A Novel and Robust Protocol for Differentiation of SH-SY5Y Neuroblastoma Cells into Neuron Like Cells

ABSTRACT

Introduction:

Human neuroblastoma cell line SH-SY5Y is a frequently used experimental cellular model in a variety of neuropsychiatric and neurodegenerative disorders. It is crucial to use a culture protocol that supports the fully differentiation of SH-SY5Y into neuron-like phenotype for the consistency of the results with neurons in vivo. However, a standardized neuronal differentiation protocol for SH-SY5Y cells still does not exist. Numerous differentiation methods have been proposed in the literature, yet SH-SY5Y cells with stronger neuronal characteristics and a more favorable environment for these differentiated cells are required in order to best representation of neurons. Therefore, in the study, we aimed to establish a more successful differentiation protocol for SH-SY5Y cells based on the primary neuron culture technique, which neuronal maturation is very well defined.

Methods:

In the study, we rearranged previous SH-SY5Y differentiation protocols, combined them with our primary neuron culture protocol and created a robust and reproducible protocol for differentiation of SH-SY5Y.

Results:

Our proposed “retinoic acid+brain-derived neurotrophic factor (RA+BDNF)-induced 7 days differentiation (conalbumin- on day 4) protocol provided well developed neurites, adequate expression and localization of neuronal and synaptic markers resembling mature neurons.

Conclusion:

The differentiation protocol we present can enable researchers to obtain satisfactory and properly differentiated SH-SY5Y cells in each independent experiment, achieving the closest possible in vivo results.

INTRODUCTION

Human neuroblastoma cell line SH-SY5Y, which is a subclone of SK-N-SH cell line obtained from the bone marrow biopsy of a four-year-old neuroblastoma patient, is widely used as cellular model for neurological diseases (1). SH-SY5Y cell line has neuroblast-like features and express a range of human-specific proteins and isoforms that are not naturally found in rodent primary cultures (2). SH-SY5Y cells are derived from the sympathetic nervous system and are considered to be derived from a neuronal lineage in its immature stage. They continuously proliferate and, express immature neuronal proteins and a limited number of neuronal markers (3).

Highlights

• An improved differentiation protocol inspired by primary neuron culture was established

• The proposed protocol provided adequate expression and localization of neuronal markers

• The protocol presented well-differentiated SH-SY5Y cells resembling mature neurons

Treatment with agents like retinoic acid (RA), phorbol ester, and dibutyl cyclic adenosine monophosphate (dbcAMP) induces SH-SY5Y cells to differentiate morphologically into various mature neuron phenotypes, such as cholinergic, dopaminergic, and noradrenergic neurons (1). Several morphological and biochemical events start arising upon neuronal differentiation, including a decrease in the proliferation rate, formation of neurites and synaptophysin-positive functional synapses, and expression of mature neuronal markers comprising neuron-specific enzymes, neurotransmitters, and their receptors. Consequently, they resemble primary neurons phenotypically (2,3). It has been reported that the differentiation markers expressed by SH-SY5Y cells may vary depending on the protocol (4).

The differentiation protocols for SH-SY5Y cells are diverse, with their own advantages and disadvantages. The most commonly used differentiation agent is RA, a vitamin A derivative with properties inhibiting growth and promoting cellular differentiation. Treating SH-SY5Y cells with a culture medium containing 1% serum and 10 µM RA drives SH-SY5Y cells toward a catecholaminergic phenotype, while using additional agents such as phorbol esters may induce a mature dopaminergic phenotype (2). Nevertheless, treatment with solely RA can only differentiate approximately 20% of SH-SY5Y cells into neurons (5). In addition to RA, application of other chemicals such as herbimycin A (herb-A), 12-O-tetradecanoyl-phorbol-13 acetate (TPA), and dbcAMP or neurotrophic factors for instance nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) can enhance neuronal differentiation of SH-SY5Y (3).

SH-SY5Y cells are an important resource since they can be handled easily in experimental conditions requiring large numbers of cells, especially for the in vitro models of neuropsychiatric and neurodegenerative diseases. Using a robust and reproducible differentiation protocol is vital for the consistency of the results with in vivo, thus cells that show more strongly neuron characteristics are needed. According to our experience in SH-SY5Y cells, the differentiation percentages and reproducibility of the best-known differentiation protocols are relatively low. Given that, we rearranged previous differentiation protocols, combined them with our primary neuronal culture protocol, and created a robust and reproducible SH-SY5Y differentiation protocol using agents that regulate neuronal maturation.

METHODS

SH-SY5Y Culture and Differentiation

SH-SY5Y cells was purchased from ATCC (ATCC, CRL-2266) and maintained in growth medium containing 1:1 mixture of Minimum Essential Medium (MEM; Gibco, 31095029) and Ham’s F-12 Nutrient Mix (Gibco, 21765029) supplemented with 10% fetal bovine serum (FBS; Gibco, 10270), 1% sodium pyruvate (Sigma, P5280–256), 1% MEM non-essential amino acids solution (Gibco, 11140050) and 1% Penstrep (Sigma, P4333) until differentiation. Differentiation was initiated when the cell confluency reached 40–50%. We developed a basal differentiation medium using a combination of components from our primary neuron culture protocol (6–8) which comprises of neurobasal medium (Gibco, 12348017) containing 1:50 B27 supplement (Gibco, 17504044), 9% sodium chloride (NaCI₂; Sigma, S3014), 1% L-glutamine (Gibco, 25030–024), 100 mM putrescine (Sigma, P7505), 40 µM progesterone (Sigma, P6149), 40 µg/ml insulin (Gibco, 12585014), 20 mg/ml conalbumin (Sigma, C7786) and 1% FBS. RA (Sigma, R2625) and/or BDNF (Sigma, B3795) differentiation agents were also applied with basal differentiation medium. Differentiation was done with four distinct conditions to determine which one is the most effective: basal differentiation medium, basal differentiation medium containing 10 µM RA, basal differentiation medium containing 50 ng/ml BDNF, and basal differentiation medium containing both 10 µM RA and 50 ng/ml BDNF. Differentiation duration was tested for 4 and 7 days. In the 7-day long differentiation experiments, the media of all groups was replaced with serum-free differentiation media on day 4 to limit cell proliferation. Additionally, on the 4^th day of differentiation, conalbumin was either removed or not from differentiation media. Experimental design was given in Figure 1. Morphology of differentiated SH-SY5Y cells were assessed by phase-contrast microscopy and neuronal characteristics of the differentiated cells were followed by Tau (neuronal cell marker), microtubule-associated protein 2 (MAP2; neuronal cell marker), tyrosine hydroxylase (TH; catecholaminergic neuron marker) and synaptophysin (synaptic marker) expressions. Since a cell line was used in the study, ethical committee approval was not required. The study was conducted in accordance with the Helsinki Declaration.

Figure 1.

Experimental design (Created with BioRender. com).

Immunofluorescent Labeling of Differentiated SH-SY5Y Cells

On day 4 and day 7, differentiated cells were fixed with 3.7% paraformaldehyde for 20 minutes and then blocked with 30% normal goat serum in 0.02% T-PBS for one hour at room temperature. The cells were incubated overnight at +4°C with primary antibodies for Tau (Thermo Fisher Scientific, 13–6400, dilution: 1/1000), MAP2 (Thermo Fisher Scientific, MA5–12826, dilution: 1/500), TH (Thermo Fisher Scientific, 701949, dilution: 1/75) and synaptophysin (Thermo Fisher Scientific, MA5–14532, dilution: 1/100). The cells were then incubated in dark for 1 hour at room temperature with corresponding secondary antibodies labelled with Alexa Flour 488 (Thermo Fisher Scientific, A11034, dilution: 1/200) and Alexa Flour 568 (Abcam, ab175473, dilution: 1/200). Overlay images obtained using Lionheart FX Automatic Fluorescent Microscope with Gen5 software (BioTek, Winooski, USA). Florescence intensities was used for corrected total cell fluorescence (CTCF) analysis. At least ten images of random areas from each group were analyzed for target protein expression. CTCF was calculated with the formula CTCF=integrated density − (area of selected cell×mean fluorescence of background readings) as previously described (9).

Statistical Analysis

CTCF values were compared using GraphPad InStat DTCG 3.06 (GraphPad Software, Inc. San Diego USA). Comparisons were performed depending on the data distribution and the difference between the SDs. Comparisons were done with one-way ANOVA followed by Tukey Kramer Multiple Comparison test where data normally distributed or with Kruskal-Wallis followed by Dunn’s Multiple Comparison test where data were not normally distributed. All data are presented as the means (SD). p<0.05 were considered statistically significant.

RESULTS

Morphological Analysis

Undifferentiated SH-SY5Y cells had polygonal soma with few short processes. SH-SY5Y cells that differentiated for 4 days with any condition presented small rounder soma and much longer processes than the undifferentiated cells. There was not any remarkable difference between the groups. When differentiation was continued for 7 days, we observed that neuronal processes gradually retracted and the morphology of the cells shifted to a spindle-like shape after the 4th day of the differentiation. Thereupon, we tried to find out what lead to this by removing one component from the medium at a time and revealed that it was due to conalbumin. Therefore, we continued the experiments with two distinct 7-day differentiation protocols; one was carried out in the presence of conalbumin for 7 days, and the other was performed by removing conalbumin on the 4th day. Phase-contrast imaging showed that SH-SHY5Y cells differentiated with “RA+BDNF-induced 7 days differentiation (conalbumin- on day 4)” protocol exhibited more similar appearance to mature neuron morphology compared to the cells differentiated with other protocols, since they had multipolar neuron-like soma, multiple long and branched neurites and connections resembling neural network (Figure 2).

Figure 2.

Phase contrast micrographs of different differantiation protocols (Magnification: 10×).

Neuronal Characteristics

Characteristics of differentiated cells were evaluated by immunofluorescent labeling of neuronal markers Tau, MAP2, synaptophysin and dopaminergic neuron marker TH. In the cells differentiated for 4 days, Tau expression was higher in RA+BDNF group than other groups (p<0.05, p<0.001, p<0.001, respectively), and MAP2 expression was higher in both basal differentiation medium and RA+BDNF groups compared to BDNF group (p<0.001, p<0.001, respectively). Basal differentiation medium group had increased TH expression than RA and RA+BDNF groups (p<0.05, p<0.05, respectively). Moreover, synaptophysin was mostly localized in nucleus and less in cytoplasm in basal differentiation medium and BDNF groups, whereas RA group had only nuclear localization and RA+BDNF group had high cytoplasmic, low nuclear and neurite localization of synaptophysin (Figure 3).

Figure 3.

a–c. Neuronal characteristics of 4 days differentiated SH-SY5Y cells: Immunofluorescent labeling of tyrosine hydroxylase (TH) (green), tau (red) and DAPI (blue) (a). Immunofluorescent labeling of synaptophysin (green), MAP2 (red) and DAPI (blue) (b). Corrected total cell fluorescence analysis of TH, Tau and MAP2 (c). *p<0.05, **p<0.001

When the cells differentiated with 7 days differentiation (conalbumin+) protocol, Tau expression was higher in BDNF group compared to the other groups (p<0.001, p<0.001, p<0.001, respectively), while MAP2 expression did not differ between the groups. Tyrosine hydroxylase expression was increased in basal differentiation group than RA and RA+BDNF groups (p<0.001, p<0.05, respectively), and in BDNF group than RA group (p<0.01). Synaptophysin had a high cytoplasmic, low nuclear localization in all groups (Figure 4).

Figure 4.

a–c. Neuronal characteristics of SH-SY5Y cells differentiated with 7 days differentiation (conalbumin+) protocol: Immunofluorescent labeling of tyrosine hydroxylase (TH) (green), tau (red) and DAPI (blue) (a). Immunofluorescent labeling of synaptophysin (green), MAP2 (red) and DAPI (blue) (b). Corrected total cell fluorescence analysis of TH, Tau and MAP2 (c). *p<0.05, **p<0.001

In the cells differentiated with 7 days differentiation (conalbumin- on day 4) protocol, the expression of Tau did not differ in groups. However, MAP2 expression was found to be higher in basal differentiation medium group than the other groups (p<0.001, p<0.001, p<0.05, respectively) and in RA+BDNF group compared to BDNF group (p<0.001). Tyrosine hydroxylase expression was decreased in RA group than basal differentiation medium and BDNF groups (p<0.001, p<0.001, respectively) and in RA+BDNF group compared to BDNF group (p<0.05). Basal differentiation medium and BDNF groups had high nuclear, low cytoplasmic localization of synaptophysin, whereas RA group had a high cytoplasmic, low nuclear and neurite localization. In RA+BDNF group synaptophysin mostly localized in cytoplasm and neurites and less in nucleus (Figure 5).

Figure 5.

a–c. Neuronal characteristics of SH-SY5Y cells differentiated with 7 days differentiation (conalbumin- on day 4) protocol: Immunofluorescent labeling of tyrosine hydroxylase (TH) (green), tau (red) and DAPI (blue) (a). Immunofluorescent labeling of synaptophysin (green), MAP2 (red) and DAPI (blue) (b). CTCF analysis of TH, Tau and MAP2 (c). *p<0.05, **p<0.001

When the expressions of neuronal markers were compared between protocols, Tau was found to be lower in the cells differentiated with 7 days differentiation (conalbumin+) protocol in basal differentiation medium, RA and RA+BDNF groups compared to the other protocols. Yet in BDNF group, the cells differentiated with 7 days differentiation (conalbumin- on day 4) protocol had lower Tau expression. MAP2 was higher in the cells differentiated with 7 days differentiation (conalbumin- on day 4) protocol in basal differentiation medium and RA+BDNF groups, while lower in the cells differentiated with 4 days differentiation in BDNF group compared to the other protocols. Furthermore, TH expression was decreased in the cells differentiated with 7 days differentiation (conalbumin- on day 4) protocol in basal differentiation medium, RA and RA+BDNF groups (Figure 6).

Figure 6.

a–d. The comparison of the expression patterns of neuronal markers in different protocols: Comparision of TH expression levels in different treatment periods (a). Comparision of tau expression levels in different treatment periods (b). Comparison of Map2 expression levels in different treatment periods (c). The sizes of nuclei was measured by Gen5 software from micrographs in Lionheart Cell Imaging System. In representetive micrographs red represents tau and blue is nucleus dye (DAPI) (d1–3). *p<0.05, **p<0.01, ***p<0.001

Additionally, morphological analyses showed that the cells with smaller nuclei expressed high levels of neuronal markers. When the groups were evaluated with this approach, the proportion of cells with small nuclei was found to be relatively lower (71%) in the “RA+BDNF-induced 7 days differentiation (conalbumin- on day 4)” protocol. However, despite this relatively lower number of cells with smaller nuclei, the differentiated cells had high neuronal markers expression, except for TH. The immunofluorescent labeling results of each experimental condition were summarized in Table 1. Based on the significant expression levels of tau and MAP2, high peripheric and neurite localization of synaptophysin, and the morphological analysis, the most appropriate differentiation protocol was selected as “RA+BDNF-induced 7 days differentiation (conalbumin- on day 4)” (Table 2).

Table 1.

Comparison of the protocols according to expression pattern and morphological analysis

Groups Differentiation periods	Basal differentiation medium	RA	BDNF	RA+BDNF
Differentiation duration: 4 days	High tyrosine hydroxylase expression Slightly higher tau expression Moderate MAP2 expression High nuclear, low cytoplasmic localization of synaptophysin ωCell count=278±33 *82% of cells have smaller nuclei	Moderate tyrosine hydroxylase expression Moderate tau expression Moderate MAP2 expression Nuclear localization of synaptophysin ωCell count=257±51 *68% of cells have smaller nuclei	Moderate tyrosine hydroxylase expression Moderate tau expression Low MAP2 expression High nuclear, low cytoplasmic localization of synaptophysin ωCell count=173±26 *88% of cells have smaller nuclei	Moderate tyrosine hydroxylase expression Extremely high tau expression Moderate MAP2 expression High cytoplasmic, low nuclear and neurite localization of synaptophysin ωCell count=102±18 *66% of cells have smaller nuclei
Differentiation duration: 7 days (Conalbumin+)	High tyrosine hydroxylase expression Low tau expression Moderate MAP2 expression High cytoplasmic, low nuclear localization of synaptophysin ωCell count=433±83 *83% of cells have smaller nuclei	Moderate/Low tyrosine hydroxylase expression Low tau expression Moderate MAP2 expression High cytoplasmic, low neurite localization of synaptophysin ωCell count=349±105 *65% of cells have smaller nuclei	Moderate tyrosine hydroxylase expression Slightly higher tau expression Moderate MAP2 expression High cytoplasmic, low nuclear localization of synaptophysin ωCell count=780±129 *86% of cells have smaller nuclei	Moderate tyrosine hydroxylase Low tau expression Moderate MAP2 expression High cytoplasmic, low neurite localization of synaptophysin ωCell count=354±43 *80% of cells have smaller nuclei
Differentiation duration: 7 days (Conalbumin- on day 4)	Moderate tyrosine hydroxylase expression Moderate tau expression Extremely high MAP2 High nuclear, low cytoplasmic localization of synaptophysin ωCell count=762±170 *94% of cells have smaller nuclei	Extremely low tyrosine hydroxylase expression Moderate tau expression Moderate MAP2 expression High cytoplasmic, low nuclear and neurite localization of synaptophysin ωCell count=344±71 *96% of cells have smaller nuclei	Moderate tyrosine hydroxylase expression Moderate/Low tau expression Low MAP2 expression High nuclear, low cytoplasmic localization of synaptophysin ωCell count=392±70 *98% of cells have smaller nuclei	Low tyrosine hydroxylase expression Slightly higher tau expression High MAP2 expression High cytoplasmic and neurite, low nuclear localization of synaptophysin ωCell count=311±26 *71% of cells have smaller nuclei

ω The cell count was determined using 10x magnification micrographs by Gen5 software.

^*The morphological analyses showed that the cells with smaller nuclei express high levels of neuronal markers (Figure 5). Because of that the nuclei size was used as an evolution parameter. The sizes of nuclei were measured by Gen5 software. The upper levels of 95% confidence intervals (CI) of nuclei sizes were designated as the cutoff values in order to determine the percentage of bigger nuclei which sizes higher than the cutoff values. The higher level of the 95% CI of the nuclei sizes of 4 day-long differentiated groups was designated as the cutoff value. According to the measurements smaller nuclei than the size 17.032 mm was accepted as a possible marker for differentiation. BDNF: Brain-derived neurotrophic factor; MAP2:microtubule-associated protein 2; RA: Retinoic acid.

Table 2.

RA+BDNF-induced 7 days differentiation (conalbumin- on day 4) protocol

Initial Culture Condition
1. Seed SH-SY5Y cells into a 6-well plate containing warm (37°C) growth medium with 10% confluence.
2. Change growth medium every 2–3 days.
3. When the cell confluency reaches 40–50% begin differentiation procedure.
Differentiation Procedure
1. Prepare basal differentiation medium composed of neurobasal medium containing 1:50 B27 supplement, 9% NaCI2, 1% L-glutamine, 100 mM putrescine, 40 µM progesterone, 40 µg/ml insulin, 20 mg/ml conalbumin and 1% FBS.
2. Add 10 µM RA and 50 ng/ml BDNF to the basal differentiation medium after filtration.
3. Discard growth medium and add warm basal differentiation medium containing RA and BDNF onto plated cells.
4. Incubate the cells at 37°C in the incubator supplied with 5% of CO2.
5. On day 4, prepare fresh basal differentiation medium without FBS and conalbumin. Add 10 µM RA and 50 ng/ml BDNF after filtration.
6. Discard old differentiation medium and add newly prepared one onto the cells.
7. Incubate the cells at 37°C in the incubator supplied with 5% of CO2 until day 7.

BDNF: Brain-derived neurotrophic factor; FBS: Fetal bovine serum; RA: Retinoic acid.

DISCUSSION

Researchers have generally limited access to central nervous system cells. Therefore, neural stem cells, primary neuronal cells or immortalized cell lines are usually preferred for in vitro studies of neuroscience. Yet, the low number of differentiated cells obtained from human neural stem cells is always a problem. In addition, there is the issue of species used for primary neuronal cultures since they are not human origin. With these limitations, there is still a need for human-derived immortalized cell lines, especially in experimental setups that require large amounts of cells. Human neuroblastoma SH-SY5Y cells are the most widely used cell lines in this regard since they express immature neuronal markers and can be differentiated into neuron-like cells. They usually express several mature neuronal markers depending on the differentiation protocol (10–12).

RA is the most widely used agent for differentiation of SH-SY5Y cells. It is stated that RA can enable SH-SY5Y cells to acquire neuron-like morphology, biochemical and electrical properties and promotes a cholinergic phenotype (13,14). Some studies have demonstrated that using additional agents such as neurotrophic factors like BDNF and NGF enhances the differentiating properties of RA and induces higher neuronal morphological characteristics and neuronal markers (15,16). BDNF enhances neurogenesis, promotes survival and growth of neurons and is involved in synaptic plasticity (17). Most of the current protocols in the literature consider using only one or two of these differentiation agents.

In the studies that use SH-SY5Y cells as neuronal model, it is crucial to establish and validate a culture protocol that supports the fully differentiation of the cells into neuron-like phenotype and provides more suitable environment for these differentiated cells. Neuronal maturation, which includes processes of neurite elongation and synapse formation, is a complex process and has been well defined in primary neuron culture technique that our group has also applied for many years. Recent studies have shown that SH-SY5Y cells cultured in primary neuron or neural stem cell culture medium, such as neurobasal medium with B27 supplement, in addition to RA treatment had further improved neuronal differentiation (5). Given that, we combined previous differentiation protocols with our primary neuron culture protocol (6–8), and created a new differentiation protocol using the agents regulate neuronal maturation, such as neurobasal medium, B27, putrescine, progesterone, insulin and conalbumin.

Media supplemented with insulin, transferrin, selenium, putrescine, or pyruvate are preferred for primary neuronal cultures to promote healthy neuronal outgrowth and differentiation (18–20). B27 supplemented neurobasal medium, the most commonly used culture medium for primary neuron cultures, supports the survival and growth of neurons (21,22). Putrescine is a polyamine plays a vital role in cell growth. Polyamines control neuronal differentiation and promote axon regeneration (23,24). Progesterone is a gonadal steroid hormone can regulate neurite outgrowth and survival, differentiation, plasticity, and regeneration (25,26). Insulin is essential for axonal growth and neuronal survival (27,28). Conalbumin belongs to transferrin iron-binding glycoproteins family. Studies have shown that transferrin is related to antibacterial activity and the regulation of iron absorption, immune response, anti-inflammatory response, antioxidant properties, and neurotrophic effects (29,30). Aizenman and de Vellis have shown in vitro growth requirement for transferrin of cortical neurons and its importance in neuronal survival (20).

Our “RA+BDNF-induced 7 days differentiation (conalbumin- on day 4)” protocol provided high number of multipolar neuron-like cells with smaller soma, well-developed neurites, adequate expression of neuronal markers and neurite localization of synaptophysin. These characteristics indicate that differentiation induced functional properties resembling mature neurons. In our laboratory, “RA+BDNF-induced 7 days differentiation (conalbumin- on day 4)” protocol is routinely used (repeated at least in 20 independent experiments) (31, 32). In each experiment, the cells showed consistent marker expressions and morphological properties. The differentiation protocol we have presented may enable researchers to differentiate SH-SY5Y cells satisfactorily and uniformly in each independent experiment. However, the protocols that provide higher TH expression and peripheral localization of synaptophysin can also be selected according to the desired cell characteristics or experimental setup.