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Excel file with all data from "Comparison and Optimization of hiPSC Forebrain Cortical Differentiation Protocols"

Comparison of Embryoid Body Formation.

<p>A, B) Embryoid bodies were either formed by dissociating iPSCs (using dispase and trituration) or by AggreWell plate technology, followed by culture in non-adherent flasks. B) Quantification of aggregate size from manually-formed or 3,000- or 8,000-cell aggregates. Mean diameter for manually formed aggregates = 118.3 µm; mean diameter for 3,000 cells/aggregate = 183.1 µm; mean diameter for 8,000 cells/aggregate = 195.2 µm. Scale bars = 200 µm. Data are represented as mean ± SEM, from 4 independent differentiations, n = 21–43. Significance determined by one-way ANOVA with a Tukey’s post-test: ***, p<0.0001. F-tests between groups showed significantly different variances, with p<0.05 between manual vs. 3,000 cells/aggregate and manual vs. 8,000 cells/aggregate. C) Immunostaining of day 40 (D40) neurons, following differentiation using either manual formation or AggreWell plates. TOPRO, nuclear marker. Scale bars = 100 µm. Representative images are shown. D) qPCR was performed using RNA harvested from day 40 cultures. Data normalized to <i>GAPDH</i> expression. Manual n = 10, AggreWell n = 10. Data are represented as mean ± SEM. Significance was determined by student's t-test: ***, p<0.0001.</p

Probe sequences for NanoString assay.

<p>Probe sequences for NanoString assay.</p

Comparison of NPC isolation methods.

<p>A) Schematic indicating the time course of differentiation and the techniques used to isolate neural progenitor cells (NPCs). Human iPSCs were differentiated for 17 days. NPCs were isolated by manual scraping of non-NPCs under a microscope (manual selection), using a proprietary neural rosette selection reagent (rosette selection), or by FACS for CD184+/CD44−/CD271−/CD24+ cells (FACS). B) Representative bright field images are shown for selection of rosettes using rosette selection reagent. White arrows indicate rosette structures to be isolated. After use of the reagent, rosettes are isolated. Scale bars = 100 µm. C) Immunostaining for various cell fate markers at day 18 after isolation at day 17. Asterisks in the bottom panel show Sox2+Nestin− cells. Scale bars = 100 µm. D) Day 17 NPCs were either manually selected or dissociated using accutase and processed for cell sorting. Manually selected or PSANCAM+ cells were plated on Matrigel for 23 days in neural differentiation media and immunostained at day 40 for neuronal markers. Scale bars = 100 µm. E) Day 17 cells were dissociated and subjected to FACS. CD184+/CD44−/CD271−/CD24+ cells (“NPCs”) and all other cells (“non-NPCs”) were plated on Matrigel and maintained in neural progenitor media for 20 days prior to immunostaining. Scale bar = 50 µm. F, G) RNA was harvested from cells at day 17 after isolation and used in the NanoString assay. Expression profiles of selected NPC fate markers (F) or other cell fate markers (G) are shown. Gene expression was normalized to the geometric mean of seven housekeeping genes. Data are represented as mean ± SEM. Data are from 5–6 independent differentiations and 3 lines, n = 6–30. Significance is shown compared to “manual selection.” Statistics were calculated using two-way ANOVA with Holm-Sidak multiple comparisons correction: *, p<0.05; **, p<0.01; ***, p<0.001.</p

Astrocyte Co-culture Increases Neuronal Maturation and Endogenous Astrocytes Arise at Later Time-Points in Differentiation.

<p>A) Schematic of differentiation up to 100 days. For astrocyte co-culture, astrocytes were added to neuronal cultures at ∼day 24 of differentiation. Endogenous astrocytes gradually emerged over the course of 100 days, after day 40. B) Day 42 and day 100 neuronal cultures were immunostained and imaged for GFAP. Scale bars = 50 µm. C) After 40–50 (D40) or 100 days (D100), cells were lysed, RNA extracted, and expression of 150 genes analyzed using the NanoString platform. A subset of neuronal markers (C) and synaptic markers (D) are shown. Data are from at least 6 independent differentiations (3 lines). For day 40–50 n = 29–38, for day 100 n = 15–19. E) Neuron cultures with or without astrocytes were immunostained and imaged using confocal microscopy at day 40. Insets in right column show VGLUT1 staining along the length of a neuronal process. Representative images are shown. Scale bars = 50 µm. F) qPCR was performed using RNA harvested from day 40 cultures. Data normalized to <i>GAPDH</i> expression. For neurons alone n = 20 for MAP2, TBR1, CUX1, GAD1, n = 37 for SYP, n = 38 for VGLUT1; for astrocyte co-culture n = 17 for GAD1, n = 18 for MAP2, TBR1, CUX1, n = 27 for SYP, n = 25 for VGLUT1. For C–F, data are represented as mean ± SEM. Significance determined by student’s t-test: **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

Key comparisons of methods tested.

<p>Key comparisons of methods tested.</p

Comparison of Plating Substrates.

<p>Aggregates were plated on either Matrigel or poly-o/laminin (POL) coated plates at days 7 or 24. A, B) Aggregates plated at day 7 (D7) and imaged at day 10 (D10) on Matrigel (A) formed typical neuroepithelial structures, while aggregates plates on POL (B) failed to adhere after two days. C, D) Aggregates were plated on either Matrigel or POL coated plates for final differentiation on day 24 (D24) and imaged at day 40 (D40). Aggregates plated on Matrigel (C) exhibited an increased density of processes, while aggregates plates on POL (D) displayed increased cell body migration from the plated aggregate. E, F) Neural aggregates were dissociated at day 24 and plated on either Matrigel (E) or POL (F). G) Aggregates were plated on either Matrigel (top row) or POL (bottom row) at day 24 and allowed to mature until day 40, followed by immunostaining and confocal microscopy for neuronal markers. Scale bars = 100 µm. Representative images are shown. H) qPCR was performed using RNA harvested from day 40 cultures. Data normalized to <i>GAPDH</i> expression. Matrigel n = 10, POL n = 10. I) Aggregates were single-cell dissociated and plated on either Matrigel (top row) or POL (bottom row) at day 24 and allowed to mature until day 40, followed by immunostaining and confocal microscopy for neuronal markers. Scale bars = 100 µm. Representative images are shown. J) qPCR was performed using RNA harvested from day 40 cultures. Data normalized to <i>GAPDH</i> expression. Matrigel n = 22, POL n = 22. For H and I, significance determined by student’s t-test: **, p<0.01; ***, p<0.001. Data are represented as mean ± SEM.</p

Number of iPSC lines, differentiations and well numbers contributing to each figure.

<p>*10/10 differentiations without dissociation failed. 3/5 differentiations with dissociation yielded MAP2+ cells.</p><p>Number of iPSC lines, differentiations and well numbers contributing to each figure.</p

Monolayer Differentiation of hiPSCs.

<p>A) Time course of differentiation using dual-SMAD inhibition. iPSC colonies were dissociated from mouse embryonic fibroblasts at day 1 (D1) and plated as a monolayer. Small molecules and growth factors were added as indicated. This protocol was performed in at least 6 independent lines; representative images from the most efficient differentiations are shown. B) Bright-field images spanning differentiation from the earliest time-point day 0 (D0) to day 40 (D40). Scale bars = 50 µm. C) Cells were immunostained at various time-points during neuronal differentiation. Confocal images at days 0, 7, 11, 27 and 40. Scale bars = 100 µm. TOPRO, nuclear marker. D) qPCR analysis of markers over differentiation. Data normalized to <i>GAPDH</i>. For Oct 4: iPS n = 3, D1 n = 3, D7 n = 3, D11 n = 6, D40 n = 5; MAP2: iPS n = 3, D1 n = 4, D7 n = 4, D11 n = 6, D40 n = 5; Tbr1: n = 2, D1 n = 3, D7 n = 3, D11 n = 5, D40 n = 5. Data are represented as mean ± SEM.</p

Effects of Neural Progenitor Cell Maintenance and Expansion on Differentiation Efficiency.

<p>A) Schematic indicating the time course of differentiation and the techniques used to maintain/differentiate neural progenitor cells (NPCs) after NPC isolation with neural rosette selection reagent at day 17. B) qPCR analysis of MAP2 expression after 16 days of differentiation of day 24 aggregates or passage 1 or 2 monolayer NPCs. Data normalized to <i>GAPDH</i>. Data are represented as mean ± SEM, n = 11–20. C) Immunostaining of day 40 (D40) neurons, following differentiation from either day 24 aggregates or passage 2 NPCs. Scale bars = 100 µm. Representative images are shown. D) NanoString analysis of cell fate markers of neural aggregates plated at day 19 or 35, after 16 days of plating in neural differentiation media, normalized to the geometric mean of seven housekeeping genes. Data are represented as mean ± SEM, n = 6. For B and D, significance was determined by student's t-test: *, p<0.05; **, p<0.01; ***, p<0.001.</p