Supplemental material – Maussion et al.
Testing ChIP antibodies in adult frontal cortex:
For each antibody, we performed a targeted ChIP assay in brain tissue using qPCR primers targeting a genomic region reported to associate with the protein, as well as Western blots. In the case of TCF7L2 and EHMT1, well known ChIP-grade antibodies are available which are widely used; for SATB2, we tested two different antibodies and selected the one that gave the stronger (lower Ct value) signal.
Assessing expression of well known markers in proliferating and differentiating NSCs
We performed qPCR using well-defined markers for proliferating and differentiating neural stem cells from both cell lines and human brain, as well as other comparison tissues (Supplemental Figure 1D and 1E). We found that both Musashi and Nestin were strongly expressed in both NSC lines at the proliferating stage (Supplemental Figure 1D), and that markers that define a broader stem cell state such as Nanog and Oct4, were not expressed, at least compared to levels detected in undifferentiated induced pluripotent stem cells (iPSC). While both NSCs were similar for these four markers, we did observe a notable difference in PAX6 expression between proliferating NSCs, with much more expression in iPSC-NPCs than in FBCs. In differentiating NSCs, we selected many markers to understand whether genes known to be expressed in more mature cells were present. In FBCs, we found high expression of DAT (SLC6A3), in line with the ventral medial diencephalic origin of these cells, as well as GFAP and SLC1A2. In iPSC-NPCs, there was high expression of VGLUT2 (SLC17A6) and TPH2, at last compared to the FBCs. As expected, all mature markers were more highly expressed in adult brain, and also in fetal brain, suggesting that the stage we selected as differentiating (30 days) for the NSCs does not represent cells that have completed differentiation. We interpret these data to mean that we have two distinct NSC lines that nonetheless reflect the diversity of developing brain cells, and that the two developmental stages selected can be distinguished by the expression of well known genes.
Electrophysiological characterization of NSCs
In order to physiologically characterize the FBCs and iPSC-NPCs, we used whole-cell patch clamp in cells cultured for 30 days post-differentiation. We recorded the membrane voltage Vm at rest (I=0), immediately after establishing the whole cell configuration. All iPSC-NPCs had a negative resting membrane potential (-28.50 ± 1.874 mV, n=26), and a high input resistance (1.558 ± 0.1582 GΩ, n=26). For the FBCs, we registered resting potentials of -50.22 ± 3.48 mV, n=10, and input resistances of 93.38 ± 29.28 MΩ, n=10, suggesting that at rest these NSCs show different properties. We also used the current-clamp recording mode to test the occurrence of action potentials in response to somatic current injections. In iPSC-NPCs (Figure 1F-1I), single overshooting action potentials were generated in response to depolarizing current pulses. While no iPSC-NPC exhibited spontaneous action potentials, a few cells fired a rebound action potential at the end of a hyperpolarizing voltage step. Voltage-clamp recordings from iPSC-NPC allowed the observation that these cells express functional voltage-gated sodium and potassium channels, which mediated appreciable inward and outward currents; specific toxins such as TTX (200 nM) and TEA (50 µM) prevented the appearance of these currents that were expected upon depolarization. In the case of FBCs (Figure 1J-1L), we were unable to observe any physiological properties characteristic of neurons, suggesting FBCs are mostly glial, or that are unable to become neurons. We conclude that in iPSC-NPCs, but not in FBCs, basic intrinsic physiological properties permit manifestation of excitability similar to that observed in neurons. These data suggest that these two NSC lines are fundamentally different with respect to their physiological properties.
Figures
Figure S1. ChIP quality control experiments and NSC characterization. A) Testing of antibodies targeting SATB2 protein in Western blots using human brain extract; we found a single band at 82Kda using an anti-SATB2 antibody from Epitomics (2819-1). We also tested anti-SATB2 antibodies using extracts from FBCs and targeting a region bound by SATB2 protein (CTIP2A4). The graph represents raw qPCR data curves for Input DNA (i.e., DNA/protein that was not immunoprecipitated, but was treated similarly in all other ways), IgG control (i.e., DNA/protein that was immunoprecipitated with a non-specific antibody), and two different antibodies that target SATB2. The closer a curve is to the Input curve, the better its ability to bind to SATB2. B) Western blot of anti-TCF7L2 antibody from Millipore (17-10109) which detects a single band at 68KDa. The graph shows raw qPCR curves demonstrating the ability of the antibody to bind to TCF7L2 at the positive control region within Axin2. C) Western blot evidence that the anti-EHMT1 Abcam antibody (ab41969 ) can detect a single 180Kda band. Graph shows qPCR data where primers targeting a region of the Mageb16 gene can be amplified after anti-EHMT1 immunoprecipitation. D) qPCR characterization of proliferating neural stem cell markers. E) qPCR characterization of differentiating neural stem cell markers. F) Sample phase image of 8330 cells in culture at D30 post-differentiation. G) Experimental protocol and representative recording of action potentials in current-clamp mode, induced by somatic current injection (ΔI=20 pA, holding potential Vhold = -60mV) from an iPSC-NPC at D30 post-differentiation. H) Experimental voltage pulse-step protocol (top) and representative voltage-clamp recording traces (from a holding potential Vhold = -60 mV), from a iPSC-NPC at D30 post-differentiation. Expanded views of Na currents (dashed boxes, insert). I) Average Na and K currents recorded from iPSC-NPCs at D30 post-differentiation (n=23, from 3 lines), plotted as a function of step voltage amplitudes. Data presented as mean±s.e.m. J) Sample phase image of FBCs in culture at D30 post-differentiation. K) Experimental protocol and representative recording of membrane voltage traces in current-clamp mode, induced by somatic current injection (ΔI=20 pA, from a holding potential Vhold = -60mV) from a FBCs at D30 post-differentiation) Experimental voltage pulse-step protocol (top) and representative voltage-clamp recording traces (from a holding potential Vhold = -60 mV), from a FBC at D30 post-differentiation.
Figure S2: Analysis of Housekeeping gene stability across cell line and tissues. Gene expressions of TBP, NONO, ACTB and GAPDH in iPSC-NSC-pro; iPSC-NSC-dif, FBC-pro, FBC-dif, adult brain and fetal brain.
Figure S3. Transcription factor expression across tissues. Gene expressions of (A) EHMT1, (B) SATB2, and (C) TCF7L2 in RPKM obtained from the GTEx database.