EGF/bFGF Promotes Survival, Migration and Differentiation of GFP-Labeled Rhesus Monkey Neural Stem Cells Xenografted into the Rat Brain

Background: Stem cell replacement therapy is considered a promising treatment for diseases of the central nervous system. Improving the ratio of surviving transplanted cells and increasing the ratio of cells that differentiate into functional neuronal cells are the most important issues related to research on neuroregenerative medicine. Epidermal growth factor (EGF) and basic broblast growth factor (bFGF) have been reported to promote the proliferation and differentiation of neural stem cells (NSCs) in vitro, but whether they have the same effect in vivo is unclear. Methods: In this study, NSCs derived from rhesus monkey embryonic stem cells (ESCs) were resuspended in medium with or without EGF/bFGF and xenotransplanted into the rat striatum. Results: No behavioral abnormalities or teratoma formation were observed in the recipient engrafted rats. GFP-labeled cells exhibited a higher survival rate and longer migration in the EGF/bFGF group than in the control group at 2 months after transplantation. Moreover, the percentages of Tuj1 + neurons and Map2 + neurons in the EGF/bFGF group were signicantly higher than those in the control group, while the percentages of astrocytes and oligodendrocytes were signicantly lower in the EGF/bGFG group than in the control group. Conclusions: These ndings indicate that EGF/bFGF can promote protrusion of nerve bers and the survival and neuronal differentiation of transplanted NSCs in the recipient brain, suggesting that EGF/bFGF has a potential application for stem cell therapy.


Introduction
Neural stem cell (NSC) therapy holds great promise for the treatment of neurological diseases (such as traumatic brain injury, neurodegenerative diseases and spinal cord injury). Our previous studies showed that engrafted NSCs can survive, differentiate into neurons [1] and functionally integrate into the host neural circuit [2]. However, the clinical applications of NSC therapy are limited by some critical issues, such as poor survival rates, low differentiation rates, and uncontrollable and unexpected differentiation of engrafted cells in the host brain [3][4][5][6]. Interestingly, extracellular signals that originate from the local microenvironment and the presence of growth factors are bene cial for grafted NSCs at the implantation site [7].
Epidermal growth factor (EGF) and basic broblast growth factor (bFGF) are well known to be mitogenic and angiogenic agents [8,9]. NSCs from embryonic stem cells (ESCs) or the subventricular zone (SVZ) express EGF and bFGF receptors. Therefore, EGF and bFGF are needed to maintain the differentiation potential of these cells in long-term culture [10][11][12]. These two factors have also been reported to have effects on the expansion, migration and differentiation of endogenous neural progenitors in the both normal and injured brain [13][14][15]. These ndings suggest that EGF and bFGF can serve as benign extracellular factors that promote the survival and differentiation of grafted NSCs and increase the neural regeneration capacity [11,12].
However, the cellular responses to EGF and bFGF are different both in vitro and in vivo. For example, EGF and bFGF have differential and site-speci c effects on progenitor cells in vivo [16] and induce distinct activation time courses of Ras and the mitogen activated protein kinase (MAPK) cascade [17]. This evidence suggests that both EGF and bFGF might have differential effects on the fate of engrafted NSCs in the host brain and that the two factors are necessary for the essential role of engrafted NSCs in NSC therapy. These ndings provide a strong impetus for investigating the in uence of combined treatment with EGF/bFGF on the survival and differentiation of NSCs after grafting.
In the past, chronic exposure to growth factors was found to raise the risk of uncontrolled tumorigenesis, which is a signi cant side effect of clinical therapies. Given these ndings, the present study was carried out to investigate the in uence of short-term exposure to EGF/bFGF on the survival, migration, and differentiation of transplanted NSCs and the protrusion of nerve bers in the recipient brain. Mechanical passaging of undifferentiated colonies was performed every 5-7 days by cutting the colonies into large clumps using a ame-pulled Pasteur pipette 2 .
After aggregation, EBs were selected and cultured in NP media (DMEM/F12, 1× ITS-x; 2 ng/ml heparin (Sigma-Aldrich)) in a 4-well plate coated with extracellular matrix (Sigma-Aldrich) for 3 to 4 days until rosettes (NSCs) appeared. The rosettes were mechanically passaged and cultured in NP media containing 20 ng bFGF.

Preparation of cells for transplantation
On the day of transplantation, NSCs (Nestin + /Sox1 + /Pax6 + /Map2 -/Tuj1 -, Fig. 1) were trypsinized into single-cell suspensions. Following three washes in 0.01 M PBS, the cells were resuspended to a concentration of approximately 100,000 cells/μl in 0.01 M PBS or 0.01 M PBS containing EGF (Sigma-Aldrich)/bFGF. All cell suspensions were kept on ice before transplantation, and cell viability and total cell number were estimated before and after the transplantation procedure.

Animals and transplantation
Eighteen six-month-old male Sprague-Dawley rats were divided randomly into two groups. The rats were anesthetized with sodium pentobarbital (0.3% in saline, 35-45 mg/kg, intraperitoneal (i.p.)) and placed in a stereotactic device (Stoelting, United States). An incision was made along the midline to expose the skull, and a 2-mm hole was drilled. A total of 5 μl of a suspension containing 1×10 5 cells per μl of 0.01 M PBS or 0.01 M PBS with 1 ng/μl EGF/bFGF was injected into the rat striatum over a 5-min period with a 5μl Hamilton syringe. After injection, the needle was held in place for 2 minutes. The following stereotaxic coordinates were used to target the rat striatum: -2 mm anteroposterior from bregma, 4 mm mediolateral, and 6 mm dorsoventral (the schematic location is shown in Fig. 2A). Following NSC transplantation, all animals received daily i.p. injections of 10 mg/kg cyclosporine A (Sigma-Aldrich). Two months after transplantation, the rats were sacri ced by deep anesthesia with pentobarbital (100 mg/kg, i.p.) for brain tissue collection. All animals were injected i.p. with the immunosuppressant cyclosporine A at a dose of 10 mg/kg 2 days prior to transplantation and then injected with the same drug daily at a dose of 10 mg/kg after surgery until sacri ce.

Immunohistochemistry
The rats were transcranially perfused with saline followed by 4% paraformaldehyde (PFA, Aladdin), and then the brain of each rat was removed from the skull and immersed in 4% PFA for four hours. Tissues were cryoprotected in increasing concentrations of sucrose (10%, 20%, and 30%) and then cut into 20-μmthick slices on a cryostat. The sections were used for uorescence analysis with an Olympus FV1000 uorescence microscope (Olympus, Japan). Sections with GFP + cells were stained with lineage-speci c phenotype markers (Map2, β-tubulin-, O4, and GFAP) as previously reported 2 (table 1, table 2).

Quanti cation and statistical analysis of the survival and differentiation of grafted cells
Cells on every ve 20-μm-thick sections near the transplant site for a total of 20 slides per animal was counted by using ImageJ software. All statistical analyses were performed in a double-blinded manner and carried out by using GraphPad Prism 5 software. Student's t test was used to analyze the results of immunohistochemistry to determine the percentage of NSCs that differentiated into neurons in the two groups (the EGF + /bFGF + group and the EGF -/bFGFgroup). The data are presented as the means ± SEMs, and the level of signi cance was set at P < 0.05.

Results
Neural progenitor cell identi cation LYON-ES cells differentiated into neural rosettes, expanded and could be subcultured while retaining their characteristic morphological and immunocytochemical properties. These cells expressed high levels of the neuroepithelial markers Nestin (Fig. 1A), Sox1 (Fig. 1B) and Pax6 (Fig. 1C) but did not express the neuronal markers Map2 (Fig. 1D) and Tuj1 (Fig. 1E). Neural progenitor cells subsequently differentiated into neurons and glial cells. The viability of Nestin + cells used for transplantation was over 95%.

Survival and migration of the grafted NSCs
The behavior of grafted neural progenitor cells in vivo was assessed two months after transplantation.
No behavioral abnormities or teratomas were observed in either the EGF + /bFGF + or EGF -/bFGFgroup. GFP + cells in the brain sections were analyzed using a confocal laser scanning microscope to evaluate the differences in the survival of the grafted NSCs between the rats in the EGF + /bFGF + group and those in the vehicle control group. This analysis revealed that there were more GFP + cells in the EGF + /bFGF + group (Fig. 2B) than in the vehicle control group (Fig. 2C). Moreover, the GFP + cells in the EGF + /bFGF + group (Fig. 3A, C) formed more nerve bers than those in the vehicle control group (Fig. 3B, D).
Although the majority of GFP + cells in the two groups remained in situ, some engrafted cells migrated away from the sites as individual cells or clusters. The distance that the grafted cells migrated away from the implantation sites at two months after the graft in the EGF + /bFGF + group was farther than that in the vehicle control. In the EGF + /bFGF + group, some grafted cells migrated into the hippocampus (Fig. 2D) and substantia nigra (Fig. 2F), while in the control group, no GFP + cells were found in the hippocampus (Fig. 2E), and fewer cells migrated into the substantia nigra than in the EGF + /bFGF + group (Fig. 2G).
By analyzing nerve bers, we found that many nerve bers from the grafted cells (Fig. 3A) protruded into the host brain. The number of bers in the experimental group was signi cantly higher than that in the control group (Fig. 3A, B). In addition, bundles of nerve bers were observed to extend in the same direction in the EGF + /bFGF + group but not in the control group.

Differentiation of grafted NSCs
Transplanted NSCs can differentiate into cells of all three neural lineages (astrocytes, oligodendrocytes and neurons), which can be identi ed by colabeling transplanted cells with GFP and other neuronal cell type-speci c markers. For both groups, cells were labeled with neuronal markers (Tuj1 and Map2), a glia marker (GFAP) and a oligodendrocyte marker (O4), and it was found that the grafted neural progenitors differentiated into neurons (Fig. 4A, B, C, D), glial cells (Fig. 4E, F) and oligodendrocytes (Fig. 4G, H).
Detailed quantitative analysis of the phenotypes of NSCs grafted into rat brains was performed by colabeling the transplanted cells with GFP and other nerve cell type-speci c markers. The results revealed that the percentage of cells that differentiated into glial cells was higher than the percentage of cells that differentiated into neurons in both engrafted groups. A small proportion of the grafted cells were Map2 + (11.87% and 16.37% in the vehicle control and EGF + /bFGF + groups, respectively; Fig. 5). A total of 19.67% and 33.47% grafted cells were Tuj1 + -in the vehicle control and EGF + /bFGF + groups, respectively (Fig. 5).
In the vehicle control and EGF + /bFGF + groups, 44.13% and 35.23% of grafted cells, respectively, were GFAP + (Fig. 5), and some of the grafted cells were O4 + (35.23% and 20.1% for the vehicle control and EGF + /bFGF + groups, respectively; Fig. 5). Nerve cell type-speci c differentiation of the engrafted cells was signi cantly different between the two groups.

Discussion
Transplanted cells exhibit poor survival rates, low differentiation rates, and uncontrollable and unexpected differentiation, which limits the clinical applications of cell transplantation. Growth factors can regulate cell proliferation and determine cell fate. However, the effects of growth factors on exogenously transplanted NSCs in the recipient brain are complicated and depend on the speci c signaling pathways activated in the cells. In this study, the effects of short-term combined treatment with EGF/bFGF on the fate of transplanted NSCs in the recipient brain were evaluated by adding EGF/bFGF to suspensions of NSCs before transplantation. Compared with vehicle control, EGF/bFGF increased the survival rate, migration distance, and neuronal differentiation of transplanted NSCs and enhanced the protrusion of nerve bers from these cells in the rat brain.
Postmortem histological evaluation of the brains of recipient rats revealed that more GFP + cells were observed in the EGF + /bFGF + group than in the vehicle control group. This suggested that EGF/bFGF promoted the survival of the grafted NSCs. In addition, the morphology of the GFP + cells was more neuron-like in the EGF + /bFGF + group than in the vehicle control group (Fig. 3C, D). These ndings are consistent with previous studies, showing that bFGF, EGF and nerve growth factor (NGF) can promote the survival of cultured neurons in vitro [19] and the proliferation of newborn neurons in the adult rat brain in vivo [20] and transplanted progenitors in recipients [20][21][22][23][24][25]. The mechanisms underlying these effects are unclear. It is believed that the primary function of growth factors is the regulation of metabolic glucose uptake and thus the maintenance of mitochondrial homeostasis and activation of anabolic pathways required for cell growth [26].
The increased survival rate of the grafted NSCs was attributed to the combined treatment effects of EGF/bFGF in the recipient brain, which can overcome the poor survival rate of transplanted cells and promote the effect of stem cell replacement therapy [4,6,27,28].
In this study, EGF/bFGF was found to promote the migration of grafted NSCs. Some grafted cells migrated into the hippocampus and substantia nigra in the EGF + /bFGF + group, while only very few cells were found in these regions in the control group. These ndings are consistent with in vivo studies showing that EGF and bFGF induce the migration of neural progenitors in the SVZ of adult animals towards the olfactory bulb or throughout the injured brain [16,29]. The speci c mechanisms underlying this effect need to be further elucidated.
Immunohistochemical analysis showed higher expression of neuronal markers and lower expression of glial markers in the EGF + /bFGF + treatment group than in the vehicle control group, suggesting that EGF/bFGF promotes neuronal differentiation of grafted NSCs. These ndings are consistent with earlier studies showing that growth factors (e.g., EGF and bFGF) promote the neuronal differentiation of cultured stem cells, endogenous progenitors and transplanted neural progenitors [30][31][32]. The mechanisms underlying this neuronal differentiation might be associated with the MAPK/Erk signaling pathway because MAPK has been shown to be activated indirectly by extracellular growth factors [33]. It is important to note that an increase in the neuronal differentiation rate of engrafted NSCs is bene cial for stem cell therapy because neurons are more bene cial to the injured brain than glial cells. However, if transplantation is performed to improve the environment for host neurons, it may be preferable for the transplanted cells to differentiate into glial cells (due to neuroprotective and anti-in ammatory effects).
The cell survival and neuronal differentiation of transplanted NSCs in the EGF + /bFGF + group were signi cantly higher than those of transplanted NSCs in the vehicle control group. However, the speci c mechanisms involved in these effects have not been identi ed. Furthermore, it would be informative to investigate the effects of growth factors on transplanted cells in models of brain diseases.

Conclusion
EGF and bFGF can promote the survival, migration and neuronal differentiation of grafted NSCs in the host brain and may have potential therapeutic applications in stem cell therapy.  Tables   Table 1 Primary antibodies used for immunocytochemistry and immunohistochemistry    Double immunohistochemistry for quantitative analysis of different cell types 2 months after transplantation. Short-term treatment with EGF/bFGF significantly increased the percentage of transplanted NSCs that differentiated into neurons in the host brain. Map2, Tuj1, GFAP, and O4 represent mature neurons, neurons, astrocytes and oligodendrocytes, respectively. All values are represented as the mean ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001.