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. 2014 Dec 4;4:7283.
doi: 10.1038/srep07283.

The Homeobox Gene DLX4 Promotes Generation of Human Induced Pluripotent Stem Cells

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Free PMC article

The Homeobox Gene DLX4 Promotes Generation of Human Induced Pluripotent Stem Cells

Naritaka Tamaoki et al. Sci Rep. .
Free PMC article

Abstract

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by defined transcription factors has been a well-established technique and will provide an invaluable resource for regenerative medicine. However, the low reprogramming efficiency of human iPSC is still a limitation for clinical application. Here we showed that the reprogramming potential of human dental pulp cells (DPCs) obtained from immature teeth is much higher than those of mature teeth DPCs. Furthermore, immature teeth DPCs can be reprogrammed by OCT3/4 and SOX2, conversely these two factors are insufficient to convert mature teeth DPCs to pluripotent states. Using a gene expression profiles between these two DPC groups, we identified a new transcript factor, distal-less homeobox 4 (DLX4), which was highly expressed in immature teeth DPCs and significantly promoted human iPSC generation in combination with OCT3/4, SOX2, and KLF4. We further show that activation of TGF-β signaling suppresses the expression of DLX4 in DPCs and impairs the iPSC generation of DPCs. Our findings indicate that DLX4 can functionally replace c-MYC and supports efficient reprogramming of immature teeth DPCs.

Figures

Figure 1
Figure 1. DP31 cells can be reprogrammed to iPSCs using OCT3/4 and SOX2.
(a) We obtained a few ES-cell-like colonies from 5 × 105 DP31 cells transduced with OCT3/4 and SOX2 (OS), but we could not obtain any ES-cell-like colonies from DP75 cells. Human ES-cell-like colonies were counted at 30 days post infection. Error bars indicate ± S.D. (n = 3). (b) Genomic PCR using transgene-specific primers, with DP31 cells as a negative control, confirmed the insertion of only two transgenes in iPSCs derived from DP31 cells transduced with OS (iPS-DP31-OS) by PCR. The numbers denote different iPSC lines. We cropped the gels and blots for clarifying our presentation. The gels have been run under the same experimental conditions. (c) iPS-DP31-OS cells expressed pluripotency markers including SSEA3, TRA-1-60, TRA-1-81, and NANOG. Scale bar = 100 μm. (d) Pluripotency of iPS-DP31-OS cells was confirmed by EB-mediated differentiation and teratoma formation assay. Immunofluorescence staining showed that EB structures derived from iPS-DP31-OS cells expressed markers characteristic of the three germ layers including βIII-tubulin (ectoderm), α-smooth-muscle actin (mesoderm), and α-fetoprotein (endoderm). Nuclei were stained with Hoechst 33342. Scale bar = 100 μm. Hematoxylin- and eosin-stained sections of teratomas generated from iPS-DP31-OS cells are shown in the lower panels. The teratomas contained various tissues of all three germ layers, such as neural-tube-like structures (ectoderm), cartilage (mesoderm), and gut-like epithelial tissue (endoderm). Abbreviations: AFP, alpha-fetoprotein; α-SMA, alpha smooth muscle actin. Scale bar = 100 μm.
Figure 2
Figure 2. DPCs from immature teeth were more amenable to reprogramming than DPCs from mature teeth and showed high expression levels of endogenous DLX4.
(a) Nine DPC lines were selected for evaluation of reprogramming potency. The four Yamanaka factors (OSKM) were introduced by a retroviral system, and the induced human ES-cell-like colonies were counted at 21 days post infection. The number of human ES-cell-like colonies obtained from each DPC line was normalized against that obtained from DP31 cells. Error bars indicate ± S.D. (n = 3). Asterisks indicate statistical significance: *P < 0.05, ** P < 0.01, and *** P < 0.001 compared to DP31 values. (b) The transcript levels of endogenous DLX4 were quantified using real-time RT-PCR. DPCs from immature teeth showed significantly higher levels of endogenous DLX4 than DPCs from mature teeth. Moreover, the transcript levels of DLX4 in all immature teeth DPCs were similar to those of human ES cells (hKES). GAPDH was used as an internal control. Error bars indicate ± S.D. (n = 3). Asterisks indicate statistical significance: *P < 0.05, ** P < 0.01, and *** P < 0.001 compared to values from DP31 cells.
Figure 3
Figure 3. Ectopic expression of DLX4 promoted human iPSC generation.
(a-c) Numbers of human ES-cell-like colonies obtained from DPCs and HDFs. Human ES-cell-like colonies isolated from 5 × 105 or 5 × 104 infected cells were counted at 30 days post infection. Error bars indicate ± S.D. (n = 3). Asterisks indicate statistical significance; *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 4
Figure 4. Activation of TGF-β signal suppressed the expression level of DLX4 in DPCs and impaired the generation of iPSCs from DPCs.
(a) DP1 and DP31 cells were cultured in DPC culture medium with TGF-β1 (5 ng/mL) or with acetic acid (as a control) for 48 h. Real-time PCR analysis showed that after activation of TGF-β signal, the transcript levels of DLX4 were down-regulated in both DP1 and DP31 cells. GAPDH was used as an internal control. Error bars indicate ± S.D. (n = 3). Asterisks indicate statistical significance; ***P < 0.001 (b) Human ES-cell-like colonies isolated from 5 × 104 infected cells (OSKM) were counted at 21 days post infection. Numbers of human ES-cell-like colonies obtained from DP1 and DP31 were dramatically reduced when TGF-β1 (5 ng/mL) was added during the reprogramming process.

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