DataSheet1_Epiprofin Transcriptional Activation Promotes Ameloblast Induction From Mouse Induced Pluripotent Stem Cells via the BMP-Smad Signaling Axis.docx

The transcriptional regulation of induced pluripotent stem cells (iPSCs) holds promise for their directed differentiation into ameloblasts, which are usually lost after tooth eruption. Ameloblast differentiation is regulated by multiple signaling molecules, including bone morphogenetic proteins (BMPs). Epiprofin (Epfn), a transcription factor, is expressed in the dental epithelium, and epithelial Epfn overexpression results in ectopic ameloblast differentiation and enamel formation in mouse incisor, a striking phenotype resembling that of mice with deletion of follistatin (a BMP inhibitor). However, it remains unknown whether and how Epfn transcriptional activation promotes ameloblast induction from mouse iPSCs. Here, we generated doxycycline-inducible Epfn-expressing mouse iPSCs (Epfn-iPSCs). Ameloblasts, which are characterized by positive staining for keratin 14 and amelogenin and alizarin red S staining, were successfully derived from Epfn-iPSCs based on a stage-specific induction protocol, which involved the induction of the surface ectoderm, dental epithelial cells, and ameloblasts at stages 1, 2, and 3, respectively. Epfn activation by doxycycline at stages 2 and/or 3 decreased cell proliferation and promoted ameloblast differentiation, along with the upregulation of p-Smad1/5/8, a key regulator of the BMP-Smad signaling pathway. Gene analysis of the BMP-Smad signaling pathway-associated molecules revealed that Epfn activation decreased follistatin expression at stage 2, but increased BMP2/4/7 expression at stage 3. Perturbations in the ameloblast differentiation process were observed when the BMP-Smad signaling pathway was inhibited by a BMP receptor inhibitor (LDN-193189). Simultaneous LDN-193189 treatment and Epfn activation largely reversed the perturbations in ameloblast induction, with partial recovery of p-Smad1/5/8 expression, suggesting that Epfn activation promotes ameloblast induction from mouse iPSCs partially by upregulating BMP-Smad activity. These results reveal the potential regulatory networks between Epfn and the BMP-Smad pathway and suggest that Epfn is a promising target for inducing the differentiation of ameloblasts, which can be used in enamel and tooth regeneration.</p

sj-docx-1-tej-10.1177_20417314221114616 – Supplemental material for Rapid and efficient generation of cartilage pellets from mouse induced pluripotent stem cells by transcriptional activation of BMP-4 with shaking culture

Supplemental material, sj-docx-1-tej-10.1177_20417314221114616 for Rapid and efficient generation of cartilage pellets from mouse induced pluripotent stem cells by transcriptional activation of BMP-4 with shaking culture by Maolin Zhang, Kunimichi Niibe, Takeru Kondo, Phoonsuk Limraksasin, Hiroko Okawa, Xinchao Miao, Yuya Kamano, Masahiro Yamada, Xinquan Jiang and Hiroshi Egusa in Journal of Tissue Engineering</p

Induction of <i>Amelx</i> expression in MSCs-<i>TetR</i>/<i>Amelx</i> by Dox treatment.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> were cultured in the growth medium in the presence (<b>+</b>) or absence (<b>-</b>) of Dox for 24–72 hours. Inducible expression of the <i>Amelx</i> gene was detected by RT-PCR (<b>a</b>) and western blot (<b>b</b>) analyses. GAPDH was used as a loading control. (<b>c</b>) MSCs-<i>TetR</i>/<i>Amelx</i> were cultured in the osteogenic induction medium in the presence (+) (black bars) or absence (-) (white bars) of Dox for 17 days in four different conditions. (Condition I: day 0–17 Dox-; Condition II: day 0–14 Dox-, day 15–17 Dox+; Condition III: day 0–14 Dox+, day 15–17 Dox-; Condition IV: day 0–17 Dox+). Expression of <i>Amelx</i> was determined by quantitative real-time RT-PCR analysis. Significant differences (*<i>P</i><0.01: ANOVA with Tukey’s multiple comparison test: n = 4) within each condition are shown.</p

Effects of forced expression of <i>Amelx</i> on ALP activity and mineralized nodule formation.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> at 3 passages were cultured in the growth medium (Con) or osteogenic induction medium (Os) in the presence (<b>+</b>) or absence (-) of Dox in 24-well plates. (<b>a</b>) ALP activity on day 7 and 14 was examined by ALP staining (bars: 100 μm). (<b>b</b>) Mineralized nodule formation on day 21, 28 and 35 was detected by von Kossa staining. (<b>c, d</b>): Calcium deposition was determined by Alizarin Red S staining on day 7, 14 and 21 (<b>c</b>) and the staining intensity was quantitatively analyzed (<b>d</b>). Significant differences (*<i>P</i><0.01: ANOVA with Tukey’s multiple comparison test: n = 9) are shown.</p

Effects of forced expression of <i>Amelx</i> at different osteogenic differentiation stages on matrix calcification of MSCs.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> at 8 passages were cultured in the growth medium (Con) or osteogenic induction medium (Os). Dox was applied to MSCs-<i>TetR</i>/<i>Amelx</i> on day 0–10 (<b>a</b>: early stage), day 10–20 (<b>b</b>: intermediate stage), or day 20–30 (<b>c</b>: late stage) of osteogenic differentiation, and Alizarin Red S staining was performed at day 10, day 20, or day 30, respectively. The staining intensity was also quantitatively analyzed. Significant differences (*<i>P</i><0.01: ANOVA with Tukey’s multiple comparison test: n = 9) are shown.</p

Establishment of a tetracycline (Tet)-controlled <i>Amelx</i> expression system in MSCs.

<p>(<b>a</b>): Schematic diagram depicting the procedure to establish a Tet-controlled <i>Amelx</i> expression system in MSCs. TetR: Tet repressor, TO: Tet operator, Dox: doxycycline (tetracycline derivative). (<b>b</b>): MSC colony (MSC-<i>TetR</i>) in culture medium containing 500 μg/mL geneticin 10 days after transduction with the pLenti3.3/<i>TetR</i> expression vector (bar; 60 μm). (<b>c</b>): Expression of TetR repressor gene in MSCs (without transduction) and MSCs-<i>TetR</i> was determined by RT-PCR. HEK293 cells subjected to the same transduction procedure (HEK-<i>TetR</i>) were used as a positive control. (<b>d, e</b>): MSCs-<i>TetR</i> were lentivirally transduced with the expression vector pLenti6.3/TO/V5/<i>Amelx</i> or plenti6.3/V5-GW/<i>GFP</i>. MSCs-<i>TetR</i>/<i>Amelx</i> (<b>d</b>) and MSCs-<i>TetR</i>/<i>GFP</i> (<b>e</b>) were selected by 10 μg/mL blastcidin S (bar; 200 μm).</p

Controllable expression of <i>osterix</i>, <i>BSP</i> and <i>osteocalcin</i> in MSCs-<i>TetR</i>/<i>Amelx</i> by Dox treatment.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> were cultured in the osteogenic induction medium in the presence (+) (black bars) or absence (-) (white bars) of Dox for 17 days in four different conditions. (Condition I: day 0–17 Dox-; Condition II: day 0–14 Dox-, day 15–17 Dox+; Condition III: day 0–14 Dox+, day 15–17 Dox-; Condition IV: day 0–17 Dox+). Expression of <i>Runx2</i> (<b>a</b>), <i>osterix</i> (<b>b</b>), <i>BSP</i> (<b>c</b>), <i>osteopontin</i> (<b>d</b>) and <i>osteocalcin</i> (<b>e</b>) was determined by a quantitative real-time RT-PCR analysis. Significant differences (**<i>P</i><0.01, *<i>P</i><0.05: ANOVA with Tukey’s multiple comparison test: n = 3) within each condition are shown.</p

Effects of forced expression of <i>Amelx</i> in MSCs-<i>tetR</i>/<i>Amelx</i> on expression of osteogenic marker genes.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> were cultured in growth medium (Con) or osteogenic induction medium (Os) in the presence (<b>+</b>) or absence (-) of Dox for 21 days. (<b>a</b>) The expression of osteogenic marker genes (<i>Runx2</i>, <i>osterix</i>, <i>osteocalcin</i>, <i>BSP</i>, and <i>osteopontin</i>) was examined by RT-PCR analysis. <i>GAPDH</i> was used as an internal control. Quantitative real-time RT-PCR analysis was performed to examine expression of <i>osterix</i> (<b>b</b>), <i>type I collagen</i> (<b>c</b>), <i>BSP</i> (<b>d</b>), and <i>DMP1</i> (<b>e</b>) genes. <i>GAPDH</i> was used as an internal control. Significant differences (*<i>P</i><0.01: ANOVA with Tukey’s multiple comparison test: n = 3) are shown.</p