High-efficient serum-free differentiation of trabecular meshwork mesenchymal stem cells into Schwann-like cells on polylactide electrospun nanofibrous scaffolds.

Cell-based therapies of the peripheral nerve injury (PNI) have provided satisfactory outcomes among which Schwann cells (SCs) are the most reliable candidate to improve repair of the damaged nerve, however, it is difficult to obtain sufficient amount of SCs for clinical applications. Trabecular meshwork-derived mesenchymal stem cells (TM-MSCs) are newly introduced neural crest originated MSCs, which may have a desirable potential for Schwann-like differentiation due to their common lineage. On the other hand, one of the challenges of cell-based therapies is usage of serum containing media which is inappropriate for clinical applications. In the present study, we investigated the differentiation potential of TM-MSCs into Schwann-like cells on polylactide (PLA) nanofibrous scaffolds in the presence or absence of serum. Our results revealed that PLA nanofibers had no negative effects on the cell growth and proliferation of TM-MSCs, and improved Schwann-like differentiation compared with tissue culture plates (TCPs). More importantly, when the cells cultured on the scaffold in the presence of serum-free media (SFM), expression mRNA levels of SC markers (S100B, GAP43, GFAP and SOX10) were significantly increased compared with those of serum-rich groups. Immunostaining of TM-MSCs cultured on serum-free PLA nanofibrous scaffolds also showed significant expression of GAP43, GFAP and SOX10 compared to those of control, indicating the efficient role of SFM in the differentiation of TM-MSCs into SCs lineage. Overall, the findings of this study revealed the differentiation potential of TM-MSCs to SC fate for the first time, and also showed the beneficial effects of SFM and PLA nanofibrous scaffolds as a promising approach for peripheral nerve regeneration.

Wnt/ß-catenin signaling pathway is involved in early dopaminergic differentiation of trabecular meshwork-derived mesenchymal stem cells.

Permanent degeneration and loss of dopaminergic (DA) neurons in substantia nigra is the main cause of Parkinson's disease. Considering the therapeutic application of stem cells in neurodegeneration, we sought to examine the neurogenic differentiation potential of the newly introduced neural crest originated mesenchymal stem cells (MSCs), namely, trabecular meshwork-derived mesenchymal stem cells (TM-MSCs) compared to two other sources of MSCs, adipose tissue-derived stem cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs). The three types of cells were therefore cultured in the presence and absence of a neural induction medium followed by the analysis of their differentiation potentials. Our results showed that TM-MSCs exhibited enhanced neural morphologies as well as higher expressions of MAP2 as the general neuron marker and Nurr-1 as an early DA marker compared to the adipose tissue-derived mesenchymal stem cells (AD-MSCs) and bone marrow-derived stem cells (BMSCs). Also, analysis of Nurr-1 immunostaining showed more intense Nurr-1 stained nuclei in the neurally induced TM-MSCs compared to those in the AD-MSCs, BMSCs, and noninduced control TM-MSCs. To examine if Wnt/beta-catenin pathway drives TM-MSCs towards a DA fate, we treated them with the Wnt agonist (CHIR, 3 µM) and the Wnt antagonist (IWP-2, 3 µM). Our results showed that the expressions of Nurr-1 and MAP2, as well as the Wnt/beta-catenin target genes, c-Myc and Cyclin D1, were significantly increased in the CHIR-treated TM-MSCs, but significantly reduced in those treated with IWP-2. Altogether, we declare first a higher neural potency of TM-MSCs compared to the more commonly used MSCs, BMSCs and ADSCs, and second that Wnt/beta-catenin activation directs the neurally induced TM-MSCs towards a DA fate.

TGFß and Wnt Signaling Pathways Cooperatively Enhance Early Dopaminergic Differentiation of the Unrestricted Somatic Stem Cells.

So far no evidence is available as to whether TGFß and Wnt signaling pathways cooperatively modulate dopaminergic differentiation of the adult stem cells. To investigate the interaction between the two pathways in early dopaminergic differentiation, we cultured the newly introduced unrestricted somatic stem cells (USSCs) in neuron differentiation media followed by treatments with inducers and inhibitors of Wnt and TGF beta pathways either alone or in combinations. Our results showed that the level of Nurr-1 as a marker for dopaminergic neuron precursors and that of the nuclear ß-catenin as the key effector of the active Wnt pathway were significantly elevated following the treatment with either TGFß or BIO (the Wnt pathway inducer). Conversely, Nurr-1 expression was significantly reduced following the combined treatments with SB431542 (the TGFß inhibitor) plus BIO or with TGFß plus Dkk1 (the specific Wnt inhibitor). Nuclear ß-catenin was also significantly reduced following combined treatments with SB431542 plus either BIO or TGFß. Altogether, our results imply that Wnt and TGFß signaling pathways cooperatively ensure the early dopaminergic differentiation of the USSC adult stem cells.

Inhibition of transforming growth factor-ß signaling pathway enhances the osteogenic differentiation of unrestricted somatic stem cells.

In recent years, extensive studies have been performed to enhance stem cell-based therapies for bone and cartilage repair. Among various sources of stem cells, cord blood-derived unrestricted somatic stem cells (USSCs) seem to be the most appropriate option for an autologous transplantation. Among different signaling pathways, the transforming growth factor-ß (TGF-ß) pathway is shown as an important regulator of proliferation and osteogenic differentiation in osteoblast progenitors as well as mesenchymal stem cells. Due to its contradictory and temporally variable effects on different cell types, we sought to investigate whether and how the TGF-ß signaling pathway regulates the osteogenic differentiation of the USSCs. Therefore, in the current study, we treated USSCs with the recombinant protein TGF-ß1 (1 ng/mL) and showed that the expression of matrix metalloproteinase 9, a well-known effector in this pathway, was significantly induced, indicating that the TGF-ß signaling pathway is active in USSCs. Then we applied a TGF-ß receptor antagonist (SB431542; 10 µM) to the osteogenic media cultured USSCs for single periods of 3.5 days within the 21-day differentiation period starting at day 0, 3.5, 7, 10.5, 14, and 17.5. The expression analysis results of the of the osteogenic marker runt-related transcription factor 2 as well as the production of bone matrix showed that SB431542 induced the osteogenic differentiation of USSCs more significantly during the early stage of differentiation, suggesting that the TGF-ß pathway temporally regulates the osteogenic differentiation of USSCs.

Altered expression of IGF-I system in neurons of the inflamed spinal cord during acute experimental autoimmune encephalomyelitis.

There is growing evidence that the impaired IGF-I system contributes to neurodegeneration. In this study, we examined the spinal cords of the EAE, the animal model of multiple sclerosis, to see if the expression of the IGF-I system is altered. To induce EAE, C57/BL6 mice were immunized with the Hooke lab MOG kit, sacrificed at the peak of the disease and their spinal cords were examined for the immunoreactivities (ir) of the IGF-I, IGF binding protein-1 (IGFBP-1) and glycogen synthase kinase 3ß (GSK3ß), as one major downstream molecule in the IGF-I signaling. Although neurons in the non EAE spinal cords did not show the IGF-I immunoreactivity, they were numerously positive for the IGFBP-1. In the inflamed EAE spinal cord however, the patterns of expressions were reversed, that is, a significant increased number of IGF-I expressing neurons versus a reduced number of IGFBP-1 positive neurons. Moreover, while nearly all IGF-I-ir neurons expressed GSK3ß, some expressed it more intensely. Considering our previous finding where we showed a significant reduced number of the inactive (phosphorylated) but not that of the total GSK3ß expressing neurons in the EAE spinal cord, it is conceivable that the intense total GSK3ß expression in the IGF-I-ir neurons belongs to the active form of GSK3ß known to exert neuroinflammatory effects. We therefore suggest that the altered expression of the IGF-I system including GSK3ß in spinal cord neurons might involve in pathophysiological events during the EAE.

Nerve growth factor prevents demyelination, cell death and progression of the disease in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE), a demyelinating disease induced in the animals parallels multiple sclerosis in human in several aspects, provides a useful model to investigate multiple sclerosis. In this study, we have therefore used this model to study functions of nerve growth factor (NGF) in EAE. NGF with considerable effects on neuron survival, proliferation and differentiation of the nervous system, is also known to act on cells of the immune system. Simultaneous upregulation of proinflammatory cytokines and increased level of NGF points at possible effects of the nerve growth factor in autoimmune diseases. To investigate roles of NGF in experimental allergic encephalomyelitis in vivo, we therefore decided to apply it intracerebroventricularly at a dose of 0.20 mg/mice prior to the induction of EAE. Our clinical observations showed that in the EAE induced animals who received NGF, severity of the disease was reduced significantly compared to that in saline treated EAE mice. Also neuropathological investigation of spinal cords revealed that in contrast to saline treated EAE mice, no signs of cell death, infiltration and demyelination can be seen in NGF treated EAE mice, suggesting that NGF may have clinical implications in multiple sclerosis.