Metabolomic and Signaling Programs Induced by Immobilized versus Soluble IFN γ in Neural Stem Cells

Abstract

Neural stem cells (NSCs) provide a strategy to replace damaged neurons following traumatic central nervous system injuries. A major hurdle to translation of this therapy is that direct application of NSCs to CNS injury does not support sufficient neurogenesis due to lack of proper cues. To provide prolonged spatial cues to NSCs IFN-γ was immobilized to biomimetic hydrogel substrate to supply physical and biochemical signals to instruct the encapsulated NSCs to be neurogenic. However, the immobilization of factors, including IFN-γ, versus soluble delivery of the same factor, has been incompletely characterized especially with respect to activation of signaling and metabolism in cells over longer time points. In this study, protein and metabolite changes in NSCs induced by immobilized versus soluble IFN-γ at 7 days were evaluated. Soluble IFN-γ, refreshed daily over 7 days, elicited stronger responses in NSCs compared to immobilized IFN-γ, indicating that immobilization may not sustain signaling or has altered ligand/receptor interaction and integrity. However, both IFN-γ delivery types supported increased βIII tubulin expression in parallel with canonical and noncanonical receptor-signaling compared to no IFN-γ. Global metabolomics and pathway analysis revealed that soluble and immobilized IFN-γ altered metabolic pathway activities including energy, lipid, and amino acid synthesis, with soluble IFN-γ having the greatest metabolic impact overall. Finally, soluble and immobilized IFN-γ support mitochondrial voltage-dependent anion channel (VDAC) expression that correlates to differentiated NSCs. This work utilizes new methods to evaluate cell responses to protein delivery and provides insight into mode of action that can be harnessed to improve regenerative medicine-based strategies.

Investigating Mechanisms of Subcutaneous Preconditioning Incubation for Neural Stem Cell Embedded Hydrogels

“…NSCs are not the only immune modulating factor in the hydrogel scaffolds that possess the ability for cross-talk with the host tissue. IFN-γ was incorporated into hydrogel groups as a neurogenic factor in either immobilized or soluble form . However, because it is a well-known pro-inflammatory cytokine, the effect of IFN-γ incorporated into hydrogel as soluble or immobilized on inflammatory responses was evaluated to ensure it did not potentiate FBR.…”

“…All work involving animals was done in compliance with the University of Akron Institutional Animal Care and Use Committee (IACUC). NSCs were isolated using a papain dissociation kit (Worthington, Lakewood, NJ) from the subventricular zone of a Fischer 344 rat (Envigo, Indianapolis, IN) or F344-Tg­(UBC-EGFP)­F455 (Rat Resource and Research Center, Columbia, MO) and cultured using the neurosphere culture system as previously described and published. ,− Cultured NSCs were used between passage numbers 3 and 11.…”

“…Central nervous system (CNS) injury, including spinal cord injury (SCI), and disease often results in long-term physical impediment ranging from sensory deficiencies to quadriplegia. − Multiple strategies have been developed to achieve regeneration following CNS injury including treatment with anti-inflammatories, modulation of chondroitin sulfate proteoglycans (CSPGs)/nerve growth cone interactions, and the delivery of stem cells. − Neural stem cells (NSCs) can replace neurons damaged or lost to CNS injury, but survival at the site is poor because of the hostile host environment that triggers apoptotic signaling in the transplanted cells. − Preconditioning NSCs to express pro-survival genes prior to application to CNS injury supports integration at the injury site by conferring to the cells the ability to tolerate the physiological stress they will experience . Stem cells are responsive to inflammatory mediators, leading to altered gene expression and signaling. , For instance, mesenchymal stem cells (MSCs) preconditioned with cytokines showed increased survival following transplantation to injury site through upregulated phosphorylation of protein kinase B (PKB), also known as AKT, extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinase (JNK) and an increased superoxide dismutase 2 (SOD2) level. , We have also recently shown that exposure of NSC to interferon gamma (IFN-γ) in vitro led to alterations in AKT/ERK/JNK signaling to support neurogenesis. , Preconditioning cells in an in vitro environment that recapitulates the eventual targeted injury site has also been shown to alter cell survival. MSCs preconditioned by incubation with traumatic brain injury (TBI) slices prior to transplantation to in vivo TBI showed improved therapeutic effect and survival .…”

Cell Biology Meets Cell Metabolism: Energy Production Is Similar in Stem Cells and in Cancer Stem Cells in Brain and Bone Marrow

“…133 Besides the spike of ROS that can activate NSCs into NPCs, branched chain amino acids, the endogenous neuroprotective bile acid tauroursodeoxycholic acid, interferon γ, and signal transducer and activator of transcription 3 (STAT3; see Box 8) have been reported recently to activate human NSCs into NPCs in vitro or in vivo in rodents. [134][135][136][137] Whether these inducers of switches of NSCs into NPCs are active in vivo in humans is not known yet.…”

Gold Nanostrip Array‐Mediated Wireless Electrical Stimulation for Accelerating Functional Neuronal Differentiation

“…[ 2 ] Neural stem cells (NSCs) hold great promise for the treatment of neurodegenerative diseases by replacing damaged neurons following injury or disease to reconstruct neural circuits and restore lost neuronal functions. [ 3 ] For NSC‐based therapy, the key to success is the efficient and rapid differentiation of NSCs into functional neurons. [ 4 ] Electrical stimulation is one of the most effective and widely used modalities for promoting neural differentiation and augmenting neurogenesis by activating voltage‐gated ion channels.…”