Estrogen treatment enhances neurogenic differentiation of human adipose derived stem cells in vitro.

OBJECTIVES: Estrogen is a sexual hormone that has prominent effects on reproductive and non-reproductive tissues. The aim of this study is to evaluate the effects of estrogen on the proliferation and neural differentiation of human adipose derived stem cells (ADSCs) during neurogenic differentiation. MATERIALS AND METHODS: Isolated human ADSCs were trans-differentiated in neural induction medium containing neurobasal medium, N2 and B27 with or without 17ß-estradiol (E2) treatment. Proliferation rate and neural differentiation of human ADSCs were assessed using MTT assay, immunostaining and real time RT- PCR analysis, respectively. RESULTS: Analysis of data show that estradiol treatment can significantly increase proliferation rate of differentiated cells (P<0.05). Immunocytochemical and real time RT-PCR analysis revealed that the expression of precursor and mature neuronal markers (nestin and MAP2) was significantly higher in the E2 treated cell cultures when compared to the untreated cell cultures (P<0.05). CONCLUSION: According to our findings, estrogen can promote proliferation and neuronal differentiation of human ADSCs.

Genetically modified murine adipose-derived mesenchymal stem cells producing interleukin-2 favor B16F10 melanoma cell proliferation.

Adipose-derived mesenchymal stem cells (ADSCs) are attractive tools for cancer gene therapy due to their intrinsic tropism to the tumor environment. Interleukin-2 (IL2) is recognized as a key regulatory molecule, which enhances the activity and growth of the immune effector cell function. High-Dose IL2 Therapy is an option for treatment of malignant melanoma but has frequent, often serious and sometimes life-threatening side effects. Here we investigated the effect of genetically modified ADSCs (GM-ADSCs) expressing IL2 in immunocompetent mouse models of subcutaneous and lung metastatic melanoma. Prior to in vivo studies, we demonstrated that IL2 produced by GM-ADSCs may act as a growth factor for melanoma cells due to the increased viability and reduced apoptosis of melanoma cells after in vitro treatment. Subcutaneous co-injection of IL2-expressing ADSCs with melanoma cells significantly enhanced the melanoma tumor growth. Furthermore, histological analysis of subcutaneous tumors for IL2 and Melan-A (a melanocytic differentiation marker) confirmed that most of cells in melanoma/IL2-ADSC co-injected tumors are melanoma cells, not IL2-ADSCs. In pulmonary metastases model, melanoma cells were injected intravenously and 10 days later mice were treated by systematical injection of GM-ADSCs. Intravenously injected IL2-ADSCs engrafted into melanoma lung tumors but were unable to reduce melanoma lung metastases. Besides, administered IL2-ADSCs significantly reduced systemic CD4+ cells and did not impact the total survival of lung metastases melanoma bearing mice. In conclusion, this study showed that IL2-producing ADSCs can favor B16F10 melanoma cell proliferation. Therefore, therapies utilizing IL2 have to be taken into careful consideration.

PhiC31/PiggyBac modified stromal stem cells: effect of interferon γ and/or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on murine melanoma.

BACKGROUND: TRAIL and IFNγ are promising anti-cancer cytokines and it has been shown that IFNγ may sensitize cancer cells to TRAIL. Adipose derived mesenchymal stem cells (ADSCs) are attractive vehicles for delivering anti-cancer agents. In this study, we evaluated the therapeutic potential of PhiC31 (φC31) recombinase and/or piggyBac transposase (pBt) modified ADSCs expressing either TRAIL, IFNγ, or co-expressing TRAIL/IFNγ in mouse models of melanoma. METHODS: The expression and bioactivity of mouse IFNγ and TRAIL in φC31 and pBt modified cells were confirmed. We examined the effects of modified ADSCs on signal intensity of red fluorescence protein expressed by melanoma cells in subcutaneous tumors or established lung metastases and on survival (6 mice per group). We also conducted a flow cytometric analysis of systemic CD4(+)CD25(+)FOXP3(+) T regulatory cells (Tregs) and histological analysis of melanoma tumors. Data were analyzed by Student t test, ANOVA, and log-rank tests. All statistical tests were two-sided. RESULTS: We demonstrated non-viral DNA-integrating vectors can be used for stable transgene expression. IFNγ inhibited melanoma cell growth in vitro probably via IFNγ-induced JAK/STAT1 signaling pathway activation. Murine TRAIL induced apoptosis in the human cell lines CAOV-4 and Ej-138, while MCF7 and B16F10 cells appeared to be insensitive to TRAIL. Treatment of melanoma cells with IFNγ did not influence their response to TRAIL. In contrast, results from in vivo studies showed that IFNγ-expressing ADSCs, engrafted into tumor stroma, inhibited tumor growth and angiogenesis, prevented systemic increase of Tregs, increased PD-L1 expression and CD8+ infiltration (but not interleukin-2+ cells), and prolonged the survival of mice (68 days, 95% confidence interval [CI] = 52 to 86 days compared to 36 days, 95% CI = 29 to 39 days for control, P < .001). CONCLUSIONS: For the first time, we employed DNA integrating vectors for safe and stable modification of MSCs. Our data indicate potential of non-virally modified IFNγ-expressing ADSCs for treatment of melanoma through direct effects of IFNγ. This study may have a significant role in the management of cancer in the future.

Effect of leukemia inhibitory factor on the myelinogenic ability of Schwann-like cells induced from human adipose-derived stem cells.

The Schwann cells (SCs) may be obtain from nerve biopsies for autologous transplantation. However, it is difficult to obtain sufficient amount of SCs for clinical applications. Human adipose-derived stem cells (ADSCs) can be induced to differentiate into Schwann-like cells (S-like cells) and used for autologous transplantation. However, effect of leukemia inhibitory factor (LIF) on the myelinogenic ability of SC-like cells induced from human ADSC is not investigated yet. The aim of this study was to evaluate of the effect of exogenous LIF on myelinogenic potential of differentiated cells in vitro. ADSCs were harvested from human fat tissue and characterized using flow cytometry. Human ADSCs were treated for sphere formation and LIF was added to terminal differentiation medium. GFAP/S100ß and MBP markers were used to confirm differentiation of human ADSCs, and myelinogenic ability of SC-like cells, respectively, using both immunostaining and real-time RT-PCR analysis. The analysis for GFAP(+)/S100ß(+) revealed that LIF can increase both differentiated cells rates and the percentage of myelinating SC-like cells (p < 0.05). Our data showed that SC-like cells induced from human ADSCs were able to generate myelin when exposed to LIF and these cells could be a potential source for the treatment of peripheral and central axonal injuries.

Efficient transdifferentiation of human adipose-derived stem cells into Schwann-like cells: A promise for treatment of demyelinating diseases.

BACKGROUND: Schwann cells (SCs) can provide a suitable option for treatment not only diseases of peripheral nervous system (PNS), but also diseases of central nervous system (CNS). It is difficult to obtain sufficient large number of SCs for clinical purpose because of their restricted mitotic activity, and by sacrificing one or more functioning nerves with the consequence of loss of sensation. So, providing an alternative source for transplantation is desired. The aim of this study was isolation, characterization of human adipose derived stem cells (ADSCs), and transdifferentiation into Schwann-cells. MATERIALS AND METHODS: After isolation of ADSCs by mechanical and enzymatic digestion of adipose samples, characterization human ADSCs using flow cytometry was carried out. Human ADSCs were sequentially treated with various factors for neurosphere formation and terminal differentiation into Schwann-like cells. We used Schwann cell markers, GFAP and S100 to confirm the effectiveness of the differentiation of human ADSCs using Immunostaining and real time RT-PCR techniques. RESULTS: Flow cytometry analysis of ADSC showed isolated stem cells were positive for CD90 and CD44 markers of mesenchymal stem cells, but for CD45 and CD34 markers were negative. Dual immunofluorescence staining and real time RT-PCR analysis for GFAP and S100 markers were revealed that approximately 90% of differentiated cells expressed co-markers. CONCLUSION: We indicated that human ADSCs have a suitable option to induce Schwann-like cells for autologous transplantation, offer promise for treatment in demyelinating diseases.

Stability of neural differentiation in human adipose derived stem cells by two induction protocols.

There are some evidences for suggesting that adipose derived stem cells (ADSCs) can be differentiated to the fate of neural cell type. ADSCs can be expanded rapidly in vitro and can be obtained by a less invasive method. In this study, we attempted to compare the stability of neural differentiation in human ADSCs by using two induction protocols. Isolated ADSCs were induced into neural-like cells using diverse effects of two specific procedures. For protocol 1, ADSCs were induced by chemical induction. In protocol 2, ADSCs were treated for sphere formation. Then, the singled cells were cultured in neurobasal media supplemented with special components. Differentiated ADSCs were evaluated for Nestin, MAP2 and GFAP expression by immunocytochemistry and semi quantitative RT-PCR techniques. Moreover, MTT assay was employed to detect cell viability and proliferation. Immunocytochemical analysis of both protocols demonstrated that ADSCs had large expression of the neural-specific markers. In RT-PCR, protocol 1 showed the highest percentage of MAP2 expression, but with time passing, the neural like state was reversible. Protocol 2 found with express of Nestin at week 1, however MAP2 and GFAP expression increased after 3 weeks. The neural-like cells produced by protocol 1 led to the further cell death. Comparative analysis showed that neural-like cell differentiation of ADSCs in chemical induction protocol was rapid but transitory, while it was approximately steady in neurosphere formation protocol.