E-MTAB-6680
Transcriptomic profiling of human neuromesodermal-like cells and spinal cord progenitors derived in vitro from H9 ES cell line
Human ES (H9) cells were directed towards a neuromesodermal progenitor-like cell state and these cells were then subsequently differentiated towards a neural cell fate. Human ES cells (H9) were differentiated into neuromesodermal progenitor-like cells by culturing in Neurobasal/1x N2/1x B27 medium (N2/B27) supplemented with 20 ng/ml bFgf and 3 µM CHIR99021 for 3 days and exposure to dual SMAD inhibition (dSMADi) (Noggin 50 ng/ml and the TGFb receptor type 1 inhibitor SB431542 10 µM) during day 3 (D3). Transcriptome analysis was then carried out following a selection procedure to enrich for NMP-like cells (sD3/NMP-like). This involved use of a hES (H9) cell line engineered with CRISPR-Cas9 to express GFP under the control of the endogenous Nkx1.2 promoter. At the end of day 3 cells were selected for high GFP expression, as high Nkx1.2 transcription is characteristic of NMP cell populations in mouse and chick embryos. These cells were then lysed and RNA extracted for RNASeq.   Humans ES cells (H9) differentiated into NMP-like cells as above (without selection) were also allowed to develop further on day 4 (in the presence of dSMADi and Retinoic acid (RA) 100nM, in N2/B27) and then in RA alone until end of day 8 (D8). These cells were then lysed and RNA extracted for RNASeq.
Human ES (H9) cells were directed towards a neuromesodermal progenitor-like cell state and these cells were then subsequently differentiated towards a neural cell fate. Human ES cells (H9) were differentiated into neuromesodermal progenitor-like cells by culturing in Neurobasal/1x N2/1x B27 medium (N2/B27) supplemented with 20 ng/ml bFgf and 3 µM CHIR99021 for 3 days and exposure to dual SMAD inhibition (dSMADi) (Noggin 50 ng/ml and the TGFb receptor type 1 inhibitor SB431542 10 µM) during day 3 (D3). Transcriptome analysis was then carried out following a selection procedure to enrich for NMP-like cells (sD3/NMP-like). This involved use of a hES (H9) cell line engineered with CRISPR-Cas9 to express GFP under the control of the endogenous Nkx1.2 promoter. At the end of day 3 cells were selected for high GFP expression, as high Nkx1.2 transcription is characteristic of NMP cell populations in mouse and chick embryos. These cells were then lysed and RNA extracted for RNASeq.   Humans ES cells (H9) differentiated into NMP-like cells as above (without selection) were also allowed to develop further on day 4 (in the presence of dSMADi and Retinoic acid (RA) 100nM, in N2/B27) and then in RA alone until end of day 8 (D8). These cells were then lysed and RNA extracted for RNASeq.