Consistent with these reports, we observed that SPC-01 cell transplantation promoted functional recovery after SCI. (S2A and B) and nestin (S2C), the early oligodendroglial marker Olig2 (S2D), and the astroglial marker GFAP (S2E). Seventeen weeks after transplantation, SPC-01 cells were positive for CNPase (S2F). scrt219-S4.tiff (559K) GUID:?58B7C39B-55C5-43D9-BA74-E5619121A5A8 Additional file 5: Figure S3 Orthogonal projection for Figure?6 images. scrt219-S5.tiff (17M) GUID:?B4C17BAE-F362-464E-8FB9-A38FA8D755D2 Abstract Introduction A growing number of studies have highlighted the potential of stem cell and more-differentiated neural cell transplantation as intriguing therapeutic approaches for neural repair after spinal cord injury (SCI). Methods A conditionally immortalized neural stem cell line derived ODM-203 from human fetal spinal cord tissue (SPC-01) was used to treat a balloon-induced SCI. SPC-01 cells were implanted into the lesion 1 week after SCI. To determine the feasibility of tracking transplanted stem cells, a portion of the SPC-01 cells was labeled with poly-L-lysine-coated superparamagnetic iron-oxide nanoparticles, and the animals grafted with labeled cells underwent magnetic resonance imaging. Functional recovery was evaluated by using the BBB and plantar tests, and lesion morphology, endogenous axonal sprouting and graft survival, and differentiation were analyzed. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted SPC-01 cells on endogenous regenerative processes. Results Transplanted animals displayed significant motor and sensory improvement 2 months after SCI, when the cells robustly survived in the lesion and partially filled the lesion cavity. qPCR revealed the increased expression of rat and human neurotrophin and motor neuron genes. The grafted cells were immunohistologically positive for glial fibrillary acidic protein (GFAP); however, we found 25% of the cells to be positive for Nkx6.1, an early motor neuron marker. Spared white matter and the robust sprouting of growth-associated protein 43 (GAP43)+?axons were found in the host tissue. Four months after SCI, the grafted cells matured into Islet2+ and choline acetyltransferase (ChAT)+ neurons, and the graft was grown through with endogenous neurons. Grafted ODM-203 cells labeled with poly-L-lysine-coated superparamagnetic nanoparticles before transplantation were detected in the lesion on T2-weighted images as hypointense spots that correlated with histologic staining for iron and the human mitochondrial marker MTCO2. Conclusions The transplantation of SPC-01 cells produced significant early functional improvement after SCI, suggesting an early neurotrophic action associated with long-term restoration of the host tissue, making the cells a promising candidate for future cell therapy in patients with SCI. MRI by using poly-L-lysine-coated superparamagnetic iron-oxide nanoparticles. Third, we showed that the transplantation of SPC-01 cells into the lesioned rat spinal cord improves functional outcome by partially bridging the spinal cord lesion and providing trophic support to the spared axons in the injured tissue. Methods Human fetal neural stem cells SPC-01 The human spinal cord cell line (SPC-01) was generated from 10-week-old human fetal spinal cord. Fetal tissue was obtained from Advanced Bioscience Resources (Alameda, CA, USA) after normal terminations and in accordance with nationally (UK and/or USA) approved ethical and legal guidelines [19,20]. Cells were prepared by mechanical and enzymatic dissociation of the fetal spinal cord cervical region into a single-cell suspension. Subsequently, cells were conditionally immortalized with the detection, the SPC-01 cells were transduced with green fluorescent protein (GFP). The GFP-expressing SPC-01 cells were generated by using a lentiviral vector containing a ubiquitous chromatin opening element (UCOE) to prevent silencing on engraftment, COL4A6 as previously described [21]. Transduced SPC-01_GFP+ cells were frozen, stored in liquid nitrogen, and used throughout the whole study. SPC-01-GFP+ cells were routinely cultured in tissue-culture flasks freshly coated with laminin (Sigma, St. Louis, MO, USA; 20 g/ml in DMEM:F12) for 1 hour at 37C. Growth media comprising DMEM:F12 supplemented with HSA (0.03%) (Baxter Healthcare Ltd., Norfolk, UK); L-glutamine (2 mtest for independent samples, if the two samples had equal variances. If they had unequal variances, the MannCWhitney test was used for evaluation. A value <0.05 was considered statistically significant. All behavioral tests were performed by two independent blind observers. Histologic and immunohistochemical analysis To analyze the volume of the spared white and gray matter and the extent of axonal sprouting, animals with SCI only (and (((and were determined by quantitative real-time reverse transcription polymerase chain reaction (qPCR) in a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) by using TaqMan Gene Expression Master Mix (catalog number 392938) and TaqMan Gene Expression Assays 4331182 (Rn02531967_s1/Bdnf/, Rn01511601_m1/Vegfa/, Rn01533872_m1/Ngf/, Rn01521847_m1/Sort1/, Hs01010223_m1/BDNF-AS1/, Hs00900055_m1/VEGFA/, Hs00171458_m1/NGF/, Hs00361760_m1/SORT1/, Hs00232355_m1/NKX6-1/, Hs00377575_m1/ISL2/, Hs00907365_m1/MNX1/, Hs00300531_m1/SYP/, Hs00252848_m1/CHAT). The qPCR was carried out in a final volume of 20 l containing 500 ng of extracted RNA. The following thermal profile was used: a single cycle of reverse transcription for 30 minutes at 50C and 15 minutes at 95C for reverse ODM-203 transcriptase inactivation and DNA polymerase activation, followed by 40 cycles of denaturation at 95C for 15 seconds and annealing and extension at 60C for 1 minute. The results were.