A robust culture system to generate neural progenitors with gliogenic competence from clinically relevant induced pluripotent stem cells for treatment of spinal cord injury
Yasuhiro KamataMiho IsodaTsukasa SanosakaReo ShibataShuhei ItoToshiki OkuboMunehisa ShinozakiMitsuhiro InoueIkuko KoyaShinsuke ShibataTomoko ShindoMorio MatsumotoMasaya NakamuraHideyuki OkanoNarihito NagoshiJun Kohyama
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Cell-based therapy targeting spinal cord injury (SCI) is an attractive approach to promote functional recovery by replacing damaged tissue. We and other groups have reported the effectiveness of transplanting neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) in SCI animal models for neuronal replacement. Glial replacement is an additional approach for tissue repair; however, the lack of robust procedures to drive iPSCs into NS/PCs which can produce glial cells has hindered the development of glial cell transplantation for the restoration of neuronal functions after SCI. Here, we established a method to generate NS/PCs with gliogenic competence (gNS/PCs) optimized for clinical relevance and utilized them as a source of therapeutic NS/PCs for SCI. We could successfully generate gNS/PCs from clinically relevant hiPSCs, which efficiently produced astrocytes and oligodendrocytes in vitro. We also performed comparison between gNS/PCs and neurogenic NS/PCs based on single cell RNA-seq analysis and found that gNS/PCs were distinguished by expression of several transcription factors including HEY2 and NFIB. After gNS/PC transplantation, the graft did not exhibit tumor-like tissue formation, indicating the safety of them as a source of cell therapy. Importantly, the gNS/PCs triggered functional recovery in an SCI animal model, with remyelination of demyelinated axons and improved motor function. Given the inherent safety of gNS/PCs and favorable outcomes observed after their transplantation, cell-based medicine using the gNS/PCs-induction procedure described here together with clinically relevant iPSCs is realistic and would be beneficial for SCI patients.Purpose: Two-dimensional (2D)-based cell culture systems, limited by their inherent heterogeneity and scalability, are a bottleneck in the production of high-quality cells for downstream biomedical applications. Finding the optimal conditions for large-scale stem cell culture while maintaining good cellular status is challenging. The aim of this study was to assess the effects of three-dimensional (3D) culture on the viability, proliferation, self-renewal, and differentiation of human induced pluripotent stem cells (IPSCs). Patients and Methods: Various culture conditions were evaluated to determine the optimal conditions to maintain the viability and proliferation of human IPSCs in a 3D environment: static versus dynamic culture, type of adhesion protein added to alginate (Matrigel™ versus gelatin), and the addition of Y-27632t on long-term 3D culture. The proliferation ability of the cells was evaluated via the MTS proliferation assay; the expression levels of the pluripotency markers Nanog and Oct3/4, PAX6 as an ectoderm marker, and laminin-5 and fibronectin as markers of extracellular matrix synthesis were assessed; and HIF1α and HIF2α levels were measured using quantitative reverse transcription polymerase chain reaction. Results: Using a high-aspect-ratio vessel bioreactor with a gentle, low-sheer, and low-turbulence environment with sufficient oxygenation and effective mass transfer of nutrients and waste, we verified its ability to promote cell proliferation and self-renewal. The findings showed that human IPSCs have the ability to maintain pluripotency in a feeder-free system and by inhibiting ROCK signaling and using hypoxia to improve single-cell viability in 3D culture. Furthermore, these cells demonstrated increased self-renewal and proliferation when inoculated as single cells in 3D alginate beads by adding RI during the culture period. Conclusion: Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs. Keywords: stem cell culture, large-scale expansion, HARV bioreactor, self-renewal
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Introduction: In murine myocardial injury model, the favorable effects of iCMs transplantation are not completely understood. Exosomes secreted from transplanted cells may mediate beneficial effect...
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Since the first studies of human embryonic stem cells (hESCs) and, more recently, human induced pluripotent stem cells (hiPSCs), the stem-cell field has been abuzz with the promise that these pluripotent populations will one day be a powerful therapeutic tool. Although it has been proposed that hiPSCs will supersede hESCs with respect to their research and/or clinical potential because of the ease of their derivation and the ability to create immunologically matched iPSCs for each individual patient, recent evidence suggests that iPSCs in fact have several underappreciated characteristics that might mean they are less suitable for clinical application. Continuing research is revealing the similarities, differences and deficiencies of various pluripotent stem-cell populations, and suggests that many years will pass before the clinical utility of hESCs and hiPSCs is realized. There are a plethora of ethical, logistical and technical roadblocks on the route to the clinical application of pluripotent stem cells, particularly of iPSCs. In this Essay, we discuss what we believe are important issues that should be considered when attempting to bring hiPSC-based technology to the clinic.
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Severe spinal cord injury (SCI) induces massive proliferation of spinal cord neural stem cells (NSCs), which are considered a promising cell source for therapeutic neural repair. However, most injury-activated spinal cord NSCs differentiate into astrocytes, rather than neurons, in the lesion site as a result of the injury-derived microenvironment. In this mini-review, we introduce the concept of tuning injury-activated endogenous NSCs by implantation of functionalized collagen scaffolds for severe SCI repair. The current state of in situ modulation of migration, scar formation, neuronal differentiation, and functional integration of injury-activated NSCs and their progeny by implantation of elaborately modified collagen scaffolds is demonstrated. Moreover, challenges and perspectives for utilizing injury-activated NSCs for regenerative repair of severe SCI are also discussed.
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Reprogramming of differentiated somatic cells into a pluripotent state (induced pluripotent stem cells, or iPSCs) has opened a veritable treasure trove of opportunities for regenerative medicine and basic research on human diseases. It is now possible to generate iPSCs from human patients suffering
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