Odontogenic and Chondrogenic Differentiation of Isolated Stem Cells Derived from Bone Marrow, Adipose Tissue and Oral Mucosa: In Vitro Study
Heba El Sayed Tarek Abd El Salam;
Abstract
Oral mucosal stem cells have attracted tremendous interest recently in tissue engineering and regenerative medicine due to their accessibility and ease of isolation in comparison with the major stem cells sources, bone marrow and adipose tissue.
This research focuses on studying and comparing stem cells isolated from bone marrow, adipose tissue and oral mucosa with regard to their characterization, proliferation and capability to differentiate into odontogenic and chondrogenic lineages.
Samples were collected from the three sources. BMSCs were centrifuged to obtain a cell pellet, while ASCs and OMSCs were treated with collagenase before centrifugation to allow release of the cells.
Cells were cultured in complete culture medium for 14 - 21 days. Media were changed every 2-3 days. After reaching confluence, the isolated cells were characterized by flow cytometry using CD90, CD105 and CD45.
Cells were allowed to proliferate and then the proliferation capacity was assessed using MTT assay to compare the proliferation of the three cell types.
After reaching the third passage (P3) the cells were induced for differentiation:
1- Odontogenic differentiation by placing the cells in odontogenic induction media for 21 days.
2- Chondrogenic differentiation by placing the cells in chondrogenic induction media for 30 days.
Summary
116
Odontogenic differentiation was evaluated by Alizarin Red stain and by the expression of Dentine Sialophosphoprotein (DSPP) which was assessed by RT-PCR.
Chondrogenic differentiation was evaluated by Alcian Blue stain and by the expression of collagen II which was assessed by real time PCR.
The results show that successful isolation of stem cells from bone marrow, adipose tissue and oral mucosa based on their ability to adhere to plastic plates was achieved. Cells were successfully sub-cultured and expanded up to passage three. Cells positively expressed CD90 and CD105 and negatively expressed CD45.
As for the MTT assay, the results of present study demonstrated that ASCs proliferated faster than BMSCs and OMSCs.
The results also showed that all cultured cells negatively expressed stem cell markers CD90 and CD105 as assessed by flow cytometry indicating loss of stemness.
The results showed that OMSCs induced by odontogenic medium were intensely stained by Alizarin Red at days 14 and 21 intracellularly and extracellularly. BMSCs and ASCs were also stained with Alizarin Red stain but less intense than OMSCs.
RT-PCR results indicated that all cultured cells efficiently differentiate into dentin forming cells and expressed specific DSPP gene which was expressed higher by OMSCs than BMSCs and ASCs in days 14 and 21.
This research focuses on studying and comparing stem cells isolated from bone marrow, adipose tissue and oral mucosa with regard to their characterization, proliferation and capability to differentiate into odontogenic and chondrogenic lineages.
Samples were collected from the three sources. BMSCs were centrifuged to obtain a cell pellet, while ASCs and OMSCs were treated with collagenase before centrifugation to allow release of the cells.
Cells were cultured in complete culture medium for 14 - 21 days. Media were changed every 2-3 days. After reaching confluence, the isolated cells were characterized by flow cytometry using CD90, CD105 and CD45.
Cells were allowed to proliferate and then the proliferation capacity was assessed using MTT assay to compare the proliferation of the three cell types.
After reaching the third passage (P3) the cells were induced for differentiation:
1- Odontogenic differentiation by placing the cells in odontogenic induction media for 21 days.
2- Chondrogenic differentiation by placing the cells in chondrogenic induction media for 30 days.
Summary
116
Odontogenic differentiation was evaluated by Alizarin Red stain and by the expression of Dentine Sialophosphoprotein (DSPP) which was assessed by RT-PCR.
Chondrogenic differentiation was evaluated by Alcian Blue stain and by the expression of collagen II which was assessed by real time PCR.
The results show that successful isolation of stem cells from bone marrow, adipose tissue and oral mucosa based on their ability to adhere to plastic plates was achieved. Cells were successfully sub-cultured and expanded up to passage three. Cells positively expressed CD90 and CD105 and negatively expressed CD45.
As for the MTT assay, the results of present study demonstrated that ASCs proliferated faster than BMSCs and OMSCs.
The results also showed that all cultured cells negatively expressed stem cell markers CD90 and CD105 as assessed by flow cytometry indicating loss of stemness.
The results showed that OMSCs induced by odontogenic medium were intensely stained by Alizarin Red at days 14 and 21 intracellularly and extracellularly. BMSCs and ASCs were also stained with Alizarin Red stain but less intense than OMSCs.
RT-PCR results indicated that all cultured cells efficiently differentiate into dentin forming cells and expressed specific DSPP gene which was expressed higher by OMSCs than BMSCs and ASCs in days 14 and 21.
Other data
| Title | Odontogenic and Chondrogenic Differentiation of Isolated Stem Cells Derived from Bone Marrow, Adipose Tissue and Oral Mucosa: In Vitro Study | Other Titles | تمييز الخلايا الجذعية المستمدة من نخاع العظام ٬ النسيج الدهني والغشاء المخاطي للفم إلى خلايا مكونه للسن وللغضروف: دراسة معملية | Authors | Heba El Sayed Tarek Abd El Salam | Issue Date | 2015 |
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