Human-induced pluripotent stem cells generated from intervertebral disc cells improve neurologic functions in spinal cord injury.

INTRODUCTION: Induced pluripotent stem cells (iPSCs) have emerged as a promising cell source for immune-compatible cell therapy. Although a variety of somatic cells have been tried for iPSC generation, it is still of great interest to test new cell types, especially those which are hardly obtainable in a normal situation. METHODS: In this study, we generated iPSCs by using the cells originated from intervertebral disc which were removed during a spinal operation after spinal cord injury. We investigated the pluripotency of disc cell-derived iPSCs (diPSCs) and neural differentiation capability as well as therapeutic effect in spinal cord injury. RESULTS: The diPSCs displayed similar characteristics to human embryonic stem cells and were efficiently differentiated into neural precursor cells (NPCs) with the capability of differentiation into mature neurons in vitro. When the diPSC-derived NPCs were transplanted into mice 9 days after spinal cord injury, we detected a significant amelioration of hindlimb dysfunction during follow-up recovery periods. Histological analysis at 5 weeks after transplantation identified undifferentiated human NPCs (Nestin(+)) as well as early (Tuj1(+)) and mature (MAP2(+)) neurons derived from the transplanted NPCs. Furthermore, NPC transplantation demonstrated a preventive effect on spinal cord degeneration resulting from the secondary injury. CONCLUSION: This study revealed that intervertebral discs removed during surgery for spinal stabilization after spinal cord injury, previously considered a "waste" tissue, may provide a unique opportunity to study iPSCs derived from difficult-to-access somatic cells and a useful therapeutic resource for autologous cell replacement therapy in spinal cord injury.

Recapitulation of in vivo-like paracrine signals of human mesenchymal stem cells for functional neuronal differentiation of human neural stem cells in a 3D microfluidic system.

Paracrine signals produced from stem cells influence tissue regeneration by inducing the differentiation of endogenous stem or progenitor cells. However, many recent studies that have investigated paracrine signaling of stem cells have relied on either two-dimensional transwell systems or conditioned medium culture, neither of which provide optimal culture microenvironments for elucidating the effects of paracrine signals in vivo. In this study, we recapitulated in vivo-like paracrine signaling of human mesenchymal stem cells (hMSCs) to enhance functional neuronal differentiation of human neural stem cells (hNSCs) in three-dimensional (3D) extracellular matrices (ECMs) within a microfluidic array platform. In order to amplify paracrine signaling, hMSCs were genetically engineered using cationic polymer nanoparticles to overexpress glial cell-derived neurotrophic factor (GDNF). hNSCs were cultured in 3D ECM hydrogel used to fill central channels of the microfluidic device, while GDNF-overexpressing hMSCs (GDNF-hMSCs) were cultured in channels located on both sides of the central channel. This setup allowed for mimicking of paracrine signaling between genetically engineered hMSCs and endogenous hNSCs in the brain. Co-culture of hNSCs with GDNF-hMSCs in the 3D microfluidic system yielded reduced glial differentiation of hNSCs while significantly enhancing differentiation into neuronal cells including dopaminergic neurons. Neuronal cells produced from hNSCs differentiating in the presence of GDNF-hMSCs exhibited functional neuron-like electrophysiological features. The enhanced paracrine ability of GDNF-hMSCs was finally confirmed using an animal model of hypoxic-ischemic brain injury. This study demonstrates the presented 3D microfluidic array device can provide an efficient co-culture platform and provide an environment for paracrine signals from transplanted stem cells to control endogenous neuronal behaviors in vivo.

Multiscale, hierarchically patterned topography for directing human neural stem cells into functional neurons.

Various biophysical and biochemical factors are important for determining the fate of neural stem cells (NSCs). Among biophysical signals, topographical stimulation by micro/nanopatterns has been applied to control NSC differentiation. In this study, we developed a hierarchically patterned substrate (HPS) platform that can synergistically enhance the differentiation of human NSCs (hNSCs) by simultaneously providing microscale and nanoscale spatial controls to facilitate the alignment of the cytoskeleton and the formation of focal adhesions. The multiscale HPS was fabricated by combining microgroove patterns (groove size: 1.5 µm), prepared by a conventional photolithographic process, and nanopore patterns (pore diameter: 10 nm), prepared from cylinder-forming block copolymer thin films. The hNSCs grown on the HPS exhibited not only a highly aligned, elongated morphology, but also a greatly enhanced differentiation into neuronal and astrocyte lineages, compared to hNSCs on a flat substrate (FS) or single-type patterned substrates [microgroove patterned substrate (MPS) and nanopore patterned substrate (NPS)]. Interestingly, the application of the HPS directed hNSC differentiation toward neurons rather than astrocytes. The expression of focal adhesion proteins in hNSCs was also significantly increased on the HPS compared to the FS, MPS, and NPS, likely a result of the presence of more focal contact points provided by nanopore structures. Inhibition of both ß1 integrin-mediated binding and the intracellular Rho-associated protein kinase pathway of hNSCs eliminated the beneficial effects of the HPS on focal adhesion formation and actin filament alignment, which subsequently reduced hNSC differentiation. More importantly, hNSCs on the HPS differentiated into functional neurons exhibiting sodium currents and action potentials. The multiscale, hierarchically patterned topography would be useful for the design of functional biomaterial scaffolds to potentiate NSC therapeutic efficacy.