論文の公開元へTransplantation of rat cranial bone-derived mesenchymal stem cells promotes functional recovery in rats with spinal cord injury
概要
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Transplantation of rat cranial
bone‑derived mesenchymal stem
cells promotes functional recovery
in rats with spinal cord injury
Yuyo Maeda1*, Takashi Otsuka2, Masaaki Takeda1, Takahito Okazaki1, Kiyoharu Shimizu1,
Masashi Kuwabara1, Masahiro Hosogai1, Louis Yuge2 & Takafumi Mitsuhara1
Cell-based therapy using mesenchymal stem cells (MSCs) is a novel treatment strategy for spinal
cord injury (SCI). MSCs can be isolated from various tissues, and their characteristics vary based
on the source. However, reports demonstrating the effect of transplanted rat cranial bone-derived
MSCs (rcMSCs) on rat SCI models are lacking. In this study, we determined the effect of transplanting
rcMSCs in rat SCI models. MSCs were established from collected bone marrow and cranial bones. SCI
rats were established using the weight-drop method and transplanted intravenously with MSCs at
24 h post SCI. The recovery of motor function and hindlimb electrophysiology was evaluated 4 weeks
post transplantation. Electrophysiological recovery was evaluated by recording the transcranial
electrical stimulation motor-evoked potentials. Tissue repair after SCI was assessed by calculating
the cavity ratio. The expression of genes involved in the inflammatory response and cell death in the
spinal cord tissue was assessed by real-time polymerase chain reaction. The transplantation of rcMSCs
improved motor function and electrophysiology recovery, and reduced cavity ratio. The expression of
proinflammatory cytokines was suppressed in the spinal cord tissues of the rats that received rcMSCs.
These results demonstrate the efficacy of rcMSCs as cell-based therapy for SCI.
Spinal cord injury (SCI) can cause severe damage, leading to permanent loss of mobility, incontinence, and other
functional losses. In the absence of effective treatments for SCI, surgical restabilization of the vertebral column
and rehabilitation are currently the primary therapeutic o
ptions1. Cell-based therapy using mesenchymal stem
cells (MSCs) has garnered attention as a novel approach for treating the damage caused by SCI. MSCs can be
isolated from various tissues, such as bone m
arrow2 or adipose t issue3, and possess self-renewal and multilineage differentiation potentials. Some studies have revealed that the characteristics of MSCs may vary in these
tissues4–6. In a previous study, researchers successfully established MSCs from rat and human cranial bones,
and demonstrated that they secrete abundant neurotrophic factors when transplanted in a rat model of cerebral
infarction7,8. However, reports demonstrating the effect of transplanted cranial bone-derived MSCs on rat SCI
models are lacking. In addition, the mechanism of action of cranial bone-derived MSCs in central nervous
system (CNS) disorders has not yet been investigated7–9. Motor function recovery has been evaluated only via
behavioral assessments, as the electrophysiological recovery has not been determined. Furthermore, no studies
have investigated the effect of transplanting MSCs on neurophysiological recovery over time using extended
neurophysiological evaluation. We have previously established techniques for recording extended transcranial
electrical stimulation motor-evoked potentials (tcMEPs) in r ats10.
Therefore, in the present study, we investigated the effects of transplanting rat cranial bone-derived MSCs
(rcMSCs) on the neurophysiology of an SCI rat model. Moreover, we determined the neurophysiological recovery
using extended tcMEP recordings and the mechanism underlying rcMSCs function in an SCI model. We propose
rcMSCs as a potential new source of cell therapy against SCIs.
1
Department of Neurosurgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi
1‑2‑3, Minami‑ku, Hiroshima, Japan. 2Division of Bio‑Environmental Adaptation Sciences, Graduate School of
Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. *email: d182072@hiroshima-u.ac.jp
Scientific Reports |
(2021) 11:21907
| https://doi.org/10.1038/s41598-021-01490-1
1
Vol.:(0123456789)
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Results
Flow cytometry analysis for MSC‑specific markers. MSC-specific markers in rat bone marrow mesenchymal stem cells (rbMSCs) and rcMSCs were analyzed using flow cytometry. The results were positive for
cell surface markers associated with MSCs, such as CD29, CD90, and CD44 (Supplementary Table S4 online);
in contrast, they were negative for cell surface markers associated with hematopoietic cells, such as CD34 and
CD45 (Supplementary Table S4 online). rbMSCs and rcMSCs exhibited similar characteristics, with their cell
surface markers resembling those described in previous reports7–9.
Multilineage cell differentiation. The differentiation potentials of the isolated rbMSCs and rcMSCs into
osteogenic and adipocytic cells were investigated. Neither type of MSCs exhibited positive staining with Alizarin
red S before osteogenic differentiation; however, positive cells were observed after differentiation (Supplementary Fig. S1A online), whereas staining with oil red O revealed that both types of MSCs tended to differentiate
into adipocytic cells (Supplementary Fig. S1 online).
Analysis of the genetic landscape of MSCs. The expression of genes encoding neurotrophic factors
and anti-inflammatory factors in rbMSCs and rcMSCs was analyzed using real-time polymerase chain reaction
(PCR). The expression of Bdnf, Gdnf, Ngf, and Vegf, which encode neurotrophic factors, and that of Tgfb and
Tsg6, which encode anti-inflammatory factors, was evaluated. The expression of Bdnf, Gdnf, and Vegf was significantly higher in rcMSCs than in rbMSCs (P < 0.05, P < 0.05, P < 0.05, respectively; Fig. 1a,b,d). The expression of
Ngf and of genes encoding anti-inflammatory factors did not differ significantly between rcMSCs and rbMSCs
at passage 3 (Fig. 1c,e,f).
Effects of cell transplantation in an SCI rat model: recovery of motor function. We assessed the
neurological function using the Basso–Beattie–Bresnahan (BBB) scale, and the inclined plane task score was
used to assess the functional benefits after the transplantation of rbMSCs and rcMSCs post-SCI. All rats showed
recovery of motor function in these tests after SCI. Improvements in the BBB scale score were significantly
greater in the rcMSC group than those in the phosphate-buffered saline (PBS) group on day 1 (P < 0.05; Fig. 2a)
and on days 3, 5, 7, 10, 14, 21, and 28 after injury (P < 0.01; Fig. 2a). In addition, improvements in the rcMSC
group were also higher than those in the rbMSC groups on day 3 (P < 0.05; Fig. 2a) and on days 5, 7, 10, 14, 21,
and 28 after injury (P < 0.01; Fig. 2a). Improvements in inclined plane task scores were significantly higher in the
rcMSC group than in the PBS group at 2, 3, 5, 7, 10, 14, 21, and 28 days after injury (P < 0.01; Fig. 2b). In addition,
improvements in the rcMSC group were also higher than those in the rbMSC group on day 2 (P < 0.05; Fig. 2b)
and on days 3, 5, 7, 10, 14, 21, and 28 after injury (P < 0.01; Fig. 2b). Rats in the rcMSC group exhibited more
motor function recovery than those in the other groups.
Effects of cell transplantation in an SCI rat model: electrophysiological recovery. Representa-
tive waveforms of tcMEPs pre-operation and at 1, 7, 21, and 28 days after SCI are shown in Fig. 3a–c. The waveform of tcMEPs disappeared and gradually recovered after SCI. The recovery rate of the amplitude improved
at 14, 21, and 28 days after SCI compared with that on the day after injury in the rbMSC and rcMSC groups
(Fig. 3d). In addition, the recovery rate of the amplitudes in the rcMSC group was higher than that in other
groups on days 14 and 21 (P < 0.05; Fig. 3d) and at 28 days after SCI (P < 0.01; Fig. 3d).
Effects of cell transplantation in an SCI rat model: cavity repair. We assessed cavity repair using
the cavity ratio and compared the histological recovery after the transplantation of rbMSCs and rcMSCs following SCI. Only small cavities were identified within the SCI lesions in the rcMSC group (Fig. 4a–d). The cavity
ratios of the rbMSC and rcMSC groups were significantly lower than those of the PBS group (Fig. 4e). The cavity
ratio of the rcMSC group was also significantly lower than that of the rbMSC group (P < 0.05; Fig. 4e).
Spinal cord tissue sampling and mRNA expression analysis of the spinal cord lesion site. The
mRNA expression of Bax/Bcl2 and Casp3/Gapdh did not differ significantly between the three groups (Fig. 5a,b).
Moreover, the levels of Il1b were significantly lower in the rbMSC and rcMSC groups than in the PBS group
(P < 0.05; Fig. 5c), and those of Tnfa were significantly lower in the rcMSC group than in the PBS group (P < 0.05;
Fig. 5d).
NG108‑15 cell death assay after exposure to oxidative and inflammatory stress. The survival
rate of NG108-15 cells exposed to inflammatory stress and cultured in rcMSC-conditioned medium (CM) was
significantly higher than that in the other groups (P < 0.01; Fig. 6a). Moreover, the survival rate of NG108-15
cells exposed to oxidative stress and cultured in rcMSC-CM or rbMSC-CM was significantly higher than that in
control cells (P < 0.01 and P < 0.05, respectively; Fig. 6b).
Analysis of mRNA expression in stress‑exposed NG108‑15 cells. The Bax/Bcl2 ratio and Casp3
expression in inflammatory stress-exposed NG108-15 cells did not differ significantly between the three groups
(Fig. 7a,b), whereas they were significantly lower in oxidative stress-exposed NG108-15 cells cultured in rcMSCCM than in control cells (P < 0.05 and P < 0.01, respectively; Fig. 7c,d). The Bax/Bcl2 ratio in cells cultured in
rbMSC-CM was also significantly lower than that in control cells (P < 0.05; Fig. 7c). ...
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