Cell Transplant. 2015;24(9):1781-97. doi: 10.3727/096368914X684042.
Beneficial Effect of Human Induced Pluripotent Stem Cell-Derived Neural Precursors in Spinal Cord Injury Repair.
Nataliya Romanyuk1, Takashi Amemori1, Karolina Turnovcova1, Pavel Prochazka1, Brigitte Onteniente2,3, Eva Sykova1,4, Pavla Jendelova1,4
1Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
2INSERM UMR 861, 91030 Evry cedex, France
3Université Evry-Val d’Essonne, 91030 Evry cedex, France
4Department of Neuroscience, Charles University, Second Faculty of Medicine, Prague, Czech Republic
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 one week after induction. Functional recovery was evaluated using the BBB, beam walking, rotarod and plantar tests. Lesion morphology, endogenous axonal sprouting, graft survival and iPS-NP differentiation were analyzed immunohistochemically. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted iPS-NPs on endogenous regenerative processes and also to monitor their behavior after transplantation. Human iPS-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 iPS-NPs. qPCR revealed the increased expression of human neurotrophins 8 weeks after transplantation. Simultaneously, the white and gray matter was spared in the host tissue. The grafted cells were immunohistochemically positive for doublecortin, MAP2, bIII-tubulin, GFAP and CNPase 8 weeks after transplantation. Human iPS-NPs further matured and 17 weeks after transplantation differentiated towards interneurons, dopaminergic neurons, serotoninergic neurons and ChAT-positive motoneurons. Human iPS-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.
Spinal cord injuries (SCI) still remain incurable in current clinical practice. Standard therapy focuses on early decompression and stabilization of the injured spine together with preventing secondary injury. At the same time, several experimental therapies are being developed in laboratories with stem cells being one of the most promising therapies for SCI. Induced pluripotent stem cells (iPSCs) offer new prospects for regenerative medicine. These cells can be reprogrammed using different reprogramming factors from mature, tissue specific cells (for example skin cells, or fibroblast cells) and tailored for individual patients. They can differentiate into any human cell in the body. Therefore, immature neural precursors derived from human iPSCs (iPSC-NPs) appear to be an ideal source for transplantation therapy in SCI. Here, we examined the therapeutic potential of iPS-NPs by transplanting them into a rat model of SCI. We used neural precursors, which were differentiated from iPSCs (IMR90) that were established from fetal human lung fibroblasts by the lentiviral transduction of four reprogramming factors (oct4, nanog, lim28 and sox2).
As a model of spinal cord injury we used a compression lesion in rats. Animals were grafted 1 week after injury, at the time when inflammation decreases and the glial scar is still not fully developed. This is the best time window for engraftment.
Figure 1: Animals grafted with iPS-NPs performed better in the flat beam test (A), spared white and gray matter (B) and showed enhanced axonal sprouting (C). Cells injected into spinal cord lesion survived well (D) and differentiated mainly into neurons (E) and less to astrocytes (F). Four months after grafting we found motoneurons (G), serotonergic (H), GABAergic (I) and dopaminergic neurons (J). Scale bar D = 500 mm, E,F = 50mm, G-I = 20mm. Significance * p< 0.05.
We were first interested in cell survival and differentiation, since neural precursors can differentiate into neurons, astrocytes as well as oligodendrocytes. We killed animals 2 and 4 months after transplantation and we observed robust survival for the whole experimental period (Fig 1D). Two months after grafting the cells were, however, rather immature, though they differentiated mainly into neurons and less into astrocytes (Fig 1E,F). Two months later we were able to detect dopaminergic, serotonergic, GABAergic neurons and motoneurons of human origin (Fig, 1G-I).
Our second question was related to the functional effect of grafted cells. We tested animals using several behavioral tests. The treated animals scored much better than the untreated ones even in tests that required weight support and stepping, such as walking across a flat beam (Fig 2A). These changes already appeared 2 weeks after grafting, so it was obvious that this is not the result of cell differentiation and maturation, which appears much later. We analyzed the expression of human neurotrophic growth factors and we found that the grafted cells upregulate NGF, FGF8, and GDNF, compared to the mRNA levels before transplantation. This trophic support resulted in increased axonal sprouting, which was detected as an increased number of GAP43+ fibers in the lesion and sparing of the white and grey matter (Fig 1B,C). We also found that the cells of human origin (astrocytes and neurons) closely communicated with the host cells, making a contribution to tissue restoration. Our findings are in agreement with our previous study on a rat stroke model in which the same grafted iPS-NPs slowly matured (over 17 weeks) into a striatal neuron phenotype (D2 receptor-, DARPP32- and calretinin-positive cells), showing that the cells are to a certain extent able to adopt a tissue-specific phenotype (1). We also observed a paracrine effect of the grafted cells, since grafted animals performed better on the tape removal test when compared to their lesioned only counterparts.
In conclusion, iPSC-NPs can be implanted into an animal model of SCI, where they robustly survive in the lesion, express neurotrophins, and facilitate the sprouting of endogenous GAP43-positive axons. All of these actions lead to rapid improvements in locomotor functions and to the sparing of the white and gray matter in a relatively short time. In the long term, transplanted iPS-NPs can slowly mature into region-specific and mature neurons and can participate to some extent in tissue reconstruction.
Figure 2: Animal performing flat beam test
The study was supported from the Norwegian Financial Mechanism 2009-2014 and the Ministry of Education, Youth and Sports under Project Contract no. MSMT-28477/2014; project 7F14057 and by the grant of MEYS LH 12024. We thank Kip Bauersfeld for critical reading of the text.
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