cell biochemistry and function Cell Biochem Funct (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3074

Effect of T3 hormone on neural differentiation of human adipose derived stem cells† Shahnaz Razavi1*, Fatemeh Sadat Mostafavi1, Mohammad Mardani1, Hamid Zarkesh Esfahani2, Mohammad Kazemi3 and Ebrahim Esfandiari1 1

Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran 3 Department of Genetic, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 2

Human adult stem cells, which are capable of self-renewal and differentiation into other cell types, can be isolated from various tissues. There are no ethical and rejection problems as in the case of embryonic stem cells, so they are a promising source for cell therapy. The human body contains a great amount of adipose tissue that contains high numbers of mesenchymal stem cells. Human adipose-derived stem cells (hADSCs) could be easily induced to form neuron-like cells, and because of its availability and abundance, we can use it for clinical cell therapy. On the other hand, T3 hormone as a known neurotropic factor has important impressions on the nervous system. The aim of this study was to explore the effects of T3 treatment on neural differentiation of hADSCs. ADSCs were harvested from human adipose tissue, after neurosphere formation, and during final differentiation, treatment with T3 was performed. Immunocytochemistry, real-time RT-PCR, Western blotting techniques were used for detection of nestin, MAP2, and GFAP markers in order to confirm the effects of T3 on neural differentiation of hADSCs. Our results showed an increase in the number of glial cells but reduction in neuronal cells number fallowing T3 treatment. Copyright © 2014 John Wiley & Sons, Ltd. key words—neural induction; hADSCs; MAP-2; GFAP; T3

INTRODUCTION Human neurodegenerative diseases are caused by a loss of neurons and glia in the CNS; it is known that this tissue after injury has a limited capacity for replacement of damaged areas, and neurogenesis is restricted to limited regions of the brain;1,2 therefore, cells capable of neuronal differentiation have broad potential for cellular therapies,3,4 as a result of their ability to self-renew, long-term expansion in vitro, and to differentiate along multiple lineage pathways.5,6 Embryonic stem cell (ESC) and neural stem cell (NSC) are essentially proposed for neuronal cellular therapies;7,8 however, their use is limited by various ethical, political, immunological and logistical constraints and by their tumorigenicity trait. Compared with ESCs, adult stem cells

*Correspondence to: Shahnaz Razavi, Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81744-176, Iran.E-mail: [email protected] † Almost all of the previous studies about the effects of T3 hormone on stem cells were performed on NSCs, and there is no evidence how this hormone affects neural differentiation of other stem cells; also, ADSCs offer significant advantages over other types of stem cells; therefore, the objective of this study was to show the potential for differentiation of ADSCs into neuron-like cells and to assess the influence of thyroid hormone T3 on their differentiation.

Copyright © 2014 John Wiley & Sons, Ltd.

have diminished self-renewal capability and multipotency, but they are immunocompatible, there are no ethical issues related to their use and problems such as histocompatibility and inadequate tissue supply never will have seen.9 For the first time, scientists characterized stem cells in the BM, and until now, most of the experiments have been performed on this kind of stem cells. BM procurements, particularly in volumes larger than a few millilitres, are very painful, frequently requiring anaesthesia, and yield low numbers of mesenchymal stem cells (MSCs) upon processing.10 For the first time, Zuk et al., had suggested that adipose-derived stem cell (ADSC) might possess the ability to differentiate into neuron-like cell.11 Neurogenic potential of ADSCs is markedly higher than that of bone marrow stromal cells.12 Adipose tissue is the most abundant and accessible source of adult stem cells, as a result of the minimal invasive collection procedure, ability of derived cells for differentiation into different cell lineages, as well as their safe autologous transplantation.13 The microenvironment of MSCs strictly regulates their differentiation and functions, and to date, differentiation of hADSCs into neuron-like cells has been promoted by adding various factors, cytokines or antioxidants.14–16 Thyroid hormones, growth hormone, prolactin, insulin and Received 12 August 2014 Revised 11 October 2014 Accepted 13 October 2014

s. razavi parathyroid hormone-related protein are developmental growth and differentiation factors.17 Thyroid hormones have several critical and important roles in various parts of our body during development time and adulthood. T3 is the main and functional form of thyroid gland hormones that is essential for some of the physiological processes in mammalian species; its activation regulates vertebrate development and physiology.18,19This hormone is critical in developmental events of key tissues, including the CNS, as well as having important regulatory effects on oxygen consumption and metabolic rate.20 At the cellular level, it acts on specific cells, and during specific phases of development, this effect is known to be a major factor in regulating the timing of proliferation and differentiation decisions in a number of neural cellular populations.21,22 Previous studies implicated that T3 levels are highest during the period of most rapid brain growth and development which in humans occurs during 2 years after birth, and lack of this hormone in these stages causes severe neurological damages.23 Besides the well-established multiple roles of thyroid hormone in early neurogenesis, T3 is an essential component of the endocrine environment that activates NSC’s growth, migration and apoptosis. Previous studies have found that transient exposure of NSCs to T3 in vitro initiates a stable switch to a glial fate through a direct, instructive mechanism;24 this has not been demonstrated with ADSCs; therefore, potential effect of thyroid hormone on ADSCs differentiation, proliferation and survival needs to be characterized. Because T3 is the main and functional form of thyroid gland hormones, we did our experiment under the effect of this hormone. Almost all off the previous studies about the effects of T3 hormone on stem cells were performed on NSCs, and there is no evidence how this hormone affects neural differentiation of other stem cells; also, ADSCs offer significant advantages over other types of stem cells; therefore, the objective of this study was to show the potential for differentiation of ADSCs into neuron-like cells and to assess the influence of thyroid hormone T3 on their differentiation.

ET AL.

I in PBS for 30 min at 37 °C with gentle agitation. Then, the enzyme neutralized by adding DMEM/10% FBS in a 1:1 ratio. After centrifugation for 10 min at 1600 rpm and removal of the supernatant, the cellular pellet was washed and then plated in a T25 flask containing DMEM : F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin, in an incubator with a humidified atmosphere containing 5% CO2 at 37 °C. After 24 h, the flasks were washed with PBS and their medium entirely changed. In this way, non-adherent cells were removed; the flasks reached almost 80–90% of confluency. Then, adherent ADSCs were expanded by serial passage to improve the purity of the preparation and to generate a homogeneous cell population. In this study, cells were passaged at the ratio of 1:3 in every passage, and cells of passages 3–5 were used in our experiments. All chemicals, except where specified otherwise, were purchased from Sigma-Aldrich, St. Louis, MO, USA. Neural induction of human ADSCs The neural induction procedure was modified from Ahmadi et al. study.25 This procedure had two steps: induction of ADSCs into neurosphere-like structures and final differentiation into neural cells. For the first step, briefly, we dissociated human ADSCs (80–90% confluence) with 0.25% trypsin-0.02% EDTA (Gibco, BRL, Paisley, UK) and then plated them on plastic tissue culture plates in a concentration of 1 × 106 in DMEM : F12 supplemented with 20 ng/ml human EGF, 20 ng/ml b-FGF, and 2% B27 (1:50,Gibco) at 37 °C and 5% CO2. Fresh medium was added every 3–4 days. After 7 days, neurospheres were dissociated and singled by pipetting in Trypsin/EDTA. Next, the singled cells re-plated in laminin-coated 24-well chamber slides in differentiation medium consisting of neurobasal medium, 5% FBS, 1% penicillin, 1% L-glutamine, 1% N2, 1% NEAA, 2% B27, 1% nystatin. The cells were incubated for 1 week under these conditions. For thyroid hormone treatment T3, a 50 nM final concentration was added in treated group26 whereas in control culture, T3 was absent (the growth factors and supplements are all from Gibco BRL, Paisley, UK).

MATERIALS AND METHODS Isolation and culture of human ADSCs

Characterization of human ADSCs

All procedures were conducted according to Isfahan University of Medical Sciences, Medical Faculty Ethic Committee approval. Human adipose tissue was obtained from subcutaneous abdominal fat of three patients, 27–45 years old who have referred to Alzahra Hospital of Isfahan for abdominoplastic surgery after receiving informed consent and cultured as previously described.25 Briefly, samples of fat tissue were washed extensively with sterile PBS in order to remove contaminating debris and red blood cells. Careful surgical removal of the connective tissue surrounding the parenchyma performed and mechanical digested aspirates were treated with 0.01% collagenase type

ADSCs were examined for surface markers by flow cytometry in order to determine their stemness. The fifth passage of ADSCs was trypsinized, centrifuged and resuspended to concentration of about 1 × 105 cells for each test. After that, the cells were washed twice with 1% BSA/PBS and incubated with antibodies against positive (CD44, CD90 and CD105) and negative (CD14, CD34 and CD45) markers (Chemicon, Temecula, CA, USA) for 30 min. Primary antibodies were directly conjugated with FITC or PE. Negative control staining was performed using a FITC-conjugated mouse IgG isotype and a PE-conjugated mouse IgG isotype antibody. Flow cytometry was performed with a FACScan flow cytometry (Becton-Dickinson, San Jose, CA).

Copyright © 2014 John Wiley & Sons, Ltd.

Cell Biochem Funct (2014)

neural induction of human-adscs influenced by t3 MTT assay Cell growth and viability of neural induced ADSCs in the presence or absence of T3 hormone was also studied by the MTT biochemical approach, based on the reduction of MTT into formazan crystals by the action of the mitochondrial dehydrogenase enzymes present in viable cells. In order to achieve this purpose, 3 × 103 cells/well, were seeded on 24-well plates and grown in the presence or absence of T3 hormone at 50 nM concentration. MTT powder was dissolved in PBS at 5 mg/ml. After seeding the cells on 24-well plate, supernatant was discarded, and differentiated cells were washed with PBS; then, treatment with a solution at a dilution 1:10 of the MTT stock solution was performed. After 4 h at 37 °C, until blue precipitation could be seen in cells, the formed formazan crystals were dissolved in 200 μl DMSO solution, giving a spectrophotometrically measurable purple solution. After transferring to a 96-well plate and incubating it for 1 h at RT in the dark, absorbance was read by a microplate reader (Hiperion MPR 4+, Germany) at a wavelength of 540 nm. Absorbance values correspond to the number of viable cells. Immunocytochemistry To characterize neural differentiation of hADSCs and to evaluate the effect of 50 nM T3 hormone on this procedure, after 2 weeks, we used immunocytochemical staining method. In order to perform this method, the following steps were performed: First, cells were grown on laminin-coated coverslips in 24-well plates for 7 days; then, cell cultures were fixed for 20 min with 4% paraformaldehyde in PBS at RT and blocked for 45 min at RT with blocking solution (PBS containing 10% goat serum and 0.2% Triton X-100). Then, cells were incubated overnight at 4 °C with primary antibodies diluted in PBS/0.1% Triton X-100 and 1% goat serum in the dark. The following antibodies were used: mouse anti-glial fibrillary acidic protein (GFAP) (1:300; Abcam, Cambridge, MA, USA), mouse anti-nestin (1:300; Abcam, Cambridge, MA, USA) and mouse anti-microtubuleassociated protein 2 (MAP-2) (1:300; Abcam, Cambridge, MA, USA). After washing with PBS, the slides were exposed to secondary antibody; rabbit anti-mouse FITC (1:500; Abcam, Cambridge, MA, USA) conjugated secondary antibody was incubated at RT for 2 h. For nuclear counter staining, we used DAPI (1:1000, Sigma). Negative control included the omission of primary antibodies from the reaction series in each experiment. Experiments were performed in triplicate, and for final analysis, cells were observed using fluorescence microscope (Olympus BX51, Japan). To perform quantitative analysis, the number of immunopositive cells was counted in several non-overlap fields in a minimum total of 100 cells per slide by ImageJ software. Also, this software was used for merging the pictures of the cells and their nuclei. Western blot A Western blot is a laboratory analysis technique that is used for detecting specific proteins; it uses three elements Copyright © 2014 John Wiley & Sons, Ltd.

to accomplish this task: (1) separation by size using electrophoresis, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. Our aim was to compare neural differentiation of ADSCs in the absence and presence of T3 hormone by detecting some neural proteins. Using tripsin/EDTA, the cells were separated after being washed twice with cold PBS. Then, cell lysis buffer (1×: 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin) (Cell Signaling Technology, USA) was added to obtain cell lysate according to manufacturer’s protocols. The protein sample was quantified with Bradford assay and was next added to 4× sampling buffer. The sample was boiled for 5 min. After electrophoresis, the separated proteins were blotted onto a nitrocellulose membrane. For the next stage of the test, the sample was treated with blocking buffer (5% skim milk and 0.1% TWEEN 20 in PBS) overnight. During this stage to the end of the experiment, the samples were shacked gently; after washing three times with washing buffer (1% BSA and 0.05% TWEEN 20 in PBS), an antibody is introduced to the sample, with the goal of tagging the proteins with antigens which that antibody locks on to. At RT, primary antibodies [GFAP (1:500), nestin (1:500), MAP-2 (1:500)] was allowed to react for 2 h. Again, washing buffer was used three times for 5 min each. Finally, the transferred protein was complexed with an enzyme-labelled antibody conjugated with HRP as a probe with a 1:1000 dilution. Once the proteins have been tagged in this way, they can be easily identified. An appropriate substrate TMB is then added to the enzyme, and together, they produce a detectable product such as a chromogenic precipitate on the membrane. Real-time RT-PCR PCR is the most powerful technique for detection of small amounts of nucleic acids; using real-time reverse transcription polymerase chain reaction (RT-PCR), we can quantify gene expression, so in the present study, we decided to use real-time RT-PCR technique for measuring the gene expression levels, First, RNA samples were prepared after 2 weeks from beginning of the transdifferentiation procedure. For this purpose, neural-induced cells cultured in 6-well plates were tripsinized, and after centrifugation, total cellular RNA was isolated using the RNeasy mini, RNA isolation kit (Qiagen). This technique consists of two different basic protocols: conversion of isolated RNA into complementary DNA (cDNA) by reverse transcription and the actual PCR reaction and its subsequent analysis. The RNA was reverse transcribed using RevertAid first strand cDNA synthesis kit (Fermentase) with oligo-dT primers (Table 1). The real-time PCR was performed with gene specific primers and the SYBRGreen PCR Master mix (Qiagen) using a thermal cycler rotor-gene 6000 (Qiagen). The gene of interest was normalized against the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The Cell Biochem Funct (2014)

s. razavi Table 1.

ET AL.

The primer sequences (forward, reverse) that were used in real-time RT-PCR technique

Gene Nestin MAP-2 GFAP GAPDH

Forward (top) Reverse (bottom) 5′-AACAGCGACGGAGGTCTCTA-3′ 5′-TTCTCTTGTCCCGCAGACTT-3′ 5′-TCAGAGGCAATGACCTTACC-3′ 5′-GTGGTAGGCTCTTGGTCTTT-3′ 5′-CCTCTCCCTGGCTCGAATG-3′ 5′-GGAAGCGAACCTTCTCGATGTA-3′ 5′-ACCACAGTCCATGCCATCAC-3′ 5′-TCCACCACCCTGTTGCTGTA-3′

expression level of each target gene was calculated as 2-ΔΔCt, as previously described. Statistical analysis The experiments were replicated at least three times. Data were presented as standard error of mean (mean ± SEM). One-way ANOVA followed by Tukey’s post hoc test multiple group comparison was used to analyze group differences on the data collected from MTT, immunocytochemistry and real-time RT-PCR. To analyze data, SPSS version 19 was applied, and significant differences were obtained. A value of (*p ≤ 0.05), (**p ≤ 0.01) and (***p ≤ 0.001) were considered statistically significant.

RESULTS Phenotypic characterization of ADSCs The primary ADSCs after 24 h adhered to the plastic surfaces of tissue culture flasks and exhibited a heterogeneous population of spindle-shaped cells morphologically. ADSCs propagated rapidly in vitro, and within three passages after initial plating of the primary culture, cells constituted a homogenous population and became more uniform and grew in a more spindle-shaped, typical fibroblast-like morphology (Figure 1A).

Size (bp)

Real-time RT-PCR program

220

94 °C—20 s, 59 °C—30 s, 72 °C—30 s 35 cycles

321

94 °C—20 s, 57 °C—30 s, 72 °C—30 s 45 cycles

161

94 °C—20 s, 59 °C—30 s, 72 °C—30 s 40 cycles

452

94 °C—20 s, 60 °C—30 s, 72 °C—30 s 25 cycles

Morphological changes during neuronal differentiation Morphological changes of the differentiated cells after 2 weeks were observed by phase contrast microscopy. Our neural induction protocol involved two steps: conversion of ADSCs into neurosphere-like structures and final differentiation to neural cells. ADSCs were induced toward the neurogenic lineage through neurosphere formation (Figure 1B). After detachment of the spheres, terminal neural induction was performed and dissociated neurospheres were cultured. Up to this stage, there were no differences between our groups, but as soon as the cells were plated and treatment with T3 hormone began, the differentiated hADSCs that were untreated (control group) were somehow different from those that were treated with T3 hormone; within 1 week after neurosphere formation, in the control group, most of the cells showed a neuron-like phenotypes in morphology. These cells were distinctively distinguished by relatively small cell bodies with neuron-like processes; cells in this group began to adhere and spread across the growth surface, forming long chains of cellular processes (Figure 1C). These changes suggest that the hADSCs differentiated into neuron-like cells. On the other hand, at the same time of differentiation, in T3-treated group cells adhered and spread over the plates’ floor and most of the cells presented with neuronal morphology including a small cell body and long extensions, but their processes were shorter compared with the cells of the control group (Figure 1D); moreover,

Figure 1. Phase contrast image of (A) stem cells derived from human adipose tissue; as we can see, almost the entire field is filled with elongated fibroblastlike cells. (B) Neurospheres dissociated from the tissue culture dish plastic substrate after 7 days culturing, surrounded by some fibroblast-like cells; almost all of the cells participate in neurosphere formation. (C) Differentiated cells derived from hADSCs 2 weeks after neural induction. We can see bipolar and multipolar cells with elongated processes. (D) Differentiated cells that were treated with T3 hormone during 1 week after neurosphere formation. Scale bars in A = 150 μm, B = 200 μm and in C, D = 100 μm Copyright © 2014 John Wiley & Sons, Ltd.

Cell Biochem Funct (2014)

neural induction of human-adscs influenced by t3 as is indicated in Figure 1C and 1D, at the same time of differentiation and in the similar fields, the number of the cells in the group treated with T3 hormone is higher than the control group. It is obvious that the proliferation rate is increased in the group treated with T3 hormone. All observations to assessment the changes in cell morphology before and after treatment were performed using bright field and phase contrast microscopy. Cell surface markers of ADSCs Flow-cytometric analysis was performed to confirm adipose tissue-derived MSCs. Cells from the third subculture were collected and tested for CD44, CD90 and CD105 expressions, which are markers specific to mesenchymal stem cells. The test results in cell culture were positive for CD44 and CD90 more than 95%. The test was negative for antibodies CD45, CD34 and CD14, which are specific markers to hematopoietic stem cells. Thus, it was confirmed that the sample of cells that had been grown consisted purely of MSCs, with no hematopoietic cells (data was not shown). Cell viability We examined the ability of T3 hormone to promote survival of neural induced ADSCs using an MTT assay. After using 50 nM of T3 hormone during neural induction, MTT assay demonstrated that the cell viability and proliferation rate of differentiated cells (absorbance value: 0.446 ± 0.154, cell numbers: 6000 ± 78) were higher than untreated differentiated cells (absorbance value: 0.256 ± 0.048, cell numbers: 2000 ± 24), and we observed a significant difference in the mean of absorbance value between T3-treated group compared with control group after 2 weeks post induction (**p < 0.01). To characterize neuronal and glial differentiation after 2 weeks of neural-induced human adipose-derived stem cells and in order to know how thyroid hormone T3 effects on this procedure, the cells were stained with the markers against nestin, MAP-2 and GFAP (Figures 2 and 3). Immunofluorescence analyses demonstrated that expressions of these markers changed obviously in our control and 50 nM T3-treated groups. Our results show that the mean percentage of the cells expressing nestin marker in the group treated with T3 hormone (54.61 ± 3.59%) was higher than the mean of this item in control group (36.11 ± 4.41%); this difference between our treat and control groups was significant (**p < 0.01); vice versa, expression of MAP-2 was significantly downregulated with T3 treatment (***p < 0.001), it means that the mean percentage of the differentiated cells expressing MAP-2 marker in the groups treated with T3 hormone (36.11 ± 4.3%) was lower than the mean of these cells in control groups (81.11 ± 4.57%). Unlike MAP-2, expression of GFAP showed significantly upregulation with treatment time (***p < 0.001), while the mean percentage of the cells expressing GFAP marker in control group was low (28.27 ± 3.58%); cells in the other group showed high expression of GFAP in more than 50% of them after T3 treatment for 1 week (75.44 ± 6%). Copyright © 2014 John Wiley & Sons, Ltd.

Figure 2. Comparative analysis between the mean percentages of immunoreactive positive cells for some neural cell’s markers in differentiated cells derived from hADSCs with or without T3 treatment. As we can see, there is a significant differences between these two kinds of cell populations in the expression of neural markers; in the presence of T3 hormone, we observed significant upregulation of nestin (**p < 0.01) and GFAP (***p < 0.001) but significant downregulation of MAP-2 (***p < 0.001)

The results of the current study indicate that using T3 hormone in the procedure of neural induction of ADSCs leads to production of more glial cells compared with neurons. Western blotting We used this technique for detecting some neural-specific proteins (nestin as neural progenitor cell marker, MAP-2 as mature neurons marker and GFAP as glial cells marker), and our aim was to compare neural differentiation of ADSCs in the absence and presence of T3 hormone. Moreover, we monitored the affectivity of our neural induction protocol with comparing the results of protein expression between ADSCs and neural-induced cells after 2 weeks of differentiation. Based on the results of this work, expression of neural cells’ markers that was mentioned in the preceding texts, after using of specific differentiation conditions and T3 hormone, undergo a series of changes (Figure 4A) that were more significant for MAP-2 and GFAP. Both of the induced differentiation groups showed expression of the GFAP protein. The group with T3 treatment showed markedly higher expression of this protein compared with the untreated group. For nestin, both groups (untreated with T3, treated with T3) showed expression of this protein, and the expression was somehow higher in the group with T3 treatment. While the expression of GFAP protein upregulated in response to our hormonal treat, MAP-2 protein expression downregulated and the results were weakly positive. In other words, following this treatment differentiation of stem cells significantly promoted toward glial linage that is an important item in neurons’ behaviour and function. Real-time RT-PCR analysis Real-time RT-PCR analysis was used to evaluate the effects of exogenous T3 hormone on differentiation potential and neurogenic ability of ADSCs at messenger RNA (mRNA) levels of differentiated cells. We examined expression of nestin, MAP-2 and GFAP markers, at week 2 post Cell Biochem Funct (2014)

s. razavi

ET AL.

Figure 3. Immunocytochemistry of neural differentiated cells from hADSCs in the presence of T3 hormone. Cells were positive for (A) nestin, (B) MAP-2 and (C) GFAP markers. Cell nuclei were counterstained with DAPI (blue), Scale bars: nestin = 50 μm, MAP-2 = 50 μm, GFAP = 50 μm

induction, to confirm the effectiveness of our differentiation protocol on human ADSCs and T3 hormone on neurogenic potential of differentiated cells (Figure 4B). Real-time RT-PCR analysis demonstrated that differentiated cells in control and T3-treated group after 2 weeks were positive for the expression of all three markers evaluated, but there were some differences in the expression of these three genes between control and T3-treated groups. The results from real-time RT-PCR technique show that the average gene expression levels of nestin in the group treated with T3 hormone (15.15 ± 1.92) was higher than the average of this gene expression in control group (12.74 ± 1.18), alhough we can see an increase in the expression of this gene in the group treated with T3 hormone; this difference was not significant. As it is clear in Figure 4B that the average gene expression levels of MAP-2 are downregulated with T3 treatment; this gene expression decreased from (9.7 ± 2.63) in the control group to (5.4 ± 0.15) in the group treated with T3 hormone, but this reduction was not significant. Unlike MAP-2 gene, expression of GFAP showed upregulation with treatment time, the mean average of this gene expression was low (1.62 ± 0.22) in control group while this was high (9.63 ± 3.72) in the group treated with T3 hormone; this increase was significant (*p < 0.05). DISCUSSION In this study, using special techniques such as MTT in order to assess cell proliferation and viability, immunocytochemistry for lineage-specific neural markers, real-time RT-PCR Copyright © 2014 John Wiley & Sons, Ltd.

and Western blot analysis to detect neural markers at the levels of mRNA and protein, neurogenic differentiation of ADSCs was demonstrated; it was found that T3 treatment during this process altered the expression of some neural markers and resulted in upregulation of GFAP, which is known as a marker of glials, while downregulation of MAP-2, neural marker related to neurons. Likewise, this hormone had no toxic effects and increased proliferation and viability of differentiated cells. Different protocols and a variety of results have been described with respect to the induction of neural cells. In the present study, a modified neural induction protocol from Ahmadi et al. study in 201225 was used. This protocol induced important and long-lasting (up to 14 days) morphological and phenotypic changes in adipose MSCs, resembling the typical features of neuronal differentiation, thus indicating that cell plasticity is a common feature of ADSCs.27 Related to our experiment, hADSCs expressed neuronal progenitor marker nestin, similar to several other stem and progenitor cell populations,11 indicating that it cannot be used as a marker for putative neurogenic potential. Also, the low levels of MAP-2 and GFAP expression in noninduced ADSCs are in agreement with the study performed by Jang et al. in 2010; because of these results, it could be suggested that ADSCs may retain a native potential for neural differentiation.15 After neuronal induction, human ADSCs demonstrated GFAP, MAP-2 and nestin along the cell body and neuritelike processes. Expression of these genes in this experiment was measured 2 weeks after neural induction, and low levels Cell Biochem Funct (2014)

neural induction of human-adscs influenced by t3

Figure 4. (A) Western blot analysis in order to detect neural cell proteins nestin, MAP-2 and GFAP expressions in two groups of neural-induced cells derived from human ADSCs: one received T3 treatment during the second week of differentiation process and the other did not treat with this hormone. Both cultures expressed these three proteins, and it seems that the expression of nestin is a little increased following our treatment, while the reduction in MAP-2 expression and the increase in GFAP expression are more prominent. (B) Comparative analysis of the mentioned cell populations’ neural markers was examined by realtime RT-PCR. While we can see downregulation in the expression of MAP-2 and a little upregulation in nestin gene’s expression, there were no significant differences in statistical analysis (p > 0.05); however, we observed an increase in the expression of GFAP gene expression in the treated cells that was significant (*p < 0.05)

of GFAP and high levels of MAP-2 expression were determined, although both of them expressed in higher levels compare with non-induced cells. In contrast, nestin expressed in lower levels. Our results were the same as the results related to the previous studies that were performed on ADSCs.25 Zhang et al. in 2009 investigated the contribution of astrocytes to the activities of NSCs after T3 treatment. T3-treated hippocampal astrocyte cultures promoted hippocampal NSCs survival by increasing the proliferation and decreasing the cell death. Also, the migration of neuroblasts from both SVZ and hippocampus was significantly increased. Furthermore, astrocytes displayed different expression patterns for genes that are implicated in the regulation of neurogenesis.28 Martinez and Gomes in 2005 demonstrated a gliamediated effect of thyroid hormone on cerebellar neurons. They have reported that cerebellar astrocytes treated with T3 secrete EGF, which directly induces neuronal proliferation and indirectly induces neurite outgrowth by increasing synthesis of extracellular matrix proteins.29 They performed another study in 2011 on rats, which suggested that by inducing EGF secretion, T3 promote neuronal migration Copyright © 2014 John Wiley & Sons, Ltd.

through glial process elongation; their findings offer new clues to the molecular mechanism of T3 function in cerebellar development and provide a better understanding of some neuroendocrine disorders associated with migration deficits.30 Using both T3 and EGF in order to induce neural induction in the present study, despite the lack of information related to the rate of migration of neural-induced cells, could be suggested that cell migration is likely to be increased; nevertheless, in the future studies, this should also be considered. Zhang et al. in 2009 demonstrated that T3 has a substantial impact on vasculature development in the brain, and in this way, it is able to be effective on neurogenesis.28 Because of the results of our study, neural-induced ADSCs exhibit enhanced proliferation, survival and glial differentiation in response to T3 hormone that is in agreement with the study performed by Desouza et al. in 2005; their results support a role for thyroid hormone in the regulation of adult hippocampal neurogenesis.31 Also, Uchida et al. in 2005 investigated the generation of new neurons following T3 deficiency. Cell proliferation in the hippocampus was markedly decreased in the hypothyroid mice while in T3-treated animals, it was equivalent to that of wild types.32 Cell Biochem Funct (2014)

s. razavi Martinez and Gomez in 2005 demonstrated that neuron– astrocyte coculture potentiates the effect of T3 on neuronal proliferation that may suggest that this contact may cooperate with astrocyte soluble factors to enhance neuronal population. These data reveal an important role of astrocytes as mediators of T3-induced cerebellar development.29 Ambrogini et al., in 2005 after searching about the total number and size of granule cells in dentate gyrus of hypothyroid rats, found that new neurons exhibit a delay in neuronal differentiation and a very immature morphology.33 In addition Calza et al. in 2002 explored the possibility of promoting myelination in experimental rat models of chronic demyelination through T3 administration. Their treatment reduced proliferation and nestin immunoreactivity and upregulated expression of markers for oligodendrocyte progenitors and mature oligodendrocytes (MBP);34 our results were somehow different and nestin expression, although not significant, but upregulated. This could be because of the differences such as stem cell types, methods of isolation and differentiation and duration of induction. In the present study, we used the parameters of morphology and phenotypic changes to evaluate MSC differentiation into neuron-like cells. Limited studies have been used functional assays such as electrophysiological analysis to determine the identity of differentiated neuronal cells;35,36 although, in order to confirm existence of functional neural cells, performing such evaluations seems indispensable. Expression of the oligodendrocyte markers were not examined in this study, although previous studies indicate the positive effects of T3 hormone on oligodendrocytes and their function that may open new and effective solutions in treating demyelinating diseases of the CNS. Fernandez et al. in 2004 worked on oligodendrocyte development and myelination under T3 treatment; their findings indicated that hypothyroidism affects NSCs and OPCs proliferation and maturation; they observed a delay in their differentiation.37 In addition, Schoonover et al. in 2004 demonstrated that reduction in myelin formation is a main sign of congenital hypothyroidism; although the total number of axons is unchanged, the number of myelinating oligodendrocytes and myelinated axons is significantly reduced,38 while Baas et al. in 2002 found that addition of T3 hormone to OPCs is sufficient to trigger their terminal differentiation; at cellular levels, T3 acts on glial and neuronal cell lines and controls proliferation, apoptosis, migration and differentiation of these cells.39

CONCLUSIONS Regarding this study, it seems that we can use adipose tissue as an alternative autologous source of stem cell for BM in order to investigate agents that consider having positive expected effects on neural cells’ activities. According to our results, T3 hormone promotes proliferation, survival and glial differentiation of neural differentiating ADSCs. Considering the critical functions of astrocytes in nervous system, T3 hormone usage in the future could be suggested Copyright © 2014 John Wiley & Sons, Ltd.

ET AL.

in order to produce astrocytes that promote proliferation, differentiation and migration of NSCs in a correct way, toward damaged areas of the CNS in vivo.

CONFLICT OF INTEREST We declare no conflict of interest.

ACKNOWLEDGEMENT The authors are grateful to Iranian Council of Stem Cell Technology, Isfahan University of Medical Sciences for financial support (Grant No. 189068).

REFERENCES 1. Magavi SS, Leavitt BR, Macklis JD. Induction of neurogenesis in the neocortex of adult mice. Nature 2000; 405(6789): 951–955. 2. Temple S, Alvarez-Buylla A. Stem cells in the adult mammalian central nervous system. Curr Opin Neurobiol 1999; 9(1): 135–141. 3. Gage FH. Mammalian neural stem cells. Science 2000; 287(5457): 1433–1438. 4. Rakic P. Adult neurogenesis in mammals: an identity crisis. J Neurosci 2002; 22(3): 614–618. 5. Jiang Y, Henderson D, Blackstad M, Chen A, Miller RF, Verfaillie CM. Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci U S A 2003; 100(Suppl 1): 11854–11860. 6. Yu J, Thomson JA. Pluripotent stem cell lines. Genes Dev 2008; 22(15): 1987–1997. 7. Zuk PA. The adipose-derived stem cell: looking back and looking ahead. Mol Biol Cell 2010; 21(11): 1783–1787. 8. Jung D-I, Ha J, Kang B-T, et al. A comparison of autologous and allogenic bone marrow-derived mesenchymal stem cell transplantation in canine spinal cord injury. J Neurol Sci 2009; 285(1): 67–77. 9. Kang S-K, Shin M-J, Jung JS, Kim YG, Kim C-H. Autologous adipose tissue-derived stromal cells for treatment of spinal cord injury. Stem Cells Dev 2006; 15(4): 583–594. 10. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284(5411): 143–147. 11. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002; 13(12): 4279–4295. 12. Kang SK, Putnam LA, Ylostalo J, et al. Neurogenesis of Rhesus adipose stromal cells. J Cell Sci 2004; 117(18): 4289–4299. 13. Pachón-Peña G, Yu G, Tucker A, et al. Stromal stem cells from adipose tissue and bone marrow of age-matched female donors display distinct immunophenotypic profiles. J Cell Physiol 2011; 226(3): 843–851. 14. Anghileri E, Marconi S, Pignatelli A, et al. Neuronal differentiation potential of human adipose-derived mesenchymal stem cells. Stem Cells Dev 2008; 17(5): 909–916. 15. Jang S, Cho H-H, Cho Y-B, Park J-S, Jeong H-S. Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin. BMC Cell Biol 2010; 11(1): 25. 16. Ashjian PH, Elbarbary AS, Edmonds B, et al. In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast Reconstr Surg 2003; 111(6): 1922–1931. 17. Sanders EJ, Harvey S. Peptide hormones as developmental growth and differentiation factors. Dev Dyn 2008; 237(6): 1537–1552. 18. Santisteban P, Bernal J. Thyroid development and effect on the nervous system. Rev Endocr Metab Disord 2005; 6(3): 217–228. 19. Williams G. Neurodevelopmental and neurophysiological actions of thyroid hormone. J Neuroendocrinol 2008; 20(6): 784–794. Cell Biochem Funct (2014)

neural induction of human-adscs influenced by t3 20. Oppenheimer J, Schwartz H, Mariash C, Kinlaw W, Wong N, Freake H. Advances in our understanding of thyroid hormone action at the cellular level. Endocr Rev 1987; 8(3): 288–308. 21. Harpavat S, Cepko CL. Thyroid hormone and retinal development: an emerging field. Thyroid 2003; 13(11): 1013–1019. 22. Billon N, Tokumoto Y, Forrest D, Raff M. Role of thyroid hormone receptors in timing oligodendrocyte differentiation. Dev Biol 2001; 235(1): 110–120. 23. Heuer H, Mason CA. Thyroid hormone induces cerebellar purkinje cell dendritic development via the thyroid hormone receptor α1. J Neurosci 2003; 23(33): 10604–10612. 24. Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay R. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev 1996; 10(24): 3129–3140. 25. Ahmadi N, Razavi S, Kazemi M, Oryan S. Stability of neural differentiation in human adipose derived stem cells by two induction protocols. Tissue Cell 2012; 44(2): 87–94. 26. Fernandez M, Paradisi M, Del Vecchio G, Giardino L, Calza L. Thyroid hormone induces glial lineage of primary neurospheres derived from non-pathological and pathological rat brain: implications for remyelination-enhancing therapies. Int J Dev Neurosci 2009; 27(8): 769–778. 27. Krampera M, Marconi S, Pasini A, et al. Induction of neural-like differentiation in human mesenchymal stem cells derived from bone marrow, fat, spleen and thymus. Bone 2007; 40(2): 382–390. 28. Zhang L, Cooper-Kuhn CM, Nannmark U, Blomgren K, Kuhn HG. Stimulatory effects of thyroid hormone on brain angiogenesis in vivo and in vitro. J Cereb Blood Flow Metab 2009; 30(2): 323–335. 29. Martinez R, Gomes FCA. Proliferation of cerebellar neurons induced by astrocytes treated with thyroid hormone is mediated by a cooperation between cell contact and soluble factors and involves the epidermal growth factor-protein kinase a pathway. J Neurosci Res 2005; 80(3): 341–349.

Copyright © 2014 John Wiley & Sons, Ltd.

30. Martinez R, Eller C, Viana NB, Gomes FC. Thyroid hormone induces cerebellar neuronal migration and Bergmann glia differentiation through epidermal growth factor/mitogen-activated protein kinase pathway. Eur J Neurosci 2011; 33(1): 26–35. 31. Desouza LA, Ladiwala U, Daniel SM, Agashe S, Vaidya RA, Vaidya VA. Thyroid hormone regulates hippocampal neurogenesis in the adult rat brain. Mol Cell Neurosci 2005; 29(3): 414–426. 32. Uchida K, Yonezawa M, Nakamura S, Kobayashi T, Machida T. Impaired neurogenesis in the growth-retarded mouse is reversed by T3 treatment. Neuroreport 2005; 16(2): 103–106. 33. Ambrogini P, Cuppini R, Ferri P, et al. Thyroid hormones affect neurogenesis in the dentate gyrus of adult rat. Neuroendocrinology 2005; 81(4): 244–253. 34. Calza L, Fernandez M, Giuliani A, Aloe L, Giardino L. Thyroid hormone activates oligodendrocyte precursors and increases a myelinforming protein and NGF content in the spinal cord during experimental allergic encephalomyelitis. Proc Natl Acad Sci 2002; 99(5): 3258–3263. 35. Li GR, Sun H, Deng X, Lau CP. Characterization of ionic currents in human mesenchymal stem cells from bone marrow. Stem Cells 2005; 23(3): 371–382. 36. Ning H, Lin G, Lue TF, Lin C-S. Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells. Differentiation 2006; 74(9): 510–518. 37. Fernandez M, Pirondi S, Manservigi M, Giardino L, Calzà L. Thyroid hormone participates in the regulation of neural stem cells and oligodendrocyte precursor cells in the central nervous system of adult rat. Eur J Neurosci 2004; 20(8): 2059–2070. 38. Schoonover CM, Seibel MM, Jolson DM, et al. Thyroid hormone regulates oligodendrocyte accumulation in developing rat brain white matter tracts. Endocrinology 2004; 145(11): 5013–5020. 39. Baas D, Legrand C, Samarut J, Flamant F. Persistence of oligodendrocyte precursor cells and altered myelination in optic nerve associated to retina degeneration in mice devoid of all thyroid hormone receptors. Proc Natl Acad Sci 2002; 99(5): 2907–2911.

Cell Biochem Funct (2014)