Human iPS to neuron (Down's syndrome) 2: Difference between revisions
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|TCOverview=Human induced pluripotent stem cells can generate every cell type of the human body and under the appropriate conditions recapitulate central aspects of embryonic development in the dish. To interrogate the transcriptome changes associated with the earliest steps of human brain development as recapitulated with human pluripotent stem cells we generated footprint-free induced pluripotent stem cells (iPSC) from control and Down syndrome fibroblasts using episomal reprogramming [1] and were next stepwise differentiated these iPSc into neuro-ectodermal cells (day6), neural stem cells (day12) and early neuronal progenitors (day 18) using an established neuronal differentiation protocol [2].<br> | |TCOverview=Human induced pluripotent stem cells can generate every cell type of the human body and under the appropriate conditions recapitulate central aspects of embryonic development in the dish. To interrogate the transcriptome changes associated with the earliest steps of human brain development as recapitulated with human pluripotent stem cells we generated footprint-free induced pluripotent stem cells (iPSC) from control and Down syndrome fibroblasts using episomal reprogramming [1] and were next stepwise differentiated these iPSc into neuro-ectodermal cells (day6), neural stem cells (day12) and early neuronal progenitors (day 18) using an established neuronal differentiation protocol [2].<br> | ||
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References:<br> | |||
[1] Briggs, James A., et al. "Integration‐Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology." Stem Cells 31.3 (2013): 467-478.<br> | [1] Briggs, James A., et al. "Integration‐Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology." Stem Cells 31.3 (2013): 467-478.<br> | ||
[2] Chambers, Stuart M., Christopher A. Fasano, Eirini P. Papapetrou, Mark Tomishima, Michel Sadelain, and Lorenz Studer. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling." Nature biotechnology 27, no. 3 (2009): 275-280.<br> | [2] Chambers, Stuart M., Christopher A. Fasano, Eirini P. Papapetrou, Mark Tomishima, Michel Sadelain, and Lorenz Studer. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling." Nature biotechnology 27, no. 3 (2009): 275-280.<br> | ||
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Figure 1: Phase microscope images of neuronally differentiated hIPSC (C32 shown) and a graphical depiction of the timepoints where RNA was harvested for CAGE analysis.<br> | Figure 1: Phase microscope images of neuronally differentiated hIPSC (C32 shown) and a graphical depiction of the timepoints where RNA was harvested for CAGE analysis.<br> | ||
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References:<br> | |||
[1] Briggs, James A., et al. "Integration‐Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology." Stem Cells 31.3 (2013): 467-478.<br> | [1] Briggs, James A., et al. "Integration‐Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology." Stem Cells 31.3 (2013): 467-478.<br> | ||
[2] Chambers, Stuart M., Christopher A. Fasano, Eirini P. Papapetrou, Mark Tomishima, Michel Sadelain, and Lorenz Studer. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling." Nature biotechnology 27, no. 3 (2009): 275-280.<br> | [2] Chambers, Stuart M., Christopher A. Fasano, Eirini P. Papapetrou, Mark Tomishima, Michel Sadelain, and Lorenz Studer. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling." Nature biotechnology 27, no. 3 (2009): 275-280.<br> | ||
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Figure 3: CAGE analysis of DNMT3B, MAP2, PAX6, beta-III-tubulin and Oct4 expression by CAGE (TPM) in DS iPSC line C11 (A), DS iPSC line C18 (B), control iPSC line C11 (C) and control iPSc line C32 (D) during neuronal differentiation. Technical replicates shown in red, green and blue in each plot.<br> | Figure 3: CAGE analysis of DNMT3B, MAP2, PAX6, beta-III-tubulin and Oct4 expression by CAGE (TPM) in DS iPSC line C11 (A), DS iPSC line C18 (B), control iPSC line C11 (C) and control iPSc line C32 (D) during neuronal differentiation. Technical replicates shown in red, green and blue in each plot.<br> | ||
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References:<br> | |||
[1] Briggs, James A., et al. "Integration‐Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology." Stem Cells 31.3 (2013): 467-478.<br> | [1] Briggs, James A., et al. "Integration‐Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology." Stem Cells 31.3 (2013): 467-478.<br> | ||
[2] Chambers, Stuart M., Christopher A. Fasano, Eirini P. Papapetrou, Mark Tomishima, Michel Sadelain, and Lorenz Studer. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling." Nature biotechnology 27, no. 3 (2009): 275-280.<br> | [2] Chambers, Stuart M., Christopher A. Fasano, Eirini P. Papapetrou, Mark Tomishima, Michel Sadelain, and Lorenz Studer. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling." Nature biotechnology 27, no. 3 (2009): 275-280.<br> |
Revision as of 19:47, 10 December 2014
Series: | IN_VITRO DIFFERENTIATION SERIES |
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Species: | Human (Homo sapiens) |
Genomic View: | Zenbu |
Expression table: | [{{{tet_config}}} FILE] |
Link to TET: | [{{{tet_file}}} TET] |
Sample providers : | Christine Wells |
Germ layer: | {{{germ_layer}}} |
Primary cells or cell line: | {{{primary_cells}}} |
Time span: | {{{time_span}}} |
Number of time points: | {{{number_time_points}}} |
Overview |
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Human induced pluripotent stem cells can generate every cell type of the human body and under the appropriate conditions recapitulate central aspects of embryonic development in the dish. To interrogate the transcriptome changes associated with the earliest steps of human brain development as recapitulated with human pluripotent stem cells we generated footprint-free induced pluripotent stem cells (iPSC) from control and Down syndrome fibroblasts using episomal reprogramming [1] and were next stepwise differentiated these iPSc into neuro-ectodermal cells (day6), neural stem cells (day12) and early neuronal progenitors (day 18) using an established neuronal differentiation protocol [2]. |
Sample description |
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For this study three sample donors were used, two 2 control iPSC (C11 iPSC derived from CRL2429 Newborn Male Caucasian fibroblasts and C32 iPSC derived from CRL1502 12wk gestation Female Black fibroblasts) and two iPSC clones from one Down Syndrome individual (C11 and C18 from an Unknown Male Caucasian). Three replicates of each iPSc line were subjected to neuronal differentiation as described [1,2] and harvested at day 0, 6, 12, 18 of differentiation for RNA extraction (Fig 1 below). |
Quality control |
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Q-PCR validation of key neuronal marker genes was performed and published [1]. The data show upregulation of mRNA expression of the neuronal marker genes PAX6 [3] , beta-III-tubulin [4], SOX1 [5] , DCX [6], SOX9 [7], SOX2 [8] and MASH1 [9] (Fig 2C and D) and robust expression of PAX6, beta-III-Tubulin and MAP2 [10] protein expression (Fig 2A). we further expect that as cells exit from pluripotency that they will display a downregulation of the pluripotency transcription factor Oct4 [11] and the DNA methyl transferase DNMT3B [12]. |
Profiled time course samples
Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples
13457-144H1 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day00 | rep1 |
13458-144H2 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day06 | rep1 |
13459-144H3 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day12 | rep1 |
13460-144H4 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day18 | rep1 |
13461-144H5 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day00 | rep2 |
13462-144H6 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day06 | rep2 |
13463-144H7 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day12 | rep2 |
13464-144H8 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day18 | rep2 |
13465-144H9 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day00 | rep3 |
13466-144I1 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day06 | rep3 |
13467-144I2 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day12 | rep3 |
13468-144I3 | iPS differentiation to neuron, down-syndrome donor C18-CCL54 | day18 | rep3 |