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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.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.

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

Mycoplasma-specific PCR analysis and fluorescence staining for primary cultured hSCAPs and proliferation activity.I. PCR analysis for 20 samples. II. fluorescence staining of mycoplasma in hSCAPs culture: arrow indicates positive staining of mycoplasma colony. III. mycoplasma-specific fluorescence staining of hSCAPs from selected 4 samples before and after mycoplasma elimination process. IV. proliferation assay for hSCAPs from selected 4 samples before and after mycoplasma elimination process.
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Fig2: Mycoplasma-specific PCR analysis and fluorescence staining for primary cultured hSCAPs and proliferation activity.I. PCR analysis for 20 samples. II. fluorescence staining of mycoplasma in hSCAPs culture: arrow indicates positive staining of mycoplasma colony. III. mycoplasma-specific fluorescence staining of hSCAPs from selected 4 samples before and after mycoplasma elimination process. IV. proliferation assay for hSCAPs from selected 4 samples before and after mycoplasma elimination process.

Mentions: In addition, for 3D osteogenic differentiation, hSCAPs were suspended in 1.1% (w/v) alginic acid (Sigma) and 0.1% (v/v) porcine gelatin (Sigma) solution (all dissolved in PBS, pH 7.4), as described before [19]. Briefly, the cell-gel solution was passed through a peristaltic pump (EYELA) and dropped using a 25-gauge needle into sterile alginate gelation solution composing of 100 mM CaCl2 (Sigma), 10 mM HEPES (Sigma), and 0.01% (v/v) Tween (Sigma) at pH 7.4 with stirring. Approximately 10,000 cells were encapsulated in each alginate hydrogel. The hydrogels remained in gently stirred CaCl2 solution for 6–10 mins and were then washed with PBS. The hSCAPs containing hydrogels were transferred to in 10 ml vessels of HARV bioreactors (Synthecon) and the vessels was rotated at 25 rpm. Subsequently, 3D osteogenic differentiation was induced using the same osteogenic medium described above. The bioprocess is illustrated in Figure 2 II. After 3D osteogenic differentiation, the sections of hydrogels were stained with alizarin red-S staining solution according to manufacturer’s instruction.Figure 2


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)

Mycoplasma-specific PCR analysis and fluorescence staining for primary cultured hSCAPs and proliferation activity.I. PCR analysis for 20 samples. II. fluorescence staining of mycoplasma in hSCAPs culture: arrow indicates positive staining of mycoplasma colony. III. mycoplasma-specific fluorescence staining of hSCAPs from selected 4 samples before and after mycoplasma elimination process. IV. proliferation assay for hSCAPs from selected 4 samples before and after mycoplasma elimination process.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4552274&req=5

Fig2: Mycoplasma-specific PCR analysis and fluorescence staining for primary cultured hSCAPs and proliferation activity.I. PCR analysis for 20 samples. II. fluorescence staining of mycoplasma in hSCAPs culture: arrow indicates positive staining of mycoplasma colony. III. mycoplasma-specific fluorescence staining of hSCAPs from selected 4 samples before and after mycoplasma elimination process. IV. proliferation assay for hSCAPs from selected 4 samples before and after mycoplasma elimination process.
Mentions: In addition, for 3D osteogenic differentiation, hSCAPs were suspended in 1.1% (w/v) alginic acid (Sigma) and 0.1% (v/v) porcine gelatin (Sigma) solution (all dissolved in PBS, pH 7.4), as described before [19]. Briefly, the cell-gel solution was passed through a peristaltic pump (EYELA) and dropped using a 25-gauge needle into sterile alginate gelation solution composing of 100 mM CaCl2 (Sigma), 10 mM HEPES (Sigma), and 0.01% (v/v) Tween (Sigma) at pH 7.4 with stirring. Approximately 10,000 cells were encapsulated in each alginate hydrogel. The hydrogels remained in gently stirred CaCl2 solution for 6–10 mins and were then washed with PBS. The hSCAPs containing hydrogels were transferred to in 10 ml vessels of HARV bioreactors (Synthecon) and the vessels was rotated at 25 rpm. Subsequently, 3D osteogenic differentiation was induced using the same osteogenic medium described above. The bioprocess is illustrated in Figure 2 II. After 3D osteogenic differentiation, the sections of hydrogels were stained with alizarin red-S staining solution according to manufacturer’s instruction.Figure 2

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.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.

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