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Mycoplasma detection and elimination are necessary for the application of stem cell from human dental apical papilla to tissue engineering and regenerative medicine.

Kim BC, Kim SY, Kwon YD, Choe SC, Han DW, Hwang YS - Biomater Res (2015)

Bottom Line: Recently, postnatal stem cells from dental papilla with neural crest origin have been considered as one of potent stem cell sources in regenerative medicine regarding their multi-differentiation capacity and relatively easy access.In this study, mycoplama contamination was evaluated with stem cells from apical papilla which were isolated from human third molar and premolars from various aged patients undergoing orthodontic therapy.The ex-vivo expanded stem cells from apical papilla were found to express stem cell markers such as Stro-1, CD44, nestin and CD133, but mycoplama contamination was detected in almost all cell cultures of the tested 20 samples, which was confirmed by mycoplasma-specific gene expression and fluorescence staining.

View Article: PubMed Central - PubMed

Affiliation: Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul, 130-701 Republic of Korea.

ABSTRACT

Background: Recently, postnatal stem cells from dental papilla with neural crest origin have been considered as one of potent stem cell sources in regenerative medicine regarding their multi-differentiation capacity and relatively easy access. However, almost human oral tissues have been reported to be infected by mycoplasma which gives rise to oral cavity in teeth, and mycoplasma contamination of ex-vivo cultured stem cells from such dental tissues and its effect on stem cell culture has received little attention.

Results: In this study, mycoplama contamination was evaluated with stem cells from apical papilla which were isolated from human third molar and premolars from various aged patients undergoing orthodontic therapy. The ex-vivo expanded stem cells from apical papilla were found to express stem cell markers such as Stro-1, CD44, nestin and CD133, but mycoplama contamination was detected in almost all cell cultures of the tested 20 samples, which was confirmed by mycoplasma-specific gene expression and fluorescence staining. Such contaminated mycoplasma could be successfully eliminated using elimination kit, and proliferation test showed decreased proliferation activity in mycoplasma-contaminated cells. After elimination of contaminated mycoplasma, stem cells from apical papilla showed osteogenic and neural lineage differentiation under certain culture conditions.

Conclusion: Our study proposes that the evaluation of mycoplasma contamination and elimination process might be required in the use of stem cells from apical papilla for their potent applications to tissue engineering and regenerative medicine.

No MeSH data available.


Related in: MedlinePlus

Osteogenic and neural differentiation of mycoplasma-eliminated hSCAPs. I. 2D Osteogenic differentiation of mycoplasma-eliminated hSCAPs: A, B and C. Phenotypic ALPase expression after 5, 10 and 15 days of osteogenic culture, D, F and G. Alizarin red-S stained image of mineralized nodules after 20 days of osteogenic culture under light microscope, E. alizarin red-S stained image of mineralized nodules after 20 days of osteogenic culture under fluorescence microscope, II. 3D Osteogenic differentiation of mycoplasma-eliminated hSCAPs: A. alginate hydrogel, B and C: alginate hydrogel encapsulating hSCAPs, D and E. alizarin red-S stained image of mineralized nodules within alginate hydrogel after 20 days of 3D osteogenic culture under fluorescence microscope, III. 2D and 3D neural differentiation of mycoplasma-eliminated hSCAPs: A and B. microscopic images of hSCAPs in 2D neural differentiation culture. C and D. βIII tubulin (red), Cam kinase II (green) and nuclear staining (DAPI; blue), E. Schematic illustration of hSCAPs encapsulation of hSCAPs for 3D neural differentiation. F. tubular alginate hydrogel, G. live and dead image of encapsulated hSCAPs in tubular alginate hydrogel, H. microscopic images of hSCAPs in 3D neural differentiation culture, I. βIII tubulin (red), Cam kinase II (green) and nuclear staining (DAPI; blue) of differentiated hSCAPs in 3D neural differentiation culture, J. βIII tubulin (green), Cam kinase II (red) and nuclear staining (DAPI; blue) of differentiated hSCAPs in 3D neural differentiation culture, Scale bars indicate 200 μm.
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Fig3: Osteogenic and neural differentiation of mycoplasma-eliminated hSCAPs. I. 2D Osteogenic differentiation of mycoplasma-eliminated hSCAPs: A, B and C. Phenotypic ALPase expression after 5, 10 and 15 days of osteogenic culture, D, F and G. Alizarin red-S stained image of mineralized nodules after 20 days of osteogenic culture under light microscope, E. alizarin red-S stained image of mineralized nodules after 20 days of osteogenic culture under fluorescence microscope, II. 3D Osteogenic differentiation of mycoplasma-eliminated hSCAPs: A. alginate hydrogel, B and C: alginate hydrogel encapsulating hSCAPs, D and E. alizarin red-S stained image of mineralized nodules within alginate hydrogel after 20 days of 3D osteogenic culture under fluorescence microscope, III. 2D and 3D neural differentiation of mycoplasma-eliminated hSCAPs: A and B. microscopic images of hSCAPs in 2D neural differentiation culture. C and D. βIII tubulin (red), Cam kinase II (green) and nuclear staining (DAPI; blue), E. Schematic illustration of hSCAPs encapsulation of hSCAPs for 3D neural differentiation. F. tubular alginate hydrogel, G. live and dead image of encapsulated hSCAPs in tubular alginate hydrogel, H. microscopic images of hSCAPs in 3D neural differentiation culture, I. βIII tubulin (red), Cam kinase II (green) and nuclear staining (DAPI; blue) of differentiated hSCAPs in 3D neural differentiation culture, J. βIII tubulin (green), Cam kinase II (red) and nuclear staining (DAPI; blue) of differentiated hSCAPs in 3D neural differentiation culture, Scale bars indicate 200 μm.

Mentions: For 2D osteogenic differentiation of mycoplasma-eliminated hSCAPs, ostegenic differentiation of hSCAPs was induced in the presence of osteogenic supplements such as ascorbic acid, dexamethasone and β-glycerophosphate, and, as shown in Figure 3 IA-C, culture time-dependent increase of phenotypic ALP expression during osteogenic differentiation. After 20 days of osteogenic differentiation, mineralized nodules were formed in osteogenic culture of hSCAPs throughout whole culture area, which proved by strong positive staining with alizarin red-S (Figure 3 ID, F and G), and such mineralized nodules were found to have high content of calcium and phosphate, which proved by high fluorescence in alizarin red-S staining under fluorescent microscope (Figure 3 IE). In addition to 2D osteogenic differentiation of mycoplasma-eliminated hSCAPs, 3D osteogenic differentiation of hSCAPs were induced by dynamic osteogenic culture of alginate encapsulated hSCAPs in a rotating HARV bioreactor (Figure 3 II). 3D highly mineralized nodules formed by differentiated hSCAPs within alginate hydrogel were found at 20 days of dynamic osteogenic culture (Figure 3 IID and E)Figure 3


Mycoplasma detection and elimination are necessary for the application of stem cell from human dental apical papilla to tissue engineering and regenerative medicine.

Kim BC, Kim SY, Kwon YD, Choe SC, Han DW, Hwang YS - Biomater Res (2015)

Osteogenic and neural differentiation of mycoplasma-eliminated hSCAPs. I. 2D Osteogenic differentiation of mycoplasma-eliminated hSCAPs: A, B and C. Phenotypic ALPase expression after 5, 10 and 15 days of osteogenic culture, D, F and G. Alizarin red-S stained image of mineralized nodules after 20 days of osteogenic culture under light microscope, E. alizarin red-S stained image of mineralized nodules after 20 days of osteogenic culture under fluorescence microscope, II. 3D Osteogenic differentiation of mycoplasma-eliminated hSCAPs: A. alginate hydrogel, B and C: alginate hydrogel encapsulating hSCAPs, D and E. alizarin red-S stained image of mineralized nodules within alginate hydrogel after 20 days of 3D osteogenic culture under fluorescence microscope, III. 2D and 3D neural differentiation of mycoplasma-eliminated hSCAPs: A and B. microscopic images of hSCAPs in 2D neural differentiation culture. C and D. βIII tubulin (red), Cam kinase II (green) and nuclear staining (DAPI; blue), E. Schematic illustration of hSCAPs encapsulation of hSCAPs for 3D neural differentiation. F. tubular alginate hydrogel, G. live and dead image of encapsulated hSCAPs in tubular alginate hydrogel, H. microscopic images of hSCAPs in 3D neural differentiation culture, I. βIII tubulin (red), Cam kinase II (green) and nuclear staining (DAPI; blue) of differentiated hSCAPs in 3D neural differentiation culture, J. βIII tubulin (green), Cam kinase II (red) and nuclear staining (DAPI; blue) of differentiated hSCAPs in 3D neural differentiation culture, Scale bars indicate 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: Osteogenic and neural differentiation of mycoplasma-eliminated hSCAPs. I. 2D Osteogenic differentiation of mycoplasma-eliminated hSCAPs: A, B and C. Phenotypic ALPase expression after 5, 10 and 15 days of osteogenic culture, D, F and G. Alizarin red-S stained image of mineralized nodules after 20 days of osteogenic culture under light microscope, E. alizarin red-S stained image of mineralized nodules after 20 days of osteogenic culture under fluorescence microscope, II. 3D Osteogenic differentiation of mycoplasma-eliminated hSCAPs: A. alginate hydrogel, B and C: alginate hydrogel encapsulating hSCAPs, D and E. alizarin red-S stained image of mineralized nodules within alginate hydrogel after 20 days of 3D osteogenic culture under fluorescence microscope, III. 2D and 3D neural differentiation of mycoplasma-eliminated hSCAPs: A and B. microscopic images of hSCAPs in 2D neural differentiation culture. C and D. βIII tubulin (red), Cam kinase II (green) and nuclear staining (DAPI; blue), E. Schematic illustration of hSCAPs encapsulation of hSCAPs for 3D neural differentiation. F. tubular alginate hydrogel, G. live and dead image of encapsulated hSCAPs in tubular alginate hydrogel, H. microscopic images of hSCAPs in 3D neural differentiation culture, I. βIII tubulin (red), Cam kinase II (green) and nuclear staining (DAPI; blue) of differentiated hSCAPs in 3D neural differentiation culture, J. βIII tubulin (green), Cam kinase II (red) and nuclear staining (DAPI; blue) of differentiated hSCAPs in 3D neural differentiation culture, Scale bars indicate 200 μm.
Mentions: For 2D osteogenic differentiation of mycoplasma-eliminated hSCAPs, ostegenic differentiation of hSCAPs was induced in the presence of osteogenic supplements such as ascorbic acid, dexamethasone and β-glycerophosphate, and, as shown in Figure 3 IA-C, culture time-dependent increase of phenotypic ALP expression during osteogenic differentiation. After 20 days of osteogenic differentiation, mineralized nodules were formed in osteogenic culture of hSCAPs throughout whole culture area, which proved by strong positive staining with alizarin red-S (Figure 3 ID, F and G), and such mineralized nodules were found to have high content of calcium and phosphate, which proved by high fluorescence in alizarin red-S staining under fluorescent microscope (Figure 3 IE). In addition to 2D osteogenic differentiation of mycoplasma-eliminated hSCAPs, 3D osteogenic differentiation of hSCAPs were induced by dynamic osteogenic culture of alginate encapsulated hSCAPs in a rotating HARV bioreactor (Figure 3 II). 3D highly mineralized nodules formed by differentiated hSCAPs within alginate hydrogel were found at 20 days of dynamic osteogenic culture (Figure 3 IID and E)Figure 3

Bottom Line: Recently, postnatal stem cells from dental papilla with neural crest origin have been considered as one of potent stem cell sources in regenerative medicine regarding their multi-differentiation capacity and relatively easy access.In this study, mycoplama contamination was evaluated with stem cells from apical papilla which were isolated from human third molar and premolars from various aged patients undergoing orthodontic therapy.The ex-vivo expanded stem cells from apical papilla were found to express stem cell markers such as Stro-1, CD44, nestin and CD133, but mycoplama contamination was detected in almost all cell cultures of the tested 20 samples, which was confirmed by mycoplasma-specific gene expression and fluorescence staining.

View Article: PubMed Central - PubMed

Affiliation: Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul, 130-701 Republic of Korea.

ABSTRACT

Background: Recently, postnatal stem cells from dental papilla with neural crest origin have been considered as one of potent stem cell sources in regenerative medicine regarding their multi-differentiation capacity and relatively easy access. However, almost human oral tissues have been reported to be infected by mycoplasma which gives rise to oral cavity in teeth, and mycoplasma contamination of ex-vivo cultured stem cells from such dental tissues and its effect on stem cell culture has received little attention.

Results: In this study, mycoplama contamination was evaluated with stem cells from apical papilla which were isolated from human third molar and premolars from various aged patients undergoing orthodontic therapy. The ex-vivo expanded stem cells from apical papilla were found to express stem cell markers such as Stro-1, CD44, nestin and CD133, but mycoplama contamination was detected in almost all cell cultures of the tested 20 samples, which was confirmed by mycoplasma-specific gene expression and fluorescence staining. Such contaminated mycoplasma could be successfully eliminated using elimination kit, and proliferation test showed decreased proliferation activity in mycoplasma-contaminated cells. After elimination of contaminated mycoplasma, stem cells from apical papilla showed osteogenic and neural lineage differentiation under certain culture conditions.

Conclusion: Our study proposes that the evaluation of mycoplasma contamination and elimination process might be required in the use of stem cells from apical papilla for their potent applications to tissue engineering and regenerative medicine.

No MeSH data available.


Related in: MedlinePlus