Establishment of LIF-dependent human iPS cells closely related to basic FGF-dependent authentic iPS cells

Abstract

Human induced pluripotent stem cells (iPSCs) can be divided into a leukemia inhibitory factor (LIF)-dependent naïve type and a basic fibroblast growth factor (bFGF)-dependent primed type. Although the former are more undifferentiated than the latter, they require signal transduction inhibitors and sustained expression of the transgenes used for iPSC production. We used a transcriptionally enhanced version of OCT4 to establish LIF-dependent human iPSCs without the use of inhibitors and sustained transgene expression. These cells belong to the primed type of pluripotent stem cell, similar to bFGF-dependent iPSCs. Thus, the particular cytokine required for iPSC production does not necessarily define stem cell phenotypes as previously thought. It is likely that the bFGF and LIF signaling pathways converge on unidentified OCT4 target genes. These findings suggest that our LIF-dependent human iPSCs could provide a novel model to investigate the role of cytokine signaling in cellular reprogramming.

Figure 1. Expression of pluripotency markers by iPSCs prepared with LIF or bFGF.

(A) Immunofluorescence staining of iPSCs prepared with LIF or bFGF on day 8. Antibodies against NANOG and TRA1-60 were used, and DNA was counterstained with Hoechst 33342. Bar, 100 µm in (A) and (C). (B) Formation of iPSC colonies that were double-positive for NANOG and TRA1-60 with LIF, bFGF, or no cytokines. Cells (1.7×10⁴) were seeded in each well, and iPSC colonies were counted on day 10. Mean + standard deviation (SD) was obtained from three independent experiments. (C) Immunofluorescence staining of iPSCs prepared with LIF or bFGF on day 8. Antibodies against SSEA4 and NANOG were used. (D) Formation of iPSC colonies that were double-positive for SSEA4 and NANOG with bFGF, LIF, or no cytokines. Cells were seeded and iPSC colonies counted as described in (B).

Figure 2. Characterization of colony morphology, gene expression patterns, and sensitivity to an FGF receptor inhibitor in L-iPSCs obtained with M₃O-SKM.

(A) Phase contrast images of L-iPSC colonies obtained on day 90. Bar, 100 µm. (B) Immunofluorescence staining of an L-iPSC colony with antibodies against NANOG and SSEA4 on day 28. Bar, 100 µm. (C) Quantitative RT-PCR analysis of the OCT4, SOX2, KLF4 and c-MYC genes in various pluripotent stem cells on day 120. Expression levels of each gene were normalized to GAPDH. The normalized value for ESCs was defined as 1.0. Mean + SD obtained from three independent experiments are shown. (D) RT-PCR analysis of the expression of M₃O in day-3 fibroblasts transduced with M₃O-SKM, day-120 L-iPSCs, and parent fibroblasts. Expression of GAPDH mRNA was monitored as a control. Bar, 100 µm. (E) Effects of SU5402 on iPSC colony formation with LIF and bFGF. SU5402 dissolved in dimethyl sulfoxide (DMSO) was added to culture medium between day 8 and 10, and the colonies were counted on day 12. DMSO was used as a control.

Figure 3. Characterization of gene expression patterns, karyotype, and teratoma formation in L-iPSCs prepared with M₃O-SKM.

(A) Immunofluorescence staining of L-iPSCs with antibodies against OCT4, NANOG, TRA1-60 and TRA1-81 on day 120. Alkaline phosphatase staining is also shown. Bar, 100 µm. (B) Quantitative RT-PCR analysis of genes that are typically expressed in pluripotent stem cells. Expression levels of each gene were normalized to GAPDH. The normalized value for ESCs was defined as 1.0. Mean + SD obtained from three independent experiments are shown. (C) Karyotype analysis of an L-iPSC on day 120. (D) Haematoxylin and eosin staining of histological sections of teratomas derived from L-iPSCs. Bar, 200 µm.

Figure 4. Characterization of L-iPSCs by using gene expression analyses and immunofluorescence staining of H3K27me3.

(A) Quantitative RT-PCR analysis of genes that are typically expressed in mouse naïve (REX1 and STELLA) or primed (FGF5 and T) pluripotent stem cells. Expression levels of each gene were normalized to GAPDH. The normalized value for ESCs was defined as 1.0. Mean + SD obtained from three independent experiments are shown. (B) Immunofluorescence staining of H3K27me3 as an indicator for X chromosome inactivation. Parent fibroblasts were stained as a positive control. Nuclei surrounded by squares were magnified (left panels). Arrows indicate H3K27me-positive areas, corresponding to inactive X chromosomes. Bar, 50 µm.

Figure 5. Characterization of LF-iPSCs with immunofluorescence staining and a teratoma formation assay.

(A) Immunofluorescence staining and alkaline phosphatase staining of LF-iPSCs on day 140. Bar, 100 µm. (B) Haematoxylin and eosin staining of histological sections of teratomas derived from LF-iPSCs. Bar, 200 µm.

Figure 6. Genome-wide gene expression patterns comparing various pluripotent stem cells.

(A) Scatter plots comparing levels of whole transcripts obtained from two WA09 ESC cultures, two dermal fibroblast cultures, two lines each for L-iPSCs, F-iPSCs and LF-iPSCs. Two independently cultured cells were used for WA09 cells. All these iPSCs were established from fibroblasts obtained from a single adult female. Two different cultures of fibroblasts obtained from the same individual were used. (B) Cluster analysis of the genome-wide gene expression patterns of cells analyzed in (A). (C) Scatter plots comparing levels of whole transcripts between L-iPSCs and other cell types used in this study. Average values obtained from two cultures or two lines for each cell type were used.