2017 Fiscal Year Research-status Report
Characteristics of Oral Tissue-Derived MSCs from CLP Patients
| Project/Area Number |
17K11934
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
アミル リサ 東京医科歯科大学, 大学院医歯学総合研究科, 非常勤講師 (10784308)
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| Co-Investigator(Kenkyū-buntansha) |
森山 啓司 東京医科歯科大学, 大学院医歯学総合研究科, 教授 (20262206)
小林 起穂 東京医科歯科大学, 大学院医歯学総合研究科, 助教 (20596233)
門田 千穂 東京医科歯科大学, 大学院医歯学総合研究科, 日本学術振興会特別研究員 (30736658)
小川 卓也 東京医科歯科大学, 大学院医歯学総合研究科, 講師 (50401360)
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| Project Period (FY) |
2017-04-01 – 2019-03-31
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| Keywords | SHED / Mesenchymal / stromal cells / Dental tissue derived / Cleft lip and palate / Osteogenic / differentiation |
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| Outline of Annual Research Achievements |
This study aims to examine the characteristics of Dental pulp stem cells (DPSCs) and Stem cell from Human Exfoliated Deciduous teeth (SHEDs) taken from cleft lip palate patients. Ethical approval was obtained from TMDU ethics committee. 19 extracted teeth were collected from 12 patients (7 control group, 5 CLP group). Dental pulp (DP) cells and Periodontal ligament (PDL)cells were isolated and sorted by MSC analysis kit (CD34-,CD45-, CD73+, CD90+, CD105+). Cell proliferation activity was measured at day 1 and 7. No significant difference was observed between DP cells and PDL cells from CLP group compared to control group. In line with this finding, comparable result of population doubling time was observed between tested two groups. The CFU assay was performed in various cell densities. Larger CFU per mm2 area and higher intensity staining was found in DP cells and PDL cells from control group compared to CLP group. Baseline ALP, RUNX2 and OPN mRNA expression were examined. Comparable expression of osteogenic marker in DP cells and PDL cells prior to osteogenic induction. DP cells and PDL cells were cultured in osteogenic medium for 21 days, alizarin red staining was detected in group cultured in osteogenic medium whereby no calcium deposition was seen in the well cultured with standard medium. Comparable alizarin red staining was found in CLP and control group. The phenomenon was also observed in alcian blue staining for the visualization of sulfated glycosaminaglycans for chondrogenic differentiation.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The study obtained the number of samples (extracted teeth) exceeding the plan. We initially plan a minimum of 3 samples of each group (CLP and matching-control healthy group). Up to now, 19 samples from 12 patients were collected. Dental pulp cells from permanent teeth (DPSCs) and from deciduous teeth (SHEDs) were successfully isolated and expanded in cell cultures. Furthermore, PDL cells were also successfully isolated from these samples, thereby simultaneously presenting the opportunity to study the characteristic of PDL cells from CLP patients. The cells were successfully isolated and expanded in cell culture until passage 4. P1-P4 were used for the experiments or stored in cryopreservation medium for later using. The growth of the cells were analyzed in various ways such as cell metabolism by MTT assay, CFU assay and population doubling time and were performed in three independent experiments. FACS analysis was performed for immunophenotying of MSC characteristics (CD34-,CD45-, CD73+, CD90+, CD105+) as well as cell sorting. The CD34-, CD45- cells and CD73+, CD90+, CD105 cells were able to grow following cell sorting procedures. Osteogenic and chondrogenic differentiation were evaluated by calcium deposition and visualization of sulfated glycosaminoglycans with alizarin red and alcian blue staining respectively. The baseline expression of osteogenic markers were measured by RT-PCR. Further work to analyze the expression genes responsible for osteogenic, chondrogenic, and adipogenic differentiation is currently underway.
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| Strategy for Future Research Activity |
Previous study has revealed genes for identification of DPSCs. The expression of MSX1, MSX2, TBX2 and ENTPD1 mRNA levels were higher in DPSCs cells compared to MSCs from other region such as bone marrow derived MSCs or adipose tissue-derived MSCs. Msh homeobox 1 (MSX1) is a homeobox transcriptional factor, that is highly expressed in craniofacial skeletal cells during early postnatal development. Mutations in the MSX1
homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. The role of MSX1 in osteogenic differentiation in hDPSCs has been demonstrated. In human DPSCs cultures, MSX1 knockdown resulted in suppressed expression of RUNX2, ALP, BMP2, and OCN, suggests that MSX1 modulated the major signaling/transcriptional pathways regulating hard tissue differentiation to enhance osteogenic potential of hDPSCs. MSX1 regulates transcriptional activity of target genes either by directly binding to the specific DNA MSX1-binding motif (C/GTAATTG) or through interactions with other transcriptional regulators. MSX1 plays an essential role in osteogenic differentiation and calcification of hDPSCs derived from deciduous teeth. It is currently unknown the characterization of DPSCs in CLP patients compare to their matching controls. Future work will examine the genes for identification in DPSCs in CLP patients as well as control patients by microarray and RT PCR.
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| Causes of Carryover |
Since we used existing materials for the institution to which we belonged, we were able to carry out the experiments at a cost less than the necessary expenses scheduled for FY 2017, and we decided to carry forward to FY 2018. The realization of the budget will be used for future works namely identification of genes of DPSCS characteristics by microarray and RT PCR.
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