Priming Mesenchymal Stem/Stromal Cells with a Combination of a Low Dose of IFN-γ and Bortezomib Results in Potent Suppression of Pathogenic Th17 Immunity Through the IDO1-AHR Axis.

Preconditioning of mesenchymal stem/stromal cells (MSCs) with the inflammatory cytokine IFN-γ enhances not only their immunosuppressive activity but also their expression of HLA and proinflammatory genes. We hypothesized that prevention of the upregulation of inflammatory cytokines and HLA molecules in IFN-γ-primed MSCs would render these cells more immunosuppressive and less immunogenic. In this study, we discovered the following findings supporting this hypothesis: (1) activated human T cells induced the expression of IDO1 in MSCs via IFN-γ secretion and those MSCs in turn inhibited T-cell proliferation in an AHR-dependent fashion; (2) there was no difference in the expression of IDO1 and HLA-DR in MSCs after priming with a low dose (25 IU/mL) versus a high dose (100 IU/mL) of IFN-γ; (3) the transient addition of bortezomib, a proteasome inhibitor, to culture MSCs after IFN-γ priming decreased the expression of HLA-DR, inflammatory cytokine genes and Vcam1 while increasing the expression of IDO1 and the production of L-kynurenine; finally, MSCs primed with a combination of a low dose of IFN-γ and bortezomib were more effective in inhibiting Th17-mediated idiopathic pneumonia syndrome (IPS) and chronic colitis than unprimed MSCs. Our results suggest that bortezomib significantly eliminates the unfavorable effects of IFN-γ priming of MSCs (increased expression of MHC molecules and inflammatory cytokines and cell aggregation genes) and simultaneously increases their immunosuppressive activity by upregulating IDO1. Taken together, our newly established MSC priming method may contribute to MSC-based cell therapy for inflammatory diseases.

Upregulation of TrkB by forskolin facilitated survival of MSC and functional recovery of memory deficient model rats.

Mesenchymal stem cells (MSCs) are effective vectors in delivering a gene of interest into degenerating brain. In ex vivo gene therapy, viability of transplanted MSCs is correlated with the extent of functional recovery. It has been reported that BDNF facilitates survival of MSCs but dividing MSCs do not express the BDNF receptor, TrkB. In this study, we found that the expression of TrkB is upregulated in human MSCs by the addition of forskolin (Fsk), an activator of adenylyl cyclase. To increase survival rate of MSCs and their secretion of tropic factors that enhance regeneration of endogenous cells, we pre-exposed hMSCs with Fsk and transduced with BDNF-adenovirus before transplantation into the brain of memory deficient rats, a degenerating brain disease model induced by ibotenic acid injection. Viability of MSCs and expression of a GABA synthesizing enzyme were increased. The pre-treatment improved learning and memory, as detected by the behavioral tests including Y-maze task and passive avoidance test. These results suggest that TrkB expression of hMSCs elevates the neuronal regeneration and efficiency of BDNF delivery for treating degenerative neurological diseases accompanying memory loss.

Therapeutic Effect of IL1ß Priming Tonsil Derived-Mesenchymal Stem Cells in Osteoporosis.

BACKGROUND: Stem cell therapies can be a new therapeutic strategy that may rebalance anabolic and anti-resorptive effects in osteoporosis patients. Tonsil-derived mesenchymal stem cells (TMSCs) can be an alternative therapeutic source for chronic degenerative diseases including osteoporosis. MSCs acquire immune regulatory function under the inflammatory cytokines. Since interleukin (IL) 1ß is known to be one of inflammatory cytokines involved in osteoporosis progression, treatment of IL1ß with TMSCs may enhance immunomodulatory function and therapeutic effects of TMSCs in osteoporosis. METHODS: For IL1ß priming, TMSCs were cultured in the presence of the medium containing IL1ß for 1 day. Characteristics of IL1ß priming TMSCs such as multipotent differentiation properties, anti-inflammatory potential, and suppression of osteoclast differentiation were assessed in vitro. For in vivo efficacy study, IL1ß priming TMSCs were intravenously infused twice with ovariectomized (OVX) osteoporosis mouse model, and blood serum and bone parameters from micro computed tomography images were analyzed. RESULTS: IL1ß priming TMSCs had an enhanced osteogenic differentiation and secreted factors that regulate both osteoclastogenesis and osteoblastogenesis. IL1ß priming TMSCs also suppressed proliferation of peripheral blood mononuclear cells (PBMCs) and decreased expression of Receptor activator of nuclear factor kappa-Β ligand (RANKL) in PHA-stimulated PBMCs. Furthermore, osteoclast specific genes such as Nuclear factor of activated T cells c1 (NFATc1) were effectively down regulated when co-cultured with IL1ß priming TMSCs in RANKL induced osteoclasts. In OVX mice, IL1ß priming TMSCs induced low level of serum RANKL/osteoprotegerin (OPG) ratio on the first day of the last administration. Four weeks after the last administration, bone mineral density and serum Gla-osteocalcin were increased in IL1ß priming TMSC-treated OVX mice. Furthermore, bone formation and bone resorption markers that had been decreased in OVX mice with low calcium diet were recovered by infusion of IL1ß priming TMSCs. CONCLUSION: IL1ß priming can endow constant therapeutic efficacy with TMSCs, which may contribute to improve bone density and maintain bone homeostasis in postmenopausal osteoporosis. Therefore, IL1ß priming TMSCs can be a new therapeutic option for treating postmenopausal osteoporosis.

Wogonin induces differentiation and neurite outgrowth of neural precursor cells.

Wogonin is a flavonoid isolated from Scutellaria baicalensis root, and has multiple pharmacological effects, including anti-inflammatory, anti-oxidant, and anti-cancer effects. It is also neuroprotective in the brain under many stress conditions, but wogonin does not elevate neuronal cell survival. Thus, the mechanisms controlling the neuroprotective effect of wogonin are not clear. Neural precursor cells (NPCs), present in the hippocampus and subventricular zone of adult brains, replace damaged cells. In this study we investigated the biological functions underlying the neuroprotective effect of wogonin on NPCs. We initially examined survival of NPCs but found it was slightly reduced at concentrations higher than 2µg/ml. When we explored differentiation of NPCs into neuronal cells, the number of differentiated cells expressing neurofilaments was increased remarkably (fourfold) in the hippocampal NPCs treated with wogonin. Wogonin maximally elevated the expressions of presynaptic protein, synapsin I and postsynaptic protein (PSD95) at a concentration of 0.7µg/ml. Differentiated cells containing longer neurites were significantly increased in cortical NPCs, primarily cultured from rat E14 embryonic brain. Wogonin also promoted differentiation of NPCs into mature neurons in vivo. When transplanted into the adult rat hippocampus, NPCs differentiated into cells expressing NeuN, the mature neuron marker, by 4weeks after transplantation. These data indicate that wogonin induces differentiation of NPCs both in culture and in vivo, and suggest that facilitation of NPC differentiation is a biological activity by which wogonin protects neurons in damaged brain.

Distinct effect of neurotrophins delivered simultaneously by an adenoviral vector on neurite outgrowth of neural precursor cells from different regions of the brain.

For many years, it has been demonstrated that neurotrophins regulate the adult nervous system, implicating their potential as therapeutic agents for the treatment of neurodegenerative diseases. We generated adenoviral vectors encoding brain-derived neutotrophin factor (BDNF) and neurotrophin-3 (NT3) and tested either separately or together for the ability to induce differentiation of neuronal precursor cells with two different origins. Separate transduction of adenovirus delivering BDNF (BDNF-Ad) or NT3 (NT3-Ad) induced the neuronal differentiation in hippocampal and cortical precursor cells. NT3-Ad infected cells extended short neurites, whereas BDNFAd infected cells had longer neurites. In the early differentiation of hippocampal precursor cells, simultaneous infection of BDNFAd and NT3-Ad promoted further differentiation and neurite elongation compared with the separate infection of each virus. In contrast, simultaneous infection did not show the synergistic effect in the cortical precursor cells, suggesting that the neurotrophins play distinct roles in different regions of the brain. However, the numbers of neurites and spines per differentiated cells were markedly increased in cortical as well as hippocampal precursor cells, indicating the promotion of efficient neurite elongation and formation of dendritic spine, when BDNF-Ad and NT3-Ad were co-infected. These results suggest more studies in the effect of a combinatorial use of neurotrophins on different sites of brain need to be carried out to develop gene therapy protocols for neurodegenerative diseases.

Secretion of EGF-like domain of heregulinß promotes axonal growth and functional recovery of injured sciatic nerve.

Neuregulin 1 (NRG1) and epidermal growth factor receptor (ErbB) signaling pathways control Schwann cells during axonal regeneration in an injured peripheral nervous system. We investigated whether a persistent supply of recombinant NRG1 to the injury site could improve axonal growth and recovery of sensory and motor functions in rats during nerve regeneration. We generated a recombinant adenovirus expressing a secreted form of EGF-like domain from Heregulinß (sHRGßE-Ad). This virus, sHRGßE-Ad allowed for the secretion of 30-50 ng of small sHRGßE peptides per 10(7-8) virus particle outside cells and was able to phosphorylate ErbB receptors. Transduction of the concentrated sHRGßE-Ad into an axotomy model of sciatic nerve damage caused an effective promotion of nerve regeneration, as shown by histological features of the axons and Schwann cells, as well as increased expression of neurofilaments, GAP43 and S100 in the distal stump of the injury site. This result is consistent with longer axon lengths and thicker calibers observed in the sHRGßE-Ad treated animals. Furthermore, sensory and motor functions were significantly improved in sHRGßE-Ad treated animals when evaluated by a behavioral test. These results suggest a therapeutic potential for sHRGßE-Ad in treatment of peripheral nerve injury.