Differences between cellular and molecular profiles of induced pluripotent stem cells generated from mouse embryonic fibroblasts

Abstract

Induced pluripotent stem (iPS) cells are a new alternative for the development of patient-specific stem cells, and the aim of this study was to determine whether differences exist between the cellular and molecular profiles of iPS cells, generated using lentiviral vectors, compared to ES cells. The lentiviral infection efficiency differed according to the method of cell culture (adherent cells: 0.085%; suspended cells: 0.785%). Six iPS cell lines exhibited typical ES cell morphology and marker expression, but varied in their in vitro/in vivo differentiation ability. Global gene transcription analysis revealed that core pluripotency genes were expressed at lower levels in iPS cell lines compared to D3-ES cells (Pou5f1: x1.6~2.2-fold, Sox2: x2.58~10.0-fold, Eras: x1.08~2.54-fold, Dppa5a: x1.04~1.41-fold), while other genes showed higher expression in iPS cells (Lin28: x1.43~2.33-fold; Dnmt3b: x1.33~2.64-fold). This pattern was repeated in a survey of specific functional groups of genes (surface markers, cell death, JAK-STAT and P13K-AKT signaling pathways, endothelial, cardiovascular, and neurogenesis genes). Among the iPS cell lines examined, only two showed similar characteristics to ES cells. These results demonstrated that, in addition to cellular characterization, the numerical evaluation of gene expression using DNA microarrays might help to identify the stem cell stability and pluripotency of iPS cells.

FIG. 1.

Generation of induced pluripotent stem (iPS) cells from mouse embryonic fibroblast (MEF) cells using lentiviral vectors carrying four transcription factors. (A) Reprogramming protocol for MEF cells without drug selection. (B) Infected cells and embryonic stem (ES)-like colonies forming under different conditions, at 2 days and 6 days post infection, respectively. Fluoromicroscopic images of the cells indicate the expression of the Venus protein reporter gene. (C) Differences in infection efficiency according to cell treatment methods. (D) Verification of ES cell characteristics in generated iPS cell lines by examination of alkaline phosphatase activity, surface marker expression of SSEA-1, and Oct-4 expression. (E) Phase-contrast images of D3-ES cells, iPS colonies, and MEF cells. (F) RT-PCR analysis of expression of infected genes and ES marker genes in iPS cell lines. (G) Western blot analysis of gene expression in iPS cell lines.

FIG. 2.

Semiquantitative real-time PCR analysis of the expression levels of the four pluripotent-related genes in D3-ES cells, six iPS cell lines, and MEF cells. The results are expressed graphically (A) and numerically (B). MEF cells were used as a control to calculate the values of the other cell groups.

FIG. 3.

In vitro and in vivo differentiation of mouse iPS cells into the three germ layers. (A) Phase-contrast and fluorescent images of day four iPS-MEF-S-6 colonies cultured on STO feeder cells and their day eight embryoid bodies (EBs) prepared on a bacteriological plate. Fluoromicroscopic images of the cells indicate expression of the Venus protein reporter gene. (B) Immunofluorescence analysis of two differentiated iPS clones (iPS-MEF-A-1 and iPS-MEF-S-6) using cell markers for the three germ layers. (C) RT-PCR analysis of ES cell marker and differentiation marker expression in D3-ES cells, two different iPS cell lines, and their EBs. ES cell markers: Oct4, Nanog, and FGF4; endoderm markers: α-amylase and α-fetoprotein; mesoderm markers: β-enolase and Renin; ectoderm markers: βIII tubulin and Map2. (D) Histological examination of differentiated tissue structures found in teratomas formed in the testis of SCID mice following inoculation with mouse iPS cells. Endoderm tissue: gut-like epithelium; mesoderm tissue: adipose tissue, muscle and cartilage; ectoderm tissue; neural tissue and epidermis.

FIG. 4.

Global gene expression analysis of iPS cells. (A) Hierarchical clustering of D3-ES cell line, six iPS cell lines (iPS-MEF-A-1, iPS-MEF-A-2, iPS-MEF-S-3, iPS-MEF-S-4, iPS-MEF-S-5, and iPS-MEF-S-6) and MEF cells. The transcriptome of the iPS clones is very similar to that of D3-ES cells. (B) Pearson correlations were calculated from the gene clustering results and showed close correlations between iPS cell groups and D3-ES cells. (C) Scatter-plot presentation of the expression values for all probe sets derived from genome-wide microarray data indicated that there were close correlations between iPS cell groups and D3-ES cells. (D–E) Examination of twofold differences in gene expression between cell groups indicated that fewer genes were up- or down-regulated in iPS cells compared to D3-ES cells than in iPS cells or D3-ES cells compared to MEF cells.