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Affect of antidepressants on the in vitro differentiation of rat bone marrow mesenchymal stem cells into neuronal cells
European Journal of Pharmaceutical Sciences 73 (2015) 81–87
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Affect of antidepressants on the in vitro differentiation of rat bone marrow mesenchymal stem cells into neuronal cells Paulina Borkowska a,⇑, Joanna Kowalska a, Anna Fila-Danilow a, Anna Maria Bielecka b, Monika Paul-Samojedny a, Malgorzata Kowalczyk a, Jan Kowalski a a b
Department of Medical Genetics, School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia, Sosnowiec, Poland Department of Pharmacology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland
a r t i c l e
i n f o
Article history: Received 29 December 2014 Received in revised form 27 January 2015 Accepted 23 March 2015 Available online 1 April 2015 Keywords: Mesenchymal stem cells Antidepressants b-III-tubulin Differentiation Branching neurons
a b s t r a c t Background: Bone marrow is a valuable source of mesenchymal stem cells (MSCs) that can be used in regenerative medicine. MSCs are able to differentiate into cells from all three germ layers under specific conditions. The aim of the current work was to study the differentiation of rat MSCs (rMSCs) into neuronlike cells. New method: We investigated how the antidepressants imipramine, desipramine, fluoxetine and tianeptine affect the differentiation of rMSCs. Furthermore, we present differentiation cocktails using a cortex astrocyte-conditioned medium (CACM) separately or in conjunction with each of the antidepressants and investigated their additive effect on the efficiency of differentiation. We also observed how various differentiation conditions affect the number of primary dendrites and branching dendrites per cell. Results: Gene expression for an early neuronal marker (b-III-tubulin) and markers that are typical for adult neurons such as Th, Htr2A and Slc6a4 were observed. The Tubb3 and Htr2A gene expression were up-regulated, Th decreased slightly and Slc6a4 was down-regulated after differentiation We observed a two-fold higher percentage of b-III-tubulin positive cells after treatment with antidepressants and two-fold increase of neuron-like cells after using CACM with imipramine or fluoxetine simultaneously. Differentiation using imipramine or in conjunction with CACM and desipramine or fluoxetine simultaneously increased the number of cell dendrites. Comparison with existing methods: The results that were obtained are completely new and need further investigations in the nearest future. Conclusions: These results suggest that antidepressants improve differentiation efficiency of rMSCs and may be useful in the preparation of rMSCs for transplantation. Differentiation efficiency is higher after long-term exposure to antidepressants, than after a 24-h exposure. Nearly additive effect of CACM and imipramine or fluoxetine suggests a beneficial role of antidepressants after transplantation. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction The existence of neural stem cells (NSCs) in the adult human brain has been well investigated and the involvement of NSCs in regeneration processes is well known (Valenzuela et al., 2007). Abbreviations: rhBDNF, recombinant human brain derived neurotrophic factor; CACM, cortex astrocyte-conditioned medium; FBS, fetal bovine serum; rhbFGF, recombinant human basic fibroblast growth factor; rhEGF, recombinant human epidermal growth factor; rrIGF, recombinant rat insulin-like growth factor; MSCs, mesenchymal stem cells; NSCs, neural stem cells; QRT-PCR, quantitative reverse transcription polymerase chain reaction. ⇑ Corresponding author at: Medical University of Silesia, Department of Medical Genetics, Jednosci 8, 41-200 Sosnowiec, Poland. E-mail address: [email protected] (P. Borkowska). http://dx.doi.org/10.1016/j.ejps.2015.03.016 0928-0987/Ó 2015 Elsevier B.V. All rights reserved.
Due to the limited capability of isolating NSCs, mesenchymal stem cells (MSCs) appear to be the best candidates for regenerative medicine. A previous investigation showed that MSCs can differentiate into cells from all three germ layers, e.g., osteoblasts, adipocytes, chondrocytes and even neurons (Pittenger et al., 1999; Woodbury et al., 2000). MSCs are easily isolated from bone marrow, easily expanded in vitro and they seem to be ideal cells for genetic manipulation (Herman et al., 2004; Kawabata et al., 2006). The ability of MSCs to differentiate into neurons has actually been widely investigated due to their ability to be used in patients with neurodegenerative diseases (Dezawa et al., 2005). Appropriate environmental conditions are crucial to the differentiation of MSCs into neurons. A suitable cocktail of proteins in a differentiation medium has an affect on the cells that express embryonic genes (e.g. OCT3/4,
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SOX1-3) and direct them into differentiation to neurons (Herman et al., 2004; Bylund et al., 2003). The main problem in transplanting differentiated stem cells is their weak viability after transplantation. One of the reasons for this situation is in the use of toxic components, e.g., DMSO, b-mercaptoethanol or butylated hydroxyanisole in the induction or differentiation procedures (Woodbury et al., 2000; Kohyama et al., 2001). Investigations of nontoxic substances that may improve in vitro differentiation and lengthen the duration of the viability transplanted cells are essential. Previous researches have shown that antidepressants may have the necessary features (Zusso et al., 2008; Malberg et al., 2000). In our experiment, we investigated how the tricyclic antidepressants imipramine and desipramine, the selective serotonin reuptake inhibitor fluoxetine and the noncatecholaminergic antidepressant tianeptine affect the differentiation of adult rat bone marrow MSCs cells into neurons. Furthermore, we present differentiation cocktails with a cortex astrocyte-conditioned medium (CACM) used separately or in conjunction with each of antidepressants and investigated their additive effect on differentiation efficiency. We also observed how various differentiation conditions affect the number of primary dendrites and branching dendrites per cell. All of the aspects of our experiment that are presented are novel. 2. Materials and methods 2.1. Culture of rats MSCs rMSCs were isolated from the femurs and tibias of adult Wistar rats as was previously described (Woodbury et al., 2000) and then maintained in a Dulbecco’s Modified Eagle’s Medium (DMEM) – nutrient mixture F-12 (DMEM/F12; PAA) supplemented with 10% fetal bovine serum (FBS; PAA) and a 1% Antibiotic Antimycotic Solution (100X) (Sigma–Aldrich). Non-adherent cells were removed for the first time after 24 h and adherent cells were cultured in a 10 ml fresh cultured medium. Non-adherent cells were centrifuged at 400g for 10 min at 4 °C and plated in new 25 cm2 tissue-culture flasks in a 10 ml cultured medium. Non-adherent cells were removed from both flasks after 24 h and adherent cells were cultured into the confluent stage. The media were replaced every two to three days. All of the protocols related to the use of animals were approved by the Local Ethics Committee. 2.2. Neuronal induction (sphere stage) MSCs from rats were harvested by using a Trypsin–EDTA solution and plated in 25 cm2 plastic flasks at a concentration of
1 105 cells/cm2 in an induction cocktail that contained a DMEM/F12 medium supplemented with 2% B-27 (Gibco), 20 ng/ ml recombinant human basic fibroblast growth factor (rhbFGF, Sigma–Aldrich), 20 ng/ml recombinant human epidermal growth factor (rhEGF, Sigma–Aldrich), a 1% Antibiotic Antimycotic Solution (100) (Figs. 1 and 2). Thirty percent of the total volume of the induction medium and growth factors were removed three times a week. Non-adherent and adherent sphere structures could be observed after seven days. Both types of spheres were dissociated from single cells using a NeuroCult™Enzymatic Dissociation Kit for Adult CNS Tissue (Mouse and Rat) (StemCell Technologies) according to the manufacturer’s protocol.
2.3. Neuronal differentiation Single cells from the spheres were plated on poly-l-ornithine and fibronectine-coated cover slips (60,000/500 ll per well) on a 24-well culture plate. Cells were differentiated in a basic differentiation medium containing NeuroCultÒNS-A Basal Medium Rat (StemCell Technologies) with a 10% differentiation supplement (StemCell Technologies), 20 ng/ml recombinant human BDNF (rhBDNF) (R&D), 20 ng/ml recombinant rat IGF (rrIGF) (R&D) and a 1% Antibiotic Antimycotic Solution (100). 50Fifty percent of the total volume of the differentiation medium was removed three times a week. The basic differentiation medium was supplemented with one of the antidepressants in three different combinations. In the first combination, 10 lM one of the antidepressants (imipramine, desipramine, fluoxetine or tianeptine) was added to a culture for 24 h on the 13th day of differentiation (Fig. 1A). In the second, 10 lM one of the antidepressants was added three times during differentiation, on the 7th, 10th and 13th day (Fig. 1B). In the third experiment, a cortex astrocytes conditioned medium was added to the basic differentiation medium at 1/2 the volume on the 7th, 10th and 13th day of differentiation (Fig. 1C) while in fourth experiment, a cortex astrocytes conditioned medium was added to the basic differentiation medium at 1/2 the volume on the 7th, 10th and 13th day of differentiation and on the 13th day of differentiation, 10 lM one of antidepressants was added for 24 h in addition to the CACM (Fig. 1D). Controls were prepared for each of the differentiation variants. In control samples, an adequate volume of the drug’s diluent was added to the medium instead of the antidepressant. Cortex astrocytes were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and for this reason an equal volume of DMEM was added to the control in the third and fourth combinations in experiment instead of CACM.
Fig. 1. Neuronal culture schemes. Exposure to one dose of antidepressants (A). Long-term treatment with antidepressants (three doses) (B). Differentiation with seven days of CACM supplementation (C). Differentiation with seven days of CACM supplementation and one dose of antidepressants (D).
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Fig. 2. Characterization of the P1 MSCs culture (A and B). Sphere formation (C and D). Heterogeneous culture of small, rounded and some spindle-shaped cells after two days of adherence (A). Spindle-shaped cells after seven days of culture (B). Small non-adherent spheres after one day of culture in an induction medium (C). Adherent sphere after five days of culture in an induction medium (D).
2.4. Cortex astrocyte-conditioned medium (CACM) An astrocyte-conditioned medium was derived from neonatal rat (Wistar) cortex monolayer cultures that had been grown in DMEM (PAA) that was supplemented with 10% FBS and a 1% Antibiotic Antimycotic Solution (100) for 21 days. The medium was replaced twice a week. On the tenth day of the culture, microglia and oligodendrocyte precursors were eliminated by orbital shaking overnight. After 20 days of culture, the medium was replaced with fresh DMEM without FBS and after 24 h the astrocyte-conditioned medium was collected, centrifuged and immediately frozen. 2.5. Reverse transcription polymerase chain reaction Total RNA was extracted from the second passage of rMSCs, rMSCs after the induction stage and rMSCs after differentiation. Total RNA was extracted using TRIzolÒ (Invitrogen, Life Technologies) according to the manufacturer’s protocol. The RNA that was obtained was evaluated using spectrophotometry and the integrity was checked using 1.5% gel electrophoresis. The QRT-PCR was carried out using 50 ng RNA at a total volume of 20 ll. The PCR cocktail contained 10 ll TaqMan2X Master Mix (Applied Biosystems, Life Technologies), 0.5 ll 40 MultiScribe and RNAse Inhibitor Mix (Applied Biosystems, Life Technologies) and 8 ll of DEPC-treated water (Ambion, Life Technologies). PCR was performed using 0.5 ll of the appropriate 20 Target Primers and Probes (Applied Biosystems, Life technologies) (Table 1) with the following cycling parameters: 48 °C for 30 min. (1 cycle), 95 °C for 10 min. (1 cycle), 50 cycles at 95 °C for 30 s. and 60 °C for 30 s. An additional five-minute incubation at 75 °C was done after the completion of the last cycle. QRT-PCR was performed in duplicate for all of the samples from four independent experiments. Moreover, a negative control (without RNA) and a positive control (total RNA isolated from adult rat cortexes) were done. mRNA for b-actin was constituted as the
Table 1 List of the primers that were used in QRT-PCR. Gene name
Accession number
ID primers (life technologies)
Product size [bp]
b-III-Tubulin Th Slc6a4 Htr2A b-Actin
NM_139254 NM_012740 NM_013034 NM_017254 NM_031144
Rn01431594_m1 Rn00562500_m1 Rn00564737_m1 Rn00568473_m1 Rn00667869_m1
68 60 79 71 91
endogenous RNA control and as the reference gene (housekeeping gene). The amplimers were electrophoresed through a 6% polyacrylamide gel stained with ethidium bromide and analyzed and registered under ultraviolet light using the gel documentation system. QRT-PCR was carried out using the ABI PRISMÒ 7700 Sequence Detection System (Applied Biosystems, Life Technologies). mRNA expression is presented as DDCt, which was calculated as follow: the DCt of each sample equals the Ct of the target gene minus the Ct of the endogenous control. DDCt equals the DCt of the sample for the target gene minus the DCt of the control sample (Livak and Schmittgen, 2001). 2.6. Immunocytochemistry Immunostaining was carried out using the standard procedures. Cells were fixed with 4% paraformaldehyde for 20 min. and then permeabilized and blocked with 0.3% Triton X-100, 1% bovine serum albumin and 0.3% normal rabbit serum PBS for 45 min. Fixed cells were incubated with the primary antibody and a suitable normal serum overnight. Cells were incubated overnight as well. The dilution of the primary antibody for b-III-tubulin was 1:650 (R&D, MAB1195). Staining was detected using secondary conjugated antibodies (dilutions 1:7500, Rockland, 610-441-002, DyLight488). The cell nuclei were counterstained with DAPI. Cells were examined with a fluorescent microscope (Olympus, BX60).
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2.7. Cell counting The presence of immunoreacting cells was determined by counting more than 500 cells in multiple fields. Only cells that had two features – an immunoreacting positive signal and a neuron-like shape – were treated as positive. Each treatment was replicated on three cover slips from three separate experiments. All specimens were examined by two independent researchers. Data are presented as the means ±SEM of separate experiments. Statistical comparisons were done using the Student’s t-test or by ANOVA using the post hoc Newman–Keuls test. Differences were deemed statistically significant at p < 0.05. Statistical analysis was performed using Prism 4.0 and Excel MS Office 2007 software. 3. Results 3.1. Isolation and culturing of rMSCs Rats MSCs were isolated using a combined method – HistopaqueÒ separation and plastic adherence. The cell suspensions were layered on HistopaqueÒ and subsequently seeded in a tissue culture flask. After two days, many of the rounded as well as spindle-shaped cells had become attached to the base of the tissue culture flask (Fig. 2A). The majority of the adherent cells displayed a spindle-like shape (Fig. 2B). Only cells after the second passage were inducted and differentiated in this experiment. 3.2. Sphere formation After the second passage, cells were inducted and one day after the induction small non-adherent spheres were observed (Fig. 2C). The small spheres grew and when they were large enough, they started to adhere to the surface of the tissue culture flask (Fig. 2D). After seven days all types of the spheres were dissociated, plated on cover slips and differentiated. 3.3. Gene expression The expression of neuronal markers was investigated at three different points of the experiment. Total RNA was extracted from the second passage of rMSCs, rMSCs after the induction stage and rMSCs after differentiation. The expression of the Tubb3 and Htr2A genes was up-regulated during the experiment but the expression of the Th gene decreased slightly during the same time. We observed a down-regulation of the expression level of Slc6a4 after differentiation compared to cells after induction (Fig. 3). 3.4. Affect of antidepressants on the number of neuronal cells MSCs were cultured in an induction medium with 20 ng/ml EGF and 20 ng/ml bFGF for seven days and afterwards in a differentiation medium with 20 ng/ml BDNF and 20 ng/ml IGF for the following 14 days. Twenty-four hours before the end of the differentiation process, 10 lM of one of the antidepressants (imipramine, desipramine, fluoxetine or tianeptine) was added to the culture (Fig. 1A). Immunocytochemistry was done after differentiation. In our study only were those cells that were stained positive and had a shape that is characteristic for neuronal cells qualified as positive for b-III-tubulin (Fig. 4). In the control group, which had not been treated with any of the antidepressants, 5% of b-III-tubulin positive cells were observed while after treatment with antidepressants from 7% to 10% cells were positive (Fig. 5). Long-term exposure to antidepressants was examined in another experiment. A new dose one of antidepressant (10 lM) was added on the 14th, 17th and 20th days of the culture when
Fig. 3. Expression of the neuronal markers that were normalized to b-actin (DDCt) at two different points of the experiment. QRT-PCR analyses of early neuronal markers (Tubb3) as well as markers for dopaminergic neurons (Th) and serotoninergic neurons (Htr2A and Slc6a4). The results are expressed as DDCt from six independent cultures and two parallel experiments.
the differentiation medium was changed (Fig. 1B). In our experiment, the number of b-III-tubulin positive cells was significantly higher than in control. The largest increase was observed after treatment with tianeptine; furthermore, the differences between tianeptine and desipramine and between tianeptine and imipramine were statistically significant (Fig. 5). We observed that the number of b-III-tubulin positive cells was higher after long-term exposure, especially in the case of tianeptine and fluoxetine, than after a 24-h exposure. In our experiment, we also investigated how the brain’s microenvironment affects the differentiation of MSCs. After seven days of differentiation, the medium was replaced with a basic differentiation medium with 20 ng/ml BDNF and 20 ng/ml IGF at 1/2 the volume and the cortex astrocyte-conditioned medium at 1/2 the volume of the total differentiation medium. The second part of the differentiation lasted seven days (Fig. 1C). The number of b-III-tubulin positive cells was significantly higher after CACM supplementation than in the control (Fig. 6). In the next experiment, we investigated how CACM and antidepressants, which were supplemented together, affected the number of neuronal cells. In this experiment, CACM was supplemented for seven days and for the last 24 h, 10 lM of one of the antidepressants was added (Fig. 1D). The number of b-III-tubulin positive cells was significantly higher when CACM was supplemented with fluoxetine or imipramine than in the culture that was only supplemented with CACM (Fig. 6). 3.5. Affect of antidepressants on the number of dendrites In addition to the presence of neuronal markers, morphological changes are another piece of evidence that differentiation occurred. The neuronal phenotype is evidence that the differentiation of MSCs into neuronal cells exists. In this experiment, the number of primary dendrites and branching dendrites in a cell were counted. The largest increase of primary dendrites and branching dendrites were observed after long-term treatment with imipramine and the differences were statistically significant (Fig. 7). We also counted number of cells’ primary dendrites and branching dendrites after CACM usage and compared it with culture after simultaneous treatment with CACM and one dose of each antidepressant. We observed that after treatment with CACM and fluoxetine increase was statistically significant while after CACM and desipramine exposure only tendency to statistical significance
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Fig. 4. Differentiated rMSCs immunofluorescence. Cells were stained with an antibody against b-III-tubulin (green). Differences in the number of primary dendrites and branching dendrites using various differentiation schemes. Negative control (A); control, differentiation using a basic differentiation medium (B and C); long-term imipramine treatment (D); CACM treatment (E); CACM + desipramine (F and G); CACM + fluoxetine (H, I). In each experiment, the nuclei were counterstained with 40 ,6diamidino-2-phenylindole (DAPI) (blue). In our study, only cells that were positive stained and had the shape that is characteristic for neuronal cells were qualified as positive. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
was observed (Fig. 7). We also observed that in cultures that were treated with antidepressants, there was an accumulation of differentiated cells, while in control this effect was not noticed.
Fig. 5. Percentage of b-III-tubulin positive cells after differentiation using one and three doses of antidepressants. Bars represent the mean value ± S.E.M. (n = 9). Significant differences with the control are marked ⁄ for p < 0.05; t-test. Significant differences between tianeptine and each antidepressant are marked # for p < 0.05; t-test.
Fig. 6. Percentage of b-III-tubulin positive cells after differentiation using CACM and after simultaneously using CACM and an antidepressant. Bars represent the mean value ± S.E.M. (n = 9). Significant differences with the control are marked ⁄ for p < 0.05; t-test. Significant differences between CACM and CACM in conjunction with an antidepressant are marked # for p < 0.05; t-test.
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Fig. 7. Number of primary dendrites and branching dendrites per cell. Bars represent the mean value ± S.E.M. (n = 11). ⁄ Statistically significant p < 0.05; t-test or ANOVA followed by post hoc Newman–Keuls test.
4. Discussion It is well known that mesenchymal cells have a mesodermal origin and can easily differentiate into adipocytes, osteoblasts, chondrocytes, mioblasts and fibroblasts while neuronal cells have an ectodermal origin. An earlier study investigated whether OCT4 and SOX-1 gene expression was observed in mesenchymal cells (Herman et al., 2004). OCT-4 and SOX-1 are expressed in embryonic stem cells and their expression is necessary in order to maintain their pluripotency (Bylund et al., 2003; Pesce and Scholer, 2000). Sphere formation after EGF and bFGF is used in a human MSCs culture is connected with a strong decrease in OCT-4 and SOX-1 gene expression and an increase in proneuronal gene expression (Neurog 2, OTX-1), which indicates an initial differentiation to neuronal cells (Herman et al., 2004). The above-mentioned results correspond with ours. The first extremely important stage of our experiment was the neuronal induction, which should have reprogrammed the MSCs and regulated their pluripotency. In a previous experiment, we investigated whether the induction stage is the key to directing the maximal number of cells toward a specific type of differentiation (Borkowska et al., 2015) but this method is not an efficient way to achieve functional neurons. The effect that was observed is caused by the addition of bFGF whose intracerebroventricular infusion increases neurogenesis (Rai et al., 2007). Animals with lack of FGFR-1 receptor have limited abilities to neurogenesis in adulthood (Zhao et al., 2007). Furthermore, except of mitogenic properties, bFGF show differentiate properties. In following work we investigated first time how antidepressants affect the differentiation process of rMSCs. In control cultures where differentiation was done with BDNF and IGF-1 5% b-IIItubulin positive cells were observed. Previous research shown that, in in vitro culture of adult human bone marrow stromal cells where induction was done with retinol acid, BDNF increased number of b-III-tubulin positive cells (Herman et al., 2004). In another experiment, in which an induction that is typical for the dopaminergic phenotype was done (SHH + bFGF + FGF8), after BDNF addition more than 60% of Th positive cells were achieved (Trzaska et al., 2009). After antidepressants addition to differentiation cocktail 10% b-III-tubulin positive cells were observed however, mechanisms of these results is still unknown. In one of the previous experiment imipramine-treated astrocytes from adult organism were converted to neurons which presented specific neuronal proteins and neuron-like shape. Not only imipramine but also fluoxetine and venlafaxine addition were cause of cell body concentration around nucleus and it was a
beginning of branching neurons formation. In consequence neuron-like cells were come into existence from 40% flatten astrocytes cells. Newly created neurons had small, highly concentrated cell body with branching dendrites and presented some neuronal markers such as neurofilament and neuron-specific enolase, however, any notable change in the electrophysiological membrane was not observed (Cabras et al., 2010). In some of rMSCs cultures we observed Gfap gene expression and our result is compatible with others (Scintu et al., 2006; Willerth et al., 2007). We can conclude that investigated antidepressants converted early astrocytes presented in rMSCs culture to neuronal cells. In following experiment Htr2A gene expression was observed. It can suggest presence of serotoninergic neurons which are able to serotonin production. Previous researches have shown that serotonin receptors agonists affect the differentiation of MSCs into neurons (Klempin et al., 2010; Nandra and Agius, 2012). Furthermore, fluoxetine induced the proliferation and differentiation of neural progenitor cells that had been isolated from rat postnatal cerebellum and increased the expression of the serotonin transporter gene in cells (Zusso et al., 2008). The hypothesis that imipramine, desipramine and fluoxetine inhibited the production of the serotonin transporter gene, increased the serotonin concentration in a culture and as a result enhanced the differentiation of rMSCs seems too speculative. The significant increase in the percentage of neuronal cells in rMSCs after seven days of exposure to tianeptine may be connected with the fact that one of mechanisms of the action of tianeptine has an effect on neurogenesis (Uzbay, 2008). The neuroprotective properties of antidepressants on cells that were treated with such chemical substances such as LPS or MPP have been proposed in many investigations (Peng et al., 2008; Zhang et al., 2012). Antidepressants are neuroprotective against the deprivation of the N2 supplement in a culture medium (Yang et al., 2012). It seems probable that antidepressants decrease the mortality of more sensitive neuronal cells but increase their percentage from 5% to 10% at the same time. In a previous work, we observed that the number of neuronal cells increased after treatment with CACM and that simultaneous use CACM and antidepressants (except for desipramine) caused an increase in the number of neuronal cells. Other authors have observed that a hippocampal astrocyte-conditioned medium increased the number of neurons in a culture (Joannides et al., 2003); furthermore, astrocytes are able to secret endogenous substances (BDNF, NGF, NT3/4, CNTF, GDNF) that affect the differentiation of NSCs and MSCs into neuronal cells (Yang et al., 2012; Rudge et al., 1992). The endogenous substances that are secreted by cortex astrocytes create a microenvironment for differentiation that is similar to natural conditions and as a result the addition of CACM enhanced the differentiation and protection of MSCs. Nearly additive effect of the simultaneous use CACM and imipramine or fluoxetine suggests that mentioned antidepressants could play an important role in in vitro differentiation and could also play a beneficial role after neuronal cells are transplanted into an animal’s brain. In a following work, we also observe a larger number of primary dendrites and branching dendrites in the neuronal cells that had differentiated after treatment with antidepressants. Unfortunately, the limited number of cells that were compared means that further investigations are necessary.
5. Conclusions In conclusion our results suggest that (1) antidepressants improve differentiation efficiency of rMSCs and these substances may be useful in the preparation of MSCs for transplantation (2) differentiation efficiency is higher after long-term exposure to
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antidepressants, than after a 24-h exposure (3) additive effect of CACM and imipramine or fluoxetine suggests a beneficial role of some antidepressants after transplantation (4) larger number of primary dendrites and branching dendrites after exposure to antidepressants is a phenomenon but needs further investigations. 6. Conflicts of interest There are no conflicts of interest. Acknowledgement This work was supported by a Grant from Medical University of Silesia. References Borkowska, P., Fila-Danilow, A., Paul-Samojedny, M., Kowalczyk, M., Hart, J., Ryszawy, J., Kowalski, J., 2015. Differentiation of adult rat mesenchymal stem cells to GABAergic, dopaminergic and cholinergic neurons. Pharmacol. Rep. 67, 179–186. Bylund, M., Andersson, E., Novitch, B.G., Muhr, J., 2003. Vertebrate neurogenesis is counteracted by So1–3 activity. Nat. Neurosci. 6, 1162–1168. Cabras, S., Saba, F., Reali, C., Scorciapino, M.L., Sirgu, A., Talani, G., Biggio, G., Sogos, V., 2010. Antidepressant imipramine induces human astrocytes to differentiate into cells with neuronal phenotype. Int. J. Neuropsychopharmacol. 13, 603–615. Dezawa, M., Hoshino, M., Ide, C., 2005. Treatment of neurodegenerative diseases using adult bone marrow stromal cell derived neurons. Exp. Opin. Biol. Ther. 5, 427–435. Herman, A., Gastl, R., Liebau, S., Popa, M.O., Fiedler, J., Boehm, B.O., Maisel, M., Lerche, M., Schwarz, J., Brenner, R., Storch, A., 2004. Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells. 2004. J. Cell Sci. 117, 4411–4422. Joannides, A., Gaughwin, P., Scott, M., Watt, S., Compston, A., Chandran, S., 2003. Postnatal astrocytes promote neural induction from adult human bone marrow-derived stem cells. J. Hematother. Stem Cell Res. 12, 681–688. Kawabata, K., Sakurai, F., Koizumi, N., Hayakawa, T., Mizuguchi, H., 2006. Adenovirus vector-mediated gene transfer into stem cells. Mol. Pharm. 3, 95– 103. Klempin, F., Babu, H., De Pietri Tonelli, D., Alarcon, E., Fabel, K., Kempermann, G., 2010. Oppositional effects of serotonin receptors 5-HT1a,2 and 2c in the regulation of adult hippocampal neurogenesis. Front Mol. Neurosci. 3, 14. http://dx.doi.org/10.3389/fnmol.2010.00014. Kohyama, J., Abe, H., Shimazaki, T., Koizumi, A., Nakashima, K., Gojo, S., Taga, T., Okano, H., Hata, J., Umezawa, A., 2001. Brain from bone: efficient’’ metadifferentiation’’ of marrow stroma derived mature osteoblasts to neurons with Noggin or demethylating agent. Differentiation 68, 235–244. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-Ct method. Methods 25, 402–408.
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Malberg, J., Eisch, A., Nestler, E., Duman, R., 2000. Chronic antidepressant treatment increases neurogenesis in adult hippocampus. J. Neurosci. 20, 9104–9110. Nandra, K.S., Agius, M., 2012. The difference between typical and athypical antipsychotics: the effects on neurogenesis. Psychiatr. Danub. 24, 95–99. Valenzuela, M., Sidhu, K., Dean, S., Sachdev, P., 2007. Neural stem cell therapy for neuropsychiatric disorders. Acta Neuropsychiatr. 19, 11–26. Peng, C.H., Chiou, S.H., Chen, S.J., Chou, Y.C., Ku, H.H., Cheng, C.K., Yen, C.J., Tsai, T.H., Chang, Y.L., Kao, C.L., 2008. Neuroprotection by imipramine against lipopolysaccharide-induced apoptosis in hippocampus-derived neural stem cells mediated by activation of BDNF and the MAPK pathway. Eur. Neuropsychopharmacol. 18, 128–140. Pesce, M., Scholer, H.R., 2000. Oct-4: control of totipotency and germline determination. Mol. Reprod. 55, 452–457. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147. Rai, K.S., Hattiangady, B., Shetty, A.K., 2007. Enhanced production and dendritic growth of new dentatr granule cells in the middle-aged hippocampus following intracerebroventricular FGF-2 infusions. Eur. J. Neurosci. 26, 1765–1779. Rudge, J.S., Alderson, R.F., Pasnikowski, E., 1992. Expression of cilliary neurotrophic factor and the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in cultured rat hippocampal astrocytes. Eur. J. Neurosci. 4, 459–471. Scintu, F., Reali, C., Pillai, R., Badiali, M., Sanna, M.A., Argiolu, F., Ristaldi, M.S., Sogos, V., 2006. Differentiation of human bone marrow stem cells into cells with a neural phenotype: diverse effects of two specific treatments. BMC Neurosci. 7, 1–14. Trzaska, K.A., King, C.C., Li, K.Y., Kuzhikandathil, E.V., Nowycky, M.C., Ye, J.H., Rameshwar, P., 2009. Brain-derived neurotrophic factor facilitates maturation of mesenchymal stem cell-derived dopamine progenitors to functional neurons. J. Neurochem. 110, 1058–1069. Uzbay, T., 2008. Tianeptine: potential effect on neuroplasticity and novel pharmacological effects. Prog. Neuro-Psychopharmacol. Biol. Psychiatr. 32, 915–924. Willerth, S.M., Faxel, T.E., Gottlieb, D.I., Sakiyama-Elbert, S.E., 2007. The effects of soluble growth factors on embryonic stem cell differentiation inside of fibrin scaffold. Stem Cells 25, 2235–2244. Woodbury, D., Schwartz, E.J., Prockop, D.J., Black, I.B., 2000. Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364– 370. Yang, D., Chen, M., Russo-Neustadt, A., 2012. Antidepressants are neuroprotective against nutrient deprivation stress in rat hippocampal neurons. Eur. J. Neurosci. 36, 2573–2587. Zhang, F., Zhou, H., Wilson, B.C., Shi, J.S., Hong, J.S., Gao, H.M., 2012. Fluoxetine protects neurons against microglial activation-mediated neurotoxicity. Parkinsonism Relat. Disord. 18, 213–217. Zhao, M., Li, D., Shimazu, K., Zhou, Y.X., Lu, B., Deng, C.X., 2007. Fibroblast growth factor receptor-1 is required for long-term potentiation, memory consolidation and neurogenesis. Biol. Psychiatr. 62, 381–390. Zusso, M., Debetto, P., Guidolin, D., Barbierato, M., Manev, H., Giusti, P., 2008. Fluoxetine-induced proliferation and differentiation of neural progenitor cells isolated from rat postnatal cerebellum. Biochem. Pharm. 76, 391–403.
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Affect of antidepressants on the in vitro differentiation of rat bone marrow mesenchymal stem cells into neuronal cells Paulina Borkowska a,⇑, Joanna Kowalska a, Anna Fila-Danilow a, Anna Maria Bielecka b, Monika Paul-Samojedny a, Malgorzata Kowalczyk a, Jan Kowalski a a b
Department of Medical Genetics, School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia, Sosnowiec, Poland Department of Pharmacology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland
a r t i c l e
i n f o
Article history: Received 29 December 2014 Received in revised form 27 January 2015 Accepted 23 March 2015 Available online 1 April 2015 Keywords: Mesenchymal stem cells Antidepressants b-III-tubulin Differentiation Branching neurons
a b s t r a c t Background: Bone marrow is a valuable source of mesenchymal stem cells (MSCs) that can be used in regenerative medicine. MSCs are able to differentiate into cells from all three germ layers under specific conditions. The aim of the current work was to study the differentiation of rat MSCs (rMSCs) into neuronlike cells. New method: We investigated how the antidepressants imipramine, desipramine, fluoxetine and tianeptine affect the differentiation of rMSCs. Furthermore, we present differentiation cocktails using a cortex astrocyte-conditioned medium (CACM) separately or in conjunction with each of the antidepressants and investigated their additive effect on the efficiency of differentiation. We also observed how various differentiation conditions affect the number of primary dendrites and branching dendrites per cell. Results: Gene expression for an early neuronal marker (b-III-tubulin) and markers that are typical for adult neurons such as Th, Htr2A and Slc6a4 were observed. The Tubb3 and Htr2A gene expression were up-regulated, Th decreased slightly and Slc6a4 was down-regulated after differentiation We observed a two-fold higher percentage of b-III-tubulin positive cells after treatment with antidepressants and two-fold increase of neuron-like cells after using CACM with imipramine or fluoxetine simultaneously. Differentiation using imipramine or in conjunction with CACM and desipramine or fluoxetine simultaneously increased the number of cell dendrites. Comparison with existing methods: The results that were obtained are completely new and need further investigations in the nearest future. Conclusions: These results suggest that antidepressants improve differentiation efficiency of rMSCs and may be useful in the preparation of rMSCs for transplantation. Differentiation efficiency is higher after long-term exposure to antidepressants, than after a 24-h exposure. Nearly additive effect of CACM and imipramine or fluoxetine suggests a beneficial role of antidepressants after transplantation. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction The existence of neural stem cells (NSCs) in the adult human brain has been well investigated and the involvement of NSCs in regeneration processes is well known (Valenzuela et al., 2007). Abbreviations: rhBDNF, recombinant human brain derived neurotrophic factor; CACM, cortex astrocyte-conditioned medium; FBS, fetal bovine serum; rhbFGF, recombinant human basic fibroblast growth factor; rhEGF, recombinant human epidermal growth factor; rrIGF, recombinant rat insulin-like growth factor; MSCs, mesenchymal stem cells; NSCs, neural stem cells; QRT-PCR, quantitative reverse transcription polymerase chain reaction. ⇑ Corresponding author at: Medical University of Silesia, Department of Medical Genetics, Jednosci 8, 41-200 Sosnowiec, Poland. E-mail address: [email protected] (P. Borkowska). http://dx.doi.org/10.1016/j.ejps.2015.03.016 0928-0987/Ó 2015 Elsevier B.V. All rights reserved.
Due to the limited capability of isolating NSCs, mesenchymal stem cells (MSCs) appear to be the best candidates for regenerative medicine. A previous investigation showed that MSCs can differentiate into cells from all three germ layers, e.g., osteoblasts, adipocytes, chondrocytes and even neurons (Pittenger et al., 1999; Woodbury et al., 2000). MSCs are easily isolated from bone marrow, easily expanded in vitro and they seem to be ideal cells for genetic manipulation (Herman et al., 2004; Kawabata et al., 2006). The ability of MSCs to differentiate into neurons has actually been widely investigated due to their ability to be used in patients with neurodegenerative diseases (Dezawa et al., 2005). Appropriate environmental conditions are crucial to the differentiation of MSCs into neurons. A suitable cocktail of proteins in a differentiation medium has an affect on the cells that express embryonic genes (e.g. OCT3/4,
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SOX1-3) and direct them into differentiation to neurons (Herman et al., 2004; Bylund et al., 2003). The main problem in transplanting differentiated stem cells is their weak viability after transplantation. One of the reasons for this situation is in the use of toxic components, e.g., DMSO, b-mercaptoethanol or butylated hydroxyanisole in the induction or differentiation procedures (Woodbury et al., 2000; Kohyama et al., 2001). Investigations of nontoxic substances that may improve in vitro differentiation and lengthen the duration of the viability transplanted cells are essential. Previous researches have shown that antidepressants may have the necessary features (Zusso et al., 2008; Malberg et al., 2000). In our experiment, we investigated how the tricyclic antidepressants imipramine and desipramine, the selective serotonin reuptake inhibitor fluoxetine and the noncatecholaminergic antidepressant tianeptine affect the differentiation of adult rat bone marrow MSCs cells into neurons. Furthermore, we present differentiation cocktails with a cortex astrocyte-conditioned medium (CACM) used separately or in conjunction with each of antidepressants and investigated their additive effect on differentiation efficiency. We also observed how various differentiation conditions affect the number of primary dendrites and branching dendrites per cell. All of the aspects of our experiment that are presented are novel. 2. Materials and methods 2.1. Culture of rats MSCs rMSCs were isolated from the femurs and tibias of adult Wistar rats as was previously described (Woodbury et al., 2000) and then maintained in a Dulbecco’s Modified Eagle’s Medium (DMEM) – nutrient mixture F-12 (DMEM/F12; PAA) supplemented with 10% fetal bovine serum (FBS; PAA) and a 1% Antibiotic Antimycotic Solution (100X) (Sigma–Aldrich). Non-adherent cells were removed for the first time after 24 h and adherent cells were cultured in a 10 ml fresh cultured medium. Non-adherent cells were centrifuged at 400g for 10 min at 4 °C and plated in new 25 cm2 tissue-culture flasks in a 10 ml cultured medium. Non-adherent cells were removed from both flasks after 24 h and adherent cells were cultured into the confluent stage. The media were replaced every two to three days. All of the protocols related to the use of animals were approved by the Local Ethics Committee. 2.2. Neuronal induction (sphere stage) MSCs from rats were harvested by using a Trypsin–EDTA solution and plated in 25 cm2 plastic flasks at a concentration of
1 105 cells/cm2 in an induction cocktail that contained a DMEM/F12 medium supplemented with 2% B-27 (Gibco), 20 ng/ ml recombinant human basic fibroblast growth factor (rhbFGF, Sigma–Aldrich), 20 ng/ml recombinant human epidermal growth factor (rhEGF, Sigma–Aldrich), a 1% Antibiotic Antimycotic Solution (100) (Figs. 1 and 2). Thirty percent of the total volume of the induction medium and growth factors were removed three times a week. Non-adherent and adherent sphere structures could be observed after seven days. Both types of spheres were dissociated from single cells using a NeuroCult™Enzymatic Dissociation Kit for Adult CNS Tissue (Mouse and Rat) (StemCell Technologies) according to the manufacturer’s protocol.
2.3. Neuronal differentiation Single cells from the spheres were plated on poly-l-ornithine and fibronectine-coated cover slips (60,000/500 ll per well) on a 24-well culture plate. Cells were differentiated in a basic differentiation medium containing NeuroCultÒNS-A Basal Medium Rat (StemCell Technologies) with a 10% differentiation supplement (StemCell Technologies), 20 ng/ml recombinant human BDNF (rhBDNF) (R&D), 20 ng/ml recombinant rat IGF (rrIGF) (R&D) and a 1% Antibiotic Antimycotic Solution (100). 50Fifty percent of the total volume of the differentiation medium was removed three times a week. The basic differentiation medium was supplemented with one of the antidepressants in three different combinations. In the first combination, 10 lM one of the antidepressants (imipramine, desipramine, fluoxetine or tianeptine) was added to a culture for 24 h on the 13th day of differentiation (Fig. 1A). In the second, 10 lM one of the antidepressants was added three times during differentiation, on the 7th, 10th and 13th day (Fig. 1B). In the third experiment, a cortex astrocytes conditioned medium was added to the basic differentiation medium at 1/2 the volume on the 7th, 10th and 13th day of differentiation (Fig. 1C) while in fourth experiment, a cortex astrocytes conditioned medium was added to the basic differentiation medium at 1/2 the volume on the 7th, 10th and 13th day of differentiation and on the 13th day of differentiation, 10 lM one of antidepressants was added for 24 h in addition to the CACM (Fig. 1D). Controls were prepared for each of the differentiation variants. In control samples, an adequate volume of the drug’s diluent was added to the medium instead of the antidepressant. Cortex astrocytes were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and for this reason an equal volume of DMEM was added to the control in the third and fourth combinations in experiment instead of CACM.
Fig. 1. Neuronal culture schemes. Exposure to one dose of antidepressants (A). Long-term treatment with antidepressants (three doses) (B). Differentiation with seven days of CACM supplementation (C). Differentiation with seven days of CACM supplementation and one dose of antidepressants (D).
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Fig. 2. Characterization of the P1 MSCs culture (A and B). Sphere formation (C and D). Heterogeneous culture of small, rounded and some spindle-shaped cells after two days of adherence (A). Spindle-shaped cells after seven days of culture (B). Small non-adherent spheres after one day of culture in an induction medium (C). Adherent sphere after five days of culture in an induction medium (D).
2.4. Cortex astrocyte-conditioned medium (CACM) An astrocyte-conditioned medium was derived from neonatal rat (Wistar) cortex monolayer cultures that had been grown in DMEM (PAA) that was supplemented with 10% FBS and a 1% Antibiotic Antimycotic Solution (100) for 21 days. The medium was replaced twice a week. On the tenth day of the culture, microglia and oligodendrocyte precursors were eliminated by orbital shaking overnight. After 20 days of culture, the medium was replaced with fresh DMEM without FBS and after 24 h the astrocyte-conditioned medium was collected, centrifuged and immediately frozen. 2.5. Reverse transcription polymerase chain reaction Total RNA was extracted from the second passage of rMSCs, rMSCs after the induction stage and rMSCs after differentiation. Total RNA was extracted using TRIzolÒ (Invitrogen, Life Technologies) according to the manufacturer’s protocol. The RNA that was obtained was evaluated using spectrophotometry and the integrity was checked using 1.5% gel electrophoresis. The QRT-PCR was carried out using 50 ng RNA at a total volume of 20 ll. The PCR cocktail contained 10 ll TaqMan2X Master Mix (Applied Biosystems, Life Technologies), 0.5 ll 40 MultiScribe and RNAse Inhibitor Mix (Applied Biosystems, Life Technologies) and 8 ll of DEPC-treated water (Ambion, Life Technologies). PCR was performed using 0.5 ll of the appropriate 20 Target Primers and Probes (Applied Biosystems, Life technologies) (Table 1) with the following cycling parameters: 48 °C for 30 min. (1 cycle), 95 °C for 10 min. (1 cycle), 50 cycles at 95 °C for 30 s. and 60 °C for 30 s. An additional five-minute incubation at 75 °C was done after the completion of the last cycle. QRT-PCR was performed in duplicate for all of the samples from four independent experiments. Moreover, a negative control (without RNA) and a positive control (total RNA isolated from adult rat cortexes) were done. mRNA for b-actin was constituted as the
Table 1 List of the primers that were used in QRT-PCR. Gene name
Accession number
ID primers (life technologies)
Product size [bp]
b-III-Tubulin Th Slc6a4 Htr2A b-Actin
NM_139254 NM_012740 NM_013034 NM_017254 NM_031144
Rn01431594_m1 Rn00562500_m1 Rn00564737_m1 Rn00568473_m1 Rn00667869_m1
68 60 79 71 91
endogenous RNA control and as the reference gene (housekeeping gene). The amplimers were electrophoresed through a 6% polyacrylamide gel stained with ethidium bromide and analyzed and registered under ultraviolet light using the gel documentation system. QRT-PCR was carried out using the ABI PRISMÒ 7700 Sequence Detection System (Applied Biosystems, Life Technologies). mRNA expression is presented as DDCt, which was calculated as follow: the DCt of each sample equals the Ct of the target gene minus the Ct of the endogenous control. DDCt equals the DCt of the sample for the target gene minus the DCt of the control sample (Livak and Schmittgen, 2001). 2.6. Immunocytochemistry Immunostaining was carried out using the standard procedures. Cells were fixed with 4% paraformaldehyde for 20 min. and then permeabilized and blocked with 0.3% Triton X-100, 1% bovine serum albumin and 0.3% normal rabbit serum PBS for 45 min. Fixed cells were incubated with the primary antibody and a suitable normal serum overnight. Cells were incubated overnight as well. The dilution of the primary antibody for b-III-tubulin was 1:650 (R&D, MAB1195). Staining was detected using secondary conjugated antibodies (dilutions 1:7500, Rockland, 610-441-002, DyLight488). The cell nuclei were counterstained with DAPI. Cells were examined with a fluorescent microscope (Olympus, BX60).
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2.7. Cell counting The presence of immunoreacting cells was determined by counting more than 500 cells in multiple fields. Only cells that had two features – an immunoreacting positive signal and a neuron-like shape – were treated as positive. Each treatment was replicated on three cover slips from three separate experiments. All specimens were examined by two independent researchers. Data are presented as the means ±SEM of separate experiments. Statistical comparisons were done using the Student’s t-test or by ANOVA using the post hoc Newman–Keuls test. Differences were deemed statistically significant at p < 0.05. Statistical analysis was performed using Prism 4.0 and Excel MS Office 2007 software. 3. Results 3.1. Isolation and culturing of rMSCs Rats MSCs were isolated using a combined method – HistopaqueÒ separation and plastic adherence. The cell suspensions were layered on HistopaqueÒ and subsequently seeded in a tissue culture flask. After two days, many of the rounded as well as spindle-shaped cells had become attached to the base of the tissue culture flask (Fig. 2A). The majority of the adherent cells displayed a spindle-like shape (Fig. 2B). Only cells after the second passage were inducted and differentiated in this experiment. 3.2. Sphere formation After the second passage, cells were inducted and one day after the induction small non-adherent spheres were observed (Fig. 2C). The small spheres grew and when they were large enough, they started to adhere to the surface of the tissue culture flask (Fig. 2D). After seven days all types of the spheres were dissociated, plated on cover slips and differentiated. 3.3. Gene expression The expression of neuronal markers was investigated at three different points of the experiment. Total RNA was extracted from the second passage of rMSCs, rMSCs after the induction stage and rMSCs after differentiation. The expression of the Tubb3 and Htr2A genes was up-regulated during the experiment but the expression of the Th gene decreased slightly during the same time. We observed a down-regulation of the expression level of Slc6a4 after differentiation compared to cells after induction (Fig. 3). 3.4. Affect of antidepressants on the number of neuronal cells MSCs were cultured in an induction medium with 20 ng/ml EGF and 20 ng/ml bFGF for seven days and afterwards in a differentiation medium with 20 ng/ml BDNF and 20 ng/ml IGF for the following 14 days. Twenty-four hours before the end of the differentiation process, 10 lM of one of the antidepressants (imipramine, desipramine, fluoxetine or tianeptine) was added to the culture (Fig. 1A). Immunocytochemistry was done after differentiation. In our study only were those cells that were stained positive and had a shape that is characteristic for neuronal cells qualified as positive for b-III-tubulin (Fig. 4). In the control group, which had not been treated with any of the antidepressants, 5% of b-III-tubulin positive cells were observed while after treatment with antidepressants from 7% to 10% cells were positive (Fig. 5). Long-term exposure to antidepressants was examined in another experiment. A new dose one of antidepressant (10 lM) was added on the 14th, 17th and 20th days of the culture when
Fig. 3. Expression of the neuronal markers that were normalized to b-actin (DDCt) at two different points of the experiment. QRT-PCR analyses of early neuronal markers (Tubb3) as well as markers for dopaminergic neurons (Th) and serotoninergic neurons (Htr2A and Slc6a4). The results are expressed as DDCt from six independent cultures and two parallel experiments.
the differentiation medium was changed (Fig. 1B). In our experiment, the number of b-III-tubulin positive cells was significantly higher than in control. The largest increase was observed after treatment with tianeptine; furthermore, the differences between tianeptine and desipramine and between tianeptine and imipramine were statistically significant (Fig. 5). We observed that the number of b-III-tubulin positive cells was higher after long-term exposure, especially in the case of tianeptine and fluoxetine, than after a 24-h exposure. In our experiment, we also investigated how the brain’s microenvironment affects the differentiation of MSCs. After seven days of differentiation, the medium was replaced with a basic differentiation medium with 20 ng/ml BDNF and 20 ng/ml IGF at 1/2 the volume and the cortex astrocyte-conditioned medium at 1/2 the volume of the total differentiation medium. The second part of the differentiation lasted seven days (Fig. 1C). The number of b-III-tubulin positive cells was significantly higher after CACM supplementation than in the control (Fig. 6). In the next experiment, we investigated how CACM and antidepressants, which were supplemented together, affected the number of neuronal cells. In this experiment, CACM was supplemented for seven days and for the last 24 h, 10 lM of one of the antidepressants was added (Fig. 1D). The number of b-III-tubulin positive cells was significantly higher when CACM was supplemented with fluoxetine or imipramine than in the culture that was only supplemented with CACM (Fig. 6). 3.5. Affect of antidepressants on the number of dendrites In addition to the presence of neuronal markers, morphological changes are another piece of evidence that differentiation occurred. The neuronal phenotype is evidence that the differentiation of MSCs into neuronal cells exists. In this experiment, the number of primary dendrites and branching dendrites in a cell were counted. The largest increase of primary dendrites and branching dendrites were observed after long-term treatment with imipramine and the differences were statistically significant (Fig. 7). We also counted number of cells’ primary dendrites and branching dendrites after CACM usage and compared it with culture after simultaneous treatment with CACM and one dose of each antidepressant. We observed that after treatment with CACM and fluoxetine increase was statistically significant while after CACM and desipramine exposure only tendency to statistical significance
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Fig. 4. Differentiated rMSCs immunofluorescence. Cells were stained with an antibody against b-III-tubulin (green). Differences in the number of primary dendrites and branching dendrites using various differentiation schemes. Negative control (A); control, differentiation using a basic differentiation medium (B and C); long-term imipramine treatment (D); CACM treatment (E); CACM + desipramine (F and G); CACM + fluoxetine (H, I). In each experiment, the nuclei were counterstained with 40 ,6diamidino-2-phenylindole (DAPI) (blue). In our study, only cells that were positive stained and had the shape that is characteristic for neuronal cells were qualified as positive. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
was observed (Fig. 7). We also observed that in cultures that were treated with antidepressants, there was an accumulation of differentiated cells, while in control this effect was not noticed.
Fig. 5. Percentage of b-III-tubulin positive cells after differentiation using one and three doses of antidepressants. Bars represent the mean value ± S.E.M. (n = 9). Significant differences with the control are marked ⁄ for p < 0.05; t-test. Significant differences between tianeptine and each antidepressant are marked # for p < 0.05; t-test.
Fig. 6. Percentage of b-III-tubulin positive cells after differentiation using CACM and after simultaneously using CACM and an antidepressant. Bars represent the mean value ± S.E.M. (n = 9). Significant differences with the control are marked ⁄ for p < 0.05; t-test. Significant differences between CACM and CACM in conjunction with an antidepressant are marked # for p < 0.05; t-test.
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Fig. 7. Number of primary dendrites and branching dendrites per cell. Bars represent the mean value ± S.E.M. (n = 11). ⁄ Statistically significant p < 0.05; t-test or ANOVA followed by post hoc Newman–Keuls test.
4. Discussion It is well known that mesenchymal cells have a mesodermal origin and can easily differentiate into adipocytes, osteoblasts, chondrocytes, mioblasts and fibroblasts while neuronal cells have an ectodermal origin. An earlier study investigated whether OCT4 and SOX-1 gene expression was observed in mesenchymal cells (Herman et al., 2004). OCT-4 and SOX-1 are expressed in embryonic stem cells and their expression is necessary in order to maintain their pluripotency (Bylund et al., 2003; Pesce and Scholer, 2000). Sphere formation after EGF and bFGF is used in a human MSCs culture is connected with a strong decrease in OCT-4 and SOX-1 gene expression and an increase in proneuronal gene expression (Neurog 2, OTX-1), which indicates an initial differentiation to neuronal cells (Herman et al., 2004). The above-mentioned results correspond with ours. The first extremely important stage of our experiment was the neuronal induction, which should have reprogrammed the MSCs and regulated their pluripotency. In a previous experiment, we investigated whether the induction stage is the key to directing the maximal number of cells toward a specific type of differentiation (Borkowska et al., 2015) but this method is not an efficient way to achieve functional neurons. The effect that was observed is caused by the addition of bFGF whose intracerebroventricular infusion increases neurogenesis (Rai et al., 2007). Animals with lack of FGFR-1 receptor have limited abilities to neurogenesis in adulthood (Zhao et al., 2007). Furthermore, except of mitogenic properties, bFGF show differentiate properties. In following work we investigated first time how antidepressants affect the differentiation process of rMSCs. In control cultures where differentiation was done with BDNF and IGF-1 5% b-IIItubulin positive cells were observed. Previous research shown that, in in vitro culture of adult human bone marrow stromal cells where induction was done with retinol acid, BDNF increased number of b-III-tubulin positive cells (Herman et al., 2004). In another experiment, in which an induction that is typical for the dopaminergic phenotype was done (SHH + bFGF + FGF8), after BDNF addition more than 60% of Th positive cells were achieved (Trzaska et al., 2009). After antidepressants addition to differentiation cocktail 10% b-III-tubulin positive cells were observed however, mechanisms of these results is still unknown. In one of the previous experiment imipramine-treated astrocytes from adult organism were converted to neurons which presented specific neuronal proteins and neuron-like shape. Not only imipramine but also fluoxetine and venlafaxine addition were cause of cell body concentration around nucleus and it was a
beginning of branching neurons formation. In consequence neuron-like cells were come into existence from 40% flatten astrocytes cells. Newly created neurons had small, highly concentrated cell body with branching dendrites and presented some neuronal markers such as neurofilament and neuron-specific enolase, however, any notable change in the electrophysiological membrane was not observed (Cabras et al., 2010). In some of rMSCs cultures we observed Gfap gene expression and our result is compatible with others (Scintu et al., 2006; Willerth et al., 2007). We can conclude that investigated antidepressants converted early astrocytes presented in rMSCs culture to neuronal cells. In following experiment Htr2A gene expression was observed. It can suggest presence of serotoninergic neurons which are able to serotonin production. Previous researches have shown that serotonin receptors agonists affect the differentiation of MSCs into neurons (Klempin et al., 2010; Nandra and Agius, 2012). Furthermore, fluoxetine induced the proliferation and differentiation of neural progenitor cells that had been isolated from rat postnatal cerebellum and increased the expression of the serotonin transporter gene in cells (Zusso et al., 2008). The hypothesis that imipramine, desipramine and fluoxetine inhibited the production of the serotonin transporter gene, increased the serotonin concentration in a culture and as a result enhanced the differentiation of rMSCs seems too speculative. The significant increase in the percentage of neuronal cells in rMSCs after seven days of exposure to tianeptine may be connected with the fact that one of mechanisms of the action of tianeptine has an effect on neurogenesis (Uzbay, 2008). The neuroprotective properties of antidepressants on cells that were treated with such chemical substances such as LPS or MPP have been proposed in many investigations (Peng et al., 2008; Zhang et al., 2012). Antidepressants are neuroprotective against the deprivation of the N2 supplement in a culture medium (Yang et al., 2012). It seems probable that antidepressants decrease the mortality of more sensitive neuronal cells but increase their percentage from 5% to 10% at the same time. In a previous work, we observed that the number of neuronal cells increased after treatment with CACM and that simultaneous use CACM and antidepressants (except for desipramine) caused an increase in the number of neuronal cells. Other authors have observed that a hippocampal astrocyte-conditioned medium increased the number of neurons in a culture (Joannides et al., 2003); furthermore, astrocytes are able to secret endogenous substances (BDNF, NGF, NT3/4, CNTF, GDNF) that affect the differentiation of NSCs and MSCs into neuronal cells (Yang et al., 2012; Rudge et al., 1992). The endogenous substances that are secreted by cortex astrocytes create a microenvironment for differentiation that is similar to natural conditions and as a result the addition of CACM enhanced the differentiation and protection of MSCs. Nearly additive effect of the simultaneous use CACM and imipramine or fluoxetine suggests that mentioned antidepressants could play an important role in in vitro differentiation and could also play a beneficial role after neuronal cells are transplanted into an animal’s brain. In a following work, we also observe a larger number of primary dendrites and branching dendrites in the neuronal cells that had differentiated after treatment with antidepressants. Unfortunately, the limited number of cells that were compared means that further investigations are necessary.
5. Conclusions In conclusion our results suggest that (1) antidepressants improve differentiation efficiency of rMSCs and these substances may be useful in the preparation of MSCs for transplantation (2) differentiation efficiency is higher after long-term exposure to
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