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The controlled differentiation of human neural stem cells into TH-immunoreactive (ir) neurons in vitro
Neuroscience Letters 386 (2005) 105–110
The controlled differentiation of human neural stem cells into TH-immunoreactive (ir) neurons in vitro Guohua Jin ∗ , Xuefeng Tan, Meilin Tian, Jianbing Qin, Huixia Zhu, Zhen Huang, Huijun Xu Department of Anatomy and Neurobiology, The Jiangsu Key Lab of Neuroregeneration, School of Basic Medical Science, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, PR China Received 4 February 2005; received in revised form 8 March 2005; accepted 1 April 2005
Abstract The expansion of human neural stem cells in vitro might overcome the poor donor supply of human fetal neural tissue in transplantation for Parkinson’s disease. However, the differentiation of human neural stem cells into dopaminergic neurons has proven difficult. In the present study, we investigated the effects of cytokines, trophic factors of developmental striatum and Ginkgolide on differentiation of human neural stem cells (hNSCs) into TH-ir neurons. The immunoreactivity to tyrosine hydroxylase (TH), a distinctive marker for dopamine neurons was used to assess dopaminergic neuronal phenotype. We demonstrate that human neural stem cells expanded in vitro can efficiently differentiate into TH-ir neurons by induction. These stem cells might serve as a continuous, on-demand source of cells for therapeutic transplantation in patients with Parkinson’s disease. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Human neural stem cells; Dopaminergic neurons; Cytokines; Ginkgolide; Differentiation; Tyrosine hydroxylase
Parkinson’s disease (PD) is a neurodegenerative disorder in which the most predominant pathological feature is the progressive loss of mesencephalic dopaminergic neurons. Transplantation of human fetal ventral mescenphalic cells into the striatum of patients suffering from PD is emerging as a successful option in the last two decades [1,4,6,9]. There are hundreds of patients with PD who have received intrastriatal grafts of mesencephalic tissue from 6- to 9-week-old aborted human fetuses. The grafts release dopamine and become functionally integrated into the patient’s brain. Several patients have shown motor improvements after transplantation [5,7,19]. However, the application of this approach clinically has been limited, primarily because of the poor donor supply of human fetal neural tissue and numerous logistical, ethical concerns. The use of neural stem cells would overcome many of these problems. Compared with fetal tissue, the use of neural stem cells would have more significant advantages as they can readily replicate in vitro providing an almost unlimited, ∗
Corresponding author. Tel.: +86 513 5051718; fax: +86 513 5051703. E-mail address: [email protected] (G. Jin).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.065
homogenous, on-demand source of cells. However, reliably inducing the differentiation of human neural stem cells to dopaminergic neurons is far from success. Hematopoietic cytokines are known to participate in the differentiation of hematopoietic stem cells in bone marrow and an emerging research suggests that they are also present in developing brain [2,21]. Ling et al. reported that mesencephalic progenitor cells derived from rat could differentiate into DA neurons using a combination of interleukin-1 (IL-1), IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophlic factor (GDNF) [8,10]. When grafted into the striatum of DA-depleted rats, these differentiation cells can attenuate rotational asymmetry. The successful conversion of rodent neural stem cells to dopaminergic neurons has thus prompted intensive effort to develop human neural stem cells with the view to their differentiation into the dopaminergic phenotype [12]. Here the successfully controlled differentiation of hNSCs into TH-ir neurons by hematopoietic cytokines combined with trophic factors of developmental striatum and Ginkgolide in vitro is reported. Mesencephalic NSCs were obtained from an aborted human fetus of 12 weeks of gestation. After removal of
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meningeal membranes, the ventral mescenphalon (VM) tissue was cut into small pieces and washed with fresh hepesbuffered Earle’s balanced salt solution (HEBSS, Sigma). The tissue was incubated in 0.125% trypsin for 30 min, and then incubated in DNase (40 ng/ml) for 10 min at 37 ◦ C, and gently triturated into a cell suspension using a fire-polished pipette. After assessment with a hemocytometer, the cells were plated at a density of 4 × 105 cells/ml into 25 cm2 flasks containing 10 ml expansion medium which including Dulbecco’s modified eagle medium (DMEM)/F12 (1:1), a supplement of B27 (1:50, Gibco), 20 g/ml human recombinant basic fibroblast growth factor (bFGF, Sigma), 20 g/ml epidermal growth factor (EGF, Sigma) and 10 g/ml leukemia inhibitory factor (LIF, Sigma). The flasks were placed in a humidified incubator at 37 ◦ C and 5% CO2 /95% air. After floating neurospheres were formed in 2 weeks, the neurospheres were mechanically dissociated and reseeded into fresh medium. The cells were passaged every 2 weeks by gently triturating the resulting neurospheres and replating the cells under the same conditions. The BrdU incorporation assay was carried out using a BrdU labeling and detection kit (Roche) according to the manufacturer’s instructions. The striatum from E14 Sprague–Dawley rats were dissociated using trypsin, DNase and mechanical trituration. The dissociated striatal cells were plated on poly-l-lysine-coated 24-well plates at density of 2 × 105 viable cells/cm2 and were cultured in complete medium containing DMEM/F12 (1:1) and 10% fetal bovine serum (FBS, Gibco). After 2 days’ culturing, the culture medium was removed to a centrifuge tube and centrifuged at 2000 × g for 10 min and the supernatant was stored at −20 ◦ C for later use. After expansion for four passages, proliferative neurospheres were gently triturated and cultured by plating them onto 3 poly-l-lysine-coated 24-well plates at a density of 1.5 × 104 cells/well in the differentiation medium. These neurospheres were divided into 5 groups with 14 wells in each group. In IL-1␣ group, the differentiation medium contained interleukin-1␣ (IL-1␣, Sigma) (0.1 ng/ml) and 10% FBS. In united factors group, the differentiation medium contained IL-1␣ (0.1 ng/ml), 10% FBS supplemented with interleukin11 (IL-11, Sigma) (1 ng/ml), leukemia inhibitory factor (LIF, Sigma) (1 ng/ml) and glial cell line-derived factor (GDNF, Sigma) (10 ng/ml). In striatal culture medium group, the culture medium consisting of extracts of striatal culture medium, IL-1␣ (0.1 ng/ml) and 10% FBS. In Ginkgolide group, the differentiation medium contained IL-1␣ (0.1 ng/ml), 10% FBS and Ginkgolide (37.5 g/ml, China Pharmaceutical University). The control group only contained 10% FBS. The cells were cultured for 3 weeks at 37 ◦ C in a humidified atmosphere of 5% CO2 before immunostaining. Cultures were fixed in 4% paraformaldehyde, blocked with 10% normal goat serum and then incubated in primary antibodies overnight. After rinsing three times in PBS they are incubated in secondary antibody. Antibodies and dilutions were as follows: primary antibodies were rabbit polyclonal anti-Nestin 1:200 (Chemicon), rabbit polyclonal
anti-TH 1:3000 (Chemicon), mouse monoclonal anti-MAP2 1:200 (Chemicon). Secondary antibodies were goat antirabbit IgG TRITC conjugate 1:100 (Sigma), goat anti-mouse IgG FITC conjugate 1:100 (Sigma). The prior eight wells of each group were immunostained with TH, and TH/MAP-2 double-immunostaining was performed in the subsequent six wells of each group. To determine the percentage of TH-ir neurons in the cultures, the number of TH-positive neurons and the number of MAP-2-positive neurons were counted in five randomly selected microscopic visual fields per well, the total cell number were assessed under microscope in the same bright fields. Image processing was carried out for cellular analysis of cell sizes and perimeters (indirectly reflecting the branching number and the length of the processes). After a homogeneity test for variance of all data from above, analysis of variance was used for statistical analysis, and Student–Newman–Keuls for comparisons between five groups. Statistical significance was taken at P < 0.01. Data are expressed as mean ± S.E.M. The neural stem cells divided rapidly in the expansion media forming floating proliferating neurospheres after 1 week’s cultivation in vitro. The expansion procedure yielded 50–100 neurospheres per signalling of media. The spheres increased in size steadily and the cultures required passaging weekly by gently triturating the neurospheres. After four passages of expansion, the total cell number increased from 400,000 ± 2740 to 12,860,000 ± 653,000 (n = 5) (Fig. 1A–F). The proliferative cells were immunoreactive for Nestin, an intermedia filament found only in progenitor cells and reactive glia [3], and BrdU can incorporate into the neurospheres (Fig. 1G and H). After removing of mitogens and the addition of FBS, the neurospheres immediately adhered to the substrate and began to differentiate. After 3 weeks of differentiation, TH-ir cells were found in each group. In the control group, 2.15 ± 1.31 TH-ir neurons were found in each field, and the differentiational rate was 0.96 ± 0.40%, the dimensions of these cell bodies were 313.39 ± 10.39 m2 , the perimeters of these cells were 136.81 ± 15.09 m, and the percentage of TH/MAP-2 double-labeled neurons in total MAP-2-ir neurons was 5.28 ± 3.76%. In the IL-1␣ group, 11.60 ± 2.58 TH-ir neurons were found in each field, and the differentiational rate was 6.28 ± 0.69%, the dimensions of these cell bodies were 317.48 ± 10.17 m2 , the perimeters of these cells were 141.73 ± 14.38 m, and the percentage of TH/MAP-2 double-labeled neurons in total MAP-2-ir neurons was 26.82 ± 6.05%. In the united factors group, 16.30 ± 2.58 TH-ir neurons were found in each field, and the differentiational rate was 8.03 ± 1.05%, the dimensions of these cell bodies were 372.74 ± 10.60 m2 , the perimeters of these cells were 207.71 ± 13.72 m, and the percentage of TH/MAP-2 double-labeled neurons in total MAP-2-ir neurons was 42.94 ± 9.48%. In striatal culture medium group, 13.50 ± 2.31 TH-ir neurons were found in each field, and the differentiational rate was 7.05 ± 1.01%, the dimensions of these cell bodies were 373.14 ± 10.33 m2 , the perimeters of these cells were 202.04 ± 12.47 m, and
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Fig. 1. Proliferating, cloning and differentiation of human fetal mesencephalic neural stem cells in vitro. The hNSCs were proliferating when cultured in a serum-free medium supplemented with EGF, bFGF and LIF (A). Floating neurospheres of primary cultured hNSCs were formed after 2 weeks (B). A single cell from passage 2 stem cell cultures is shown 1 day after plating in 96-well plates with growth medium (C). This cell divided into two cells (D, 3 DIV) and continued to proliferate (E, 7 DIV), by 14 DIV, gave rise to a spherical clone (F). Cells in the proliferative spheres expressed Nestin (G) and BrdU can incorporate into the neurospheres (H). After differentiation, GFAP-ir astrocytes (I) and CNP-ir oligodendrocytes (J) can be seen besides neurons. Magnification bars: 20 m (A and B); 10 m (C–E), bar in (C); 20 m (F–J), bar in (F).
the percentage of TH/MAP-2 double-labeled neurons in total MAP-2-positive neurons was 35.00 ± 7.44%. In Ginkgolide group, 12.65 ± 2.37 TH-ir neurons were found in each field, and the differentiational rate was 6.61 ± 0.78%, the dimensions of these cell bodies were 386.24 ± 9.71 m2 , the perimeters of these cells were 308.91 ± 12.88 m, and the percentage of TH/MAP-2 double-labeled neurons in total
MAP-2-ir neurons was 31.18 ± 6.74% (Figs. 2 and 3). In addition to TH-ir cells, other neuronal (MAP-2-ir), astroglial (GFAP-ir) and oligodendroglial (CNP-ir) cells were also found in each cultures. Self-renewing and multipotent neural stem cells that give rise to neurons, astrocytes and oligodendrocytes have been found in the brain’s of both developing and adult rodents
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Fig. 2. Differentiation of human ventral mesencephalic neural stem cells in vitro. TH and MAP-2 double-immunostaining was performed in neural stem cells after culturing for 3 weeks on poly-l-lysine-coated coverslips. In the control group, the TH-ir neurons were few with small cell bodies and a few processes (A–C). In the IL-1␣ group, there were many more TH-ir neurons also with small cell bodies and short processes (D–F). In the united factors group, the TH-ir neurons were the most and the cells had large cell bodies and long processes (G–I). In the striatal culture medium group, there were many TH-ir neurons with large cell bodies and long processes (J–L). In the Ginkogolide group, there were also many TH-ir neurons with large cell bodies and numerous processes (M–O). All the TH-ir neurons (red in A, D, G, J, M) expressed MAP-2 (green in B, E, H, K, N, respectively). Double-positive signals are presented in (C, F, I, L, O) (yellow). Magnification bar represents 20 m, bar in (A).
[11]. Their existence has also been documented in the human brain [14,17], thus the neural stem cells could be a promising candidate for donor cells in neural transplantation. The strategy for using stem cells as donors is made up of four stages: isolation, proliferation, differentiation and finally transplantation into the brain. In order to ensure the highest success rate of transplantation, we investigated the first three stages of this strategy. The results presented in this paper showed that a high percentage of neural stem cells had been isolated from human
VM (aged 12 weeks) which were characterized by Nestin immunoreactivity. The numerous BrdU-ir cells observed in neurospheres demonstrated that active proliferation was occurring in the cultures, since the BrdU marker can only be incorporated into actively dividing cells. And the total number of stem cells was expanded 32-fold in 8 weeks in vitro by incubation in culture media containing the mitogenic factor EGF, bFGF and LIF. Production of DA neuron from rodent NSCs has been discussed fully and accurately [8,10], whereas the method and mechanisms of inducing human NSCs to
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Fig. 3. The number of TH-ir neurons (A), the differentiational rate of TH-ir neurons (B), the cell bodies’ sizes of TH-ir neurons (C), the cells’ perimeters of TH-ir neurons (D) and the percentage of TH/MAP-2 double-labeled neurons in MAP-2-ir neurons (E) in five groups. * P < 0.01, difference from control group; # P < 0.01, difference from IL-1␣ group; P < 0.01, difference from united factors group; P < 0.01, difference from striatal culture medium group. CG: the control group; IG : the IL-1␣ group; UG: the united factors group; SG: striatal culture medium group; GG: Ginkogolide group.
differentiation into DA neuron in vitro were reported in only a few papers [13,18]. Our present study showed that IL-1␣ had a evident induction effect on DA neural differentiation. The number of TH-ir cells apparently increased in the IL-1␣ group than that of the control group, but the morphology of these cells was immature with small somas and short processes. However, using IL-1␣, IL-11, LIF and GDNF as conversion factors in the media, a significantly differentiated phenotype of DA neuron was achieved, the morphologies of these
TH-ir cells were more mature, showing large somas and long processes. The results indicated that cytokines including IL1␣, IL-11, LIF and GDNF can not only induce hNSCs to differentiate into TH-ir neurons but also promote the growth of processes. The results also show that in the striatal culture medium group, the number of TH-ir cells were paralleled with the IL-1␣ group, but the cells were more mature with larger cell bodies and longer processes, which suggested that the
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developing striatum can secrete some extracellular matrix or neurotrophic factors that can facilitate the morphological development of TH-ir neurons. Extract of Ginkgo biloba (EGb) is isolated and purified by a multi-step protocol from the leaves of the Ginkgo family. The major effective ingredients of EGb are falvone glycosides (24%) and terpene lactones (6%) [16]. At present, EGb medicine is available in china and some places in the world mainly for preventing cardiovascular and neurovascular diseases. Recently, it has been reported the employment of EGb in Alzheimer’s disease (AD) with encouraging results [20]. Furthermore, our previous studies also demonstrated that Ginkgolide had neurotrophic factors effect in the development of embryonic basal forebrain neurons in culture, including the nitric oxide synthase (NOS) and acetylcholinesterase (AChE) neurons [15]. Our present study showed that in Ginkgolide group, the TH-ir neurons had largest cell bodies and longest processes, which means that Ginkgolide also had an obvious effect in promoting the development of THir neurons, especially the extending of processes. However, the exact components of Ginkgolide that can promote DA neuronal development require further studies. Considered together, these results demonstrate that selfrenewing and multipotent human neural stem cells from fetal VM can be isolated and expanded in vitro; IL-1␣ has a obvious effect in inducing mesencephalic hNSCs to TH-ir neurons. The utilization of IL-11, LIF, GDNF and IL-1␣ in concert has a cooperative effect on inducing hNSCs to differentiate into mature TH-ir neurons. Striatal culture medium can facilitate the morphological development of TH-ir neurons; Ginkgolide can obviously promote the development of TH-ir neurons, especially the growth of processes. As we know, TH is the first and rate-limiting enzyme in the procedure of dopamine synthesis, however, another important facet we need to know is whether the differentiated THir neurons are the functional dopaminergic neurons, whose apparent property is to be able to produce the neurotransmitter dopamine as well as the phenotypic characteristics.
Acknowledgement This study was supported by a grant (No. 200201) from the Jiangsu key laboratory of neuroregeneration, People’s Republic of China.
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The controlled differentiation of human neural stem cells into TH-immunoreactive (ir) neurons in vitro Guohua Jin ∗ , Xuefeng Tan, Meilin Tian, Jianbing Qin, Huixia Zhu, Zhen Huang, Huijun Xu Department of Anatomy and Neurobiology, The Jiangsu Key Lab of Neuroregeneration, School of Basic Medical Science, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, PR China Received 4 February 2005; received in revised form 8 March 2005; accepted 1 April 2005
Abstract The expansion of human neural stem cells in vitro might overcome the poor donor supply of human fetal neural tissue in transplantation for Parkinson’s disease. However, the differentiation of human neural stem cells into dopaminergic neurons has proven difficult. In the present study, we investigated the effects of cytokines, trophic factors of developmental striatum and Ginkgolide on differentiation of human neural stem cells (hNSCs) into TH-ir neurons. The immunoreactivity to tyrosine hydroxylase (TH), a distinctive marker for dopamine neurons was used to assess dopaminergic neuronal phenotype. We demonstrate that human neural stem cells expanded in vitro can efficiently differentiate into TH-ir neurons by induction. These stem cells might serve as a continuous, on-demand source of cells for therapeutic transplantation in patients with Parkinson’s disease. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Human neural stem cells; Dopaminergic neurons; Cytokines; Ginkgolide; Differentiation; Tyrosine hydroxylase
Parkinson’s disease (PD) is a neurodegenerative disorder in which the most predominant pathological feature is the progressive loss of mesencephalic dopaminergic neurons. Transplantation of human fetal ventral mescenphalic cells into the striatum of patients suffering from PD is emerging as a successful option in the last two decades [1,4,6,9]. There are hundreds of patients with PD who have received intrastriatal grafts of mesencephalic tissue from 6- to 9-week-old aborted human fetuses. The grafts release dopamine and become functionally integrated into the patient’s brain. Several patients have shown motor improvements after transplantation [5,7,19]. However, the application of this approach clinically has been limited, primarily because of the poor donor supply of human fetal neural tissue and numerous logistical, ethical concerns. The use of neural stem cells would overcome many of these problems. Compared with fetal tissue, the use of neural stem cells would have more significant advantages as they can readily replicate in vitro providing an almost unlimited, ∗
Corresponding author. Tel.: +86 513 5051718; fax: +86 513 5051703. E-mail address: [email protected] (G. Jin).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.065
homogenous, on-demand source of cells. However, reliably inducing the differentiation of human neural stem cells to dopaminergic neurons is far from success. Hematopoietic cytokines are known to participate in the differentiation of hematopoietic stem cells in bone marrow and an emerging research suggests that they are also present in developing brain [2,21]. Ling et al. reported that mesencephalic progenitor cells derived from rat could differentiate into DA neurons using a combination of interleukin-1 (IL-1), IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophlic factor (GDNF) [8,10]. When grafted into the striatum of DA-depleted rats, these differentiation cells can attenuate rotational asymmetry. The successful conversion of rodent neural stem cells to dopaminergic neurons has thus prompted intensive effort to develop human neural stem cells with the view to their differentiation into the dopaminergic phenotype [12]. Here the successfully controlled differentiation of hNSCs into TH-ir neurons by hematopoietic cytokines combined with trophic factors of developmental striatum and Ginkgolide in vitro is reported. Mesencephalic NSCs were obtained from an aborted human fetus of 12 weeks of gestation. After removal of
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meningeal membranes, the ventral mescenphalon (VM) tissue was cut into small pieces and washed with fresh hepesbuffered Earle’s balanced salt solution (HEBSS, Sigma). The tissue was incubated in 0.125% trypsin for 30 min, and then incubated in DNase (40 ng/ml) for 10 min at 37 ◦ C, and gently triturated into a cell suspension using a fire-polished pipette. After assessment with a hemocytometer, the cells were plated at a density of 4 × 105 cells/ml into 25 cm2 flasks containing 10 ml expansion medium which including Dulbecco’s modified eagle medium (DMEM)/F12 (1:1), a supplement of B27 (1:50, Gibco), 20 g/ml human recombinant basic fibroblast growth factor (bFGF, Sigma), 20 g/ml epidermal growth factor (EGF, Sigma) and 10 g/ml leukemia inhibitory factor (LIF, Sigma). The flasks were placed in a humidified incubator at 37 ◦ C and 5% CO2 /95% air. After floating neurospheres were formed in 2 weeks, the neurospheres were mechanically dissociated and reseeded into fresh medium. The cells were passaged every 2 weeks by gently triturating the resulting neurospheres and replating the cells under the same conditions. The BrdU incorporation assay was carried out using a BrdU labeling and detection kit (Roche) according to the manufacturer’s instructions. The striatum from E14 Sprague–Dawley rats were dissociated using trypsin, DNase and mechanical trituration. The dissociated striatal cells were plated on poly-l-lysine-coated 24-well plates at density of 2 × 105 viable cells/cm2 and were cultured in complete medium containing DMEM/F12 (1:1) and 10% fetal bovine serum (FBS, Gibco). After 2 days’ culturing, the culture medium was removed to a centrifuge tube and centrifuged at 2000 × g for 10 min and the supernatant was stored at −20 ◦ C for later use. After expansion for four passages, proliferative neurospheres were gently triturated and cultured by plating them onto 3 poly-l-lysine-coated 24-well plates at a density of 1.5 × 104 cells/well in the differentiation medium. These neurospheres were divided into 5 groups with 14 wells in each group. In IL-1␣ group, the differentiation medium contained interleukin-1␣ (IL-1␣, Sigma) (0.1 ng/ml) and 10% FBS. In united factors group, the differentiation medium contained IL-1␣ (0.1 ng/ml), 10% FBS supplemented with interleukin11 (IL-11, Sigma) (1 ng/ml), leukemia inhibitory factor (LIF, Sigma) (1 ng/ml) and glial cell line-derived factor (GDNF, Sigma) (10 ng/ml). In striatal culture medium group, the culture medium consisting of extracts of striatal culture medium, IL-1␣ (0.1 ng/ml) and 10% FBS. In Ginkgolide group, the differentiation medium contained IL-1␣ (0.1 ng/ml), 10% FBS and Ginkgolide (37.5 g/ml, China Pharmaceutical University). The control group only contained 10% FBS. The cells were cultured for 3 weeks at 37 ◦ C in a humidified atmosphere of 5% CO2 before immunostaining. Cultures were fixed in 4% paraformaldehyde, blocked with 10% normal goat serum and then incubated in primary antibodies overnight. After rinsing three times in PBS they are incubated in secondary antibody. Antibodies and dilutions were as follows: primary antibodies were rabbit polyclonal anti-Nestin 1:200 (Chemicon), rabbit polyclonal
anti-TH 1:3000 (Chemicon), mouse monoclonal anti-MAP2 1:200 (Chemicon). Secondary antibodies were goat antirabbit IgG TRITC conjugate 1:100 (Sigma), goat anti-mouse IgG FITC conjugate 1:100 (Sigma). The prior eight wells of each group were immunostained with TH, and TH/MAP-2 double-immunostaining was performed in the subsequent six wells of each group. To determine the percentage of TH-ir neurons in the cultures, the number of TH-positive neurons and the number of MAP-2-positive neurons were counted in five randomly selected microscopic visual fields per well, the total cell number were assessed under microscope in the same bright fields. Image processing was carried out for cellular analysis of cell sizes and perimeters (indirectly reflecting the branching number and the length of the processes). After a homogeneity test for variance of all data from above, analysis of variance was used for statistical analysis, and Student–Newman–Keuls for comparisons between five groups. Statistical significance was taken at P < 0.01. Data are expressed as mean ± S.E.M. The neural stem cells divided rapidly in the expansion media forming floating proliferating neurospheres after 1 week’s cultivation in vitro. The expansion procedure yielded 50–100 neurospheres per signalling of media. The spheres increased in size steadily and the cultures required passaging weekly by gently triturating the neurospheres. After four passages of expansion, the total cell number increased from 400,000 ± 2740 to 12,860,000 ± 653,000 (n = 5) (Fig. 1A–F). The proliferative cells were immunoreactive for Nestin, an intermedia filament found only in progenitor cells and reactive glia [3], and BrdU can incorporate into the neurospheres (Fig. 1G and H). After removing of mitogens and the addition of FBS, the neurospheres immediately adhered to the substrate and began to differentiate. After 3 weeks of differentiation, TH-ir cells were found in each group. In the control group, 2.15 ± 1.31 TH-ir neurons were found in each field, and the differentiational rate was 0.96 ± 0.40%, the dimensions of these cell bodies were 313.39 ± 10.39 m2 , the perimeters of these cells were 136.81 ± 15.09 m, and the percentage of TH/MAP-2 double-labeled neurons in total MAP-2-ir neurons was 5.28 ± 3.76%. In the IL-1␣ group, 11.60 ± 2.58 TH-ir neurons were found in each field, and the differentiational rate was 6.28 ± 0.69%, the dimensions of these cell bodies were 317.48 ± 10.17 m2 , the perimeters of these cells were 141.73 ± 14.38 m, and the percentage of TH/MAP-2 double-labeled neurons in total MAP-2-ir neurons was 26.82 ± 6.05%. In the united factors group, 16.30 ± 2.58 TH-ir neurons were found in each field, and the differentiational rate was 8.03 ± 1.05%, the dimensions of these cell bodies were 372.74 ± 10.60 m2 , the perimeters of these cells were 207.71 ± 13.72 m, and the percentage of TH/MAP-2 double-labeled neurons in total MAP-2-ir neurons was 42.94 ± 9.48%. In striatal culture medium group, 13.50 ± 2.31 TH-ir neurons were found in each field, and the differentiational rate was 7.05 ± 1.01%, the dimensions of these cell bodies were 373.14 ± 10.33 m2 , the perimeters of these cells were 202.04 ± 12.47 m, and
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Fig. 1. Proliferating, cloning and differentiation of human fetal mesencephalic neural stem cells in vitro. The hNSCs were proliferating when cultured in a serum-free medium supplemented with EGF, bFGF and LIF (A). Floating neurospheres of primary cultured hNSCs were formed after 2 weeks (B). A single cell from passage 2 stem cell cultures is shown 1 day after plating in 96-well plates with growth medium (C). This cell divided into two cells (D, 3 DIV) and continued to proliferate (E, 7 DIV), by 14 DIV, gave rise to a spherical clone (F). Cells in the proliferative spheres expressed Nestin (G) and BrdU can incorporate into the neurospheres (H). After differentiation, GFAP-ir astrocytes (I) and CNP-ir oligodendrocytes (J) can be seen besides neurons. Magnification bars: 20 m (A and B); 10 m (C–E), bar in (C); 20 m (F–J), bar in (F).
the percentage of TH/MAP-2 double-labeled neurons in total MAP-2-positive neurons was 35.00 ± 7.44%. In Ginkgolide group, 12.65 ± 2.37 TH-ir neurons were found in each field, and the differentiational rate was 6.61 ± 0.78%, the dimensions of these cell bodies were 386.24 ± 9.71 m2 , the perimeters of these cells were 308.91 ± 12.88 m, and the percentage of TH/MAP-2 double-labeled neurons in total
MAP-2-ir neurons was 31.18 ± 6.74% (Figs. 2 and 3). In addition to TH-ir cells, other neuronal (MAP-2-ir), astroglial (GFAP-ir) and oligodendroglial (CNP-ir) cells were also found in each cultures. Self-renewing and multipotent neural stem cells that give rise to neurons, astrocytes and oligodendrocytes have been found in the brain’s of both developing and adult rodents
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Fig. 2. Differentiation of human ventral mesencephalic neural stem cells in vitro. TH and MAP-2 double-immunostaining was performed in neural stem cells after culturing for 3 weeks on poly-l-lysine-coated coverslips. In the control group, the TH-ir neurons were few with small cell bodies and a few processes (A–C). In the IL-1␣ group, there were many more TH-ir neurons also with small cell bodies and short processes (D–F). In the united factors group, the TH-ir neurons were the most and the cells had large cell bodies and long processes (G–I). In the striatal culture medium group, there were many TH-ir neurons with large cell bodies and long processes (J–L). In the Ginkogolide group, there were also many TH-ir neurons with large cell bodies and numerous processes (M–O). All the TH-ir neurons (red in A, D, G, J, M) expressed MAP-2 (green in B, E, H, K, N, respectively). Double-positive signals are presented in (C, F, I, L, O) (yellow). Magnification bar represents 20 m, bar in (A).
[11]. Their existence has also been documented in the human brain [14,17], thus the neural stem cells could be a promising candidate for donor cells in neural transplantation. The strategy for using stem cells as donors is made up of four stages: isolation, proliferation, differentiation and finally transplantation into the brain. In order to ensure the highest success rate of transplantation, we investigated the first three stages of this strategy. The results presented in this paper showed that a high percentage of neural stem cells had been isolated from human
VM (aged 12 weeks) which were characterized by Nestin immunoreactivity. The numerous BrdU-ir cells observed in neurospheres demonstrated that active proliferation was occurring in the cultures, since the BrdU marker can only be incorporated into actively dividing cells. And the total number of stem cells was expanded 32-fold in 8 weeks in vitro by incubation in culture media containing the mitogenic factor EGF, bFGF and LIF. Production of DA neuron from rodent NSCs has been discussed fully and accurately [8,10], whereas the method and mechanisms of inducing human NSCs to
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Fig. 3. The number of TH-ir neurons (A), the differentiational rate of TH-ir neurons (B), the cell bodies’ sizes of TH-ir neurons (C), the cells’ perimeters of TH-ir neurons (D) and the percentage of TH/MAP-2 double-labeled neurons in MAP-2-ir neurons (E) in five groups. * P < 0.01, difference from control group; # P < 0.01, difference from IL-1␣ group; P < 0.01, difference from united factors group; P < 0.01, difference from striatal culture medium group. CG: the control group; IG : the IL-1␣ group; UG: the united factors group; SG: striatal culture medium group; GG: Ginkogolide group.
differentiation into DA neuron in vitro were reported in only a few papers [13,18]. Our present study showed that IL-1␣ had a evident induction effect on DA neural differentiation. The number of TH-ir cells apparently increased in the IL-1␣ group than that of the control group, but the morphology of these cells was immature with small somas and short processes. However, using IL-1␣, IL-11, LIF and GDNF as conversion factors in the media, a significantly differentiated phenotype of DA neuron was achieved, the morphologies of these
TH-ir cells were more mature, showing large somas and long processes. The results indicated that cytokines including IL1␣, IL-11, LIF and GDNF can not only induce hNSCs to differentiate into TH-ir neurons but also promote the growth of processes. The results also show that in the striatal culture medium group, the number of TH-ir cells were paralleled with the IL-1␣ group, but the cells were more mature with larger cell bodies and longer processes, which suggested that the
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developing striatum can secrete some extracellular matrix or neurotrophic factors that can facilitate the morphological development of TH-ir neurons. Extract of Ginkgo biloba (EGb) is isolated and purified by a multi-step protocol from the leaves of the Ginkgo family. The major effective ingredients of EGb are falvone glycosides (24%) and terpene lactones (6%) [16]. At present, EGb medicine is available in china and some places in the world mainly for preventing cardiovascular and neurovascular diseases. Recently, it has been reported the employment of EGb in Alzheimer’s disease (AD) with encouraging results [20]. Furthermore, our previous studies also demonstrated that Ginkgolide had neurotrophic factors effect in the development of embryonic basal forebrain neurons in culture, including the nitric oxide synthase (NOS) and acetylcholinesterase (AChE) neurons [15]. Our present study showed that in Ginkgolide group, the TH-ir neurons had largest cell bodies and longest processes, which means that Ginkgolide also had an obvious effect in promoting the development of THir neurons, especially the extending of processes. However, the exact components of Ginkgolide that can promote DA neuronal development require further studies. Considered together, these results demonstrate that selfrenewing and multipotent human neural stem cells from fetal VM can be isolated and expanded in vitro; IL-1␣ has a obvious effect in inducing mesencephalic hNSCs to TH-ir neurons. The utilization of IL-11, LIF, GDNF and IL-1␣ in concert has a cooperative effect on inducing hNSCs to differentiate into mature TH-ir neurons. Striatal culture medium can facilitate the morphological development of TH-ir neurons; Ginkgolide can obviously promote the development of TH-ir neurons, especially the growth of processes. As we know, TH is the first and rate-limiting enzyme in the procedure of dopamine synthesis, however, another important facet we need to know is whether the differentiated THir neurons are the functional dopaminergic neurons, whose apparent property is to be able to produce the neurotransmitter dopamine as well as the phenotypic characteristics.
Acknowledgement This study was supported by a grant (No. 200201) from the Jiangsu key laboratory of neuroregeneration, People’s Republic of China.
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