Cell fusion enhances mesendodermal differentiation of human induced pluripotent stem cells

Abstract

Human induced pluripotent stem cells (iPS cells) resemble embryonic stem cells and can differentiate into cell derivatives of all three germ layers. However, frequently the differentiation efficiency of iPS cells into some lineages is rather poor. Here, we found that fusion of iPS cells with human hematopoietic stem cells (HSCs) enhances iPS cell differentiation. Such iPS hybrids showed a prominent differentiation bias toward hematopoietic lineages but also toward other mesendodermal lineages. Additionally, during differentiation of iPS hybrids, expression of early mesendodermal markers-Brachyury (T), MIX1 Homeobox-Like Protein 1 (MIXL1), and Goosecoid (GSC)-appeared with faster kinetics than in parental iPS cells. Following iPS hybrid differentiation there was a prominent induction of NODAL and inhibition of NODAL signaling blunted mesendodermal differentiation. This indicates that NODAL signaling is critically involved in mesendodermal bias of iPS hybrid differentiation. In summary, we demonstrate that iPS cell fusion with HSCs prominently enhances iPS cell differentiation.

FIG. 1.

Human induced pluripotent stem (iPS) hybrids are pluripotent. (A) Experimental design of cell fusion and iPS hybrid generation. (B) Phase-contrast images of undifferentiated iPS hybrids and parental iPS cells. Scale bar=200 μm. (C) M-FISH analysis of iPS hybrid indicating a normal tetraploid karyotype 92,XXXY (above: MCK karyogram; below: inverted DAPI picture). (D) Scatter plot analysis of global gene expression of undifferentiated iPS hybrids and parental iPS cells. (E) Bidirectional hierarchical cluster analysis of gene expression in hematopoietic stem cells (HSCs), undifferentiated iPS cells, and iPS hybrids. Hybrid_1, 2, and 3 represented data from different cord blood samples. Color code: blue, low expression; yellow, high expression. (F) ChIP–quantitative polymerase chain reaction (qPCR) analysis of H3K4me3 and H3K27me3 of OCT4, NANOG, and CD45 promoters in HSCs, undifferentiated iPS cells, and iPS hybrids (n=3).

FIG. 2.

iPS hybrids show differentiation bias toward mesendoderm. (A) Embryoid body (EB) assay of iPS hybrids and parental iPS cells (day 7). Note cystic EBs in iPS hybrids. Scale bar=200 μm. (B) Cluster analysis of gene expression in iPS hybrids and iPS cells of (A) by reverse transcription-qPCR (days 7 and 14). Hybrid_1–3 as in Fig. 1E. (C) Cluster analysis of transcriptomes of iPS hybrids and iPS cells during differentiation as in (A) (days 7 and 14). (D) Gene set enrichment analysis (GSEA) of differentiating iPS hybrids and iPS cells (days 2 and 4; P<0.0001). (E) Bidirectional hierarchical cluster analysis of gene expression during neural differentiation. (F) Bidirectional hierarchical cluster analysis of gene expression of early and late-passage iPS hybrid_1 (P10 and P35, respectively) during differentiation (days 7 and 14) as in (B). Color code in (B), (E), and (F) as in Fig. 1. Data are presented as mean of three independent experiments.

FIG. 3.

Upregulation of early mesendoderm markers in iPS hybrids. (A) Expression kinetics of T, MIXL1, and GSC in differentiating iPS hybrids (Hybrid_1, 2, and 3) and parental iPS cells. Please note the prominent peak of T, MIXL1, and GSC expression in iPS hybrids at day 2 compared with parental iPS cells (*P<0.05). (B) ChIP-qPCR analysis of H3K4me3 and H3K27me3 in T, MIXL1, and GSC promoter regions of differentiating iPS hybrids as in (A) (n=3). (C) NODAL expression in differentiating iPS hybrids and parental iPS cells (differential expression at days 2 and 4, *P<0.05). (D) ChIP-qPCR analysis of H3K4me3 and H3K27me3 kinetics in NODAL promoter region of differentiating iPS hybrids as in (B). (E) Western blot analysis of p-SMAD2 in differentiating iPS hybrids and iPS cells as in (A). Data are presented as mean±SD of three independent experiments. Gene expression at day 0 was arbitrarily set 1.

FIG. 4.

Mesendodermal differentiation bias of iPS hybrids is blocked by ACTIVIN/NODAL inhibitor SB431542 (SB). (A) Western blot analysis of p-SMAD2 with or without SB (EB assay, day 2, 20% FCS). (B) Phase-contrast images of EBs with or without SB as in (A) at day 7. Scale bar=200 μm. (C) Frequencies of cystic EBs in differentiation culture with and without SB treatment (day 7; **P<0.001). (D) Expression kinetics of T, MIXL1, and GSC in differentiating iPS hybrids with or without SB as in (A). Please note that SB treatment efficiently blunts T, MIXL1, and GSC expression in iPS hybrids at day 2 (*P<0.05). (E) Bidirectional hierarchical cluster analysis of gene expression during differentiation as in (A). (F) Cluster analysis of gene expression upon neural differentiation with or without SB as in (A) but under serum-free condition. Color code in (E) and (F) as in Fig. 1. Data are presented as mean of three independent experiments.