Improving Efficiency of Direct Pro-Neural Reprogramming: Much-Needed Aid for Neuroregeneration in Spinal Cord Injury.

Spinal cord injury (SCI) is a medical condition affecting ~2.5-4 million people worldwide. The conventional therapy for SCI fails to restore the lost spinal cord functions; thus, novel therapies are needed. Recent breakthroughs in stem cell biology and cell reprogramming revolutionized the field. Of them, the use of neural progenitor cells (NPCs) directly reprogrammed from non-neuronal somatic cells without transitioning through a pluripotent state is a particularly attractive strategy. This allows to "scale up" NPCs in vitro and, via their transplantation to the lesion area, partially compensate for the limited regenerative plasticity of the adult spinal cord in humans. As recently demonstrated in non-human primates, implanted NPCs contribute to the functional improvement of the spinal cord after injury, and works in other animal models of SCI also confirm their therapeutic value. However, direct reprogramming still remains a challenge in many aspects; one of them is low efficiency, which prevents it from finding its place in clinics yet. In this review, we describe new insights that recent works brought to the field, such as novel targets (mitochondria, nucleoli, G-quadruplexes, and others), tools, and approaches (mechanotransduction and electrical stimulation) for direct pro-neural reprogramming, including potential ones yet to be tested.

Epigenetic Clock and Circadian Rhythms in Stem Cell Aging and Rejuvenation.

This review summarizes the current understanding of the interaction between circadian rhythms of gene expression and epigenetic clocks characterized by the specific profile of DNA methylation in CpG-islands which mirror the senescence of all somatic cells and stem cells in particular. Basic mechanisms of regulation for circadian genes CLOCK-BMAL1 as well as downstream clock-controlled genes (ССG) are also discussed here. It has been shown that circadian rhythms operate by the finely tuned regulation of transcription and rely on various epigenetic mechanisms including the activation of enhancers/suppressors, acetylation/deacetylation of histones and other proteins as well as DNA methylation. Overall, up to 20% of all genes expressed by the cell are subject to expression oscillations associated with circadian rhythms. Additionally included in the review is a brief list of genes involved in the regulation of circadian rhythms, along with genes important for cell aging, and oncogenesis. Eliminating some of them (for example, Sirt1) accelerates the aging process, while the overexpression of Sirt1, on the contrary, protects against age-related changes. Circadian regulators control a number of genes that activate the cell cycle (Wee1, c-Myc, p20, p21, and Cyclin D1) and regulate histone modification and DNA methylation. Approaches for determining the epigenetic age from methylation profiles across CpG islands in individual cells are described. DNA methylation, which characterizes the function of the epigenetic clock, appears to link together such key biological processes as regeneration and functioning of stem cells, aging and malignant transformation. Finally, the main features of adult stem cell aging in stem cell niches and current possibilities for modulating the epigenetic clock and stem cells rejuvenation as part of antiaging therapy are discussed.

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates.

BACKGROUND: The development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI. AIM: To investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs). METHODS: Seven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group (n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control (n = 3) was injected identically with the equivalent volume of vehicle. RESULTS: Follow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones. CONCLUSION: Our data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes.

Chitosan-g-oligo(L,L-lactide) Copolymer Hydrogel Potential for Neural Stem Cell Differentiation.

We evaluated the applicability of chitosan-g-oligo(L,L-lactide) copolymer (CLC) hydrogel for central nervous system tissue engineering. The biomechanical properties of the CLC hydrogel were characterized and its biocompatibility was assessed with neural progenitor cells obtained from two different sources: H9-derived neural stem cells (H9D-NSCs) and directly reprogrammed neural precursor cells (drNPCs). Our study found that the optically transparent CLC hydrogel possessed biomechanical characteristics suitable for culturing human neural stem/precursor cells and was noncytotoxic. When seeded on films prepared from CLC copolymer hydrogel, both H9D-NSC and drNPC adhered well, expanded and exhibited signs of spontaneous differentiation. While H9D-NSC mainly preserved multipotency as shown by a high proportion of Nestin+ and Sox2+ cells and a comparatively lower expression of the neuronal markers ßIII-tubulin and MAP2, drNPCs, obtained by direct reprogramming, differentiated more extensively along the neuronal lineage. Our study indicates that the CLC hydrogel may be considered as a substrate for tissue-engineered constructs, applicable for therapy of neurodegenerative diseases. Impact statement We synthetized a chitosan-g-oligo(L,L-lactide) hydrogel that sustained multipotency of embryonic-derived neural stem cells (NSCs) and supported differentiation of directly reprogrammed NSC predominantly along the neuronal lineage. The hydrogel exhibited no cytotoxicity in vitro, both in extraction and contact cytotoxicity tests. When seeded on the hydrogel, both types of NSCs adhered well, expanded, and exhibited signs of spontaneous differentiation. The biomechanical properties of the hydrogel were similar to that of human spinal cord with incised pia mater. These data pave the way for further investigations of the hydrogel toward its applicability in central nervous system tissue engineering.