Personal tools

Human iPS to neuron (Down's syndrome) 2

From FANTOM5_SSTAR

Jump to: navigation, search

Series:IN_VITRO DIFFERENTIATION SERIES
Species:Human (Homo sapiens)
Genomic View:Zenbu
Expression table:TET
Link to TET:FILE
Sample providers :Christine Wells
Germ layer:ectoderm
Primary cells or cell line:primary cells
Time span:18 days
Number of time points:4


Overview

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].

References:
[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.
[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.

Sample description

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).

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.

References:
[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.
[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.

Quality control

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].



Figure 2: Immunofluorescent detection of neuronal marker proteins beta-III-tubulin, MAP2 and PAX6 in neuronally differentiated hIPSc cultures (A) and Q-PCR mRNA expression quantification of key neuronal genes (B and C) in these samples. Reproduced from [1].

Sample culture information:

hIPS cell culture

Human iPS cells were cultured under feeder-free culture conditions on Matrigel (1:50 dilution BD Biosciences) with mouse embryonic fibroblast conditioned DMEM/F12 culture medium supplemented with 20% KnockOut serum replacement (KOSR), 0.1 mM non-essential amino acids, 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol in the presence of 100 ng/ml human bFGF. Medium was exchanged daily. These cells were next FACS sorted for the cell surface marker TRA160, to ensure isolation of RNA from 100% undifferentiated cells at timepoint 0.

Neural differentiation of hIPS cells

hiPSC cultures were grown for ~5 days after mechanical passage in conditioned medium (as above), and changed directly into KOSR supplemented with 10 μM SB431542 (Sigma) and 5 μM dorsomorphin (Stemgent) for the first 6 and 12 days of differentiation, respectively, with media changes every 2 days to initiate neural conversion. KOSR was gradually substituted with N2B27 medium (Neurobasal medium supplemented with Glutamax, N2 and B27 supplements (all from Gibco)): 25%, 50%, 75% and 100% N2B27 in KOSR on days 4, 6, 8 and 10 respectively. Neurospheres were formed on day 6 of differentiation by 10 min incubation in 1 mg/ml Collagenase IV (Gibco) at 37°C and dislodging of large pieces of colonies by use of a cell scraper and P1000 pipette. Neuralized colony fragments were seeded into Ultra-low Cluster plates (Costar) where they aggregated into tight spheres. Neurospheres were expanded in Ultra-low Cluster plates until day 12 and seeded onto Matrigel (BD), and N2B27 media was changed every 3 days. Adherent cultures were passaged every 4-5 days by cell dissociation buffer (Sigma) at a 1:2 – 1:3 ratio until day 18.

CAGE data

Figure 3 A-D shows the CAGE expression of DNMT3B, MAP2, PAX6, beta-III-tubulin and Oct4 in the two two DS iPSC (A and B) and control iPSC (C and D) subjected to neuronal differentiation. The expected down regulation of the pluripotency markers and upregulation of neuronal identity genes is observed.

Figure 3A


Figure 3B


Figure 3C


Figure 3D

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.

References:
[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.
[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.
[3] Heins, Nico, Paolo Malatesta, Francesco Cecconi, Masato Nakafuku, Kerry Lee Tucker, Michael A. Hack, Prisca Chapouton, Yves-Alain Barde, and Magdalena Götz. "Glial cells generate neurons: the role of the transcription factor Pax6." Nature neuroscience 5, no. 4 (2002): 308-315.
[4] Reubinoff, Benjamin E., Pavel Itsykson, Tikva Turetsky, Martin F. Pera, Etti Reinhartz, Anna Itzik, and Tamir Ben-Hur. "Neural progenitors from human embryonic stem cells." Nature biotechnology 19, no. 12 (2001): 1134-1140.
[5] Pevny, Larysa H., Shantini Sockanathan, Marysia Placzek, and Robin Lovell-Badge. "A role for SOX1 in neural determination." Development 125, no. 10 (1998): 1967-1978.
[6] Gleeson, Joseph G., Peter T. Lin, Lisa A. Flanagan, and Christopher A. Walsh. "Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons." Neuron 23, no. 2 (1999): 257-271.
[7] Cheung, Martin, and James Briscoe. "Neural crest development is regulated by the transcription factor Sox9." Development 130, no. 23 (2003): 5681-5693.
[8] Ellis, Pam, B. Matthew Fagan, Scott T. Magness, Scott Hutton, Olena Taranova, Shigemi Hayashi, Andrew McMahon, Mahendra Rao, and Larysa Pevny. "SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult." Developmental neuroscience 26, no. 2-4 (2005): 148-165.
[9] Sommer, Lukas, Nirao Shah, Mahendra Rao, and David J. Anderson. "The cellular function of MASH1 in autonomic neurogenesis." Neuron 15, no. 6 (1995): 1245-1258.
[10] Hu, Bao-Yang, Jason P. Weick, Junying Yu, Li-Xiang Ma, Xiao-Qing Zhang, James A. Thomson, and Su-Chun Zhang. "Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency." Proceedings of the National Academy of Sciences 107, no. 9 (2010): 4335-4340.
[11] Nichols, Jennifer, Branko Zevnik, Konstantinos Anastassiadis, Hitoshi Niwa, Daniela Klewe-Nebenius, Ian Chambers, Hans Schöler, and Austin Smith. "Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4." Cell 95, no. 3 (1998): 379-391.
[12] Okano, Masaki, Daphne W. Bell, Daniel A. Haber, and En Li. "DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development." Cell 99, no. 3 (1999): 247-257.

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



13457-144H1iPS differentiation to neuron, down-syndrome donor C18-CCL54day00rep1
13458-144H2iPS differentiation to neuron, down-syndrome donor C18-CCL54day06rep1
13459-144H3iPS differentiation to neuron, down-syndrome donor C18-CCL54day12rep1
13460-144H4iPS differentiation to neuron, down-syndrome donor C18-CCL54day18rep1
13461-144H5iPS differentiation to neuron, down-syndrome donor C18-CCL54day00rep2
13462-144H6iPS differentiation to neuron, down-syndrome donor C18-CCL54day06rep2
13463-144H7iPS differentiation to neuron, down-syndrome donor C18-CCL54day12rep2
13464-144H8iPS differentiation to neuron, down-syndrome donor C18-CCL54day18rep2
13465-144H9iPS differentiation to neuron, down-syndrome donor C18-CCL54day00rep3
13466-144I1iPS differentiation to neuron, down-syndrome donor C18-CCL54day06rep3
13467-144I2iPS differentiation to neuron, down-syndrome donor C18-CCL54day12rep3
13468-144I3iPS differentiation to neuron, down-syndrome donor C18-CCL54day18rep3