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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

Tooth-like structures formed from H1-ESC line and hiPSC lines in 3 weeks.(A) Left two columns: tooth-like structures after and before isolated from the surrounding tissues from H1-ESC and ifhU1-iPSC groups (tooth is outlined by a dotted line); right three columns: HE and immunohistochemical staining (IHC) of relative tooth sections (H1-ESC: sagittal section; ifhU1-iPSC: cross section), showing the tooth-like structures containing dental pulp (dp), dentin (d), enamel space (es), and a layer of ameloblasts (Am, high magnification in the blue box) in both groups. Positive Amel (black arrows) expresses in these ameloblasts with the papillary layer ( black arrowheads at high magnification in the green box). Scale bar: the upper and bottom figures in the same column share the same scale bar as 500, 500, 400, 100, 100 μm from left to right orderly. (B) IHC (top, HLA-I in cytoplasm) and IF (bottom, hNA in nucleus) staining in cross sections of the ifhU1-iPSCs derived tooth: human iPSC derived cells and tissues including ameloblast layer (arrowheads), cyst epithelial cells (arrows) and epithelium-derived cyst (*). DAPI is shown in blue. Scale bars: 200 μm. (C) Image of a piece of bone detected from E14.5 mouse dental mesenchyme being transplanted under kidney capsule for 3 weeks with positive BSP expression. Scale bars: 1000, 100, 100 μm orderly. (D) Nano-indentation analyses, Left: representative images of fractured enamel and dentin surfaces in adult human tooth group (human), group of 3-week mouse tooth from E14.5 tooth germs (Mouse-WT), regenerative tooth groups from H1-ESCs and ifhU-iPSCs; right: hardness and elastic modulus of enamel and dentin in above groups (each group: n=3). (E) Raman spectroscopy analyses of enamel and dentin from human, mouse, hESCs, and ifhU-iPSCs showed great similarity for all groups on Raman peaks. (F) Efficiencies of tooth-like structures for H1-ESC line and 8 iPSC lines.
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Fig2: Tooth-like structures formed from H1-ESC line and hiPSC lines in 3 weeks.(A) Left two columns: tooth-like structures after and before isolated from the surrounding tissues from H1-ESC and ifhU1-iPSC groups (tooth is outlined by a dotted line); right three columns: HE and immunohistochemical staining (IHC) of relative tooth sections (H1-ESC: sagittal section; ifhU1-iPSC: cross section), showing the tooth-like structures containing dental pulp (dp), dentin (d), enamel space (es), and a layer of ameloblasts (Am, high magnification in the blue box) in both groups. Positive Amel (black arrows) expresses in these ameloblasts with the papillary layer ( black arrowheads at high magnification in the green box). Scale bar: the upper and bottom figures in the same column share the same scale bar as 500, 500, 400, 100, 100 μm from left to right orderly. (B) IHC (top, HLA-I in cytoplasm) and IF (bottom, hNA in nucleus) staining in cross sections of the ifhU1-iPSCs derived tooth: human iPSC derived cells and tissues including ameloblast layer (arrowheads), cyst epithelial cells (arrows) and epithelium-derived cyst (*). DAPI is shown in blue. Scale bars: 200 μm. (C) Image of a piece of bone detected from E14.5 mouse dental mesenchyme being transplanted under kidney capsule for 3 weeks with positive BSP expression. Scale bars: 1000, 100, 100 μm orderly. (D) Nano-indentation analyses, Left: representative images of fractured enamel and dentin surfaces in adult human tooth group (human), group of 3-week mouse tooth from E14.5 tooth germs (Mouse-WT), regenerative tooth groups from H1-ESCs and ifhU-iPSCs; right: hardness and elastic modulus of enamel and dentin in above groups (each group: n=3). (E) Raman spectroscopy analyses of enamel and dentin from human, mouse, hESCs, and ifhU-iPSCs showed great similarity for all groups on Raman peaks. (F) Efficiencies of tooth-like structures for H1-ESC line and 8 iPSC lines.

Mentions: We then harvested D7 epithelial sheets and recombined them with the mouse dental mesenchyme before transplantation into mouse subrenal capsule (Additional file 1). After 3 weeks, we observed tooth-like structures with the fibrous cysts in the kidney (Figure 2A). We isolated individual tooth-like structures by removing them from the fibrous cysts and the surrounding bone (Figure 2A, left columns). We found that the tooth-like structures always appeared with the presence of fibrous cysts. The tooth-like structure contained dental pulp, dentin, enamel space, and enamel organ (Figure 2A, middle columns). The enamel organs have elongated ameloblasts with a ruffled border-like structure and papillary layer (Figure 2A, middle columns). We also observed the expression of Ameloblastin (Amel) located in the layer of ameloblasts and its papillary layer (Figure 2A). We confirmed the human origin of the epithelial component in cross sections of recombinant tooth prior to isolation by immunostainings with human specific antibodies against human leukocyte antigen-I (HLA-I) and human nucleus antigen (hNA) (Figure 2B). Both antibodies stained negatively in the dental pulp, cartilage, surrounding bone-like structures, which were developed from mouse dental mesenchyme (Figure 2B). As expected, both human specific antibodies stained positively for the ameloblasts (Figures 2B1 and 2B4), papillary layer besides (Figures 2B2 and 2B4), and squamous epithelial cells in the cyst (Figure 2B3). Furthermore, positive HLA-I staining was localized in the cytoplasm (Figures 2B1-2B3), while hNA was complementarily localized in the nucleus (Figures 2B4 and 2B5). As control, without recombination with hESCs or ifhU-iPSCs derived epithelial sheets, mouse dental mesenchymes transplanted under identical conditions formed bone-like structures instead (n=10/10), as confirmed by positive staining of bone sialoprotein (BSP) in the whole bone-like structure embedded with osteocytes (Figure 2C).Figure 2


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)

Tooth-like structures formed from H1-ESC line and hiPSC lines in 3 weeks.(A) Left two columns: tooth-like structures after and before isolated from the surrounding tissues from H1-ESC and ifhU1-iPSC groups (tooth is outlined by a dotted line); right three columns: HE and immunohistochemical staining (IHC) of relative tooth sections (H1-ESC: sagittal section; ifhU1-iPSC: cross section), showing the tooth-like structures containing dental pulp (dp), dentin (d), enamel space (es), and a layer of ameloblasts (Am, high magnification in the blue box) in both groups. Positive Amel (black arrows) expresses in these ameloblasts with the papillary layer ( black arrowheads at high magnification in the green box). Scale bar: the upper and bottom figures in the same column share the same scale bar as 500, 500, 400, 100, 100 μm from left to right orderly. (B) IHC (top, HLA-I in cytoplasm) and IF (bottom, hNA in nucleus) staining in cross sections of the ifhU1-iPSCs derived tooth: human iPSC derived cells and tissues including ameloblast layer (arrowheads), cyst epithelial cells (arrows) and epithelium-derived cyst (*). DAPI is shown in blue. Scale bars: 200 μm. (C) Image of a piece of bone detected from E14.5 mouse dental mesenchyme being transplanted under kidney capsule for 3 weeks with positive BSP expression. Scale bars: 1000, 100, 100 μm orderly. (D) Nano-indentation analyses, Left: representative images of fractured enamel and dentin surfaces in adult human tooth group (human), group of 3-week mouse tooth from E14.5 tooth germs (Mouse-WT), regenerative tooth groups from H1-ESCs and ifhU-iPSCs; right: hardness and elastic modulus of enamel and dentin in above groups (each group: n=3). (E) Raman spectroscopy analyses of enamel and dentin from human, mouse, hESCs, and ifhU-iPSCs showed great similarity for all groups on Raman peaks. (F) Efficiencies of tooth-like structures for H1-ESC line and 8 iPSC lines.
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Related In: Results  -  Collection

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Fig2: Tooth-like structures formed from H1-ESC line and hiPSC lines in 3 weeks.(A) Left two columns: tooth-like structures after and before isolated from the surrounding tissues from H1-ESC and ifhU1-iPSC groups (tooth is outlined by a dotted line); right three columns: HE and immunohistochemical staining (IHC) of relative tooth sections (H1-ESC: sagittal section; ifhU1-iPSC: cross section), showing the tooth-like structures containing dental pulp (dp), dentin (d), enamel space (es), and a layer of ameloblasts (Am, high magnification in the blue box) in both groups. Positive Amel (black arrows) expresses in these ameloblasts with the papillary layer ( black arrowheads at high magnification in the green box). Scale bar: the upper and bottom figures in the same column share the same scale bar as 500, 500, 400, 100, 100 μm from left to right orderly. (B) IHC (top, HLA-I in cytoplasm) and IF (bottom, hNA in nucleus) staining in cross sections of the ifhU1-iPSCs derived tooth: human iPSC derived cells and tissues including ameloblast layer (arrowheads), cyst epithelial cells (arrows) and epithelium-derived cyst (*). DAPI is shown in blue. Scale bars: 200 μm. (C) Image of a piece of bone detected from E14.5 mouse dental mesenchyme being transplanted under kidney capsule for 3 weeks with positive BSP expression. Scale bars: 1000, 100, 100 μm orderly. (D) Nano-indentation analyses, Left: representative images of fractured enamel and dentin surfaces in adult human tooth group (human), group of 3-week mouse tooth from E14.5 tooth germs (Mouse-WT), regenerative tooth groups from H1-ESCs and ifhU-iPSCs; right: hardness and elastic modulus of enamel and dentin in above groups (each group: n=3). (E) Raman spectroscopy analyses of enamel and dentin from human, mouse, hESCs, and ifhU-iPSCs showed great similarity for all groups on Raman peaks. (F) Efficiencies of tooth-like structures for H1-ESC line and 8 iPSC lines.
Mentions: We then harvested D7 epithelial sheets and recombined them with the mouse dental mesenchyme before transplantation into mouse subrenal capsule (Additional file 1). After 3 weeks, we observed tooth-like structures with the fibrous cysts in the kidney (Figure 2A). We isolated individual tooth-like structures by removing them from the fibrous cysts and the surrounding bone (Figure 2A, left columns). We found that the tooth-like structures always appeared with the presence of fibrous cysts. The tooth-like structure contained dental pulp, dentin, enamel space, and enamel organ (Figure 2A, middle columns). The enamel organs have elongated ameloblasts with a ruffled border-like structure and papillary layer (Figure 2A, middle columns). We also observed the expression of Ameloblastin (Amel) located in the layer of ameloblasts and its papillary layer (Figure 2A). We confirmed the human origin of the epithelial component in cross sections of recombinant tooth prior to isolation by immunostainings with human specific antibodies against human leukocyte antigen-I (HLA-I) and human nucleus antigen (hNA) (Figure 2B). Both antibodies stained negatively in the dental pulp, cartilage, surrounding bone-like structures, which were developed from mouse dental mesenchyme (Figure 2B). As expected, both human specific antibodies stained positively for the ameloblasts (Figures 2B1 and 2B4), papillary layer besides (Figures 2B2 and 2B4), and squamous epithelial cells in the cyst (Figure 2B3). Furthermore, positive HLA-I staining was localized in the cytoplasm (Figures 2B1-2B3), while hNA was complementarily localized in the nucleus (Figures 2B4 and 2B5). As control, without recombination with hESCs or ifhU-iPSCs derived epithelial sheets, mouse dental mesenchymes transplanted under identical conditions formed bone-like structures instead (n=10/10), as confirmed by positive staining of bone sialoprotein (BSP) in the whole bone-like structure embedded with osteocytes (Figure 2C).Figure 2

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