Takashi Amemori

HPMA-RGD Hydrogels Seeded with Mesenchymal Stem Cells Improve Functional Outcome in Chronic Spinal Cord Injury

Chronic spinal cord injury (SCI) is characterized by tissue loss and a stable functional deficit. While several experimental therapies have proven to be partly successful for the treatment of acute SCI, treatment of chronic SCI is still challenging. We studied whether we can bridge a chronic spinal cord lesion by implantation of our newly developed hydrogel based on 2-hydroxypropyl methacrylamide, either alone or seeded with mesenchymal stem cells (MSCs), and whether this treatment leads to functional improvement. A balloon-induced compression lesion was performed in adult 2-month-old male Wistar rats. Five weeks after injury, HPMA-RGD hydrogels [N-(2-hydroxypropyl)-methacrylamide with attached amino acid sequences--Arg-Gly-Asp] were implanted into the lesion, either with or without seeded MSCs. Animals with chronic SCI served as controls. The animals were behaviorally tested using the Basso–Beattie-Breshnahan (BBB) (motor) and plantar (sensory) tests once a week for 6 months. Behavioral analysis showed a statistically significant improvement in rats with combined treatment, hydrogel and MSCs, compared with the control group (P < 0.05). Although a tendency toward improvement was found in rats treated with hydrogel only, this was not significant. Subsequently, the animals were sacrificed 6 months after SCI, and the spinal cord lesions evaluated histologically. The combined therapy (hydrogel with MSCs) prevented tissue atrophy (P < 0.05), and the hydrogels were infiltrated with axons myelinated with Schwann cells. Blood vessels and astrocytes also grew inside the implant. MSCs were present in the hydrogels even 5 months after implantation. We conclude that 5 weeks after injury, HPMA-RGD hydrogels seeded with MSCs can successfully bridge a spinal cord cavity and provide a scaffold for tissue regeneration. This treatment leads to functional improvement even in chronic SCI.

Co-transplantation of olfactory ensheathing glia and mesenchymal stromal cells does not have synergistic effects after spinal cord injury in the rat

BACKGROUND AIMS Olfactory ensheathing glia (OEG) and mesenchymal stromal cells (MSC) are suitable candidates for transplantation therapy of spinal cord injury (SCI). Both facilitate functional improvement after SCI by producing trophic factors and cytokines. In this study, the co-transplantation of both types of cells was studied to clarify their additive and/ or synergistic effects on SCI. METHODS A balloon-induced compression lesion was used to produce SCI in rats. OEG, MSC or both OEG and MSC (3 x 10(5) cells of each cell type) were implanted by intraspinal injection 1 week after SCI. The effect of transplantation was assessed using behavioral, electrophysiologic and histologic methods. RESULTS Hindlimb function was examined with Basso, Beattie and Bresnahan (BBB) and Plantar tests. Improvement was found in all three groups of transplanted rats with different time-courses, but there was no significant difference among the groups at the end of the experiment. Motor-evoked potentials after SCI decreased in amplitude from 7 mV to 10 microV. Linear regression analysis showed a modest recovery in amplitude following transplantation, but no change in the control rats. Histologic findings showed that the white and gray matter were significantly spared by transplantation after SCI. CONCLUSIONS Functional improvement was achieved with transplantation of OEG and/or MSC, but the co-transplantation of OEG and MSC did not show synergistic effects. The poor migration of OEG and MSC might prevent their concerted action. Pre-treatment with a Rho antagonist and a combination of intraspinal and intravenous injection of the cells might be beneficial for SCI therapy.

Human conditionally immortalized neural stem cells improve locomotor function after spinal cord injury in the rat

IntroductionA 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).MethodsA conditionally immortalized neural stem cell line derived 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.ResultsTransplanted 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 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.ConclusionsThe 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.

Beneficial Effect of Human Induced Pluripotent Stem Cell-Derived Neural Precursors in Spinal Cord Injury Repair

Despite advances in our understanding and research of induced pluripotent stem cells (iPSCs), their use in clinical practice is still limited due to lack of preclinical experiments. Neural precursors (NPs) derived from a clone of human iPSCs (IMR90) were used to treat a rat spinal cord lesion 1 week after induction. Functional recovery was evaluated using the BBB, beam walking, rotarod, and plantar tests. Lesion morphology, endogenous axonal sprouting, graft survival, and iPSC-NP differentiation were analyzed immunohistochemically. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted iPSC-NPs on endogenous regenerative processes and also to monitor their behavior after transplantation. Human iPSC-NPs robustly survived in the lesion, migrated, and partially filled the lesion cavity during the entire period of observation. Transplanted animals displayed significant motor improvement already from the second week after the transplantation of iPSC-NPs. qPCR revealed the increased expression of human neurotrophins 8 weeks after transplantation. Simultaneously, the white and gray matter were spared in the host tissue. The grafted cells were immunohistochemically positive for doublecortin, MAP2, bIII-tubulin, GFAP, and CNPase 8 weeks after transplantation. Human iPSC-NPs further matured, and 17 weeks after transplantation differentiated toward interneurons, dopaminergic neurons, serotoninergic neurons, and ChAT-positive motoneurons. Human iPSC-NPs possess neurotrophic properties that are associated with significant early functional improvement and the sparing of spinal cord tissue. Their ability to differentiate into tissue-specific neurons leads to the long-term restoration of the lesioned tissue, making the cells a promising candidate for future cell-based therapy of SCI.

Alzheimer’s Disease: Mechanism and Approach to Cell Therapy

Alzheimer’s disease (AD) is the most common form of dementia. The risk of AD increases with age. Although two of the main pathological features of AD, amyloid plaques and neurofibrillary tangles, were already recognized by Alois Alzheimer at the beginning of the 20th century, the pathogenesis of the disease remains unsettled. Therapeutic approaches targeting plaques or tangles have not yet resulted in satisfactory improvements in AD treatment. This may, in part, be due to early-onset and late-onset AD pathogenesis being underpinned by different mechanisms. Most animal models of AD are generated from gene mutations involved in early onset familial AD, accounting for only 1% of all cases, which may consequently complicate our understanding of AD mechanisms. In this article, the authors discuss the pathogenesis of AD according to the two main neuropathologies, including senescence-related mechanisms and possible treatments using stem cells, namely mesenchymal and neural stem cells.

Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons

IntroductionThe use of immortalized neural stem cells either as models of neural development in vitro or as cellular therapies in central nervous system (CNS) disorders has been controversial. This controversy has centered on the capacity of immortalized cells to retain characteristic features of the progenitor cells resident in the tissue of origin from which they were derived, and the potential for tumorogenicity as a result of immortalization. Here, we report the generation of conditionally immortalized neural stem cell lines from human fetal spinal cord tissue, which addresses these issues.MethodsClonal neural stem cell lines were derived from 10-week-old human fetal spinal cord and conditionally immortalized with an inducible form of cMyc. The derived lines were karyotyped, transcriptionally profiled by microarray, and assessed against a panel of spinal cord progenitor markers with immunocytochemistry. In addition, the lines were differentiated and assessed for the presence of neuronal fate markers and functional calcium channels. Finally, a clonal line expressing eGFP was grafted into lesioned rat spinal cord and assessed for survival, differentiation characteristics, and tumorogenicity.ResultsWe demonstrate that these clonal lines (a) retain a clear transcriptional signature of ventral spinal cord progenitors and a normal karyotype after extensive propagation in vitro, (b) differentiate into relevant ventral neuronal subtypes with functional T-, L-, N-, and P/Q-type Ca2+ channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity.ConclusionsWe propose that these cells represent a useful tool both for the in vitro study of differentiation into ventral spinal cord neuronal subtypes, and for examining the potential of conditionally immortalized neural stem cells to facilitate functional recovery after spinal cord injury or disease.

A comparative study of three different types of stem cells for treatment of rat spinal cord injury.

Three different sources of human stem cells—bone marrow-derived mesenchymal stem cells (BM-MSCs), neural progenitors (NPs) derived from immortalized spinal fetal cell line (SPC-01), and induced pluripotent stem cells (iPSCs)—were compared in the treatment of a balloon-induced spinal cord compression lesion in rats. One week after lesioning, the rats received either BM-MSCs (intrathecally) or NPs (SPC-01 cells or iPSC-NPs, both intraspinally), or saline. The rats were assessed for their locomotor skills (BBB, flat beam test, and rotarod). Morphometric analyses of spared white and gray 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 iPSC-NP-grafted animals, which also displayed the highest amount of preserved white and gray matter. Grafted iPSC-NPs and SPC-01 cells significantly increased the number of growth-associated protein 43 (GAP43+) axons, reduced astrogliosis, downregulated Casp3 expression, and increased IL-6 and IL-12 levels. hMSCs transiently decreased levels of inflammatory IL-2 and TNF-α. These findings correlate with the short survival of hMSCs, 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 a dense structure of the implant, whereas iPSC-NPs displayed a more neuronal phenotype with a loose structure of the graft. We concluded that the BBB scores of iPSC-NP- and hMSC-injected rats were superior to the SPC-01-treated group. The iPSC-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.