Important result in 2018
Transplantation of neural precursors generated from spinal progenitor cells reduces inflammation in spinal cord injury via NF-κB pathway inhibition
Transcription factors from the NFkB family are involved in a number of processes throughout cellular life. Mainly, they include immune responses including inflammation, cell survival, growth and development.
In our projects, we focused on determining levels of NFκB activation after experimental compression spinal cord trauma and changes in its activation after in situ transplantation of neural precursors (SPC-01) as well as on identifying mechanisms behind functional improvements induced by SPC-01 cells.
We found that activation of the NFκB pathway in the first month after injury has a bimodal character with the highest levels of activity at days 3 and 28, which reflected the host cellular response at the injury site and also correlated with infiltration waves of peripheral immune cells, which were determined by other research groups. Previously, a subacute administration of cells at 1 week after injury has been shown to be the most beneficial in transplantation therapies. In our work we showed that one of the reasons for this may be the observed decrease in NFκB activity at 7 days after the induction of injury. Administration of stem cells inhibited the pro-inflammatory NF-κB pathway with reduced TNF-α levels, and thus ameliorated the development of spinal cord injury.
Stem cells inhibit activation of inflammatory pathway in spinal cord injury
Immunohistochemical staining with hematoxylin and DAB (NF-kB p65) of spinal cord from uninjured rats (A, A1, A2), or 28 days after injury from rats treated with saline (B, B1, B2) or 28 days from rats transplanted with SPC-01 (C, C1, C2). Black arrows point to nuclei void of NF-kB p65 expression, red arrows highlight nuclei with translocated p65 and green arrows indicate cells with nuclei negative to p65 surrounded by p65+ cytoplasm (A1, A2, B1, B2, C1, C2). In the lesion center of spinal cords, a significantly lower NF-κB p65 activity was observed at 28 days in the group treated with SPC-01 cells (D). Transplantation of SPC-01 cells resulted in marked reduction of TNF-α levels 10 and 14 days after injury when compared with animals injected with saline only (E). Rats transplanted with SPC-01 cells displayed a significantly smaller cavity size than rats treated with saline (F). Reduction in gliosis was observed in the central parts of spinal cord lesion in rats transplanted with SPC-01 (G).
Important results in 2017
1. Grafted mesenchymal stem cells labeled with iron oxide or cobalt-zinc-iron nanoparticles and oxidative stress in vivo
To assess the efficacy of stem cell therapy, magnetic resonance imaging (MRI) combined with a contrast label appears to be an effective noninvasive technique for the tracking of transplanted stem cells in living organisms. The application of MRI in vivo requires the use of a safe contrast agent, that is, the achievement of a sufficient level of cell labeling for MRI and, simultaneously, biocompatibility of the label with stem cells and the host tissue without any side effects on their biological properties and functions.
Rat mesenchymal stem cells (rMSCs) labeled with 1) poly-l-lysine-coated superparamagnetic iron oxide nanoparticles or 2) silica-coated cobalt-zinc-iron nanoparticles were implanted into the left brain hemisphere of rats, to assess their effects on the levels of oxidative damage to biological macromolecules in brain tissue. Animals were sacrificed 24 hours or 4 weeks after the treatment, and the implantation site along with the surrounding tissue was isolated from the brain. The comet assay with enzymes of excision DNA repair (endonuclease III and formamidopyrimidine-DNA glycosylase) was used to analyze breaks and oxidative damage to DNA in the brain tissue. Oxidative damage to proteins and lipids was determined by measuring the levels of carbonyl groups and 15-F 2t -isoprostane (enzyme-linked immunosorbent assay). In histological sections, the expression of glial fibrillary acidic protein and CD68 was analyzed to detect astrogliosis and inflammatory response.
MRI displayed implants of labeled cells as extensive hypointense areas in the brain tissue. The signal was clearly visible within 4 weeks after implantation of rMSCs. No increase of oxidative damage to DNA, lipids, or proteins over the control values was detected in any sample of brain tissue from the treated animals. Also, immunohistochemistry did not indicate any serious tissue impairment around the graft.
The transplantation of magnetically labeled cells into the rat brain did not show any damage to the macromolecules of the nerve tissue (A-C) or induced any inflammatory reaction (D-F). Magnetically labeled cells, unlike unlabeled (G), were well visible at MRI. (H, I).
Novotna B, Herynek V, Rossner P Jr, Turnovcova K, Jendelova P.: The effects of grafted mesenchymal stem cells labeled with iron oxide or cobalt-zinc-iron nanoparticles on the biological macromolecules of rat brain tissue extracts. Int J Nanomedicine. 2017 Jun 20;12:4519-4526
2. A green tea polyphenol epigallocatechin-3-gallate enhances neuroregeneration after spinal cord injury by altering levels of inflammatory cytokines
Spinal cord injury (SCI) is a debilitating condition which is characterized by an extended secondary injury due to the presence of inflammatory local milieu. Epigallocatechin gallate (EGCG) appears to possess strong neuroprotective properties. Here, we evaluated the beneficial effect of EGCG on recovery from SCI. Male Wistar rats were given either EGCG or saline directly to the injured spinal cord and thereafter a daily IP injection. The results demonstrated that EGCG-treated rats displayed a superior behavioral performance in a flat beam test, higher axonal sprouting and positive remodelation of glial scar. Cytokine analysis revealed a reduction in IL-6, IL2, MIP1α and RANTES levels on days 1 and 3, and an upregulation of IL-4, IL-12p70 and TNFα 1 day following SCI in EGCG-treated rats. Treatment with EGCG was effective in decreasing the nuclear translocation of subunit p65 (RelA) of the NF-κB dimer, and therefore canonical NF-κB pathway attenuation. A significant increase in the gene expression of growth factors (FGF2 and VEGF), was noted in the spinal cord of EGCG-treated rats.
The image A shows transveral section of the spinal cord lesion stained with hematoxyline and NF-κB - DAB staining after EGCG treatment. The nuclear translocation of subunit p65 (RelA) of the NF-κB dimer (black arrows), nuclei stained with hematoxylin without NF-κB (red arrow) and cells with NF-κB in cell cytoplasm (green arrows). Bar A = 500 μm, Bar A1 = 20 μm. EGCG treatment significantly supports axonal sprouting in spinal cord lesion (B). Locomotor improvement (flat beam test) was observed in animals treated with EGCG when compared with control (C). Levels of proinflammatory cytokines 1 and 3 days after SCI in EGCG and saline-treated groups (D).
Important result in 2016
Stem Cell Comparison Study in Treatment of Experimental Spinal Cord Injury Model
Three different sources of human stem cells – bone marrow mesenchymal stem cells (MSCs), and two neural progenitors (NPs) derived from immortalized spinal fetal cell line (SPC 01), or induced pluripotent stem cells (iPS-NPs) – were compared in the treatment of a balloon-induced spinal cord compression lesion in rats. Morphometric analyses of spared white and grey matter, axonal sprouting and glial scar formation, as well as qPCR and Luminex assay were conducted to detect endogenous gene expression, while inflammatory cytokine levels were performed to evaluate the host tissue response to stem cell therapy. The highest locomotor recovery was observed in iPS-NP-grafted animals, which also displayed the highest amount of preserved white and grey matter. Grafted iPS-NPs and SPC-01 cells significantly increased the number of GAP43+ axons, reduced astrogliosis, downregulated Casp 3 expression and increased IL-6 and IL-12 levels. hMSCs transiently decreased levels of inflammatory IL-2 and TNF-a. These findings correlate with the hMSCs short survival, while NPs survived for 2 months and matured slowly into glia and tissue specific neuronal precursors. SPC-01 cells differentiated more in astroglial phenotypes with dense structure of the implant, whereas iPS-NPs displayed a more neuronal phenotype with loose structure of the graft. The iPS-NP treatment of spinal cord injury (SCI) provided the highest recovery of locomotor function due to robust graft survival, and its effect on tissue sparing, reduction of glial scarring and increased axonal sprouting.
While both grafted neural precursors survived well in the injured spinal cord for 2 months, bone marrow mesenchymal stem cells administered intrathecally were found only on the spinal cord surface 14 days after application. Both types of neural precursors differentiated into astrocytes, oligodendrocytes and neurons. Application of all three cell types improved the motor skills of experimental animals. Neural precursors from iPSC spared the gray matter and promoted the growth of axons at the site of injury.
Ruzicka J, Machova-Urdzikova L, Gillick J, Amemori T, Romanyuk N, Karova K, Zaviskova K, Dubisova J, Kubinova S, Murali R, Sykova E, Jhanwar-Uniyal M, Jendelova P. A Comparative Study of Three Different Types of Stem Cells for Treatment of Rat Spinal Cord Injury. Cell Transplant. 2017 Apr 13;26(4):585-603. Epub 2016 Nov 2.