Profiling of embryonic stem cell differentiation

Abstract

Embryonic stem (ES) cells have been shown to recapitulate normal developmental stages. They are therefore a highly useful tool in the study of developmental biology. Profiling of ES cell-derived cells has yielded important information about the characteristics of differentiated cells, and allowed the identification of novel marker genes and pathways of differentiation. In this review, we focus on recent results from profiling studies of mouse embryos, human islets, and human ES cell-derived differentiated cells from several research groups. Global gene expression data from mouse embryos have been used to identify novel genes or pathways involved in the developmental process, and to search for transcription factors that regulate direct reprogramming. We introduce gene expression databases of human pancreas cells (Beta Cell Gene Atlas, EuroDia database), and summarize profiling studies of islet- or human ES cell-derived pancreatic cells, with a focus on gene expression, microRNAs, epigenetics, and protein expression. Then, we describe our gene expression profile analyses and our search for novel endoderm, or pancreatic, progenitor marker genes. We differentiated mouse ES cells into mesendoderm, definitive endoderm (DE), mesoderm, ectoderm, and Pdx1-expressing pancreatic lineages, and performed DNA microarray analyses. Genes specifically expressed in DE, and/or in Pdx1-expressing cells, were extracted and their expression patterns in normal embryonic development were studied by in situ hybridization. Out of 54 genes examined, 27 were expressed in the DE of E8.5 mouse embryos, and 15 genes were expressed in distinct domains in the pancreatic buds of E14.5 mouse embryos. Akr1c19, Aebp2, Pbxip1, and Creb3l1 were all novel, and none has been described as being expressed, either in the DE, or in the pancreas. By introducing the profiling results of ES cell-derived cells, the benefits of using ES cells to study early embryonic development will be discussed.

Figure 1. A schematic drawing of M15 cell-mediated signaling events

Signaling molecules involved in the in vitro differentiation process mediated by M15 are shown. Abbreviations: BIO - 6-bromoindirubin-3'-oxime; BMP7 - bone morphogenetic protein 7; DAPT - N-(N-(3,5-difluorophenacetyl)-L-alanyl)-S-phenylglycine t-butyl ester; Dex - dexamethasone; ES - embryonic stem; FGF - fibroblast growth factor; HGF - hepatocyte growth factor; Pdx1 - pancreatic and duodenal homeobox 1.

Figure 2. Microarray analyses of ES cell-derived cells

(A) ES cells and ES cell-derived cells were isolated based on the expression of cell surface antigens, as previously described [48]. We isolated ES cells (ES), ectoderm (ECT), mesendoderm (MES), lateral plate mesoderm (LPM), paraxial mesoderm (PAM), and DE at day 5 (D5), day 7 (D7), and day 8 (D8, DE Pdx1-, DE Pdx1+). (B) Clustering of gene expression in ES, ECT, LPM, PAM, MES, D5 DE, D7 DE, D8 DE Pdx1-, and D8 DE Pdx1+ cell lineages. Each line indicates an individual gene. Red lines indicate genes with high expression and green lines indicate genes with low expression in the DE lineages. The y-axis represents normalized values of the expression levels. Abbreviations: D - day; DE - definitive endoderm; ES - embryonic stem; ECT - ectoderm; LPM - lateral plate mesoderm; MES - mesendoderm; PAM - paraxial mesoderm; Pdx1 - pancreatic and duodenal homeobox 1.

Figure 3. Numbers of endoderm-specific candidate genes

Summary of the numbers of genes selected for further analyses by whole mount in situ hybridization (top). The numbers of genes expressed in gut endoderm at E8.5 (middle) or the pancreatic bud at E14.5 (bottom) are shown. Blue circle: genes expressed at >5-fold in D5 DE. Red circle: genes expressed at >5-fold in D8 DE Pdx1+. Green circle: genes expressed at >5-fold in D7 DE versus D5 DE. Brown circle, genes expressed at >2-fold in D8 DE Pdx1+ versus D8 DE Pdx1-. (A, B) The names of genes expressed only in E8.5 gut tube, but not in E14.5 pancreas bud are listed in (A). The names of genes expressed in both E8.5 AIP and E14.5 pancreas bud are listed in (B). More information is detailed in Tables 1 and 2.

Figure 4. The co-expression of candidate genes with insulin, glucagon, or Pdx1 in the E14.5 pancreatic bud

C2cd4b was co-expressed with insulin, but not glucagon, in the trunk (upper panels). Hipk2 was co-expressed with glucagon, but not insulin, in the epithelium. Akr1c19 was co-expressed with Pdx1 or insulin in the epithelium. Scale bar: 100 μm.