Results: 5

1.
Figure 3

Figure 3. From: A robust method to derive functional neural crest cells from human pluripotent stem cells.

NC cells from the LSB-short method can differentiate into multiple NC lineages. NC cells spontaneously differentiate into A: smooth muscle cells (SMAa+), B: peripheral neurons (peripherin+), C: Schwann cells (GFAP+) (iPS-3 cells, representative images). Scale bar = 100 μm.

Faith R Kreitzer, et al. Am J Stem Cells. 2013;2(2):119-131.
2.
Figure 2

Figure 2. From: A robust method to derive functional neural crest cells from human pluripotent stem cells.

The LSB-short method generates NC cells in a robust and reproducible manner and with high efficiency. A: Flow cytometry analyses for HNK1 and p75 expression in NC cells differentiated from a representative iPS cell line, iPS-1 (n = 3), performed in live cells without fixation to reduce non-specific p75 expression. B: NC differentiations from iPS-1, iPS-2, and H9 cells produce high percentages of NC cells, as indicated by the increase in p75 staining. C: Quantification of p75+ expression by flow cytometry across multiple cell lines indicates that the LSB-short method is robust and highly reproducible. Each black shape in the graphs indicates an independent NC differentiation. For all experiments, undifferentiated iPS-1 cells were used as a negative control.

Faith R Kreitzer, et al. Am J Stem Cells. 2013;2(2):119-131.
3.
Figure 1

Figure 1. From: A robust method to derive functional neural crest cells from human pluripotent stem cells.

Modified NC differentiation methods, LSB-long and LSB-short, generate NC cells from human pluripotent stem cells via a neural precursor stage. A: Schematic of LSB-long method, which takes 13-14 days to complete. B: PAX6+ neural precursor cells appear as early as day 5 using the LSB-long method (iPS-1, representative image). C: By day 10-11 of the long method, a small number of p75+ NC cells were observed next to large regions of neural precursor (PAX6+) cells (H9, representative image). D: Schematic of LSB-short method showing a shorter NC differentiation phase. E: With the short method, PAX6+ neural precursor cells were observed at day 2 of NC differentiation (iPS-1, representative image). F: NC cells co-expressed HNK1/p75 (iPS-5, representative image). G: At day 5 of the short method, AP2a-expressing NC cells predominated in the culture wells, which contained almost no PAX6+ neural precursor cells (iPS-1, representative image). H: Day 5 NC cells from the short method also expressed SOX10, a marker of migrating NC cells (H9, representative image). Scale bar = 100 μm.

Faith R Kreitzer, et al. Am J Stem Cells. 2013;2(2):119-131.
4.
Figure 5

Figure 5. From: A robust method to derive functional neural crest cells from human pluripotent stem cells.

NC cells migrate to appropriate chemoattractants. A, B: Schematics of Boyden chamber experiments. A: NC cell chemotaxis potential was quantified in Boyden chambers by loading cells in the top well and chemoattractant (SDF1, Fgf8b, BMP2, or Wnt3a) in the bottom well. B: NC cell chemokinesis potential was quantified by loading cells on the top and chemoattractant on both the top and bottom as shown. C: Representative image of the bottom filter of the migration chamber showing that Draq5-labeled cells (red) migrated through the filter from the upper chamber in response to drug loaded into the bottom of the lower chamber. D: Schematic of how migrating NC cells are visualized by excitation of Draq5 nuclear dye and emission detection is quantified. E: NC cells migrated to all chemoattractants tested. F: NC cells do not display chemokinetic behavior in response to chemoattractants, except for BMP2. iPS-1, n = 3 experiments performed in triplicate. One-way ANOVA, ***p < 0.001, **p < 0.01.

Faith R Kreitzer, et al. Am J Stem Cells. 2013;2(2):119-131.
5.
Figure 4

Figure 4. From: A robust method to derive functional neural crest cells from human pluripotent stem cells.

Activation of migratory NC expression program. iPS-derived NC cells (iPS NCCs) analyzed in the context of well-described NC markers and previously reported in vitro and in vivo NC transcriptome datasets. A: Various markers for distinct NC sub-populations are compared between the iPS NCC method and a previous method to derive NC stem cells (NCSC). These include markers for neural plate border specifiers (NPBS), NC specification, and NC migration, and distinct markers for cranial, trunk and cardiac NC cells. Red = upregulation; green = downregulation. B: Differential gene-expression changes in iPS NCCs relative to iPS cells are visualized upon a previously developed model of NC cell differentiation (WikiPathway WP2064 revision 47071). Differentially expressed genes are colored red or blue (up or downregulation, respectively) from AltAnalyze. C, D: Comparison of differentially expressed genes from RNA transcriptome profiles of human embryo–derived NC trunk and ES-derived NC cells to iPS NCCs. For ES-derived NC cells, RNA-Seq of single samples was performed resulting in lower stringency results (see Materials and Methods). E: Hierarchically clustered heatmap of alternative exons differing between NCCs and ES or iPS cells from three distinct differentiation methods using the software AltAnalyze. Blue indicates exon-exclusion and red exon-inclusion relative to pluripotent stem cell profiles.

Faith R Kreitzer, et al. Am J Stem Cells. 2013;2(2):119-131.

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