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Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells.

Cai J, Zhang Y, Liu P, Chen S, Wu X, Sun Y, Li A, Huang K, Luo R, Wang L, Liu Y, Zhou T, Wei S, Pan G, Pei D - Cell Regen (Lond) (2013)

Bottom Line: Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30% for 8 different iPSC lines, comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures, possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, 190 Kai Yuan Avenue, Science Park, Guangzhou, 510530 P.R.China.

ABSTRACT

Background: Tooth is vital not only for a good smile, but also good health. Yet, we lose tooth regularly due to accidents or diseases. An ideal solution to this problem is to regenerate tooth with patients' own cells. Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells (ifhU-iPSCs).

Results: We first differentiated ifhU-iPSCs to epithelial sheets, which were then recombined with E14.5 mouse dental mesenchymes. Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30% for 8 different iPSC lines, comparable to H1 hESC. We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures, possessing physical properties such as elastic modulus and hardness found in the regular human tooth.

Conclusion: Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

No MeSH data available.


Related in: MedlinePlus

hESC/iPSC derived epithelial lineages.(A) Representative the epithelial differentiation process of hESCs or hiPSCs through supplement of RA and BMP4 in N2 medium for 7 days, then either changing into DSFM directly or passaging at a split ratio of 1:3 for culture continuously. Scale bar corresponds to 200 μm. (B) qPCR of representative experiment showing the down regulation of ESC-specific transcription factor (Oct4) and up regulation of keratinized epithelial markers (K18, p63, K19, CD29, K14). (C) Western blot for Oct4, p63, and K18 of lysates from H1 or ifhU1-iPSCs derived epithelial cells; GAPDH is the loading control. (D) Phase contrast captures and immunofluorescence staining [IF: p63 (red), K14 (green), DAPI (blue)] of epithelial sheets derived from H1 and ifhU1-iPSCs at D7 and D21. Scale bars correspond to 2000 and 200 μm, respectively. (E) Scanning electron microscope (SEM) and transmission electron microscopy (TEM) images of H1-ESCs and ifhU1-iPSCs derived epithelial sheets at D7 and D14. White arrowheads indicate desmosomes between epithelial cells of the sheet. Scale bars in SEM and TEM correspond to 20 and 0.5 μm respectively.
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Fig1: hESC/iPSC derived epithelial lineages.(A) Representative the epithelial differentiation process of hESCs or hiPSCs through supplement of RA and BMP4 in N2 medium for 7 days, then either changing into DSFM directly or passaging at a split ratio of 1:3 for culture continuously. Scale bar corresponds to 200 μm. (B) qPCR of representative experiment showing the down regulation of ESC-specific transcription factor (Oct4) and up regulation of keratinized epithelial markers (K18, p63, K19, CD29, K14). (C) Western blot for Oct4, p63, and K18 of lysates from H1 or ifhU1-iPSCs derived epithelial cells; GAPDH is the loading control. (D) Phase contrast captures and immunofluorescence staining [IF: p63 (red), K14 (green), DAPI (blue)] of epithelial sheets derived from H1 and ifhU1-iPSCs at D7 and D21. Scale bars correspond to 2000 and 200 μm, respectively. (E) Scanning electron microscope (SEM) and transmission electron microscopy (TEM) images of H1-ESCs and ifhU1-iPSCs derived epithelial sheets at D7 and D14. White arrowheads indicate desmosomes between epithelial cells of the sheet. Scale bars in SEM and TEM correspond to 20 and 0.5 μm respectively.

Mentions: We first must devise a way to obtain dental epithelia from hESCs or ifhU-iPSCs and decided on a stage-specific approach based on retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) in N2 medium [24]. To this end, we obtained epithelial cells with keratinocyte-like morphology at D7 (Figure 1A). When passaged with a ratio at 1:3, these cells became definitive keratinocytes in a defined keratinocyte serum-free medium (DSFM) at D42 (Figure 1A). However, these cells survive poorly after passage. We then allowed the cells to grow as epithelial sheets without passage, and they became denser at D14, and detached slightly with some cell death at D21 (Figure 1A). The differentiating cells at D7, 14, 21, 28 were harvested and investigated for the expression of pluripotent and keratinocyte progenitor’s markers by qPCR and Western blot. During epithelial differentiation, H1-ESCs and ifhU-iPSCs behaved similarly as shown with markers examined at RNA level (Oct4, K18, p63, K19, CD29, and K14), showing up regulated expressions in epithelial markers and down-regulation of pluripotent marker Oct4 (Figure 1B). Similar trends were also observed for protein expression (Oct4, K18 and p63, Figure 1C). Moreover, the expression of p63 and K14 were verified by immunofluorescence. p63 was detected earlier at D7 and continuously expressed at D21, while K14 expression was detected later at D21 (Figure 1D). As a result, we obtained homogenous layers of epithelial cells as sheets from both hESCs and ifhU-iPSCs at D7 (Figure 1D). These sheets harvested at D7 were tenacious and flexible, showing the flat and smooth surface at the apical side as observed by scanning electronic microscopy (SEM) (Figure 1E). The sheets became rugged with prominent nuclei at D14 (Figure 1E). Under transmission electron microscopy (TEM), the desmosomes could be observed clearly between the epithelial cells at both D7 and D14 (Figure 1E). Together, these results suggest that the epithelial sheets generated at D7 from hESCs or ifhU-iPSCs have desired properties for being induced for tooth regeneration.Figure 1


Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells.

Cai J, Zhang Y, Liu P, Chen S, Wu X, Sun Y, Li A, Huang K, Luo R, Wang L, Liu Y, Zhou T, Wei S, Pan G, Pei D - Cell Regen (Lond) (2013)

hESC/iPSC derived epithelial lineages.(A) Representative the epithelial differentiation process of hESCs or hiPSCs through supplement of RA and BMP4 in N2 medium for 7 days, then either changing into DSFM directly or passaging at a split ratio of 1:3 for culture continuously. Scale bar corresponds to 200 μm. (B) qPCR of representative experiment showing the down regulation of ESC-specific transcription factor (Oct4) and up regulation of keratinized epithelial markers (K18, p63, K19, CD29, K14). (C) Western blot for Oct4, p63, and K18 of lysates from H1 or ifhU1-iPSCs derived epithelial cells; GAPDH is the loading control. (D) Phase contrast captures and immunofluorescence staining [IF: p63 (red), K14 (green), DAPI (blue)] of epithelial sheets derived from H1 and ifhU1-iPSCs at D7 and D21. Scale bars correspond to 2000 and 200 μm, respectively. (E) Scanning electron microscope (SEM) and transmission electron microscopy (TEM) images of H1-ESCs and ifhU1-iPSCs derived epithelial sheets at D7 and D14. White arrowheads indicate desmosomes between epithelial cells of the sheet. Scale bars in SEM and TEM correspond to 20 and 0.5 μm respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230506&req=5

Fig1: hESC/iPSC derived epithelial lineages.(A) Representative the epithelial differentiation process of hESCs or hiPSCs through supplement of RA and BMP4 in N2 medium for 7 days, then either changing into DSFM directly or passaging at a split ratio of 1:3 for culture continuously. Scale bar corresponds to 200 μm. (B) qPCR of representative experiment showing the down regulation of ESC-specific transcription factor (Oct4) and up regulation of keratinized epithelial markers (K18, p63, K19, CD29, K14). (C) Western blot for Oct4, p63, and K18 of lysates from H1 or ifhU1-iPSCs derived epithelial cells; GAPDH is the loading control. (D) Phase contrast captures and immunofluorescence staining [IF: p63 (red), K14 (green), DAPI (blue)] of epithelial sheets derived from H1 and ifhU1-iPSCs at D7 and D21. Scale bars correspond to 2000 and 200 μm, respectively. (E) Scanning electron microscope (SEM) and transmission electron microscopy (TEM) images of H1-ESCs and ifhU1-iPSCs derived epithelial sheets at D7 and D14. White arrowheads indicate desmosomes between epithelial cells of the sheet. Scale bars in SEM and TEM correspond to 20 and 0.5 μm respectively.
Mentions: We first must devise a way to obtain dental epithelia from hESCs or ifhU-iPSCs and decided on a stage-specific approach based on retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) in N2 medium [24]. To this end, we obtained epithelial cells with keratinocyte-like morphology at D7 (Figure 1A). When passaged with a ratio at 1:3, these cells became definitive keratinocytes in a defined keratinocyte serum-free medium (DSFM) at D42 (Figure 1A). However, these cells survive poorly after passage. We then allowed the cells to grow as epithelial sheets without passage, and they became denser at D14, and detached slightly with some cell death at D21 (Figure 1A). The differentiating cells at D7, 14, 21, 28 were harvested and investigated for the expression of pluripotent and keratinocyte progenitor’s markers by qPCR and Western blot. During epithelial differentiation, H1-ESCs and ifhU-iPSCs behaved similarly as shown with markers examined at RNA level (Oct4, K18, p63, K19, CD29, and K14), showing up regulated expressions in epithelial markers and down-regulation of pluripotent marker Oct4 (Figure 1B). Similar trends were also observed for protein expression (Oct4, K18 and p63, Figure 1C). Moreover, the expression of p63 and K14 were verified by immunofluorescence. p63 was detected earlier at D7 and continuously expressed at D21, while K14 expression was detected later at D21 (Figure 1D). As a result, we obtained homogenous layers of epithelial cells as sheets from both hESCs and ifhU-iPSCs at D7 (Figure 1D). These sheets harvested at D7 were tenacious and flexible, showing the flat and smooth surface at the apical side as observed by scanning electronic microscopy (SEM) (Figure 1E). The sheets became rugged with prominent nuclei at D14 (Figure 1E). Under transmission electron microscopy (TEM), the desmosomes could be observed clearly between the epithelial cells at both D7 and D14 (Figure 1E). Together, these results suggest that the epithelial sheets generated at D7 from hESCs or ifhU-iPSCs have desired properties for being induced for tooth regeneration.Figure 1

Bottom Line: Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30% for 8 different iPSC lines, comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures, possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, 190 Kai Yuan Avenue, Science Park, Guangzhou, 510530 P.R.China.

ABSTRACT

Background: Tooth is vital not only for a good smile, but also good health. Yet, we lose tooth regularly due to accidents or diseases. An ideal solution to this problem is to regenerate tooth with patients' own cells. Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells (ifhU-iPSCs).

Results: We first differentiated ifhU-iPSCs to epithelial sheets, which were then recombined with E14.5 mouse dental mesenchymes. Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30% for 8 different iPSC lines, comparable to H1 hESC. We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures, possessing physical properties such as elastic modulus and hardness found in the regular human tooth.

Conclusion: Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

No MeSH data available.


Related in: MedlinePlus