学位の種別:課程博士University of Tokyo(東京大学

He Haiping

何 海萍

UTokyo Repository

論文の内容の要旨 
 
 
論文題目 Biological characterization and potential application of umbilical cord Wharton' s 
jelly-derived mesenchymal stem cells 
（臍帯ワルトンゼリー由来間葉系幹細胞の生物学的性状とその応用に関する研究） 
 
 
氏名  何 海萍 
 
 
【Introduction】Umbilical cord (UC) Wharton’s jelly (WJ) is a rich source of mesenchymal stem cells 
(MSCs) as well as bone marrow (BM) and adipose tissue. WJ-derived MSCs (WJ-MSCs) exhibit the 
characteristics of MSCs as defined by the International Society for Cellular Therapy (ISCT) criteria. First, 
MSCs are plastic adherent when maintained in standard culture conditions; second, they are positive for 
CD105, CD73, HLA-class I, CD90 and negative for CD45 and HLA-DR surface molecules .The 
identification of MSCs with the use of specific markers remains discussion. There is no single specific 
marker can be used to define multipotent MSCs. Stage-specific embryonic antigen (SSEA) 4 has been 
reported as a stem cell marker in bone marrow-derived MSCs, but whether SSEA4
+ 
cells have growth and 
differentiation advantages over SSEA4
−
 cells remains controversial; Third, MSCs have the multipotent 
ability of various lineages to generate adipocytes, osteoblasts and chondrocytes. Hsieh et al reported that 
WJ-MSCs were more primitive and more similar to embryonic stem (ES) cells than BM-MSC. Fourth, 
MSCs have the distinct immunomodulatory effect. Although some conflicting data showed the 
inflammatory environment might alter the MSCs immunosuppressive effect. My research presentation is 
consisted of two parts; (1) to investigate the significance of SSEA4 in WJ-MSCs and (2) to study the 
immunosuppressive effect of WJ-MSCs on activated T cells. 
【Methods】WJ-MSCs were collected by the explant (WJe-MSCs) or collagenase methods (WJc-MSCs) 
and analyzed the characteristics by flow cytometry and reverse transcription polymerase chain reaction 
(RT-PCR). To study the significance of SSEA4 expression in WJ-MSC, I continued to culture WJ-MSCs 
until 9 passages (P9) and analyzed SSEA4 expression with gene expression of stem cells markers. In 
some experiments, I sorted WJe-MSCs by SSEA4 expression using FACS Aria and analyzed the growth 
and differentiation ability to osteocytes and adipocytes with the induction medium, respectively. To 
evaluate whether culture conditions influenced the SSEA4 expression, WJe-MSCs were cultured in the 
medium supplemented with different fetal bovine serum (FBS) concentrations.  
 Next, I evaluated the WJ-MSCs immunosuppressive effect on activated T cells in vitro and in 
xeno-GVHD mice model. Mixed lymphocyte reaction (MLR) was performed on WJ-MSCs or control. 
CFSE-labeled human peripheral blood (PB) or cord blood (CB) derived mononuclear cells (MNCs) as the 
responder cells were mixed with irradiated (50Gy) human PB or CB-derived dendrite cells (DCs) at a 
10:1 ratio. In the presence of low-dose anti-CD3 antibody, CFSE-labeled responder cells were analyzed 
by flow cytometry. In order to confirm whether the inhibitory effects of WJ-MSCs on MLR requires the 
cell-to-cell contact or not, I next performed MLR on separated WJ-MSCs using by transwell chamber, 
co-cultured MLR with WJ-MSCs supernatant, and evaluated the effect of indoleamine 2,3-dioxygenase 
(IDO), as a known critical factor of immunosuppression by using the inhibitor. In addition, to evaluate the 
influence on the immunosuppressive effect by the number of passages, I compared the suppressive effect 
on MLR by the late passage WJ-MSCs on MLR compared with those of early passage of WJ-MSCs. 
Although it is still preliminary experiments, I investigated WJ-MSCs immunosuppressive effect in 
xeno-GVHD mice, which were irradiated and injected with activated T cells transduced luciferase gene. I 
compared monitored the distribution of human activated T cells by IVIS, weight loss and 
immunohistological analysis.  
【Results】Both WJe-MSCs and WJc-MSCs were positive for CD73, CD90, CD105, and HLA-ABC 
with a small percentage of cells also positive for CD271 and negative for CD34, CD45, and HLA-DR. In 
addition, both WJe-MSCs and WJc-MSCs expressed the ES cells-related genes, Nanog, Oct4, Klf4, Rex1, 
and Sox2. I periodically monitored SSEA4, SSEA3, and CD73 expressions in WJe-MSCs and 
WJc-MSCs during P0–P9. The percentages of SSEA4+ cells at P0 were similar in WJe-MSCs and 
WJc-MSCs (n=8). However, the percentage of WJe-MSCs SSEA4
+
cells decreased after the first passage 
and recovered to the original level by P7, whereas the incidence of WJc-MSCs SSEA4
+
 cells was 
relatively stable until P9. In contrast, the percentage of SSEA3
+
 cells among both WJe-MSCs and 
WJc-MSCs was highest at P0 that declined and disappeared by P5. I sorted SSEA4
+
 and SSEA4
− 
cells 
from
 
WJe-MSCs at P4 using FACS Aria. The percentage of SSEA4
+
 cells derived from the sorted 
SSEA4
+
 WJe-MSCs decreased rapidly in the first week and then increased gradually until week 4. 
Interestingly, SSEA4
+
 MSCs were present in the SSEA4
–
 WJe-MSCs, and the incidence of SSEA4
+
 cells 
in the subsequent cultures was similar to that in the SSEA4
+
-sorted cells. RT-PCR analysis showed that 
the sorted SSEA4
+
 and SSEA4
− 
WJe-MSCs expressed Nanog, Oct4, and Klf4. Both types of cells showed 
similar growth rates and could differentiate into adipocytes and osteocytes, although osteogenic 
differentiation of WJe-MSCs require relatively high concentration of bone morphogenetic protein 2 and a 
longer culture period up to 5 weeks. I examined SSEA4/3 expression after 1 week in cultures containing 
various concentrations of FBS (0.1 to 20%). The SSEA4 expression was positively correlated with the 
FBS concentration, whereas SSEA3 expression was negatively correlated. To see that the increase in 
SSEA4 expression upon increasing FBS concentration is caused by the change in expression alone but not 
because of increased WJe-MSCs proliferation, I analyzed SSEA4 expression associated with growth 
curve with different FBS concentrations. In consistent with the prior data, SSEA4 expression was 
positively correlated with FBS concentration, while SSEA3 was inversely correlated. The higher FBS 
concentration accelerated the proliferation of WJe-MSCs with higher expression of SSEA4. Furthermore, 
when the sorted SSEA4
+
 and SSEA4
− 
WJe-MSCs were cultured in 1 to 10% FBS for a week, SSEA4 
expression in both sorted SSEA4
+
 and SSEA4
-
 WJ-MSCs was positively correlated with FBS 
concentrations, although the percentage of SSEA4 was higher in SSEA4+ cells than that in SSEA4
-
 cells. 
 Next, I studied the immunosuppressive effect of WJ-MSCs on activated T cells in mixed 
lymphocyte reaction (MLR). In MLR, I found that WJ-MSCs efficiently inhibited the responder T cells 
derived from the same donor of WJ-MSCs, triggered by autologous or allogeneic dendritic cells (DC) 
(Autologous and allogeneic MLR). The 3
rd
 party MLR also strongly suppressed allogeneic T cells 
responses triggered by allogeneic DCs indicating that immunosuppressive effect of WJ-MSCs is not 
restricted by MHC.WJ-MSCs also inhibited T cells proliferation upon PHA stimulation, whereas their 
inhibitory effects were significantly attenuated by the blockade of cell to cell contact using the transwell 
chamber. The culture supernatant of WJ-MSCs alone revealed mild inhibitory effects on MLR. These 
inhibitory effects of the WJ-MSCs were reversed by the addition of indoleamine 2, 3-dioxygenase (IDO) 
inhibitor, 1-methyltryptophan (1-MT) in a dose-dependent manner, confirming the critical role of IDO 
reported previously. Concerning the influence of passage number on immunosuppressive effect, I found 
that WJ-MSCs in the late passage (p10) had the same of immunosuppressive effect compared with that in 
the early passage (p1).  
【Discussion】To gain insight into the role of SSEA3 and SSEA4 in UC WJ-MSCs, I examined the 
SSEA3 and SSEA4 expressions on WJ-MSCs obtained by different methods and compared the 
differentiation abilities of SSEA4
+
 and SSEA4
− 
cells. I did not find any significant differences between 
WJe-MSCs and WJc-MSCs in cell numbers, MSC surface markers, or ES cell-related gene expressions at 
P0. In addition, the incidence of SSEA4
+ 
and SSEA3
+
 at P0 was similar between the two types, although 
SSEA3 disappeared rapidly in the early culture passages. The role of SSEA3 and SSEA4 in MSCs 
remains controversial. Gang et al. (Blood, 109:1743, 2007) reported that SSEA4
+
 cells proliferated 
predominantly when the culture was initiated from primary BM cells, which were mostly hematopoietic 
cell. But in my study, both sorted SSEA4
+
 and SSEA4
− 
WJ-MSCs eventually differentiated into 
osteocytes and adipocytes in a similar manner, and there were no differences in ES-marker gene 
expression between the SSEA4
+
 and SSEA4
−
 MSCs. Interestingly, SSEA4
+
 cells appeared even from the 
SSEA4
− 
MSCs, and the incidence of SSEA4
+
 cells derived from the SSEA4
− 
MSCs demonstrated a 
similar transition pattern as those derived from the SSEA4
+
 MSCs. These results suggested that the 
culture medium might include the substrate for SSEA4. Finally, I demonstrated clearly for the first time 
that SSEA4 expression was positively correlated with FBS concentration, while SSEA3 expression 
appeared to be negatively correlated with FBS concentration, regardless of cell growth or cell 
concentration. These results indicate that SSEA4 may display altered expression profiles in response to 
culture medium including FBS and may not be an essential marker of WJ-MSC pluripotency.  
 Concerning the immunomodulatory effects of MSCs, MSCs have the ability to migrate to 
inflammatory tissues and suppress adverse immune reactions. In fact, BM-derived MSCs are already 
applied for the patients with acute graft versus host disease (aGVHD) with promising efficacy. However, 
the mechanism by which WJ-MSCs exert their immunosuppressive effects is not completely understood. 
Here, I demonstrated that WJ-MSCs have the immunosuppressive effect on the stimulated T cells in 
autologous, allogeneic and 3
rd
 party MLR setting, indicating that immunosuppressive effect of WJ-MSCs 
is not restricted by MHC. In addition, I observed the inhibitory effect of separated WJ-MSCs on MLR 
using transwell chamber or co-cultured MLR with WJ-MSCs supernatant, although the 
immunosuppressive effect was less than that of cell to cell contact. The immunosuppressive effect of 
WJ-MSCs on MLR was reversed by the addition of IDO inhibitor, 1-MT in a dose dependent manner. It 
suggests that soluble IDO may play an important role in immunosuppressive effect of MLR. In previous 
papers, BM-derived MSCs secreted soluble factors such as HLA-G, TGF-β, pGE2, IDO, IL6. I think I 
need to detect the additional factors to IDO, in cell-to-cell contact between WJ-MSCs and the responder T 
cells. Furthermore, I am now exploring to administer WJ-MSCs for the treatment of xenogeneic GVHD 
in mice. But as reported previously in BM-MSCs, WJ-MSCs suppressed GVHD and prolong the 
survivals, but in some conditions, they stimulated the GVHD. Recently, some reports suggest that the 
inflammatory environment might alter the MSC from immunosuppression to immunostimulation. To 
make the steady suppressive function of MSCs, primed MSCs with IFN-γ with or without TNF-α were 
recommended recently. I thought I need to treat the WJ-MSCs before injection to mice.  
【Conclusion】WJ-MSCs could be efficiently obtained by explants method. WJ-MSCs are feasible 
alternative source of other MSCs for regenerative medicine and immunotherapy. In order to make the 
effects of WJ-MSCs more effective and safety, further investigations are necessary on the mechanisms of 
multipotency and immunomodulatory effect.  


臍帯ワルトンゼリー由来間葉系幹細胞の生物学的性状とその応用に関する研究

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臍帯ワルトンゼリー由来間葉系幹細胞の生物学的性状とその応用に関する研究

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