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Introduction

In recent decades, stem cells have attracted increasing attention due to their capacity to self-replicate or produce specific differentiated cell types and their potential as cell therapies for human disease (1). Neural stem cells (NSCs) that reside within the central nervous system (CNS) can differentiate into neurons, astrocytes or oligodendrocytes under specific conditions, which can help to develop new therapies for CNS disease (2–4). Bone marrow derived-mesenchymal stem cells (BMSCs) are multipotent stem cells that are capable of differentiating into a variety of lineages (5–7). Through stress signals, BMSCs show significant tropism for the injured site to manage the regenerative process via direct or indirect interactions (8–10). BMSCs are easily obtained and cultured and they can avoid the immune rejection through autologous transplantation. Therefore, BMSCs have emerged as ideal cellular material for cell and gene therapies in regenerative medicine (11, 12).

However, it requires more effort to achieve the aim of cell and gene therapies using NSCs or BMSCs clinically. One main unsolved problem is how to modulate NSCs or BMSCs to differentiate into the required specific types of cells, which is the premise of a safe and effective treatment. In addition, the interaction between them is not yet clear. In the present study, the Transwell co-culture system was used to mimic the in vivo environment and the differentiation of NSCs or BMSCs was observed to investigate the interaction between them.

Materials and methods

Cell culture Preparation and culture of human embryo NSCs

Primary dissociated cell cultures were prepared from the periventricular region of the telencephalon of a 15 weeks gestation, as described previously (13). A suspension of primary neural cells at a density of 5×105/ml was plated on uncoated tissue culture dishes (Corning Life Sciences, Glendale, AZ, USA) in the following growth medium: Dulbecco's modified Eagle's medium/F12 (1:1) supplemented with N2 (1:50), B27 (1:100) (Invitrogen, Life Technologies, Carlsbad, CA, USA), basic fibroblast growth factor (Millipore, Billerica, MA, USA), heparin and epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA). Medium was changed every 3–4 days. Cell aggregates were dissociated into passage culture with 0.25% trypsin/1 mmol/1 EDTA (Sigma-Aldrich) when the neurospheres were >10 cell diameters in size and replated in growth medium at a density of 5×105/ml. Cells from passages 3 to 7 were used.

Preparation and culture of human bone marrow-derived BMSCs

Bone marrow cells were harvested from volunteer donors who had provided informed consent and the study was carried out according to local ethical guidelines. Briefly, primary BMSCs were generated as previously described (14). Cultures were maintained at 37˚C in a humidified atmosphere of 5% CO2. Culture medium was replaced twice a week. BMSCs were harvested after reaching ≥90% confluence by digestion with 0.25% trypsin/1 mmol/1 EDTA and replated into passage culture at a density of 1.0×106/ml. All the BMSCs were cultured in <8 passages.

Transwell co-culture

To investigate the interaction of BMSCs and NSCs during differentiation, the Transwell chambers were used to co-culture BMSCs and NSCs and four experimental groups were established (Fig. 1). According to the experimental groups, BMSCs or NSCs were seeded onto the upper insert of a 6-well Transwell (with 0.4 µm pores; Millipore) at a density of 5×105/ml placed above the NSCs or BMSCs, with the different culture medium. After 1, 4, 7, 10 and 14 days of culture, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured in the culture medium as described below. After 14-day culture, expression of neural markers was analyzed by immunofluorescence as described below.

Figure 1. General scheme of the experimental groups. (A) Mono-culture of bone marrow derived-mesenchymal stem cells (BMSCs); (B) co-culture of neural stem cells (NSCs) and BMSCs, with BMSCs cultured in the Transwell chambers; (C) mono-culture of NSCs; and (D) co-culture of BMSCs and NSCs, with NSCs cultured in the Transwell chambers.

Identifying the differentiation by immunofluorescence

For immunofluorescence, cells cultured in the Transwell chambers were fixed with 4% paraformaldehyde and permeabilized using 0.3% Triton X-100 in phosphate-buffered saline prior to blocking in 10% normal goat serum. Subsequently, the samples were incubated with primary antibodies at 4˚C overnight. Fluorochrome-conjugated species-specific secondary antibodies were used for immunodetection. Nuclei were stained with 4′,6-diamidino-2-phenylindole (1 µg/ml; Sigma-Aldrich). The following antibodies and final dilutions were used: Rabbit anti-Nestin (1:250; cat. no. AB5922; Millipore), rabbit anti-microtubule-associated protein 2 (MAP-2) (1:200; cat. no. AB5622; Millipore) and goat anti-rabbit immunoglobulin G-cyanine 3 (1:1,000; cat. no. A10520; Invitrogen). The positive cells were counted per high-power field under a fluorescence microscope (Carl Zeiss Axiovert 200; Carl Zeiss Microscopy GmbH, Gottingen, Germany). Each sample was counted randomly in ten separated high-power fields.

Enzyme-linked immunosorbent assay (ELISA)

At the different time points of the co-culture, the media were collected and centrifuged at 10,000 x g at 4˚C for 5 min and the supernatants were stored at −20˚C. The immunoreactive levels of BDNF and NGF were measured by ELISA kits (Promega, Madison, WI, USA) according to the manufacturer's instructions. Absorbance at 450 nm was measured with a microplate reader (Bio-Rad, Hercules, CA, USA).

Statistical analysis

The results obtained were expressed as mean ± standard deviation of triplicate or quadruplicate cultures. One-way analysis of variance was performed for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results

Distribution of NSCs in human embryo brain

In the present study, t he human embryo NSCs were cultured and distributions of NSCs varied in different regions of the human embryo brain. The number of NSCs in the hippocampus was higher than other regions (P<0.05; Fig. 2).

Figure 2. Distribution of neural stem cells in human embryo brain (n=5). SVZ, subventricular zone.

Interaction between NSCs and BMSCs during differentiation

Previous studies indicated that NSCs and BMSCs alleviated the damage of brain injury as they could differentiate into neural cells following transplantation (8, 15, 16). However, the interaction between them during differentiation remains unknown. To assess the interaction, the advantage of the Transwell co-culture system was used.

NSCs and BMSCs in the co-culture system were found to differentiate into neurons. The statistical result showed that NSCs promoted BMSCs to differentiate into neurons (Fig. 3A; group A vs. group B: 36.368±7.94 vs. 42.887±6.64%; P=0.034) and NSCs (Fig. 3B; group A vs. group B: 10.368±1.83 vs. 14.775±3.33%; P=0.002). However, BMSCs had no influence on the differentiation of NSCs (Fig. 3C; group C vs. group D: 46.377±7.15 vs. 48.116±5.07%; P=0.538).

Figure 3. Differentiation of bone marrow derived-mesenchymal stem cells (BMSCs) and neural stem cells (NSCs) in different experimental groups (n=10). The ratio of (A) microtubule-associated protein 2 (MAP-2) and (B) Nestin positive cells that were differentiated from BMSCs in groups A and B; and (C) the ratio of MAP-2 positive cells that were differentiated from NSCs in groups C and D.

Secretion of BDNF and NGF in co-culture

As known, NSCs and BMSCs secrete neurotrophic factors, including BDNF and NGF (17, 18). The contribution of BDNF and NGF to a range of cell responses has been confirmed, including cell differentiation (19–21). Therefore, the level of BDNF and NGF in different experimental groups was detected. As the detection range of the ELISA kits is 7.8-500 ng/l, the concentration of BDNF and NGF at days 7, 10 and 14 was in the range (detection occurred at days 1, 4, 7, 10 and 14). The concentration of BDNF and NGF in the four groups increased with the prolonged time (P<0.05; Tables I and II). Compared to the mono-culture groups (groups A and C), a higher concentration of BDNF and NGF was observed in the two co-culture groups (groups B and D) (P<0.05; Tables I and II). In addition, there was no significant difference in the concentration of BDNF and NGF between groups B and D (P>0.05).

Table I. Concentration of brain-derived neurotrophic factor at different time points in the four experimental groups.

Table I.

Concentration of brain-derived neurotrophic factor at different time points in the four experimental groups.

Variables	7 days	10 days	14 days	F	P-value
Groups
A	8.272±0.208	9.518±0.479	10.572±0.350	45.229	<0.001
B	9.004±0.286a, b	11.046±0.551a, b	12.282±0.698a, b	47.161	<0.001
C	8.248±0.274	9.610±0.642	10.726±0.253	41.890	<0.001
D	8.763±0.438a, b	10.702±0.514a	11.838±0.575a, b	47.033	<0.001
F	6.695	9.813	14.28
P-value	0.003	0.001	0.000

a Compared to group A, P<0.05;

b compared to group B, P<0.05. Data are ng/l; n=5.

Table II. Concentration of nerve growth factor at different time points in the four experimental groups.

Table II.

Concentration of nerve growth factor at different time points in the four experimental groups.

Variables	7 days	10 days	14 days	F	P-value
Groups
A	9.808±0.329	12.146±0.865	14.852±0.502	86.139	<0.001
B	11.492±0.582a, b	14.168±0.322a, b	16.070±0.390a, b	133.383	<0.001
C	9.106±0.527	11.950±0.159	14.574±0.745	130.655	<0.001
D	10.850±0.454a, b	13.860±0.249a, b	15.828±0.451a, b	199.619	<0.001
F	24.262	27.895	9.143
P-value	0.000	0.000	0.01

a Compared to group A, P<0.05;

b compared to group B, P<0.05. Data are ng/l; n=5.

Discussion

NSCs and BMSCs possess a significant expansion and differentiation potential, placing themselves as a potential source of material for cell transplantation therapies. Previous studies identified that transplanting BMSCs and NSCs decreased the apoptosis of neurons and improved the prognosis (10, 15, 22, 23). However, the mechanisms are complicated and modulated by a variety of factors, such as cytokines, neurotrophic factors, inflammation and apoptosis (24–26). The interaction of NSCs with transplanted BMSCs and whether the interaction is beneficial to the treatment remains to be determined. Confirming these may aid in improving the understanding of the potential of cell therapy and improving the efficacy.

In the present study, the interaction between NSCs and BMSCs during differentiation was investigated and the following findings are considered: i) NSCs promoted BMSCs to differentiate into neurons and NSCs, although they do not come into direct contact; and ii) co-culture increased the level of BDNF and NGF. Similarly, certain studies also identified that NSCs induced the differentiation of BMSCs. Sanchez-Ramos et al (27) observed that the amount of neuron-specific nuclear protein-positive cells differentiated from BMSCs co-cultured with the brain tissue of fetal rat was approximately two-fold higher compared to the induced group of mono-cultured BMSCs.

In the present study, BMSCs did not affect the differentiation of NSCs; however, other studies exhibited conflicting results. Lou et al (28) reported that BMSCs induced NSCs to differentiate into more MAP-2 positive cells and Wang et al (29) also found that the interactions between MSCs and NSCs are involved in specifying neuronal fate.

Co-culture with Transwell chambers circumvents the contact of NSCs and BMSCs, however, the small soluble factors can pass through. Therefore, these small soluble factors may be involved in the interplay of NSCs and BMSCs during differentiation. NSCs and BMSCs can secrete BDNF and NGF, which play the crucial roles in cell differentiation and the concentration of the two neurotrophic factors was increased in the present study. Through binding to the tropomyosin-receptor-kinase (Trk) receptors (TrkA and TrkB), BDNF and NGF activated the downstream signaling. Previous studies indicated that BDNF and NGF modulated cell differentiation through the protein kinase B/mitogen-activated protein kinase pathway (21). BDNF has also been reported to possibly contribute to the differentiation of NSCs by triggering the Wnt/β-catenin signaling pathway (30). However, the mechanisms of BDNF and NGF in modulating the differentiation of NSCs and BMSCs requires further confirmation.

Taken together, the interaction of NSCs and BMSCs promoted BMSCs to differentiate into neurons and NSCs, which may be associated with BDNF and NGF. However, the underlying mechanism requires further clarification. To understand the interplay of NSCs and BMSCs during differentiation is likely to aid in obtaining new information for cell transplantation therapy in CNS disease.

References