized microfluidic circuits, to profile early differentiation lineages in the human embryoid body (EB) system. We presented a cellular landscape for hPSC early differentiations covering different cellular lineages, including neural cell, muscle cell, endothelial cell, stromal cell, liver cell, and epithelial cell. Through pseudotemporal analysis, we constructed the differentiation trajectories of these progenitor cells and revealed the gene expression dynamics in the process of differentiation. We reset Primed H9 into Naïve-like H9 and studied cell state transition process via scRNA-seq. We found that mesendoderm genes are enriched in Naïve-like H9. Functionally, Naïve-like H9 showed better potency for differentiation into the hematopoietic lineages. We constructed the differentiation landscape of hPSC early differentiation by scRNA-seq analysis. We offer new insights into molecular pathways of early embryonic lineages that can be harnessed for optimization of differentiation protocols. (Pipeline for C1 data demultiplex : https://github.com/bioeauty/sccpipe)
项目整体设计: scRNA-seq analysis of naïve and primed human pluripotent stem cells and embryoid bodys
Primed H9 was maintained in mTeSR1 media (STEMCELL Technologies) on tissue culture plates coated with Matrigel (BD Bioscience). H9 was reset into a naïve-like state by RSeT media (STEMCELL Technologies). EBs were differentiated in DMEM/F12 (GIBCO) supplemented with 20% FBS (GIBCO), 50 U/mL penicillin/streptomycin (GIBCO), 2 mM L-Glutamine (GIBCO), 1 x non-essential amino acids, and 100 μM ß--mercaptoethanol (Sigma).
处理方案:
-
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C1 system and C1 high-throughput integrated fluidics circuits (HT IFCs) were used to capture single cells. Immediately after cell capture, lysis, reverse transcription and preamplification were performed in the HT IFCs.; mRNA Capture Beads (VAHTS mRNA-seq v2 Library Prep Kit for Illumina, Vazyme) were used to extract mRNA from total RNA.
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Samples harvested from HT IFCs were used to create libraries for Illumina sequencing with Illumina Nextera XT DNA Library kit.; PrimeScript™ Double Strand cDNA Synthesis Kit (TaKaRa) was used to synthesize double-stranded cDNA from purified polyadenylated mRNA templates. We used TruePrep DNA Library Prep Kit V2 for Illumina (TaKaRa) to prepare cDNA libraries for Illumina sequencing.
测序信息
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poly(A)+ RNA
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PAIRED
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Forward; -
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ILLUMINA
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Illumina HiSeq 2000
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Specific; Unspecific
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文章
Mapping human pluripotent stem cell differentiation pathways using high throughput single-cell RNA-sequencing.
