Study Of Human Umbilical Cord Mesenchymal Stem Cells Transplantation In Rats Traumatic Spinal Cord Injury

Spinal cord injury (SCI) results in loss of neurons, degeneration of axons, formation of glial scar, and severe functional impairment. Human umbilical cord mesenchymal stem cells (hUC-MSCs) can be induced to form neural cells in vitro. Therefore, the stem cells may be a valuable source in the repair of spinal cord injury.Part 1 Isolation, culture, and identification of human umbilical cord mesenchymal stem cellsObjectives:To explore the isolation, culture, and expansion method of hUC-MSCs.Methods:1. Human umbilical cords were collected from full-term caesarian section births and processed within 3–6 h. Umbilical arteries and vein were removed, and the remaining tissue was diced into small fragments. The explants were transferred to culture flasks containing DMEM/F12 along with 10% fetal bovine serum. They were left undisturbed for 4–6 days to allow migration of cells from the explants. They were re-fed and passaged as necessary.2. The mesenchymal cells were trypsinized and suspended in DMEM/F12 at a concentration of 3×106/ml. A 100μl sample was incubated for 35 min at 48 oC with 15μl of various mouse anti-human antibodies against following surface markers: CD14, CD34, CD45, CD90, CD73, CD105, HLA-DR. Then, they were washed, centrifuged, and fixed in 1.5 ml of 4% paraformaldehyde. A FACScan machine was used to analyze antibody binding.Results:1. hUC-MSCs were successfully isolated from umbilical cord explants and expanded as primary cultures. The cells demonstrated a fibroblast-like or spindle-shaped morphology in confluent layers in culture. 2. Flow cytometry showed that the cells expressed high levels of matrix markers (CD90) and mesenchymal stem cells (MSCs) markers (CD73, CD105) but did not express hematopoietic lineage markers (CD34, CD45, CD14) and HLA-DR.Conclusions:1. MSCs can be successfully isolated, cultured, and expanded from human umbilical cord Wharton's jelly using routine technical approaches.2. hUC-MSCs express surface markers similar to MSCs derived from bone marrow, umbilical cord blood, and placenta.3. Wharton's jelly of umbilical cord is a rich and easily attainable source of MSCs for cell therapy.Part 2 Biological characteristics and MRI of superparamagnetic iron oxide nanoparticles labeled human umbilical cord mesenchymal stem cellsObjectives:The purpose of this study was to evaluate the influence of superparamagnetic iron oxide (SPIO) nanoparticles on hUC-MSCs and the feasibility of tracking for hUC-MSCs by noninvasive magnetic resonance imaging (MRI).Methods:1. The label (Feridex) was added to the cultures at concentrations equivalent to 5.6, 11.2, 22.4, and 44.8μg Fe/ml (diluted with DMEM/F12) and incubated for 12 or 24 h. Control cultures were incubated without SPIO.2. Prussian Blue staining was used to determinate the labeling efficiency (number of Prussian Blue labeled cells/total number of cells in the sample). Cell viability and growth curves were evaluated with tetrazolium dye 3-(4,5-dimethylthiazol-2)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) assay.3. In vitro MRI Gradient echo T2-weighted (GRE T2*WI) images [repetition time (TR) 3,200 ms; echo time (TE) 354 ms; FOV=172.5×172.5; slice thickness=3 mm] and spin echo T2-weighted (SE T2WI) images (TR 6,000 ms; TE 98 ms; FOV=220×220; slice thickness=4 mm) were applied to examine SPIO-labeled hUC-MSCs.4. In vivo MRI was used to track SPIO-labeled hUC-MSCs transplanted in rat spinal cord. Results:1. A good correlation between SPIO uptake and the iron concentration in the culture medium was observed. As the concentration of SPIO and the incubation time increased, the amount of intracellular iron increased.2. The data show that neither survival nor proliferation potential was impaired in dealing with 22.4μg Fe/ml for 24 h. However, treatment with 44.8μg Fe/ml for 24 h impairs cell survival and proliferation significantly.3. GRE T2*WI and SE T2WI images revealed the presence of a hypointense signal. A good linear correlation between the number of labeled cells and intensity of the GRE T2*WI images was observed. Similar result was obtained in the SE T2WI images.4. In vivo MRI 3 days and 14 days after injection showed a large reduction in signal intensity in the region transplanted with SPIO-labeled hUC-MSCs. The images from unlabelled hUC-MSCs showed a smaller reduction in signal intensity. Prussian Blue co-stained with nuclear fast red confirmed the presence of SPIO-labeled cells in the region of transplant site with SPIO-labeled hUC-MSCs.Conclusions:1. hUC-MSCs can be labeled efficiently without cell toxicity using SPIO at optimized low dosages.2. Noninvasive imaging of transplanted hUC-MSCs is feasible. These results may be beneficial to cell-based therapies for clinical implementation.Part 3 Transplantation of human umbilical cord mesenchymal stem cells in rats traumatic spinal cord injuryObjectives:To evaluate the effects of hUC-MSCs transplantation on functional recovery in rats traumatic spinal cord injury.Methods:1. Rats were randomly divided into 3 groups, sham operation group (n=12), control group (n=12), and hUC-MSCs group (PC, n=12). All groups were subjected to spinal cord injury by weight drop device except for the sham group. Control group received DMEM/F12 injections, but hUC-MSCs group undertook cells suspension treatments. 2. Rats from each group were examined for neurological function. Enzyme linked immunosorbent assay (Elisa) was used for evaluation of contents of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin-3 (NT-3).3. Survival, migration, and differentiation of hUC-MSCs was explored with immunofluorescence.4. The expression of GAP-43, NF-200, and GFAP was also assessed using immunohistochemistry and immunofluorescence.Results:1. Significant improvements in locomotion were observed in the hUC-MSCs groups. There is statistical significance compared with control group.2. Transplanted hUC-MSCs produced large amounts of GDNF and NT-3 in the host spinal cord, which may be beneficial to the repair of injured spinal cord.3. Transplanted hUC-MSCs survived for at least 8 weeks. However, they did not differentiate into neural cells.4. The functional recovery was accompanied by increased length of neurofilament positive fibers and increased numbers of growth cone around the lesion site. There were fewer reactive astrocytes in both the rostral and caudal stumps of the spinal cord in the hUC-MSCs group than in the control group.Conclusions:1. hUC-MSCs can survive and migrate in the host spinal cord after transplantation without differentiating into neural cells.2. hUC-MSCs can inhibit the formation of glial scar and enhance neuroregeneration, which promote the functional recovery after SCI.3. These data suggest that hUC-MSCs may possess therapeutic potential for traumatic spinal cord injury.