Poly-L-ornithine promotes preferred differentiation of neural stem/progenitor cells via ERK signalling pathway

Neural stem/progenitor cells (NSPCs) replacement therapies are the most attractive strategies to restore an injured brain. Key challenges of such therapies are enriching NSPCs and directing them differentiation into specific neural cell types. Here, three biomaterial substrates Poly-L-ornithine (PO), Poly-L-lysine (PLL) and fibronectin (FN) were investigated for their effects on proliferation and differentiation of rat NSPCs, and the underlying mechanisms were also explored. The results showed PO significantly increased NSPCs proliferation and induced preferred differentiation, compared with PLL and FN. Checking protein markers of several neural cell subtypes, it is showed PO significantly induced NSPCs expressing Doublecortin (DCX) and Olig2, one for neuroblasts and young neurons and the other for young oligodendrocytes. It is suggested the ERK signaling pathway was involving in this process because an ERK antagonist U0126 could inhibit PO’s effects mentioned above, as well as an ERK pathway agonist Ceramide C6 could enhance them. Given that both neurons and oligodendrocytes are the most vulnerable cells in many neurological diseases, PO-induced preferred differentiation into neurons and oligodendrocytes is a potential paradigm for NSPCs-based therapies.

Sun, T. et al. report that NSPCs on FN-coated dish, lose their proliferation potential after P6 29 . As a result, it is necessary to explore the outcomes caused by different substrates on the biological behaviors of NSPCs.
In this present study, three substrates (PO, PLL and FN) were tested for their ability to promote proliferation and preferred differentiation of NSPCs. Meanwhile, the likely underlying mechanism(s) was explored. The aim of this study is to investigate their different effects on the proliferation and differentiation of NSPCs and therefore look for a more suitable biomaterial candidate to mimic the physiological microenvironment for expanding NSPCs and directing their preferred differentiation. At the same time, try to elucidate the possible underlying mechanism(s), which might provide a favorable cell replacement strategy for basic and clinical research associated with various neurological diseases and injuries.

Results
Culture and immunofluorescence identification for NSPCs. For preparation of NSPCs, fresh tissues were dissected from neocortical tissues from E14.5 Sprague-Dawley rats. The suspended growth of neurospheres was notably observed after 3 days cultured in the neurosphere culture system (Fig. 1A). Meanwhile, cells expressing Nestin, a marker for NSPCs, reached to 50-60% of total cells in a neurosphere, which was consistent with the previous study 15 (Fig. 1B). In the adherent culture system, NSPCs also expressed Nestin (Fig. 1C, red) and Sox2 (Fig. 1D, green), another marker for NSPCs, after seeded on PO, PLL or FN (data not shown).

PO, PLL and PN showed no different effects on survival of NSPCs.
First, we evaluated the effects of PO, PLL and FN on the death/survival of NSPCs by using cell death/survival assays through flow cytometry. As shown in Fig. 2, though there was a tendency that there were more cell death with the prolonged culture time, no considerable difference was observed among PO, PLL and FN on day 3, 7 and 14 post cultured. The data suggested that these substrates have no different influence on the death/ survival of NSPCs.
PO significantly increased proliferation of NSPCs. Next, we investigated the effects of PO, PLL and FN on the proliferation of NSPCs. Here, NSPCs were cultured in the enrichment medium with 20 ng/ml bFGF and 20 ng/ml EGF according to the well-known standard method. First, CCK8 assay illustrated that the absorbance at 450nm was higher in NSPCs growing on PO on day 7 and 14 in comparison with PLL or FN (Fig. 3A), which implied that NSPCs growing on PO proliferated more vigorously than those on PLL or FN. Second, Ki-67 assay showed that the co-labeling percentage of Ki-67 and Nestin in NSPCs cultured on PO was significantly higher than that on PLL or FN on day 7 and 14 ( Fig. 3B, C). Third, western blotting data showed that the expression level of Nestin of NSPCs growing on PO, which was preferentially expressed in NSPCs, was markedly higher than that on PLL or FN on day 7 and 14 ( Fig. 3D, E). Together, these data indicated that PO has a better ability to promote the proliferation of NSPCs comparing with PLL or FN.

PO induced preferred neuronal and oligodendrocytic differentiation of NSPCs. To investigate
their effects on the differentiation of NSPCs, western blotting assays were performed. During this process, NSPCs were cultured in differentiation medium without 20 ng/ml bFGF and 20 ng/ml EGF. As shown in Fig. 4A, B, the expression of Doublecortin (DCX), which is preferentially expressed in neuroblasts and as the marker of young neurons, obviously increased in NSPCs growing on PO at all checking time points compared with PLL or FN. Their effects on glial cell fate, such as astrocyte or oligodendrocyte, were also examined. The expression level of glial fibrillary acidic protein (GFAP), which is widely used to identify astroglia in the brain, had no significant difference at all time points among different substrates (Fig. 4A, D). However, the expression of Olig2, expressed by young oligodendrocytes, significantly increased in NSPCs  markedly higher than that on PLL or FN on day 7 and 14. Blotting bands were analyzed using the Image Lab ™ software for relative density and normalized to β -actin controls. *P < 0.05, # P < 0.01, One-Way ANOVA followed by Tukey's post hoc test. N.S. indicates no significant difference.
Scientific RepoRts | 5:15535 | DOi: 10.1038/srep15535 cultured on PO on day 7 and 14, compared to PLL or FN (Fig. 4A,C). These data indicated that NSPCs cultured on PO were more likely to give birth to young neurons and oligodendrocytes, representing a better candidate directing neuronal or oligodendrocytic differentiation of NSPCs.

ERK activation involves in PO-induced preferred neuronal differentiation from NSPCs. ERK
has been reported to be implicated in the ECM-stimulated (including cell culture substrate) cell biological behaviors such as cell growth, proliferation and differentiation 30 . To unravel the potential mechanisms for preferential fate choice of NSPCs induced by PO, and whether ERK signaling pathway was involved in this process, the following tests were conducted. The activation of ERK was evaluated via measuring  quantitative results from (A). Bands were analyzed using the Image Lab ™ software for relative density and normalized to total ERK controls. # P < 0.01, One-Way ANOVA followed by Tukey's post hoc test.
Scientific RepoRts | 5:15535 | DOi: 10.1038/srep15535 ERK phosphorylation (p-ERK) in western blot assay. As shown in Fig 5A, B, the expression of p-ERK in NSPCs cultured on PO was remarkably higher than that on PLL or FN at all checking time points. To provide direct evidence for the involvement of ERK in PO-induced preferred neuronal/oligodendrocytic differentiation, NSPCs cultured on PO were treated with a specific ERK antagonist U0126 (10 μ M) or agonist Ceramide C6 (10 μ M). Immunostaining data showed that antagonist significantly reduced the number of DCX + cells (Fig. 6B, C) compared with the vehicle control. However, it had no obvious effect on number of GFAP + cells (Fig. 6B, D) or Olig2 + cells (Fig. 6B, E). Instead, with the addition of 10 μ M Ceramide C6 could enhance more differentiation of NSPCs growing on PO into young neurons obviously (Fig. 6B, C). However, there was no effect on the astroglial and oligodendrocytic differentiating capacity (Fig. 6B, D, E). Taken together, these data indicated that the activation of ERK signaling pathway might contribute to PO-induced preferred differentiation of NSPCs into neurons, but not oligodendrocytes.

Discussion
The utility of neural stem/progenitor cells (NSPCs), with the ability of self-renewal and differentiation into functional neural cells [4][5][6][7][8][9][10][11]31 , holds promise for cell therapy-based treatment of many neurological disorders such as Parkinson's disease 32 , traumatic brain injury 33 and ischemic attack [34][35][36] . How to expand NSPCs and induce preferred differentiation into specific neural cell lineage is an attractive field to date. The adherent culture system with substrate(s) could mimic the physiological microenvironment for culturing and highly efficient expansion of NSPCs. Among the widely used substrates for culturing NSPCs such as PO, PLL and FN, this study, to our limited knowledge, for the first time demonstrates that PO promotes proliferation of NSPCs and preferential differentiation into neuronal and oligodendrocytic cell types, compared with PLL or FN. Given that both neurons and oligodendrocytes are the most vulnerable cells in many neurological diseases and injuries, PO-induced preferred differentiation into neurons and oligodendrocytes is a favorable strategy for NSPCs-based therapies.
During last decades, PO, PLL and FN are the most widely used culturing substrates for investigating NSPCs 24 . They can provide a good physical support for the attachment of NSPCs, therefore benefit survival of NSPCs in culture conditions. Few studies compared their effects on the proliferation and preferred differentiation of NSPCs. However, recent findings make comparative study necessary. For example, some evidences indicate that PLL is likely to enhance inflammatory responses 25 . Furthermore, it's reported that adherent NSPCs on FN would lose their potentiality of proliferation after P6 29 . These evidence suggest that different biomaterials might have distinct effects on characteristics of NSPCs. In this study, we found PLL, FN, PO had no different effects on the survival of NSPCs, as reported previously 37,38 . Furthermore, we indeed found PO promoted the proliferation of NSPCs and preferential differentiation into neuronal and oligodendrocytic cells, compared to PLL or FN. In addition, previous studies have found that PO coating significantly increases the mechanical strength of the alginate microcapsule, which could provide a physical support for the attachment of NSPCs during cell-based therapy 24 and results in lower shear stress to increase biocompatibility 39 . While, Flanagan LA et al. revealed that human NSPCs grown on PO for 5 days were less well spread 18 . The reason of this discrepancy between the two results might be the difference in coating procedure, cell density and difference in species. However, it provides a clue for the combination of PO and laminin for the culture of NSPCs and even for the improvement of biomaterials. Together, these data suggest PO hold the potential serving as a better biomaterial candidate than PLL and FN for enriching neurons and/or oligodendrocytes from NSPCs to treating neurological disorders in the future.
Although these conventional biomaterials are widely employed, the underlying mechanisms whereby NSPCs binding to substrates and undergoing proliferation and differentiation remain open. Usually, substrates in ECM offer outside-in signals for cells mediated by an array of integrins on the cell surface. Especially, β 1 -integrin receptor (such as α 5 β 1 , α 3 β 1 and α 6 β 1 integrin complexes [40][41][42] ) has been shown to be crucial for NSPCs proliferation and neurogenesis 43,44 . However, the underlying mechanisms for how integrins transmit the signals across the cell membrane to cytoplasm remain elusive. Data in this research have shown the activation of ERK1/2 is required for PO-induced preferred neuronal differentiation from NSPCs. It has been reported that ERK1/2 is implicated in neuronal differentiation 30,45,46 . Therefore, it is needed to be further investigated how ERK1/2 acts with integrins to mediate PO signaling preferred neuronal differentiation. However, the PO-induced preferential differentiation into oligodendrocyte from NSPCs is not dependent on ERK1/2 activation, because the number of Olig2 + cells differentiated from NSPCs is not affected by the addition of ERK1/2 antagonist or agonist. This implies the mechanism whereby PO induced preferred oligodendrocytic differentiation is different from neuronal one. The mechanism(s) underlying PO-induced oligodendrocytic differentiation is not clear and needs to be determined in the future research.
In summary, our study has revealed pivotal insights into the activity of PO on NSPCs proliferation and differentiation, and might open new avenues for NSPCs-based therapies specifically promoting neuronal and oligodentrocytic differentiation in brain disorders.

Methods
Animal. This study was performed in accordance with the China's animal welfare legislation for the care and use of animals and approved by the Third Military Medical University Chongqing, China. We did our best to minimize the number of animals and decrease their suffering. E14.5 Sprague-Dawley rats were sacrificed after anesthetized with pentobarbital (60 mg/kg intraperitoneally).
Cell Proliferation Assay. Cell proliferation was assessed by Cell Counting Kit-8, that determines the cell viability in cell proliferation through WST reduction assay (Dojindo, CK04-05), which detects dehydrogenase activities of viable cells. 100 μ l of cell suspension (~10000 cells/well) was dispensed in a 96-well cell culture cluster with different pre-coated substrates, and they were incubated with 10% (v/v) WST solution for 2.5 h at 37 °C. Then, the absorbance of the culture medium at a test wavelength of 450 nm was determined using a microplate reader and a reference wavelength of 630 nm as well.
NSPCs adhered to different precoated dishes were dissociated using StemPro ® Accutase ® Cell Dissociation Reagent (Life Technologies, A1110501), and the dissociated cell suspensions were seeded in 24-well cell culture plates (2-5 × 10 5 cells/ml) for fluorescence to visualise the percentage of Ki-67 + cells as described above.
In vitro cytotoxicity assay. Lactate dehydrogenase (LDH) releasing levels were measured to evaluate cytotoxicity. LDH, released from injured cells in the medium, indicated the integrity of the cell membrane. Neurosphere culture supernatants were collected on day 3, 7 and 14. Cells were lysed with 2% Triton X-100 (Sigma, ×100) for 15 min to release all LDH from the cytoplasm after collecting the last supernatants. Cell lysis, served as positive controls, determined the maximal LDH release. LDH release was detected using the absorbance of the culture medium at a test wavelength of 450 nm with a microplate reader (Nanjing Jiancheng bioengineering institute, A020-2) according to the manufacturer's instructions and it was shown as a rate of LDH released in the medium to total cellular (n = 4).
Annexin-V Assay. Flow cytometry was used to detect Annexin-V staining via an Annexin V-FITC Apoptosis Detection Kit (Beyotime). Adherent cells dissociated using StemPro ® Accutase ® Cell Dissociation Reagent (Life Technologies, A1110501) were collected and processed in accordance with the manufacturer recommendation. After 10 minutes of incubation with Annexin V-FITC at room temperature, cells were resuspended and incubated in binding buffer. Then Propidium Iodide was added into cell suspension for 5 minutes on ice. Cells were analyzed using a FACScan flow cytometer (Becton Dickinson).

Statistical methods.
All data were showed as mean ± SEM and statistical analyses were carried out with SPSS v17.0 (SPSS Inc, Chicago, IL). Unpaired Student's t test or one-way ANOVA followed by Tukey's post hoc test was used to define statistical significance, and p < 0.05 was considered statistically significant.