Cultivation, screening, identification and transplantation of Muse cell from human umbilical cord-derived for spinal cord injury in rats.
10.3969/j.issn.1003-0034.2019.04.007
- Author:
Zi-Kuan LENG
;
Zheng-Chao GAO
;
Xi-Jing HE
1
,
2
;
Ying-Jie ZHAO
;
Li-Jun SUN
;
Jing-Jing ZHAI
;
Jian-Zhong XU
Author Information
1. Department of Orthopaedics, the Second of Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi, China
2. xijing_h@vip.tom.com.
- Publication Type:Journal Article
- Keywords:
Cell culture techniques;
Cell transplantation;
Spinal cord injuries;
Umbilical cord
- MeSH:
Alprostadil;
Animals;
Cell Differentiation;
Cells, Cultured;
Humans;
Mesenchymal Stem Cells;
Rats;
Spinal Cord Injuries;
Umbilical Cord;
Wharton Jelly
- From:
China Journal of Orthopaedics and Traumatology
2019;32(4):327-334
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To investigate multilineage-differentiating stress-enduring (Muse) by immunomagnetic bead screening from Wharton's jelly mesenchymal stromal cells(WJ-MSCs), and explore transplantation of Muse cell for safety and effectivensess of sub acute cord injury in rats.
METHODS:Donated Wharton's Jelly-mesenchymal stromal cells (WJ-MSCs) were successfully derived from a human umbilical cord by a series of procedures namely physical isolation of Wharton's Jelly from cord membrane, collagenase and trypsin treatment and density gradient centrifugation. Magnetic activated cell sorting was performed to specifically select SSEA3+ Muse cells, and flow cytometry and immunocytochemistry were used to identify further. In vivo, spinal cord contusion injury model in rats was induced by NYU-III impactor, and were randomly divided and equally into four groups, namely group A (sham), group B (control), group C (Non-Muse cells transplantation) and group D (Muse cells transplantation). Laminectomy was conducted in group A but no spinal cord contusion injury. Laminectomy and cord injury were performed in group B, C and D, 10 g trip rod was freely falling down from 12.5 mm. Two weeks later, group B, C and D were received PBS injection, Non-Muse cells transplantation and Muse cells transplantation respectively, four-point injection were performed in each cord with totally 4×10⁵ cells. BBB scores were evaluated on 1 day, 1, 2, 3, 4, 5 and 6 week after injury. Four weeks after cell transplantation, the rats were sacrificed, and immunohistochemistry were carried out to observe survival, migration and differentiation of the injected cells.
RESULTS:The expression of CD105, CD90 and CD73 were over 99.5% in the derived WJ-MSCs population, but CD45 and CD14 were lower than 0.5%, positive rate of SSEA3+ was 1.46% under flow cytometer, However, after MACS sorting, the percentage of 92.0% Muse cells expressed SSEA3 and CD105, and immunohistochemistry results of SSEA3 showed typically membrane morphology with special processes. In vivo, BBB scores was 21 in group A at different time points. One-way ANOVA and LSD analysis showed that BBB scores in group C and D were significantly higher than that in group B (=0.004, 0.002), but there was no significantly difference between group C and D. Further intra-group paired t test showed that BBB score was significantly higher at 4 weeks than that 3 weeks in group C (=0.005). However, in group D, BBB scores were significantly higher at 4 and 6 week than those at 3 and 5 weeks, values were 0.005 and 0.016 respectively. Immunohistochemistry results showed that both Muse cells and Non-Muse cells could survive for 4 weeks in rats and they migrated from the four-point injection to injury site. But there showed more Muse cells survival than Non-Muse cells in the cord.
CONCLUSIONS:Immunomagnetic bead screening is efficient to select large number of purified SSEA3+ Muse cells. Muse cells could survive and target-migrate in injured cord to improve BBB scores continuously. Muse cells are a novel kind of seed cells in the spinal cord injury treatment.
