Kato Toshiki

加藤 俊貴

English

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Glucocorticoids Impair the Tissue Regenerative
Function of Mesenchymal Stem Cells
著者 Kato Toshiki
著者別名 加藤 俊貴
発行年 2018
その他のタイトル ステロイド治療は間葉系幹細胞の組織修復能を低下
させる
学位授与大学 筑波大学 (University of Tsukuba)
学位授与年度 2018
報告番号 12102甲第8768号
URL http://hdl.handle.net/2241/00153962
    
 
 
 
 
論文の要旨 
Abstract of thesis 
 
Glucocorticoids are widely used as one of popular anti-inflammatory drugs, whereas it is shown 
that the strong immunomodulatory action causes undesirable effects including osteonecrosis of the 
femoral head (ONFH). In order to treat these disorders, mesenchymal stem cells (MSCs) are 
considered as promising cells, because of their multi-differentiation potential and the ability to produce 
cytokines that recruit various types of cells for tissue regeneration. However, little information has 
been known regarding the effect of glucocorticoid therapy on the regenerative function of MSCs. 
Based on these backgrounds, in this thesis, the applicant aimed to investigate (1) the proliferative and 
self-renewal abilities of glucocorticoids on the function of bone regeneration in MSCs, (2) the 
importance of wnt/β-catenin signaling in vitro, and (3) the contribution of Dkk-1, an antagonist of wnt/β-
catenin signaling in vivo. 
 
(1) The proliferative and self-renewal abilities of glucocorticoids on the function of bone regeneration 
in MSCs 
To uncover the glucocorticoid action on the nature of bone marrow (BM) derived-MSCs, the 
applicant conducted colony formation assay using primary BM that was aspirated from traumatic or 
氏     名 加藤 俊貴 
学 位 の 種 類       E 博士（ 人間生物学  ） 
A学 位 記 番 号       E 博甲第   ８７６８   号 
A学位授与年月       E 平成 ３０年 ４月 ３０日 
A学位授与の要件       E 学位規則 第４条第１項該当（昭和２８年４月１日文部省令第９号） 
審 査 組 織 グローバル教育院 
学位論文題目  Glucocort icoids Impair the Tissue Regenerative Function of 
Mesenchymal Stem Cells 
(ステロイド治療は間葉系幹細胞の組織修復能を低下させる ) 
 （職名） （学位） （氏名） 
主   査 筑波大学教授 農学博士 深水 昭吉 
副   査 筑波大学教授（グローバル教育院） Ph.D. Bernd Fleischmann 
副   査 筑波大学教授（グローバル教育院） 博士（医学） 矢田 幸博 
副   査 筑波大学准教授 博士（薬学） 鈴木 裕之 
 steroid-induced ONFH patients, and found that BM derived from steroid-induced ONFH patients 
contained a lower number of colony-forming unit fibroblasts than that from traumatic ONFH patients. 
Moreover, the applicant showed less and unstable proliferative ability of BM-MSCs derived from 
steroid-induced ONFH patients, compared with BM-MSCs derived from traumatic ONFH patients.  
 
(2) The importance of wnt/β-catenin signaling in vitro 
In order to investigate the differences in the characteristics between BM and adipose tissue (AT) 
derived MSCs, the applicant next isolated and analyzed AT-MSCs derived from steroid-induced 
ONFH patients (sAT-MSC). Although their proliferative potential was found to be not impaired, the 
applicant demonstrated decreased osteogenic potential of sAT-MSCs, compared with AT-MSCs 
derived from traumatic ONFH patients (nAT-MSC). The applicant also illustrated that alkaline 
phosphatase (ALP), a regulator of the calcification of extracellular matrix, was downregulated in sAT-
MSCs and overexpression of ALP restored the osteogenic ability of sAT-MSCs in vitro. To identify the 
downregulation mechanism of ALP in sAT-MSCs, the applicant focused on wnt/β-catenin signaling, 
because it plays an important role in bone development. The applicant found that Dickkopf1 (Dkk-1), 
one of the known antagonists of wnt/β-catenin signaling, was upregulated in sAT-MSCs, where, in 
this situation, the impaired osteogenic potential was rescued when Dkk-1 shRNA (shDkk-1) was 
transfected. In accordance with these observations, the applicant showed the reduced ability of 
osteogenic differentiation and the increased expression of Dkk-1 leading to the downregulation of ALP 
in nAT-MSCs, where it was chronically exposed to dexamethasone in vitro to mimic the 
microenvironment of sAT-MSCs. 
 
(3) The contribution of Dkk-1, an antagonist of wnt/β-catenin signaling in vivo 
In addition to the osteogenic differentiation ability in vitro, the applicant clarified that sAT-MSCs 
possessed lower bone regenerative ability compared with nAT-MSCs using the murine calvarial defect 
model. Based on this result, the applicant also revealed that the reduced bone regenerative capacity 
in sAT-MSCs was caused by elevated Dkk-1 along with the impaired osteogenic differentiation ability 
in vitro. To prove the presence of transplanted AT-MSCs in the repaired bone regions, the applicant 
performed immunohistological analysis using anti-human osteipontin (hOPN) antibody, a marker of 
mature osteoblast, and found hOPN-positive cells in the repaired bone region with nAT-MSCs, mock-
transfected nAT-MSCs, and shDkk-1-transfected sAT-MSCs. On the other hand, the applicant 
observed no positive cells in sAT-MSCs and mock-transfected sAT-MSCs at the transplanted regions. 
Finally, the applicant found that the level of Dkk-1 in plasma was elevated in steroid-induced ONFH 
patients compared with traumatic ONFH patients. 
 
In this thesis, the applicant demonstrated that the proliferative potential of BM-MSCs was 
impaired by chronic glucocorticoid treatment, whereas it did not affect proliferation in AT-MSCs 
derived from steroid-induced ONFH. Intriguingly, the applicant uncovered that the downregulation of 
 wnt/β-catenin signaling due to highly expressed Dkk-1 impaired the osteogenic ability of sAT-MSCs, 
observed that Dkk-1 was elevated in the plasma from steroid-induced ONFH patients, and suggested 
that Dkk-1 induction is a key promoter of AT-MSCs as a useful therapy source. (653 words) 
 
 
審査の要旨 
 Abstract of assessment result 
 
【批評 Review】 
The applicant provided several lines of evidence regarding the effect of glucocorticoids on the 
function of bone regeneration in MSCs, and found potential biological significance and contribution of 
Dkk-1 as one of the known antagonists of wnt/β-catenin signaling in the bone regenerative capacity 
of AT-MSCs in vitro and in vivo. However, it still remains unclear how Dkk-1 is induced in response to 
and regulated by glucocorticoid. To address these questions, further studies will be required using the 
cell culture methods that the applicant successfully developed. 
 
【最終試験の結果 Result】 
The final examination committee conducted a meeting as a final examination on 5th March, 
2018. The applicant provided an overview of dissertation, addressed questions and comments raised 
during Q&A session. All of the committee members reached a final decision that the applicant has 
passed the final examination. 
 
【結論 Conclusion】 
Therefore, the final examination committee approved that the applicant is qualified to be 
awarded Doctor of Philosophy in Human Biology. 


ステロイド治療は間葉系幹細胞の組織修復能を低下させる

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