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.

Decellularized Wharton's jelly extracellular matrix as a promising scaffold for promoting hepatic differentiation of human induced pluripotent stem cells.

Liver tissue engineering as a therapeutic option for restoring of damaged liver function has a special focus on using native decellularized liver matrix, but there are limitations such as the shortage of liver donor. Therefore, an appropriate alternative scaffold is needed to circumvent the donor shortage. This study was designed to evaluate hepatic differentiation of human induced pluripotent stem cells (hiPSCs) in decellularized Wharton's jelly (WJ) matrix as an alternative for native liver matrix. WJ matrices were treated with a series of detergents for decellularization. Then hiPSCs were seeded into decellularized WJ scaffold (DWJS) for hepatic differentiation by a defined induction protocol. The DNA quantitative assay and histological evaluation showed that cellular and nuclear materials were efficiently removed and the composition of extracellular matrix was maintained. In DWJS, hiPSCs-derived hepatocyte-like cells (hiPSCs-Heps) efficiently entered into the differentiation phase (G1) and gradually took a polygonal shape, a typical shape of hepatocytes. The expression of hepatic-associated genes (albumin, TAT, Cytokeratin19, and Cyp7A1), albumin and urea secretion in hiPSCs-Heps cultured into DWJS was significantly higher than those cultured in the culture plates (2D). Altogether, our results suggest that DWJS could provide a proper microenvironment that efficiently promotes hepatic differentiation of hiPSCs.

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.

Extremely Low Frequency Electromagnetic Field in Mesenchymal Stem Cells Gene Regulation: Chondrogenic Markers Evaluation.

There is little evidence demonstrating the effects of electromagnetic fields (EMFs) generated within the biological entity and the effect of extrinsic fields on cellular programing. Taking the path of the more studied stimuli into attention, mechanical forces, it could be understood that nonchemical factors play a consequential role in transcriptional regulatory networks. Cartilaginous tissue consists of collagen protein that is considered as a piezoelectric substrate and is influenced by electric fields making chondrogenic specific genes an exciting candidate for bioelectromagnetic studies. As electromagnetic properties highly depend on the frequencies applied, this study delves into the ability of two EMFs with the frequency of 25 Hz and 50 Hz in inducing SOX9 and COL2 gene expressions in a three-dimensional (3D) mesenchymal stem cell (MSC)-alginate construct. Cell-alginate beads were divided into six groups and treated for a time period of 21 days. To determine the results, qualitative and quantitative data were both reviewed. On observation of real-time polymerase chain reaction (PCR) data, it was apparent that TGF-ß1 treatment had a greater COL2 and SOX9 gene expression impact on MSCs compared to pulsed electromagnetic field (PEMF) treatments alone. COL2 was shown to have a greater transcriptional tendency to PEMF, whereas under defined electromagnetic parameters applied in this study, no significant difference was detected in SOX9 gene expressions compared to the control group. PEMF co-treatments enhanced the deposition of extracellular matrix molecules, as the matrix-rich beads were positively stained by Alcian blue. This genre of study is the venue for the control and healing of connective tissue defects.

Direct conversion of human fibroblasts into dopaminergic neural progenitor-like cells using TAT-mediated protein transduction of recombinant factors.

Recent progress in the generation of induced neural progenitor cells (iNPCs) holds tremendous potential for regenerative medicine. However, a major limitation is the lack of a reliable source for cell replacement therapy in neurological diseases such as Parkinson's disease (PD). Here, we show that the combination of small molecules (SM) and TAT-mediated protein transduction of SOX2 and LMX1a in a 3D sphere culture directly convert human fibroblasts to induced dopaminergic neural progenitor-like cells (iDPCs). The generated iDPCs expressed various NPC markers (SOX2, PAX6, NESTIN, OLIG2) and midbrain progenitor markers (EN1, LMX1a, FOXA2, WNT1) as detected by immunostaining and real-time PCR. Following differentiation, the majority of cells expressed neuronal dopaminergic markers as indicated by co-expression of TH with NURR1, and/or PITX3. We found that SOX2 and LMX1a TAT-mediated protein transduction in the combination of SM could directly convert human fibroblasts to self-renewal iDPCs. In conclusion, to our best knowledge, this is the first report of generation of safe DPCs and may suggest an alternative strategy for cell therapy for the treatment of neurodegenerative disorders.

Induced neural lineage cells as repair kits: so close, yet so far away.

Transdifferentiation or direct reprogramming of somatic cells into neural lineage cells has provided an invaluable new tool to advance the regenerative neural medicine. Here, we provide an overview of the various strategies currently available for producing of induced neural lineage cells in vitro as well as the direct reprogramming of neural cells in vivo. We also discussing some of the challenges faced in harnessing the potential of induced neural lineage cells for biomedical applications.

Lithium induces follicular atresia in rat ovary through a GSK-3ß/ß-catenin dependent mechanism.

Lithium chloride (LiCl) is a drug used to treat bipolar disorder, but has side effects in the female reproductive system. Although lithium is known to decrease folliculogenesis and induce follicular atresia in rodent ovaries, its cellular and molecular effects in the ovary have not yet been addressed. To investigate these effects, 23-day-old immature female rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG), followed by injections of 250 mg/kg LiCl every 12 hr for four doses. Ovaries were removed 40 and 48 hr after PMSG administration and prepared for histology, immunohistochemistry, Western blotting, and DNA laddering analysis. Our results showed that in the ovaries of LiCl-treated rats, few antral but more atretic follicles were present compared to those of the control rats. The induction of atresia by LiCl was further confirmed by the presence of DNA fragmentation, accompanied by a reduced level of 17ß-estradiol in the serum. At the cellular level, lithium significantly decreased the number of proliferating cell nuclear antigen (PCNA)-positive cells and conversely increased the number of TUNEL-positive cells in the granulosa layer of the antral follicles. At the molecular level, lithium increased the level of phosphorylated glycogen synthase kinase-3ß, and unexpectedly decreased the expression of active (stabilized) ß-catenin. Altogether, our results indicate that lithium disrupts the balance between proliferation and apoptosis in granulosa cells, leading to follicular atresia possibly through the reduction in both the stabilized ß-catenin and 17ß-estradiol synthesis.

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.

Inhibition of GSK-3ß enhances neural differentiation in unrestricted somatic stem cells.

GSK-3ß is a key molecule in several signalling pathways, including the Wnt/ß-catenin signalling pathway. There is increasing evidence suggesting Wnt/ß-catenin signalling is involved in the neural differentiation of embryonic, somatic and neural stem cells. However, a large body of evidence indicates that this pathway maintains stem cells in a proliferative state. To address this controversy, we have investigated whether the Wnt/ß-catenin pathway is present and involved in the neural differentiation of newly introduced USSCs (unrestricted somatic stem cells). Our results indicate that the components of Wnt/ß-catenin signalling are present in undifferentiated USSCs. We also show that the treatment of neurally induced USSCs with BIO (6-bromoindirubin-3'-oxime), a specific GSK-3ß inhibitor and Wnt activator, for 5 and 10 days results in increased expression of a general neuronal marker (ß-tubulin III). Moreover, the expression of pGSK-3ß and stabilized ß-catenin increased by BIO in neurally induced USSCs, indicates that the Wnt pathway is activated and functional in these cells. Thus, inhibition of GSK-3ß in USSCs enhances their neural differentiation, which suggests a positive role of the Wnt/ß-catenin signalling pathway towards neural fate.

In vitro study of alginate-gelatin scaffolds incorporated with silica NPs as injectable, biodegradable hydrogels.

Porous substrates composed of biodegradable polymers and nanoparticles have found extensive use as three-dimensional (3D) scaffolds to regenerate damaged tissues through the incorporation of cells or growth factors. Here, injectable thermally responsive hydrogels based on SiO2 nanoparticles (NPs), alginate, and gelatin biopolymers, with possible utilization for cartilage tissue engineering, are introduced. The nanocomposites contain different amounts of SiO2 NPs for reinforcement and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) for chemical crosslinking of polymer chains in the 3D hydrogel network. The cross-sectional structure of the hydrogels containing 0.25, 1.5, and 3.0% SiO2 NPs was observed by FE-SEM, confirming porous morphology with interconnected pores. Based on the rheometer analyses, by increasing the amount of SiO2 NPs, the mechanical strength of the gels can be found. In addition, in vitro biodegradation studies show that the hydrogels without SiO2 are more unstable than the hydrogels containing SiO2 NPs. In vitro biocompatibility of the products tested by MTT assay indicates that cell viability and attachment depend on the presence of SiO2 NPs.

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.

DKK1 expression is suppressed by miR-9 during induced dopaminergic differentiation of human trabecular meshwork mesenchymal stem cells.

Wnt/ß-catenin pathway has been recently identified as one of the key players in dopaminergic (DA) neuron differentiation. DKK1, the potent inhibitor of the Wnt/ß-catenin pathway, is expressed in a precisely controlled manner in ventral midbrain during brain development, however the molecular mechanism underlying this regulation is still unknown. Here we show that human trabecular meshwork mesenchymal stem cells (TM-MSCs) can be used as an efficient tool for in vitro differentiation of DA neurons. After differentiating TM-MSCs to DA neuron-like cells, ß-catenin protein accumulation was increased in the nucleus, indicating the increased activity of Wnt/ß-catenin pathway in the time-window of DA differentiation. Interestingly, DKK1 transcript level was reduced dramatically after DA induction in TM-MSCs which was accompanied by an increase in the in silico-predicted MIR9 and MIR101 levels. Measuring DKK1 expression level after overexpressing either MIR9 or MIR101 and performing luciferase assay alongside, revealed that both miR-9 and miR-101 suppress DKK1 expression and that miR-9 exerts a direct inhibitory effect on 3'UTR regulatory region. Therefore miR-9 and miR-101 might explain, at least in part, the underlying regulatory mechanism of DKK1 reduction and resulting Wnt/ß-catenin pathway activation during DA neuron differentiation process.

Osteogenic differentiation of Wharton's jelly-derived mesenchymal stem cells cultured on WJ-scaffold through conventional signalling mechanism.

Wharton's jelly-derived extracellular matrix (WJ-ECM) has attracted researcher's attention for its biomedical applications. Previously, we fabricated a biomimetic spongy scaffold from decellularized WJ-ECM and, in this study, we sought to examine the osteogenic inductive potential of this scaffold and its underlying mechanism. To address this question, mesenchymal stem cells (MSCs) were isolated from WJ using a mechanical method and cultured on the scaffold, under dynamic condition, for over 21 days in the presence or absence of osteogenic medium. The status of signalling pathways involved in the osteogenic differentiation and the expression profile of integrins in the WJ-derived MSCs (WJ-MSCs) were examined. WJ-MSCs displayed differentiation capacities and expressed surface antigens, characteristics of MSCs. Histologically, WJ-MSCs seeded on the scaffold showed a proper cellular attachment, penetration and migration. They also exhibited a higher degree of alkaline phosphatase activity, calcium deposition and osteogenic gene expression, than those cultured in 2D condition. The expression of Wnt, BMP and TGF-ß signalling target genes together with that of α2, αv and ß1 integrins was increased in WJ-MSCs in both presence and absence of osteogenic induction medium. Taken together, our results demonstrate that WJ-derived scaffold induces osteogenic differentiation of WJ-MSCs, possibly through activating integrins and subsequently conventional intracellular signalling pathways.

Fabrication of a co-culture micro-bioreactor device for efficient hepatic differentiation of human induced pluripotent stem cells (hiPSCs).

Primary hepatocytes, as the gold standard cell type for in vitro models, lose their characteristic morphology and functions after few days. There is an urgent need to develop physiologically relevant models that recapitulate liver microenvironment to obtain mature hepatocyte from stem cells. We designed and fabricated a micro-bioreactor device mimicking the physiological shear stress and cell-cell interaction in liver sinusoid microenvironment. Induced pluripotent stem cells (iPSCs) were co-cultured with human umbilical vein endothelial cells (HUVECs) in the micro-bioreactor device with continuous perfusion of hepatic differentiation medium (100 µL/h). Simulation results showed that flow field inside our perfusion device was uniform and shear stress was adjusted to physiological condition (<2 dyne/cm2). IPSCs-derived hepatocytes (iPSCs-Heps) that were cultured in micro-bioreactor device showed a higher level of hepatic markers compared to those in static condition. Flow cytometry and immunocytochemistry analysis revealed iPSCs cultured in the device sequentially acquired characteristics of definitive endodermal cells (SOX17 positive), hepatoblasts (AFP positive) and mature hepatocyte (ALB positive). Moreover, the albumin and urea secretion were significantly higher in micro-bioreactor device than those cultured in culture dishes during experiment. Thus, based on our results, we propose our micro-bioreactor as a beneficial device to generate mature hepatocytes for drug screening and basic research.

Fabrication of a three dimensional spongy scaffold using human Wharton's jelly derived extra cellular matrix for wound healing.

The Wharton's jelly (WJ) contains significant amounts of extracellular matrix (ECM) components and rich source of endogenous growth factors. In this study, we designed a new biomimetic spongy scaffold from decellularized WJ-derived ECM and used it as a skin substitute. Histological analysis and biochemical assays showed that bio-active molecules preserved in the fabricated scaffolds and that the scaffolds have highly interconnected porous structure. Cytotoxicity and mechanical evaluation of the scaffold indicated that it is non-toxic and has appropriate mechanical properties. MTT assay, SEM and histological analysis of human fibroblast, seeded on the scaffolds, confirmed cellular viability, attachment, penetration and proliferation. The effectiveness of WJ-derived scaffolds in the regeneration of full-thickness wound was assessed through an in vivo experiment. Our results demonstrated that the scaffolds were well integrated into the mouse tissue and absorbed the exudates after one week. Unlike the controls, in WJ group there were not only complete wound closing and disappearance of the scab, but also complete reepithelialization, newly generated epidermal layers and appendages after 12days of implantation. Taken together, our results indicate that WJ-derived scaffolds are able to improve attachment, penetration and growth of the fibroblast cells and speed up the healing processes, which would offer a proper skin graft for wound healing.

The synergistic effect of nano-hydroxyapatite and dexamethasone in the fibrous delivery system of gelatin and poly(l-lactide) on the osteogenesis of mesenchymal stem cells.

Recently, electrospun nanofibrous scaffolds are vastly taken into consideration in the bone tissue engineering due to mimicking the natural structure of native tissue. In our study, surface features of nanofibers were modified through simultaneous electrospining of the synthetic and natural polymers using poly l-lactide (PLLA) and gelatin to fabricate the hybrid scaffold (PLLA/gelatin). Then, hydroxyapatite nanoparticles (nHA) were loaded in electrospun PLLA nanofibers (PLLA,nHA/gelatin) and also dexamethasone (DEX) was incorporated in these fibers (PLLA,nHA,DEX/gelatin) in the second experiment. Fabricated nanofibrous composite scaffolds were characterized via SEM, FTIR spectroscopy, contact angle, tensile strength measurements, DEX release profile and MTT assay. After seeding adipose derived mesenchymal stem cells, osteoinductivity and osteoconductivity of fabricated scaffolds were analyzed using common osteogenic markers such as alkaline phosphatase activity, calcium depositions and gene expression. These results confirmed that all properties of nanofibers were improved by modifications. Moreover, osteogenic differentiation of stem cells increased in PLLA,nHA/gelatin group in comparison with PLLA/gelatin. The sustained release of DEX was obtained from PLLA,nHA,DEX/gelatin which subsequently led to more osteogenic differentiation. Taken together, PLLA,nHA,DEX/gelatin showed significant potential to support the stem cell proliferation and ostogenic differentiation, and can be a good candidates for tissue engineering and regenerative medicine applications.

Brief azacytidine step allows the conversion of suspension human fibroblasts into neural progenitor-like cells.

In recent years transdifferentiation technology has enabled direct conversion of human fibroblasts to become a valuable, abundant and accessible cell source for patient-specific induced cell generation in biomedical research. The majority of transdifferentiation approaches rely upon viral gene delivery which due to random integration with the host genome can cause genome instability and tumorigenesis upon transplantation. Here, we provide a simple way to induce neural progenitor-like cells from human fibroblasts without genetic manipulation by changing physicochemical culture properties from monolayer culture into a suspension in the presence of a chemical DNA methyltransferase inhibitor agent, Azacytidine. We have demonstrated the expression of neural progenitor-like markers, morphology and the ability to spontaneously differentiate into neural-like cells. This approach is simple, inexpensive, lacks genetic manipulation and could be a foundation for future chemical neural transdifferentiation and a safe induction of neural progenitor cells from human fibroblasts for clinical applications.

Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach.

BACKGROUND: A number of studies generated induced neural progenitor cells (iNPCs) from human fibroblasts by viral delivering defined transcription factors. However, the potential risks associated with gene delivery systems have limited their clinical use. We propose it would be safer to induce neural progenitor-like cells from human adult fibroblasts via a direct non-genetic alternative approach. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have reported that seven rounds of TAT-SOX2 protein transduction in a defined chemical cocktail under a 3D sphere culture gradually morphed fibroblasts into neuroepithelial-like colonies. We were able to expand these cells for up to 20 passages. These cells could give rise to cells that expressed neurons and glia cell markers both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: These results show that our approach is beneficial for the genetic material-free generation of iNPCs from human fibroblasts where small chemical molecules can provide a valuable, viable strategy to boost and improve induction in a 3D sphere culture.

mRNA translation during oocyte maturation plays a key role in development of primordial germ cells in Xenopus embryos.

It is believed that cytoplasmic localization in the egg is necessary for development of primordial germ cells (PGCs) in Xenopus embryos. In this study, we sought to determine if translation of maternal mRNA during oocyte maturation is involved in the development of PGCs. Donor oocytes were collected from both stimulated (those who receive gonadotropin) and unstimulated females, artificially matured and fertilized using a host transfer technique. Using chloramphenicol (50 microM and 500 microM RNA), RNA translation was inhibited during oocyte maturation. Our results showed that in unstimulated embryos treated with 50 microM chloramphenicol, there was a significant reduction in the number of PGCs reaching genital ridges. In stimulated embryos, however, the number of PGCs was unchanged unless a higher concentration (500 microM) of chloramphenicol was used. From these results it is suggested that maternal mRNA translation during oocyte maturation plays a key role in development of PGCs.

Activation of TGFß1 signaling enhances early dopaminergic differentiation in unrestricted somatic stem cells.

So far there is increasing evidence for the involvement of transforming growth factor beta TGFß (transforming growth factor) in differentiation and maintenance of midbrain dopaminergic neurons. Considering that USSCs (unrestricted somatic stem cells) have the potentials to differentiate into neuron-like cells and even dopaminergic neurons and that no evidence available on the role of TGFß signaling in dopaminergic differentiation of these cells, we investigated the presence of TGFß signaling components in USSCs and their involvement on USSCs differentiation into early dopaminergic neurons. Our results showed that components of TGFß signaling were present and functional in undifferentiated USSCs, after which the neurally induced USSCs treated with TGFß1 for 3 days resulted in increased expression of ß-tubulin III (a general neuronal marker) and Nurr-1 (an early dopaminergic marker) at both mRNA and protein levels. Consistently, TGFß inhibition in culture medium by using SB431542 in the presence or absence of TGFß1, significantly decreased the expression of both neural markers. We therefore suggest that activation of TGFß signaling-pathway in neurally induced USSCs enhances neural differentiation with an early dopaminergic phenotype which points at the positive role of the TGFß signaling pathway toward dopaminergic fate.