学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 岡 明, 東京大学教授 高橋 尚人, 東京大学特任准教授 池田 祐一, 東京大学講師 正本 庸介, 東京大学准教授 大津 真University of Tokyo(東京大学

MUKAI TAKEO

向井 丈雄

UTokyo Repository

論文の内容の要旨 
論文題目 Neurotropism and neurorestorative mechanisms in umbilical cord 
derived-mesenchymal stromal cells 
（ 臍帯由来間葉系細胞の神経向性と神経修復機序に関する検討）  
氏名  向井 丈雄 
 
 Neonatal brain damage during the perinatal period is the cause of cerebral palsy later in life, 
and is a condition which places a great burden on not only the infant but also the family who raise. 
Those who present central nervous symptoms such as consciousness disorder, breathing / nursing 
disorder and spasms etc. in the neonatal period are collectively referred to as having "neonatal 
encephalopathy", and this is a condition that is by no means rare, and is incident in approximately 2 
people per 1000 births in Japan. There are expectations for cell therapies with neuroprotective action 
accompanied by neurotrophic factor secretions for "neonatal encephalopathy" that do not have 
effective treatments once they have manifested.  
Here I focused on umbilical cord-derived mesenchymal cells (MSC) as a new treatment for 
suppressing the onset of cerebral palsy. Among the sources of MSC, the human umbilical cord (UC) is 
a rich source of MSC, and they have advantages as a source; (1) not physically invasive for the donor 
and have no ethical issues, because the umbilical cord, which is the source of the cells, has been 
discarded as medical waste; (2) easy to sample, store and transport because they are an abundant 
source associated with childbirth; (3) have pluripotency to various tissue cells including the nervous 
system; (4) thought to be useful as a cell therapy for cerebral palsy because they have low 
immunogenicity while having sufficient immunosuppressive ability. Recently many clinical trials 
using UC-MSCs have been conducted for the purpose of immunomodulatory, anti-inflammatory and 
tissue repair. Among them, clinical studies on neurological disorders have already been conducted, and 
the target diseases include spinocerebellar degeneration, spinal cord injury, traumatic encephalopathy, 
cerebral palsy in adolescence and early adulthood. Based on the above, by clarifying the usefulness of 
UC-MSCs and its mechanism of action as a subject for neonatal encephalopathy, it is thought that 
novel cell therapy using UC-MSCs is effective in preventing the onset of cerebral palsy and lead to the 
improvement of QOL for children with cerebral palsy, as well as their families. 
In addition, because supplemented fetal bovine serum (FBS) in the medium with which 
UC-MSCs are cultured introduces the possibility of xenogeneic antigens and infections including 
bovine spongiform encephalopathy, lower antigenic and safer medium is needed for clinical use. 
Therefore, in this study, I used the serum-free new medium, RM medium, for the culture of UC-MSCs 
for clinical use as an alternative to FBS containing α-MEM medium, and I investigated the restorative 
effect of UC-MSCs cultured with RM medium in a neonatal brain injured model. 
Therefore the objectives of this study are to investigate neurotropism of UC-MSCs including 
potentials of neurogenic differentiation and migration towards injured cells, to evaluate the 
neurorestorative effect and the mechanisms of UC-MSCs in a neonatal brain injuries in vivo, and 
finally to prove validity of the mechanisms in vitro.  
At first I differentiated UC-MSCs by culturing in neurogenic differentiation medium. 
UC-MSCs shifted to a neuronal-like bipolar morphology, with long thin processes and a depressed 
proliferation rate, in response to culturing in neurogenic differentiation medium for about 72 hours. 
Then I revealed UC-MSCs can form neurospheres by culturing on spheroid plates supplemented with 
epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), and the expression of 
neural biomarkers was compared amongst cells derived from four groups: (1) undifferentiated 
UC-MSCs, (2) differentiated UC-MSCs, (3) undifferentiated UC-MSC-neurospheres, and (4) 
differentiated UC-MSC-neurospheres. I found that there were more neural markers expression in 
differentiated UC-MSCs and differentiated UC-MSC-neurospheres compared to their undifferentiated 
counterparts. Next I examined the migration ability of UC-MSCs toward normal or glucose-depleted 
neurogenic cells using Transwell migration assays because MSCs are known to migrate toward sites of 
tissue damage. The number of migrating cells co-cultured with glucose-depleted SH-SY5Y cells was 
significantly higher compared to that with normal SH-SY5Y cells in all groups. These results in vitro 
suggest UC-MSCs have neurotropism including neurogenic differentiation, neurosphere formation and 
migration towards injured cells. 
Then, I investigated the restorative effects of UC-MSCs cultured with serum-free medium in 
the neonatal IVH model in vivo. IVH is a common complication in preterm neonates and is 
responsible for high mortality and subsequent disabilities such as cerebral palsy, though there are no 
effective therapies available at present. I first established an animal model of neonatal IVH using 
newborn postnatal day 5 B6 Albino mice. The mice in the IVH group showed significantly decreased 
traveled distance and rearing number compared to those in the control group, and both UC-MSCs 
cultured with αMEM and serum-free RM improved behavioral outcome of IVH mice. Moreover the 
RM group exhibited significant behavioral improvement compared to the control group. 
Histopathological analysis revealed that the IVH group exhibited more GFAP-positive glial cells, 
decreased white matter thickness (hypomyelination), and increased TUNEL-positive apoptotic cells in 
the periventricular area than control group. UC-MSCs cultured with RM significantly attenuated these 
periventricular reactive gliosis, hypomyelination, and periventricular cell death observed after IVH. As 
for tracing the UC-MSCs in IVH mice, the luciferase gene was transfected into UC-MSCs and 
performed in vivo imaging analysis. In the mice injected with UC-MSCs, prominent luminescent 
signals were detected first in the lung, spreading to the neck and head, and no luminescent signals 
were detected 21 days after injection. Furthermore, human Alu genes were detected 1 day after 
UC-MSC injection mostly in lungs and brains. However, Alu elements were not detected 3 weeks after 
UC-MSC injection. Moreover, HLA Class 1 and CD105 double positive cells were observed in the 
perivascular region of the mouse brain in the UC-MSCs injected mice but disappeared 21 days after 
injection. To assess the key factors in the repair of damaged tissue in the IVH + UC-MSCs group, 
mouse sera and cerebrospinal fluid (CSF) were collected, and human brain-derived neurotrophic factor 
(BDNF), nerve growth factor (NGF), and hepatocyte growth factor (HGF) were measured using the 
multiplex beads immunoassay. Human BDNF and HGF were detected in IVH + UC-MSCs group. 
RT-PCR results also revealed BDNF and HGF were elevated in mice brain tissue of the IVH + 
UC-MSCs group. BDNF and HGF have been reported to be able to permeate across blood brain 
barrier (BBB). In addition, it has been reported that IVH induces BBB disruption and increased BBB 
permability. Therefore IVH surgery in my study might induce increased permeability of BBB followed 
by elevation of BDNF and HGF although I couldn’t detect them in all mice treated with UC-MSCs in 
my hands. As for mechanisms that UC-MSCs relieved gliosis and hypomyelination, BDNF has been 
reported to improve hypomyelination via Erk phosphorylation or TrkB signaling. And also HGF has 
been reported to influence the development and growth of oligodendrocytes, as well as the 
proliferation of myelin-forming Schwann cells. In addition, it has been reported HGF reduces gliosis 
by suppressing MCP-1 induction. These mechanisms of BDNF and HGF might alleviate gliosis and 
hypomyelination observed after IVH. 
Finally I investigated whether HGF and BDNF secreted from UC-MSCs contribute to 
neurogenesis, proliferation and viability in injured nerve system in vitro. I firstly confirmed secretion 
of HGF and BDNF from UC-MSCs from three different UCs, and inhibition of them by neutralizing 
antibodies. In order to investigate the effect of HGF and BDNF alone or both of them secreted from 
UC-MSCs on neurogenesis, I examined GAP-43 expression in injured neurons after oxygen glucose 
depletion (OGD). The results revealed that co-culture with UC-MSCs increased GAP-43-positive 
immature neurons and restored neurite outlength. However, addition of neutralizing antibodies of HGF 
and BDNF attenuated this improvement. Next I investigated the effect of HGF and BDNF on cell 
proliferation of cortical neurons after OGD. Proportion of Histone H3-positive cells, and incorporation 
of BrdU significantly decreased after OGD, and co-culture with UC-MSCs improved them. Addition 
of neutralizing antibodies of HGF and BDNF attenuated the cell proliferation. Interestingly the effects 
of expression of GAP-43, neurites outlength and cell proliferation were reverted partially by the 
addition of neutralizing antibodies, and there was no synergistic effect by the addition of them. I also 
evaluated caspase activity in cortical neurons after OGD, using fluorescent-labeled inhibitor of 
caspases (FLICA), and the results exhibited the ratio of the number of apoptotic and necrotic cells to 
the total number of cells exhibited that co-culture with UC-MSCs significantly improved apoptosis 
and necrosis of cortical neuron after OGD, whereas addition of neutralizing antibodies increased both 
of apoptotic and necrotic cells. These results indicate that HGF and BDNF secreted from UC-MSCs 
play an important role for neurogenesis, cell proliferation and viability after OGD, but they are not all 
secreted from UC-MSCs which contribute to cell proliferation and viability, and there are also other 
factors secreted from UC-MSCs which can contribute to neurogenesis. Both of HGF and BDNF are 
multi-functional growth factor expressed in both of adult and fetal central nervous system, and are 
reported to play an important role for anti-apoptosis and neurogenesis via activating 
phosphatidylinositol 3-kinase/Akt pathway and MAP-kinase pathway. Consistently, in our study, 
inhibition of HGF and BDNF decreased the extent of neurogenesis and cell viability. The results in our 
study indicate that HGF and BDNF secreted from UC-MSCs contribute to neurogenesis, cell 
proliferation and survival in injured nerve system, and that there are also other factors secreted from 
UC-MSCs which contribute to neurogenesis, proliferation and viability of neurons. 
Further studies are needed to examine the elevation of cytokines after IVH and determine the 
dose protocol of UC-MSC administration. The development of IVH results in an inflammatory 
response, and it has been reported that UC-MSCs have immunomodulatory properties. Therefore, 
UC-MSCs may play a key role in management of these inflammatory cytokines. In addition, inhibition 
experiment in vitro suggested the necessity of further study to prove the presence of other important 
neurotrophic factors secreted from UC-MSCs. As another problem, the concentrations of secreted 
HGF and BDNF demonstrated variability that was dependent on UC-MSC lot. Lot-to-lot variation in 
these secreted neurotrophic factors is an important issue considering their potential for clinical 
application. 
These results suggest that UC-MSCs ameliorate neuronal injury followed by functional 
improvement by secretion of neurotrophic factors such as BDNF and HGF rather than neuronal 
differentiation and eternal cell replacement, and that intravenous injection of UC-MSCs may be 
feasible treatment for neonatal brain injuries. 


臍帯由来間葉系細胞の神経向性と神経修復機序に関する検討

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臍帯由来間葉系細胞の神経向性と神経修復機序に関する検討

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