Efficient isolation and gene expression profiling of small numbers of neural crest stem cells and developing Schwann cells

Abstract

Schwann cells develop from multipotent neural crest stem cells and are important for neuronal survival, maintenance of axonal integrity, and myelination. We used transgenic mice expressing green fluorescent protein in a tissue-specific manner to isolate viable, pure populations of neural crest stem cells and developing Schwann cells, which are not readily accessible by microdissection. Starting with the minute amounts of RNA obtained, a two-round amplification procedure was used to achieve reproducible DNA array hybridizations. We validated our screening procedure by comparisons with the literature and by in situ hybridization. Stage-to-stage comparisons and hierarchical clustering for neural crest and five stages of Schwann cell development suggest a wealth of candidates for genes involved in stem cell regulation and in early Schwann cell development. The combination of methods applied in this study should be generally useful for isolating and profiling other stem cell and difficult to isolate cell populations.

Figure 1.

Expression of GFP in the developing PNS of Plp-GFP transgenic mice. A, At E9.5, GFP⁺ cells are detected in the developing somites (arrow). B, At E12.5, GFP⁺ cells are found in the DRG (drg) and around the sciatic nerve (sn) growing into the hindlimb. C, At E16, GFP⁺ cells are found within the sciatic nerve. Scale bars: A, 0.5 mm; B, C, 200 μm. D, When analyzed by FACS, sciatic nerve cell suspensions obtained from transgenic mice at E16 show a distinct population of GFP⁺ cells. E, Quantification of survival assays and immunohistochemical characterization of sorted cells. Expression of p75 (red diamonds) and Sox10 (green squares) is maintained by ∼90% of cells at each developmental stage, whereas expression of the O4 antigen (black triangles) begins around E14. At E14 a subpopulation of sorted cells acquires the ability to survive 24 hr in culture without supplementation with growth factors, whereas at later stages almost all cells survive (yellow circles). F, Reproducibility of microarray hybridizations. Probes derived from RNA of sorted cells were hybridized to Affymetrix GeneChips, and expression indices were determined for all the genes. At each developmental stage, three or four replicate experiments were compared in all pairwise combinations by calculating Pearson's correlation coefficient for the expression indices. All genes on the chips were included in the comparisons. The filled circles show the correlation coefficients (r), with three or six values for three or four replicas, respectively, and the horizontal lines indicate the means of these values.

Figure 2.

Expression profiles of genes in SC development. Profiles of three genes known to be involved in SC development are shown: peripheral myelin protein 22 (A), early growth response 2 (B), and fatty acid binding protein 7 (C). Profiles of three genes not previously known to be expressed in the early SC lineage are shown: putative neuronal cell adhesion molecule (punc) (D), prion protein (E), and reelin (F). Affymetrix probe-set identification numbers are above the graphs. Mean expression indices ± SD are shown.

Figure 3.

Expression of candidate genes assessed by in situ hybridization. A, Expression of punc mRNA is detected at E9.5 in migrating NCSCs of the trunk (arrow), which were identified by expression of Sox10 on nearly adjacent sections (B). C, Serpine 2 is found in E12 peripheral nerves (pn, arrow); hybridization with the serpine 2 sense control probe is shown in D. E, Reelin is expressed in E16 peripheral nerves. Location of peripheral nerves was determined by hybridization of a probe for MBP mRNA to semi-adjacent sections (F). Scale bar, 100 μm. NCSC, Neural crest stem cells; nt, neural tube; drg, DRG; pn, peripheral nerve.

Figure 4.

A, Number of genes differentially expressed with an LCB of ≥1.5-fold between developmental stages (additional criteria: change in expression index ≥100; p ≤ 0.05 by modified t test; genes called present on at least 50% of arrays used for the comparison). B, Categories of genes differentially expressed between E9.5 and E12 with LCB ≥3×, or ≥2× if the mean expression index was >1000 for at least one stage. C, Genes differentially expressed between developmental stages were grouped into functional categories. The most prominent groups regulated through SC development are indicated in blue for genes in these groups mainly downregulated or in red if mainly upregulated. Diagram of developmental stages adapted from Jessen and Mirsky (1999). D, Hierarchical clustering of expression patterns of genes called present for at least one developmental stage. Columns represent expression levels at the various developmental stage; rows correspond to individual genes. Expression levels are relative to the mean across stages, normalized to have a mean of 0 and an SD of 1. Expression levels are color coded relative to the mean: blue for values less than the mean and red for values larger than the mean. The scale indicates SDs above or below the mean. Clusters discussed in Results are numbered 1-5.