Neural differentiation footprints of mesenchymal stem cell by fetal brain extract

Stem cells has brought promising hope to improve impairment in different organs especially those with limited intrinsic regeneration capacity like nervous system. With the use of mesenchymal stem cells' (MSC) capacity to differentiate toward neural cells, this study aimed to examine the potential of fetal rat brain extract (FBE) as a biological inducer to mimic natural differentiation environment.


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Keywords: mesenchymal stem cells; neural differentiation; fetal brain extract Background Stem cells have been given new hope to treat different kinds of diseases. Cell therapy, especially with the use of these cells, has been accepted as a promising therapeutic goal (1). Stem cells can differentiate into endodermal (e.g. hepatocytes) and ectodermal (e.g. neural cells) as well as mesodermal cell lineages (2). In the nervous system, neural cells lose their function due to different reasons. Impairment of the nervous system, in turn, leads to other systems malfunction (3).
The nervous system has limited ability to repair itself (1), and the current therapies have not contributed to a significant improvement in the disease status. On the other hand, Mesenchymal Stem Cells (MSCs) spontaneously express some neural markers, such as Nestin, c-Fos, and γ enolase. This shows an intrinsic predisposition to differentiate into neural cells (4). Also, MSCs migrate toward injured sites and repair the affected tissues (5). Inherent potential of neurogenesis, axon growth stimulation, and generation of different types of neural cells from precursor ones have led researchers to use them in neurological disorders (6).
Differentiation of MSCs into the neural cells for substitution in affected tissues is a probably novel and efficient approach to resolve neurological disorders symptoms 5 (7). In this context, chemical substances have been used for neural differentiation, which have resulted in satisfactory as well as unfavorable outcomes (8)(9)(10)(11). For instance, use of growth factors solely or in different combinations has shown better outcomes (12). It is suggested that that the ideal method is to provide a condition similar to natural differentiation events in the body, which will be done by the presence of all necessary cytokines and chemokines. The clinical use of MSCs needs preclinical supplementary studies, especially in animal models (13). The present study aims to investigate the potential of fetal brain extract (FBE) as a physiologic inducer to promote neural differentiation in Bone Marrow-derived MSCs (BM-MSCs).

Methods
-Fetal brain extract preparation: This study is in accordance to institutional and national guidelines and regulations. Ethics committee in research at Shiraz University approved the experiments and confirmed that all experiments conform to the relevant regulatory standards. Rattus norvegicus were obtained from Animal House of Shiraz University of Medical Sciences, Shiraz, Iran. Light, temperature, and humidity were set according to the standard conditions for animal welfare and maintenance. The animals were fed with ordinary factory-made chow and water was available ad libitum.
Pregnant rats at the late second week of gestation were transferred to the 6 laboratory. Deep anesthesia was applied by ether and then, they were euthanized by cervical dislocation. All procedures were carried out in cold condition. The fetuses were taken out of the womb, their skulls were opened, and the whole brains were removed. Brains from all fetuses were homogenized and pooled. Dulbecco`s Modified Eagle Media (DMEM, Gibco, USA) was added to the suspension (1 ml of DMEM per 150 mg of suspension) and the mixture was centrifuged at 8600g at 4ºC for 10 minutes.
The supernatant was considered as FBE and was kept at -76ºC until use.
-Establishment of BM-MSCs at passage III: Three male adult rats weighing 110-140g were chosen. They were euthanized using the method mentioned above. Femurs and tibias of both sides were taken out and floated in PBS enriched with 1% penestreptomycine. In this study, Complete Culture Medium (CCM) is considered as 88% DMEM, 10% Fetal Calf Serum (FCS), 1% penestreptomycine, and 1% fungizone. Bone extremities were cut and flushed with DMEM. Flushing was done through both sides until the bone color changed from red to pale. Centrifugation was done at room temperature (1200rpm, 15min) and the supernatant was discarded. Then, 1 ml of CCM was added to the cell pellet and mixed. Afterwards, a T25 culture flask was filled with 5 ml of CCM and the cell suspension was added 7 to the flask. The flask was then transferred to a humidified incubator (37ºC, 5% Co 2 ). After 24 hours, the medium was removed, the cells were rinsed with PBS, and the flask was refilled with fresh CCM. After 10-12 days, the flask surface reached confluency. At this stage, the cells were passaged. The medium was discarded, the cells were rinsed with PBS, and one volume -Characterization of BM-MSCs at passage III: A confluent T75 flask at P3 was prepared. The cells were harvested by Trypsin/EDTA treatment as mentioned before. After centrifugation, the cell pellet was subjected to total RNA extraction (Column RNA extraction kit, Dena Zist Asia, Iran).
Absorbance ratio (A260/A280) and electrophoresis of RNA on agarose gel were performed to evaluate RNA quantity and quality. Subsequently, cDNA 8 was synthesized, as well (Accupower cyclescript RT premix dN6, Bioneer, South Korea). Finally, Polymerase Chain Reaction (PCR) was carried out using the reagents of cinnagen company, Iran. Epidermal Growth Factor (EGF) (final concentration of 0.01 ng/µlit for each factor). All the flasks were transferred to the incubator (37ºC, 5% Co 2 ).
-Growth curves: P3 was prepared in three 24-well plates. At 70% confluency, the culture medium was substituted with specific media composition defined 9 for each group (explained in previous step) with a modification (CCM was used instead of DMEM). After 24 hours, one well of each plate was considered, its medium was removed, and cells were rinsed with PBS, and then harvested from with Trypsin/EDTA treatment. After centrifugation, 1ml of CCM was added to the cell pellet. A certain volume of the cell suspension (e.g. 7 μlit) was mixed with the same volume of trypan blue stain. The whole mixture was placed on the neubauer slide and cells number was calculated using this formula: N is the mean number of cells in four squares of the neubauer slide and T 1 is the cell number for each well (V=1ml). After calculating the cells number for two other wells (T 2 and T 3 ), the mean of T1, T2 and T3 was accounted as the cell number on the first day. These procedures were repeated for three groups for eight consecutive days.
-Expression of neural markers: Expression of neural markers was assessed at two time points. In this regard, two T75 flasks (for days three and seven) were prepared for each group at P3. At 70% confluency, the cells were exposed to the group-specific culture media. ChAT, choline acetyl transferase; GFAP, glial fibrillary acidic protein; GaLc, galactocerebridase; TH, tyrosine hydroxylase.
-Nissl staining: Cells at P3 were established in three 6-well plates. After seven days, the medium was discarded and the cells were treated with 70% ethanol. Then, the cells were rinsed with dH 2 O, exposed to giemsa stain, and kept in an incubator at 60ᵒC for two hours. The cells were rinsed with dH 2 O again and were decolorized with 0.5% acetic acid. At the end, they were fixed with 96% and 100% ethanol, respectively. Images were captured via an invert microscope.

Results
-Extraction of MSCs from BM: Given BM contains different types of cells, diverse morphologies were seen at P0 (Fig 1). This condition changed at P1 and the following passages. The majority of the cells showed spindle-shaped or fibroblastic-like forms. However, there were still some cells with different morphologies (Fig 2).  Majority of cells in group D still showed spindle-shape morphology with higher cell density compared to group C (Fig 5).

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In the group T, cells with axon and dendrite-like processes with 2, 3, 4, or even more branches had increased remarkably. Moreover, some cells had distinct elongated processes. Furthermore, cell density was higher in comparison to the group D (Fig 6). medium. There were also cells with some projections. As the appearance of these cells was not seemed healthy, the existence of projections might be related to cell shrinkage or cytoskeletal destruction. There was no significant difference in this groups' cell density on days three and seven (Fig 7). In the group D, the number of spindle-shaped cells was more than the other forms.
Aggregation of cells with elongated processes was also seen in some fields (Fig 8). Neural-like morphology were prominent in the group T at day 7. Indeed, the processes became more developed. These processes were short in some cells, but long in some others. Spindle-shaped and flattened cells were seen, as well (Fig. 9). Middle: aggregation of cells with neural-like morphology. Down: forming of long branches was considerable in some cells.

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-Growth curves: In order to evaluate the effect of FBE on cell viability, growth curves were considered and the results have been presented in Figure   10. -Expression of neural markers: Signatures of GFAP as a specific marker of astroglial cells (14) and GaLc as a specific marker of oligodendrocytes and schwann cells (2) were sought. Expression of ChAT and TH as the identifiers of cholinergic and dopaminergic neurons, respectively (15,16) was also assessed. Moreover, PCR products of the T group were sequenced in order to ensure about the identity of ampliqons. 21 GFAP expression was not significant in the study groups on days 3 and 7.
However, a footprint might be detectable in the group D and even in the group T at the second time point (Fig. 11). On day 3, expression of GaLC in the group T was approximately equal to that in the group D and both were higher compared to the group N. It seems that this marker was considerably expressed in the group T compared to the two other groups on day 7 (Fig. 12).

Discussion
To the best of our knowledge, no unique marker has been available to characterize MSCs identity yet. Therefore, a pattern of markers expression and inexpression was sought. According to the results, presence of mesenchymal markers and absence of hematopoietic ones were confirmed by RT-PCR. A few days after being in P0, fibroblastic-like or spindle-shaped cells as the typical morphology of MSCs were dominated. As no other growth stimulators than DMEM was present, low density of cells in the group C was not an unexpected event. On the other hand, cell density was higher in the group D due to employment of two growth factors in addition to the DMEM. Proliferative capability of EGF beside neural 27 differentiating potential of bFGF could be effective in this regard (16).
Interestingly, the highest cell density belonged to the T group. This indicated that FBE was more potent even than the combined effect of the two stimulators in terms of cell multiplication. Additionally, the highest and lowest cell counts belonged to T and N groups, respectively. This implies that the FBE did not induce cell death. Besides, based on growth curve analysis and PDT, the medium in the group T provided better circumstances for growth. Additionally, the injured brain extract accelerated the differentiation of fetal stem cells compared to the control group. Another investigation revealed a decrease in Oct-4 expression as a marker for stemness and an increase in nestin and MAP2 expression as markers of neural cells over 3 days. However, cell loss occurred due to apoptosis (22). In another study, MSCs derived from human umbilical cord blood were cultured in the presence of the brain tissue extract. These cells acquired a morphology similar to that of neural cells (23). Also, brain tissue extract was used in order to advance omental stem cells toward neural lineage. It was argued that the brain extract constituted nerve growth factor, BDNF, and 30 neurotrophine3/4, which could progress neural cells formation (24). Experiments have also shown the positive therapeutic effects of MSCs in neural disorders, such as Huntington's and Parkinson's diseases, via secretion of neurotrophines and anti-inflammatory cytokines (25). After transplantation of these cells into the brain in rodents or humans, they could differentiate into diverse neural cells (hippocampus, cerebral cortex, cerebellum, olfactory bulb, etc.) (26,27).

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As development of neural cells is completed before birth, whole brain-derived extract at the fetal stage was used in the present study to take its advantages for neural differentiation. Incomplete process of differentiation might have some benefits in comparison to the complete one. Stem cells that had passed the initial steps of differentiation had a much lower risk of tumorigenesis compared to preliminary stem cells (28).

Conclusions
Although efforts are being made to generate mature functional neural cells in laboratory settings, development of a fast, simple, and inexpensive method should be considered as a priority. In this regard, it is necessary to draw a clear picture of molecular and physiologic mechanisms for neural cells' generation while available models have not yielded an acceptable number of viable and differentiated cells while reliance on endogenous repertoire of MSCs does not gain desirable outcome.

Declarations
Ethics approval and consent to participate: The present study is in accordance to the declaration of Helsinki and has approved by the Ethical Committee of Shiraz University.

Consent for publication: Not applicable
Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests.
Funding: This work was financially supported by Shiraz University. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Authors' contributions: MMGS contributed substantially to the concept and design of the study. IRJ and SZ acquired the data. IRJ, DM, AM, MJZ, and AM had roles in data analysis and interpretation. IRJ drafted the manuscript. All authors read and approved the final manuscript.