Tbx1 modulates endodermal and mesodermal differentiation from mouse induced pluripotent stem cells

Abstract

The T-box transcriptional factor (Tbx) family of transcriptional factors has distinct roles in a wide range of embryonic differentiation or response pathways. Tbx1, a T-box transcription factor, is an important gene for the human congenital disorder 22q11.2 deletion syndrome. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem-cell-based therapies. In this study, we generated iPSCs from Tbx1(-/-) and Tbx1(+/+) fibroblasts and investigated the spontaneous differentiation potential of iPSCs by detailed lineage analysis of the iPSC-derived embryoid bodies. Undifferentiated Tbx1(-/-) and Tbx1(+/+) iPSCs showed similar expression levels of pluripotent markers. The ability of the Tbx1(-/-) iPSCs to generate endodermal and mesodermal lineages was compromised upon spontaneous differentiation into embryonic bodies. Restoration of Tbx1 expression in the Tbx1(-/-) iPSCs to normal levels using an inducible lentiviral system rescued these cells from the potential of defective differentiation. Interestingly, overexpression of Tbx1 in the Tbx1(-/-) iPSCs to higher levels than in the Tbx1(+/+) iPSCs again led to a defective differentiation potential. Additionally, we observed that expression of fibroblast growth factor (FGF) 10 and FGF8 was downregulated in the Tbx1(-/-) iPSC-derived cells, which suggests that Tbx1 regulates the expression of FGFs. Taken together, our results implicated the Tbx1 level as an important determinant of endodermal and mesodermal lineage differentiation during embryonic development.

FIG. 1.

Analysis of generated Tbx1^−/− and Tbx1^+/+ induced pluripotent stem cells (iPSCs). (A) Amplification with wild-type (Tbx1^+/+)–specific primer pairs results in 216-bp products and with neomycin cassette-specific primer pairs results in 470-bp products for Tbx1^−/− iPSCs. (B) The morphology of Tbx1^−/− and Tbx1^+/+ iPSCs. Scale bars=200 μm. (C) Reverse transcription–polymerase chain reaction (RT-PCR) analysis for isolated iPSCs using ES marker genes. TC-1 ES cells were used as a positive control and mouse embryo fibroblasts (MEFs) as a negative control. (D) iPSCs were examined for the expression of alkaline phosphatase (AP), Oct4, and SSEA-1. AP (dark red) expression was detected using immunocytochemistry; SSEA-1 and Oct4 (red) expression was detected by immunofluorescence staining. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI; blue). Scale bars=200 μm (AP). (E) In vitro differentiation of the iPSCs. Immunostaining images show all three germ layers, including AFP⁺ (endoderm), alpha smooth muscle actin (α-SMA)⁺ (mesoderm), and tubulin⁺ (ectoderm) cells. Scale bars=50 μm. (F) Teratoma formation in immunodeficient mice by the iPSCs. H&E staining was performed for the teratoma. The teratoma resulting from Tbx1^−/− and Tbx1^+/+ iPSCs contained various tissues.

FIG. 2.

Role of Tbx1 in the expression of endodermal lineage genes, in vitro. (A) Morphology of embryoid bodies (EBs) after 3 days of differentiation. (B–D) Real-time quantitative RT-PCR (qRT-PCR) was performed to detect the endoderm markers Sox17 (B), Foxa2 (C), and Gata4 (D). The expression level of genes in the Tbx^+/+ at day 1 is defined as 1. (E, F) EBs from Tbx1^−/− and Tbx1^+/+ iPSCs on day 11 of differentiation were determined for the expression of AFP by flow cytometry. (E) A representative histogram of AFP expression. (F) Mean percentage of AFP⁺ cells. Values are means±SEM; *P<0.05. All experiments were independently repeated at least three times.

FIG. 3.

Role of Tbx1 in the expression of mesodermal lineage genes in vitro. The mRNA levels of the mesodermal markers Brachyury (A), Flk-1 (B), as well as early cardiogenic markers Nkx2.5 (C) and Mef2c (D) in Tbx1^−/− and Tbx1^+/+ EBs were measured using qRT-PCR. The expression level of genes in the Tbx^+/+ at day 1 or 7 is defined as 1. (E) Terminal cardiac markers (α-MHC and cTnT) were measured on day 15 of the differentiation by qRT-PCR. The expression level of genes in the Tbx^+/+ is defined as 1. (F) The incidence of spontaneously beating EBs from the iPSCs was quantified at different time points during differentiation. (G, H) The expression of α-SMA was analyzed by flow cytometry 10 days after the differentiation. (G) A representative histogram of α-SMA expression. (H) Mean percentage of α-SMA⁺ cells. Values are means±SEM; *P<0.05. All experiments were independently repeated at least three times.

FIG. 4.

Role of Tbx1 in the expression of ectodermal lineage genes in vitro. The mRNA levels of ectodermal markers Fgf5 (A), Sox1 (B), and Nestin (C) were measured by qRT-PCR during in vitro differentiation of Tbx1^−/− and Tbx1^+/+ iPSCs. The expression level of genes in the Tbx^+/+ at day 1 is defined as 1. (D, E) The expression of β III tubulin was analyzed by flow cytometry 10 days after the differentiation. (D) A representative histogram of β III tubulin expression. (E) Mean percentage of β III tubulin⁺ cells. Values are means±SEM. All experiments were independently repeated at least three times.

FIG. 5.

Effect of restoration and overexpression of Tbx1 in the endodermal differentiation in vitro. Tbx1^−/− iPSCs transduced with inducible lentivirus containing Tbx1 gene were induced to differentiate by EB cultures. The expression of Tbx1 in the Tbx1^−/− iPSCs was induced by 50 or 500 ng/mL doxycycline. Tbx1^+/+ iPSCs in the presence of phosphate-buffered saline (PBS) were used as positive controls. The expression level of Tbx1 was determined by western blot (A). qRT-PCR was performed to detect genes of the endoderm markers Sox17 (B), Foxa2 (C), and Gata4 (D). The expression level of genes in the Tbx^+/+ at day 1 is defined as 1. (E) Quantitative evaluation of endodermal induction by analysis of the AFP-positive area after immunofluorescent staining for EBs from iPSCs on day 11 of differentiation. Values are means±SEM; *P<0.05. All experiments were independently repeated at least three times.

FIG. 6.

Effect of restoration and overexpression of Tbx1 in the expression of mesodermal differentiation in vitro. Tbx1^−/− iPSCs transduced with inducible lentivirus containing Tbx1 gene were induced to differentiate by EB cultures. The expression of tbx1 in the tbx1^−/− iPSCs was induced by 50 or 500 ng/mL doxycycline. Tbx1^+/+ iPSCs in the presence of PBS were used as positive controls. The mRNA levels of the mesodermal markers Flk-1 (A), Brachyury (B), and the early cardiogenic markers Mef2c (C) and Nkx2.5 (D) were measured by qRT-PCR during in vitro differentiation. (E) Terminal cardiac markers (α-MHC and cTnT) were measured on day 15 of differentiation by qRT-PCR. (F) The incidence of spontaneously beating EBs from iPSCs was quantified at different time points during differentiation. Values are means±SEM; *P<0.05. All experiments were independently repeated at least three times.

FIG. 7.

Role of Tbx1 in the fibroblast growth factor signaling. The mRNA levels of Fgf10 (A) and Fgf8 (B) were measured by qRT-PCR during in vitro differentiation of Tbx1^−/− and Tbx1^+/+ iPSCs. The expression level of genes in the Tbx^+/+ at day 1 is defined as 1. Values are means±SEM; *P<0.05. All experiments were independently repeated at least three times.