No abstract

The acute blockade of spinal glycinergic inhibition with intrathecal strychnine (i.t. STR; a glycine antagonist) in rats induces a change in somatosensory processing which is very similar to the sensory dysesthesia of clinical neural injury pain. In the present study, the effects of i.t. STR were examined in urethane-anesthetized rats. Noxious paw pinch (PP) or tail immersion (TI) in 55 degree C water evoked a pronounced pressor response, increased heart rate (HR) and desynchronized the electroencephalogram; a non-noxious, hair deflection (HD) elicited only minor cardiovascular responses. After i.t. STR (40 micrograms), an identical HD stimulus evoked markedly enhanced cardiovascular responses, resembling those evoked by noxious stimuli, and a HD-evoked motor withdrawal was observed. Consistent STR-dependent responses were only observed if a light plane of anesthesia was maintained for the duration of the experiment. The effects of i.t. STR were dose-dependent and reversible, lasting 15-30 min. Spinal morphine (50 micrograms) completely abolished the cardiovascular responses to PP and TI, but the HD-evoked, STR-dependent cardiovascular and motor withdrawal responses remained unchanged. In contrast, the non-selective excitatory amino acid antagonist, gamma-D-glutamylglycine (DGG; 50 micrograms) was effective in suppressing both the STR-dependent cardiovascular and motor withdrawal responses. These data suggest that STR-dependent responses evoked by non-noxious stimuli are mediated by mechanisms distinct from those of conventional noxious stimuli and that i.t. STR may be useful for investigating the spinal pharmacology of somatosensory processing following the loss of spinal glycinergic inhibition.

During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH ° and ALDH MSC subsets demonstrated similar expression of stromal cell (>95% CD73 , CD90 , CD105 ) and pericyte (>95% CD146 ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH MSC or CDM produced by ALDH MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix-modifying functions (tissue inhibitor of metalloprotinase 1 & 2 (TIMP1/2)). Collectively, MSCs selected for ALDH demonstrated enhanced proangiogenic secretory functions and represent a purified MSC subset amenable for vascular regenerative applications. Stem Cells 2017;35:1542-1553.

Aims/hypothesis Novel strategies to stimulate the expansion of beta cell mass in situ are warranted for diabetes therapy. The aim of this study was to elucidate the secretome of human bone marrow (BM)-derived multipotent stromal cells (MSCs) with documented islet regenerative paracrine function. We hypothesised that regenerative MSCs will secrete a unique combination of protein factors that augment islet regeneration. Methods Human BM-derived MSCs were examined for glucose-lowering capacity after transplantation into streptozotocin-treated NOD/severe combined immunodeficiency (SCID) mice and segregated into samples with regenerative (MSC R ) vs nonregenerative (MSC NR ) capacity. Secreted proteins associated with islet regenerative function were identified using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics. To functionally validate the importance of active Wnt signalling, we stimulated the Wnt-signalling pathway in MSC NR samples during ex vivo expansion using glycogen synthase kinase 3 (GSK3) inhibition (CHIR99201), and the conditioned culture media (CM) generated was tested for the capacity to support cultured human islet cell survival and proliferation in vitro. Results MSC R showed increased secretion of proteins associated with cell growth, matrix remodelling, immunosuppressive and proangiogenic properties. In contrast, MSC NR uniquely secreted proteins known to promote inflammation and negatively regulate angiogenesis. Most notably, MSC R maintained Wnt signalling via Wnt5A/B (~2.5-fold increase) autocrine activity during ex vivo culture, while MSC NR repressed Wnt signalling via Dickkopf-related protein (DKK)1 (~2.5-fold increase) and DKK3 secretion. Inhibition of GSK3 activity in MSC NR samples increased the accumulation of nuclear β-catenin and generated CM that augmented beta cell survival (13% increases) and proliferation when exposed to cultured human islets. Conclusions/interpretation Maintenance of active Wnt signalling within human MSCs promotes the secretion of matricellular and proangiogenic proteins that formulate a niche for islet regeneration.

Touch-evoked allodynia, an important symptom of clinical neural injury pain, can be modelled acutely and reversibly in the urethane-anesthetized rat using intrathecal (i.t.) strychnine (STR). Allodynia, after i.t. STR (40 micrograms), is manifest as a significant enhancement of cardiovascular and motor responses evoked by normally innocuous brushing of the hair (hair deflection), as compared to responses evoked by either hair deflection after i.t. saline (SAL), or to i.t. STR (40 micrograms) with no tactile stimulus. The present study investigated: (1) the pharmacology of afferent neural inputs involved in STR-dependent allodynia using neonatal capsaicin and the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX); and (2) the effect of i.t. STR on responses evoked by peripheral noxious stimulation. Neonatal capsaicin (25 mg/kg, s.c., post-natal day (PND) 1, and 50 mg/kg, s.c., PND 2, 3, 4, 11, 25, 55 and 85) significantly attenuated the responses evoked by noxious mechanical, thermal or chemical stimuli, but had no effect on STR-dependent allodynia. All hair deflection-evoked, STR-dependent responses were dose-dependently inhibited by i.t. NBQX. The ED50 values and 95% confidence intervals were 10.4 micrograms (5.5-19.6) for the motor withdrawal response, 14.4 micrograms (8.6-24.0) for changes in MAP and 12.2 micrograms (6.8-21.8) for changes in HR. Cortical EEG synchrony was unchanged by i.t. NBQX confirming its spinal locus of action. Intrathecal STR neither reduced nor enhanced the responses elicited by noxious stimuli in capsaicin- or vehicle-pretreated rats. These results indicate that STR-dependent allodynia is initiated by primary afferents not normally involved in nociception (possibly A beta-fibers), and that STR-sensitive modulation in the spinal cord is selective for non-noxious sensory input. The sensitivity of STR-dependent allodynia to non-NMDA receptor antagonists, and the failure of i.t. STR to produce hyperalgesia to mechanical, thermal or chemical noxious stimuli, confirm the independence of nociceptive pathways and STR-sensitive afferent inputs in this model.

Multipotent/mesenchymal stromal cells (MSCs) exist within a variety of postnatal tissues; however, global proteomic analyses comparing tissue-specific MSC are limited. Using human bone marrow (BM)-derived MSCs as a gold standard, we used label-free mass spectrometry and functional assays to characterize the proteome, secretome, and corresponding function of human pancreas-derived MSCs (Panc-MSCs) with a classical phenotype (CD90+/CD73+/CD105+/CD45−/CD31−). Both MSC subtypes expressed mesenchymal markers vimentin, α-SMA, and STRO-1; however, expression of nestin was increased in Panc-MSCs. Accordingly, these Vimentin high /Nestin high cells were isolated from fresh human pancreatic islet and non-islet tissues. Next, we identified expression of >60 CD markers shared between Panc-MSCs and BM-MSCs, including validated expression of CD14. An additional 19 CD markers were differentially expressed, including reduced pericyte-marker CD146 expression on Panc-MSCs. Panc-MSCs also showed reduced expression of proteins involved in lipid and retinoid metabolism. Accordingly, Panc-MSCs showed restricted responses to adipogenic stimuli in vitro, although both MSC types demonstrated trilineage differentiation. In contrast, Panc-MSCs demonstrated accelerated growth kinetics and competency to proneurogenic stimuli in vitro. The secretome of Panc-MSCs was highly enriched for proteins associated with vascular development, wound healing and chemotaxis. Similar to BM-MSCs, Panc-MSCs conditioned media augmented endothelial cell survival, proliferation, and tubule formation in vitro. Importantly, the secretome of both MSC types was capable of stimulating chemotactic infiltration of murine endothelial cells in vivo and reduced hyperglycemia in STZ-treated mice following intrapancreatic injection. Overall, this study provides foundational knowledge to develop Panc-MSCs as a unique MSC subtype with functional properties beneficial in regenerative medicine for diabetes and vascular disease. K E Y W O R D S adipogenesis, multipotent stromal cells, nestin, pancreas, proteomics, regenerative medicine, secretome

No abstract

Uncompromised by chronic disease‐related comorbidities, human umbilical cord blood (UCB) progenitor cells with high aldehyde dehydrogenase activity (ALDHhi cells) stimulate blood vessel regeneration after intra‐muscular transplantation. However, implementation of cellular therapies using UCB ALDHhi cells for critical limb ischemia, the most severe form of severe peripheral artery disease, is limited by the rarity (<0.5%) of these cells. Our goal was to generate a clinically‐translatable, allogeneic cell population for vessel regenerative therapies, via ex vivo expansion of UCB ALDHhi cells without loss of pro‐angiogenic potency. Purified UCB ALDHhi cells were expanded >18‐fold over 6‐days under serum‐free conditions. Consistent with the concept that ALDH‐activity is decreased as progenitor cells differentiate, only 15.1% ± 1.3% of progeny maintained high ALDH‐activity after culture. However, compared to fresh UCB cells, expansion increased the total number of ALDHhi cells (2.7‐fold), CD34+/CD133+ cells (2.8‐fold), and hematopoietic colony forming cells (7.7‐fold). Remarkably, injection of expanded progeny accelerated recovery of perfusion and improved limb usage in immunodeficient mice with femoral artery ligation‐induced limb ischemia. At 7 or 28 days post‐transplantation, mice transplanted with expanded ALDHhi cells showed augmented endothelial cell proliferation and increased capillary density compared to controls. Expanded cells maintained pro‐angiogenic mRNA expression and secreted angiogenesis‐associated growth factors, chemokines, and matrix modifying proteins. Coculture with expanded cells augmented human microvascular endothelial cell survival and tubule formation under serum‐starved, growth factor‐reduced conditions. Expanded UCB‐derived ALDHhi cells represent an alternative to autologous bone marrow as an accessible source of pro‐angiogenic hematopoietic progenitor cells for the refinement of vascular regeneration‐inductive therapies. Stem Cells Translational Medicine 2017;6:1607–1619