Cell Biochem Biophys DOI 10.1007/s12013-014-0222-z
ORIGINAL PAPER
Differentiation of Mesenchymal Stem Cells into Neural Stem Cells Using Cerebrospinal Fluid Wei Ge • Chao Ren • Xin Duan • Deqin Geng Caiyi Zhang • Xiaoyun Liu • Hao Chen • Meirong Wan • Runlu Geng
•
Ó Springer Science+Business Media New York 2014
Abstract Optimization of a methodology for mesenchymal stem cells (MSCs) differentiation into neural stem cells (NSCs) using cerebrospinal fluid (CSF). MSCs were extracted from umbilical cord blood from healthy, fullterm, newborn infants and from the bone marrow of patients. CSF was taken from healthy adult volunteers and patients. Four groups investigated were: A (n = 8) cord blood MSC induced with healthy volunteer CSF (control group); B (n = 7): patient MSCs induced with health volunteer CSF; Group C (n = 12): patient MSCs induced with their own CSF; group D (n = 6): cord blood MSCs induced with patient CSF. Following induction, cell differentiation state was examined using microscopy, flow cytometry, and immunohistochemistry. There were significantly more clinically applicable MSCs in Groups B and C
than groups A and D (P \ 0.05) and Group B had significantly more clinically applicable MSCs than group C (P \ 0.05). The presence of NSCs was as with the MSCs. Group B had significantly more clinically applicable NSCs than all of the other groups. In addition, group B cells grew significantly faster than the other groups (P \ 0.05). Upon CSF induction, MSCs differentiated into NSCs suitable for clinical treatment. The source of the MSCs and/or CSF influenced the number of NSCs produced and the NSC growth rate. Thus, the source of MSCs and CSF should be considered before initiating a stem cell clinical treatment. Keywords Marrow Cord blood Induction Neural stem cells Differentiation
Introduction Wei Ge and Chao Ren are Co-first author. W. Ge (&) D. Geng (&) C. Zhang H. Chen R. Geng Department of Neurology, Affiliated Hospital of Xuzhou Medical College, 99 Huaihai Road West, Xuzhou 221002, Jiangsu, China e-mail: [email protected] D. Geng e-mail: [email protected] C. Ren Department of Neurology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University Medical College, Yantai 264000, Shandong Province, China X. Duan Diagnostic Radiology Center, The Cancer Hospital of Xuzhou, Xuzhou 221005, China X. Liu M. Wan Central Laboratory, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China
It is widely accepted that nerve cells are unable to repair themselves if they experience trauma, necrosis, or degeneration. This makes treating diseases of the central nervous system (CNS) over the long-term difficult. Clinical use of stem cells has been postulated as one way of overcoming these issues. Mesenchymal stem cells (MSCs) are considered as one of the most useful types of stem cells that can be used in a clinical setting [10]. MSCs possess the potential for multilineage differentiation and self-renewal can be easily sourced and have fewer ethical considerations surrounding them than embryonic stem cells. It is essential to determine an efficient and practical method for inducing MSCs differentiation into neural stem cells (NSCs), for both clinical and basic research needs. This laboratory has previously published MSC differentiation methods [9, 20, 21] and has applied for a patent for a MSC induction methodology (Patent Application Number 2008100200943). This current
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investigation examined the induction of human MSCs (from umbilical cord and bone marrow) to differentiate into NSCs using cerebrospinal fluid (CSF), with the aim of optimizing a technique that produces a sufficient number of NSCs that will meet the requirements for subsequent clinical treatment.
Materials and Methods Specimens CSF samples were taken from healthy adult volunteers and patients, while bone marrow was only taken from patients. Cord blood was taken from healthy, full-term, newborn infants. The hospital medical ethics committee approved this study. Experimental Design Four groups were used in the study: A (n = 8) comprised cord blood MSCs from healthy newborn infants that were subsequently induced with CSF from healthy volunteers; B (n = 7) involved bone marrow MSCs from patients that were induced by CSF from healthy volunteers; C (n = 12) employed bone marrow MSCs from patients that were induced with the patient’s own CSF; and D (n = 6) investigated cord blood MSCs from healthy newborn infants induced with CSF from patients. There were no significant differences in sex ratio or age between the groups (P [ 0.05). Separation, Cultivation, and Induction of MSCs The posterior superior iliac spine was the puncture site for bone marrow cell extraction. Under aseptic conditions, bone marrow (20 mL) was obtained from patients. Cord blood (20 mL) was taken from healthy newborn infants under aseptic conditions. Samples were mixed with heparin (10,000 U/L) and 20 mL of phosphate-buffered saline (PBS), centrifuged (2,5009g, 15 min) followed by removal of fat and supernatant. Cells were resuspended in an equal volume of Percoll in PBS and centrifuged (2,5009g, 25 min). Cells were washed 3 times in PBS (10 mL) and resuspended in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with 10 % fetal bovine serum (FBS; HyClone, Logan, Utah, USA). The cell suspension was seeded into uncoated, T25 culture flasks (BD FalconTM, Franklin lakes, New Jersey, USA) at a concentration of 1 9 106 cells/mL and cultures were then incubated at 37 °C/ 5 % CO2. These cells were designated as the primary generation (passage 0, P0). After 3 days, half of the culture media was changed, and 5 days later the entire culture media was changed. When cultures were 90 % confluent, they were harvested with 1 mL of 0.25 % trypsin (HyClone, Logan,
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Utah, USA) and passaged at a 1:3 dilution. This process was continued until P5. For differentiation into NSCs, MSCs from the four groups at each passage (P1–5) were digested with 1 mL of 0.25 % trypsin (HyClone, Logan, Utah, USA) and were seeded at a density of 2 9 106 cells/well (0.1 mL) on poly-L-lysine-coated (100 mg/mL; Sigma, St. Louis, Missouri, USA) coverslips in 6-well plates. Following digestion, cells were incubated in 2 mL of DMEM/F12 serum-free medium for 24 h; after 72 h, 10 lL of CSF from the appropriate source for that group was added to the culture medium every 24 h over a 72-h period. When the cell concentration reached 1 9 107 cells/mL, NSCs were harvested for clinical transplantation treatments. Identification of MSCs Cell growth and morphology of the primary MSCs and those from P1–5 were observed. Images were acquired at 25 °C using an inverted phase-contrast microscope (Moticam 3000; Motic Group Co., Ltd.). The differentiation of MSCs was examined after induction for 6, 48, and 72 h. Antigen expression was examined in separate experiments, in triplicate, as previously described, with some modifications. Cells on coverslips were fixed for 30 min with 4 % paraformaldehyde and 0.3 % glutaraldehyde, and then washed 3 times with PBS. Cells were incubated with a monoclonal mouse anti-human b-tubulin antibody (1:500 dilution; Wuhan Boster Biological Technology, Ltd., China) or a polyclonal rabbit anti-human GFAP antibody (1:1000; Wuhan Boster Biological Technology, Ltd.) at 4 °C overnight. They were then washed 3 times with PBS and incubated with either a FITC-conjugated goat antirabbit IgG (1:500; Wuhan Boster Biological Technology, Ltd.) or a TRITC-conjugated goat anti-rabbit IgG (1:500; Wuhan Boster Biological Technology, Ltd.) for 30 min at 25 °C. Following this incubation with secondary antibodies, cell were washed 3 times with PBS and then observed under a microscope (LEICA TCS-SP2, Germany LEICA Inc.). As a control, PBS was used to replace the primary antibody. Flow Cytometry P2 cells were induced for 7 days then digested with 0.25 % trypsin. Cells were resuspended to a concentration of 1 9 107 cells/mL and incubated with TRITC-conjugated anti-human CD34, CD44, and CD90 developed in goat or mice (Wuhan Boster Biological Technology, Ltd.) for 30 min at 25 °C. Cells were washed twice with PBS, and at least 400 lL of PBS was added to cells and allowed to incubate at 4 °C. Surface markers were identified by flow cytometry within 24 h of staining. As a control, cells were mock-treated with PBS in place of the primary antibody.
Cell Biochem Biophys
Fig. 1 Growth and changes in passage 0 of MSCs at (9100 magnification). Cells cultured for: a 24 h b 48 h c 72 h d 7 days e 10 days of bone marrow-derived MSCs. Cells cultured for: f 24 h g 48 h h 72 h i 14 days j 21 days of cord-derived MSCs
Cell Quantification Cells at P1 were induced after 3 days, digested with trypsin, and washed 3 times with 10 mM PBS. Cells were then resuspended in 100 lL of PBS and counted using a handheld automated cell counter (Millipore Corporation, USA). Statistical Analysis SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used to assist with statistical analysis of our results. Values were expressed as the mean ± standard deviation ðx sÞ. For comparison among the groups, we used a one-way analysis of variance (ANOVA) where a P value \0.05 was considered as statistically significant.
morphology of the MSCs in all four groups were similar, forming large colonies with 80–100 % fusion. Cells were passaged every 5 days (Fig. 1). After 24 h of induction, the NSCs from all groups displayed similar growth rate and morphological characteristics. After 3 days, a number of neurites were visible, and the cells gradually formed tapered, triangular, and irregular shapes. The cell soma at day 7 was similar to the astrocyte dendrite and axon-like structures. Cells were further elongated, filamentous and reticular as they continued to be cultured. The soma of cultures at day 45 had tumor-like protuberances and cells had undergone apoptosis by day 50. The entire process, from purification, culture and induction to apoptosis of NSCs from two sources took two months. The time to apoptosis of cord blood-derived NSCs was earlier than for bone marrow-derived NSCs (Fig. 2).
Results
Identification of MSCs
Morphological Changes Observed in MSCs Post-Induction
Pre-induction, cells were negative for the presence of btubulin and GFAP. Six hours post-induction, cells were positive for both b-tubulin and GFAP, but the proportion of these cells was very low. As the time from initial induction increased, the number of cells positive for these markers also increased. At 72 h post-induction, expression levels for b-tubulin were highest, while GFAP levels were highest after 48 h. In contrast, the control group (Group A) displayed some non-specific staining, similar to that observed in non-induced MSCs (Fig. 3).
Mesenchymal stem cells extracted from umbilical cord blood and bone marrow appeared semi-adherent 24 h postinoculation. After 48 h, all MSCs in the bone marrow groups had completely adhered to the culture surface and a ‘budding phenomenon’ was visible. Cord blood MSCs were less adherent; however, the ‘budding phenomenon’ was still visible, but to a lesser extent. The growth rate and
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Fig. 2 Changes in MSC morphology following induction (9100 magnification). a Neurospheres 7 days post-induction. b Pseudopodia extended from neurosphere 8 days after induction. c Neurospheres were semi-adherent 14 days after induction. d Cellular axons
e Dendritic cells f Astrocytes g Neuron-like cells. h Connected axons. i Cells in the process of detaching from the culture surface j Tumor-like protuberances. k Apoptotic cells 50 days after the induction of cord-derived MSCs
Stem Cell Surface Markers
able to secrete cytokines, such as brain-derived neurotrophic factor, interleukins and transforming growth factor b1, to promote the recovery of neurological function [1, 3, 5, 13]. There is a lack of standards with respect to the use of stem cells in clinical treatments [2]. However, since 2009 the Chinese government has been attempting to implement a clear policy for the clinical application of stem cells (http://www.stemcellsportal.com). Using in vitro experiments to investigate purification, amplification and differentiation of stem cells and their transplantation, it will be possible to develop standardized techniques for the clinical application of stem cells. Research into the directed differentiation of MSCs is still at an exploratory stage. At present, several are methods used to induce the differentiation of MSCs into neural cells. These include exposure to cytokines, such as NGF, EGF, and bFGF [12]; chemical induction with substances such as 2-mercaptoethanol and DMSO [7]; exposure to a combination of cytokines and chemicals [8, 18]; exposure to brain homogenate [22]; gene transfection [11]; and coculture or exposure to culture medium and Chinese medicines [6]. Cytokines are commonly used as induction agents because they play a role in promoting stem cell proliferation and differentiation through their own receptors [16]. In the last 10 years, research has been published related to the induction of MSC differentiation into neurons using CSF [10, 15, 17, 19–21, 23]. These articles suggest that MSCs can be forced to differentiate into neural cells using CSF, and that this process is tissuespecific. Therefore, the specific tissue microenvironment
More than 90 % of P2 cells were positive for CD34, CD44, and CD90. The proportion of MSCs positive for CD34 was greater in those derived from bone marrow compared with those derived from cord blood. At day 7 post-induction, the proportion of MSCs positive for CD34 was lower than P2, while the numbers of cells positive for CD90 and CD44 were increased (Fig. 4). Clinical Availability of Stem Cells The time at which MSCs were available for clinical use did not differ based on source (P [ 0.05); however, the number of MSCs among the four groups was significantly different (P \ 0.05) (Table 1).The time required to obtain clinically relevant NSCs from the four groups differed (P \ 0.05), and the NSC count within the four groups also differed (P \ 0.05) (Table 2).
Discussion It is generally believed that once nerve cells are injured, it is almost impossible to renew and/or repair them [4]. Recent research regarding the plasticity of the CNS and nerve cell regeneration has been theorized in the hopes that stem cells can be used to treat patients with neurodegenerative diseases. MSCs and NSCs induced from the CNS and nerve cells possess the ability to self-renew and differentiate into cells of multiple lineages. These are also
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Fig. 3 Immunohistochemical and fluorescent immunohistochemistry staining of MSCs before and after induction (9200 magnification, and 9400 magnification). a GFAP staining in bone marrow-derived MSCs prior to induction b GFAP staining of bone marrow-derived MSCs at 6 h post-induction c GFAP staining in bone marrow-derived MSCs at 48 h post-induction d b-tubulin staining in bone marrow-derived MSCs prior to induction e b-Tubulin staining of bone marrow-derived MSCs at 6 h post-induction f b-tubulin staining in bone marrow-derived MSCs
for 72 h post-induction g GFAP staining in cord-derived MSCs at 48 h post-induction using a FITC-labeled secondary antibody h GFAP staining of cord-derived MSCs at 48 h post-induction using a TRITClabeled secondary antibody i A merged image of (g) ? (h) j b-tubulin staining in bone marrow-derived MSCs at 72 h post-induction using a TRITC-labeled secondary antibody k GFAP staining in bone marrowderived MSCs at 72 h post-induction using a FITC-labeled secondary antibody l A merged image of (j) ? (k)
can induce MSCs to directly differentiate [14]. According to this information, an attempt was made to induce MSC differentiation in a microenvironment similar to that found
in the body. Our findings revealed that morphological changes were visible in cells at 24 h post-induction. After 3 days, a number of neurites had formed, and the cell
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Fig. 4 Flow cytometry analysis of cell surface markers. On P2 cells: a approximately 90 % of events were positive for CD34. b 99 % of cells were positive for the presence of CD45 c CD44 was present on 96 % of analyzed cells d approximately 85 % of cells analyzed were CD90-positive. On bone marrow-derived MSCs at 7 days post-
induction: e 18 % of cells were positive for CD34. f approximately 18 % were CD45-positive. g Approximately 99 % were positive for CD44 h analysis of the CD90 cell surface marker showed that 95 % of cells analyzed contained this marker
Table 1 MSCs available for subsequent clinical treatment and the time (in days) to reach listed cell count ðx sÞ
Table 2 The number of NSCs available for subsequent clinical treatment and the time (in days) to reach listed cell count ðx sÞ
Groups
Time (days)
Cell count (107 cells/mL)
Groups
Time (d)
A
6.3 ± 1
2.45
A
12.1 ± 1
2.81
B C
6.3 ± 2 6.6 ± 1
4.304 3.26m4
B C
10.0 ± 14 12.3 ± 1m
5.384 3.834m
D
6.7 ± 1
2.13m*
D
12.3 ± 1m
2.334m*
MSC mesenchymal stem cell
NSC neural stem cell
4
P \ 0.05, compared with group A
4
P \ 0.05, compared with group A
m
P \ 0.05, compared with group B
m
P \ 0.05, compared with group B
Cell count (107 cells/mL)
* P \ 0.05, compared with group C
* P \ 0.05, compared with group C
cultures gradually formed a tapered, triangular, and irregular shape. The cell soma on day 7 was similar to the dendrite and axon-like structures of astrocytes. Compared with cytokine-induced cells, those that were induced with CSF were quicker to differentiate and which also occurred in higher proportions. This has resolved numerous problems associated with the use of culture media and stimulating factors. In conclusion, CSF contains sufficient components to induce the differentiation of MSCs into NSCs. CSF also
provides a better microenvironment for the differentiation of MSCs into NSCs. The resulting neural-like cells are also produced in sufficient numbers and meet the minimum requirements for clinical treatment. However, the number of cells produced and the time taken to reach the end-point of differentiation differed between our study groups. Our results mirror those of Li-Dong et al. [10], who showed that the induction effects of CSF from different patients were inconsistent. Further research is needed to determine what factor(s) in the CSF is/are responsible for these effects.
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References 1. Conti, L., Cattaneo, E., & Papadimou, E. (2008). Novel neural stem cell systems. Expert Opinion on Biological Therapy, 8(2), 153–160. 2. Feng, C. (2008). Experimental study of mild hypothermia and neural stem cell transplantation for ischemic nerve dysfunction. Dalian: Dalian Medical University. 3. Hess, D. C., & Borlongan, C. V. (2008). Stem cells and neurological diseases. Cell Proliferation, 41(Suppl 1), 94–114. 4. Jackson, E. L., & Alvarez-Buylla, A. (2008). Characterization of adult neural stem cells and their relation to brain tumors. Cells Tissues Organs, 188(1–2), 212–224. 5. Jeong, S. W., Chu, K., Jung, K. H., Kim, S. U., Kim, M., & Roh, J. K. (2003). Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke: A Journal of Cerebral Circulation, 34(9), 2258–2263. 6. Jia, Y., Yang, Y. J., Zhou, Y., Song, Y. Z., Liu, L. X., Song, J. H., et al. (2002). The study of inducing rat bone marrow stromal cells to differentiate into neural cells by using Baicalin. Chinese Medical Journal, 82, 5. 7. Kang, X. Q., Zang, W. J., Bao, L. J., Li, D. L., Xu, X. L., & Yu, X. J. (2006). Differentiating characterization of human umbilical cord blood-derived mesenchymal stem cells in vitro. Cell Biology International, 30(7), 569–575. 8. Kogler, G., Sensken, S., Airey, J. A., Trapp, T., Muschen, M., Feldhahn, N., et al. (2004). A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. The Journal of Experimental Medicine, 200(2), 123–135. 9. Kun, Z., & De-Qin, G. (2009). Treatment of cerebrovascular disease with stem cell transplantation and gene therapy. Chinese Journal of Postgraduates of Medicine, 32, 3. 10. Li-Dong, S. (2004). Inducing bone marrow stromal cells to differentiate into nerve cells for subsequent Parkinson’s disease treatment. Suzhou: Suzhou University. 11. Lim, J. Y., Park, S. I., Oh, J. H., Kim, S. M., Jeong, C. H., Jun, J. A., et al. (2008). Brain-derived neurotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways. Journal of Neuroscience Research, 86(10), 2168–2178. 12. Low, C. B., Liou, Y. C., & Tang, B. L. (2008). Neural differentiation and potential use of stem cells from the human umbilical cord for central nervous system transplantation therapy. Journal of Neuroscience Research, 86(8), 1670–1679.
13. Okano, H. (2010). Neural stem cells and strategies for the regeneration of the central nervous system. Proceedings of the Japan Academy Series B, Physical and Biological Sciences, 86(4), 438–450. 14. Rivera, F. J., Sierralta, W. D., Minguell, J. J., & Aigner, L. (2006). Adult hippocampus derived soluble factors induce a neuronal-like phenotype in mesenchymal stem cells. Neuroscience Letters, 406(1–2), 49–54. 15. Shen, Y. X., Wang, P., & Shi, E. D. (2011). Culturing of bone marrow mesenchymal stem cells in vitro with cerebrospinal fluid. Chinese Journal of Clinical Rehabilitative Tissue Engineering Research, 15, 5. 16. Tureyen, K., Vemuganti, R., Bowen, K. K., Sailor, K. A., & Dempsey, R. J. (2005). EGF and FGF-2 infusion increases postischemic neural progenitor cell proliferation in the adult rat brain. Neurosurgery, 57(6), 1254–1263. discussion 1254–1263. 17. Wang, P. (2009). The study of isolating and culturing of bone marrow mesenchymal stem cells and the influence of cerebrospinal fluid on bone marrow mesenchymal stem cells. Suzhou: Soochow University. 18. Woodbury, D., Schwarz, E. J., Prockop, D. J., & Black, I. B. (2000). Adult rat and human bone marrow stromal cells differentiate into neurons. Journal of Neuroscience Research, 61(4), 364–370. 19. Yang, H. Y., Gu, R., Wang, W. J., & Gao, Z. L. (2009). The cerebrospinal fluid promotes the differentiation of neural stem cells after transplantation of bone marrow mesenchymal stem cells. Chinese Journal of Laboratory Diagnosis, 13(3), 300. 20. Ye, Y., Want, M. R., & Dai, R. F. (2009). Induction of human bone marrow mesenchymal stem cells differentiation into neural stem/progenitor cells using cerebrospinal fluid. International Journal of Anesthesiology and Resuscitation, 30, 5. 21. Ye, Y., Zeng, Y. M., Wan, M. R., & Lu, X. F. (2011). Induction of human bone marrow mesenchymal stem cells differentiation into neural-like cells using cerebrospinal fluid. Cell Biochemistry and Biophysics, 59(3), 179–184. 22. Yong-Zhou, S., Hui-Xian, C., Zhe, L., Xin-Sheng, L., ZhengXian, W., & Zheng-Liang, S. (2008). Effects of brain homogenate on the differentiation of rat bone mesenchymal stem cells into neuron-like cells following traumatic brain injury. Chinese Journal of Clinical Rehabilitative Tissue Engineering Research, 12, 5. 23. Zeng, K. B., Wang, X. F., & Yan, Y. (2003). The effect in growth of bone marrow stromal cells in vitro of cerebrospinal fluid. Chinese Journal of Contemporary Neurology and Neurosurgery, 11, 4.
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ORIGINAL PAPER
Differentiation of Mesenchymal Stem Cells into Neural Stem Cells Using Cerebrospinal Fluid Wei Ge • Chao Ren • Xin Duan • Deqin Geng Caiyi Zhang • Xiaoyun Liu • Hao Chen • Meirong Wan • Runlu Geng
•
Ó Springer Science+Business Media New York 2014
Abstract Optimization of a methodology for mesenchymal stem cells (MSCs) differentiation into neural stem cells (NSCs) using cerebrospinal fluid (CSF). MSCs were extracted from umbilical cord blood from healthy, fullterm, newborn infants and from the bone marrow of patients. CSF was taken from healthy adult volunteers and patients. Four groups investigated were: A (n = 8) cord blood MSC induced with healthy volunteer CSF (control group); B (n = 7): patient MSCs induced with health volunteer CSF; Group C (n = 12): patient MSCs induced with their own CSF; group D (n = 6): cord blood MSCs induced with patient CSF. Following induction, cell differentiation state was examined using microscopy, flow cytometry, and immunohistochemistry. There were significantly more clinically applicable MSCs in Groups B and C
than groups A and D (P \ 0.05) and Group B had significantly more clinically applicable MSCs than group C (P \ 0.05). The presence of NSCs was as with the MSCs. Group B had significantly more clinically applicable NSCs than all of the other groups. In addition, group B cells grew significantly faster than the other groups (P \ 0.05). Upon CSF induction, MSCs differentiated into NSCs suitable for clinical treatment. The source of the MSCs and/or CSF influenced the number of NSCs produced and the NSC growth rate. Thus, the source of MSCs and CSF should be considered before initiating a stem cell clinical treatment. Keywords Marrow Cord blood Induction Neural stem cells Differentiation
Introduction Wei Ge and Chao Ren are Co-first author. W. Ge (&) D. Geng (&) C. Zhang H. Chen R. Geng Department of Neurology, Affiliated Hospital of Xuzhou Medical College, 99 Huaihai Road West, Xuzhou 221002, Jiangsu, China e-mail: [email protected] D. Geng e-mail: [email protected] C. Ren Department of Neurology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University Medical College, Yantai 264000, Shandong Province, China X. Duan Diagnostic Radiology Center, The Cancer Hospital of Xuzhou, Xuzhou 221005, China X. Liu M. Wan Central Laboratory, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China
It is widely accepted that nerve cells are unable to repair themselves if they experience trauma, necrosis, or degeneration. This makes treating diseases of the central nervous system (CNS) over the long-term difficult. Clinical use of stem cells has been postulated as one way of overcoming these issues. Mesenchymal stem cells (MSCs) are considered as one of the most useful types of stem cells that can be used in a clinical setting [10]. MSCs possess the potential for multilineage differentiation and self-renewal can be easily sourced and have fewer ethical considerations surrounding them than embryonic stem cells. It is essential to determine an efficient and practical method for inducing MSCs differentiation into neural stem cells (NSCs), for both clinical and basic research needs. This laboratory has previously published MSC differentiation methods [9, 20, 21] and has applied for a patent for a MSC induction methodology (Patent Application Number 2008100200943). This current
123
Cell Biochem Biophys
investigation examined the induction of human MSCs (from umbilical cord and bone marrow) to differentiate into NSCs using cerebrospinal fluid (CSF), with the aim of optimizing a technique that produces a sufficient number of NSCs that will meet the requirements for subsequent clinical treatment.
Materials and Methods Specimens CSF samples were taken from healthy adult volunteers and patients, while bone marrow was only taken from patients. Cord blood was taken from healthy, full-term, newborn infants. The hospital medical ethics committee approved this study. Experimental Design Four groups were used in the study: A (n = 8) comprised cord blood MSCs from healthy newborn infants that were subsequently induced with CSF from healthy volunteers; B (n = 7) involved bone marrow MSCs from patients that were induced by CSF from healthy volunteers; C (n = 12) employed bone marrow MSCs from patients that were induced with the patient’s own CSF; and D (n = 6) investigated cord blood MSCs from healthy newborn infants induced with CSF from patients. There were no significant differences in sex ratio or age between the groups (P [ 0.05). Separation, Cultivation, and Induction of MSCs The posterior superior iliac spine was the puncture site for bone marrow cell extraction. Under aseptic conditions, bone marrow (20 mL) was obtained from patients. Cord blood (20 mL) was taken from healthy newborn infants under aseptic conditions. Samples were mixed with heparin (10,000 U/L) and 20 mL of phosphate-buffered saline (PBS), centrifuged (2,5009g, 15 min) followed by removal of fat and supernatant. Cells were resuspended in an equal volume of Percoll in PBS and centrifuged (2,5009g, 25 min). Cells were washed 3 times in PBS (10 mL) and resuspended in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with 10 % fetal bovine serum (FBS; HyClone, Logan, Utah, USA). The cell suspension was seeded into uncoated, T25 culture flasks (BD FalconTM, Franklin lakes, New Jersey, USA) at a concentration of 1 9 106 cells/mL and cultures were then incubated at 37 °C/ 5 % CO2. These cells were designated as the primary generation (passage 0, P0). After 3 days, half of the culture media was changed, and 5 days later the entire culture media was changed. When cultures were 90 % confluent, they were harvested with 1 mL of 0.25 % trypsin (HyClone, Logan,
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Utah, USA) and passaged at a 1:3 dilution. This process was continued until P5. For differentiation into NSCs, MSCs from the four groups at each passage (P1–5) were digested with 1 mL of 0.25 % trypsin (HyClone, Logan, Utah, USA) and were seeded at a density of 2 9 106 cells/well (0.1 mL) on poly-L-lysine-coated (100 mg/mL; Sigma, St. Louis, Missouri, USA) coverslips in 6-well plates. Following digestion, cells were incubated in 2 mL of DMEM/F12 serum-free medium for 24 h; after 72 h, 10 lL of CSF from the appropriate source for that group was added to the culture medium every 24 h over a 72-h period. When the cell concentration reached 1 9 107 cells/mL, NSCs were harvested for clinical transplantation treatments. Identification of MSCs Cell growth and morphology of the primary MSCs and those from P1–5 were observed. Images were acquired at 25 °C using an inverted phase-contrast microscope (Moticam 3000; Motic Group Co., Ltd.). The differentiation of MSCs was examined after induction for 6, 48, and 72 h. Antigen expression was examined in separate experiments, in triplicate, as previously described, with some modifications. Cells on coverslips were fixed for 30 min with 4 % paraformaldehyde and 0.3 % glutaraldehyde, and then washed 3 times with PBS. Cells were incubated with a monoclonal mouse anti-human b-tubulin antibody (1:500 dilution; Wuhan Boster Biological Technology, Ltd., China) or a polyclonal rabbit anti-human GFAP antibody (1:1000; Wuhan Boster Biological Technology, Ltd.) at 4 °C overnight. They were then washed 3 times with PBS and incubated with either a FITC-conjugated goat antirabbit IgG (1:500; Wuhan Boster Biological Technology, Ltd.) or a TRITC-conjugated goat anti-rabbit IgG (1:500; Wuhan Boster Biological Technology, Ltd.) for 30 min at 25 °C. Following this incubation with secondary antibodies, cell were washed 3 times with PBS and then observed under a microscope (LEICA TCS-SP2, Germany LEICA Inc.). As a control, PBS was used to replace the primary antibody. Flow Cytometry P2 cells were induced for 7 days then digested with 0.25 % trypsin. Cells were resuspended to a concentration of 1 9 107 cells/mL and incubated with TRITC-conjugated anti-human CD34, CD44, and CD90 developed in goat or mice (Wuhan Boster Biological Technology, Ltd.) for 30 min at 25 °C. Cells were washed twice with PBS, and at least 400 lL of PBS was added to cells and allowed to incubate at 4 °C. Surface markers were identified by flow cytometry within 24 h of staining. As a control, cells were mock-treated with PBS in place of the primary antibody.
Cell Biochem Biophys
Fig. 1 Growth and changes in passage 0 of MSCs at (9100 magnification). Cells cultured for: a 24 h b 48 h c 72 h d 7 days e 10 days of bone marrow-derived MSCs. Cells cultured for: f 24 h g 48 h h 72 h i 14 days j 21 days of cord-derived MSCs
Cell Quantification Cells at P1 were induced after 3 days, digested with trypsin, and washed 3 times with 10 mM PBS. Cells were then resuspended in 100 lL of PBS and counted using a handheld automated cell counter (Millipore Corporation, USA). Statistical Analysis SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used to assist with statistical analysis of our results. Values were expressed as the mean ± standard deviation ðx sÞ. For comparison among the groups, we used a one-way analysis of variance (ANOVA) where a P value \0.05 was considered as statistically significant.
morphology of the MSCs in all four groups were similar, forming large colonies with 80–100 % fusion. Cells were passaged every 5 days (Fig. 1). After 24 h of induction, the NSCs from all groups displayed similar growth rate and morphological characteristics. After 3 days, a number of neurites were visible, and the cells gradually formed tapered, triangular, and irregular shapes. The cell soma at day 7 was similar to the astrocyte dendrite and axon-like structures. Cells were further elongated, filamentous and reticular as they continued to be cultured. The soma of cultures at day 45 had tumor-like protuberances and cells had undergone apoptosis by day 50. The entire process, from purification, culture and induction to apoptosis of NSCs from two sources took two months. The time to apoptosis of cord blood-derived NSCs was earlier than for bone marrow-derived NSCs (Fig. 2).
Results
Identification of MSCs
Morphological Changes Observed in MSCs Post-Induction
Pre-induction, cells were negative for the presence of btubulin and GFAP. Six hours post-induction, cells were positive for both b-tubulin and GFAP, but the proportion of these cells was very low. As the time from initial induction increased, the number of cells positive for these markers also increased. At 72 h post-induction, expression levels for b-tubulin were highest, while GFAP levels were highest after 48 h. In contrast, the control group (Group A) displayed some non-specific staining, similar to that observed in non-induced MSCs (Fig. 3).
Mesenchymal stem cells extracted from umbilical cord blood and bone marrow appeared semi-adherent 24 h postinoculation. After 48 h, all MSCs in the bone marrow groups had completely adhered to the culture surface and a ‘budding phenomenon’ was visible. Cord blood MSCs were less adherent; however, the ‘budding phenomenon’ was still visible, but to a lesser extent. The growth rate and
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Fig. 2 Changes in MSC morphology following induction (9100 magnification). a Neurospheres 7 days post-induction. b Pseudopodia extended from neurosphere 8 days after induction. c Neurospheres were semi-adherent 14 days after induction. d Cellular axons
e Dendritic cells f Astrocytes g Neuron-like cells. h Connected axons. i Cells in the process of detaching from the culture surface j Tumor-like protuberances. k Apoptotic cells 50 days after the induction of cord-derived MSCs
Stem Cell Surface Markers
able to secrete cytokines, such as brain-derived neurotrophic factor, interleukins and transforming growth factor b1, to promote the recovery of neurological function [1, 3, 5, 13]. There is a lack of standards with respect to the use of stem cells in clinical treatments [2]. However, since 2009 the Chinese government has been attempting to implement a clear policy for the clinical application of stem cells (http://www.stemcellsportal.com). Using in vitro experiments to investigate purification, amplification and differentiation of stem cells and their transplantation, it will be possible to develop standardized techniques for the clinical application of stem cells. Research into the directed differentiation of MSCs is still at an exploratory stage. At present, several are methods used to induce the differentiation of MSCs into neural cells. These include exposure to cytokines, such as NGF, EGF, and bFGF [12]; chemical induction with substances such as 2-mercaptoethanol and DMSO [7]; exposure to a combination of cytokines and chemicals [8, 18]; exposure to brain homogenate [22]; gene transfection [11]; and coculture or exposure to culture medium and Chinese medicines [6]. Cytokines are commonly used as induction agents because they play a role in promoting stem cell proliferation and differentiation through their own receptors [16]. In the last 10 years, research has been published related to the induction of MSC differentiation into neurons using CSF [10, 15, 17, 19–21, 23]. These articles suggest that MSCs can be forced to differentiate into neural cells using CSF, and that this process is tissuespecific. Therefore, the specific tissue microenvironment
More than 90 % of P2 cells were positive for CD34, CD44, and CD90. The proportion of MSCs positive for CD34 was greater in those derived from bone marrow compared with those derived from cord blood. At day 7 post-induction, the proportion of MSCs positive for CD34 was lower than P2, while the numbers of cells positive for CD90 and CD44 were increased (Fig. 4). Clinical Availability of Stem Cells The time at which MSCs were available for clinical use did not differ based on source (P [ 0.05); however, the number of MSCs among the four groups was significantly different (P \ 0.05) (Table 1).The time required to obtain clinically relevant NSCs from the four groups differed (P \ 0.05), and the NSC count within the four groups also differed (P \ 0.05) (Table 2).
Discussion It is generally believed that once nerve cells are injured, it is almost impossible to renew and/or repair them [4]. Recent research regarding the plasticity of the CNS and nerve cell regeneration has been theorized in the hopes that stem cells can be used to treat patients with neurodegenerative diseases. MSCs and NSCs induced from the CNS and nerve cells possess the ability to self-renew and differentiate into cells of multiple lineages. These are also
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Fig. 3 Immunohistochemical and fluorescent immunohistochemistry staining of MSCs before and after induction (9200 magnification, and 9400 magnification). a GFAP staining in bone marrow-derived MSCs prior to induction b GFAP staining of bone marrow-derived MSCs at 6 h post-induction c GFAP staining in bone marrow-derived MSCs at 48 h post-induction d b-tubulin staining in bone marrow-derived MSCs prior to induction e b-Tubulin staining of bone marrow-derived MSCs at 6 h post-induction f b-tubulin staining in bone marrow-derived MSCs
for 72 h post-induction g GFAP staining in cord-derived MSCs at 48 h post-induction using a FITC-labeled secondary antibody h GFAP staining of cord-derived MSCs at 48 h post-induction using a TRITClabeled secondary antibody i A merged image of (g) ? (h) j b-tubulin staining in bone marrow-derived MSCs at 72 h post-induction using a TRITC-labeled secondary antibody k GFAP staining in bone marrowderived MSCs at 72 h post-induction using a FITC-labeled secondary antibody l A merged image of (j) ? (k)
can induce MSCs to directly differentiate [14]. According to this information, an attempt was made to induce MSC differentiation in a microenvironment similar to that found
in the body. Our findings revealed that morphological changes were visible in cells at 24 h post-induction. After 3 days, a number of neurites had formed, and the cell
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Fig. 4 Flow cytometry analysis of cell surface markers. On P2 cells: a approximately 90 % of events were positive for CD34. b 99 % of cells were positive for the presence of CD45 c CD44 was present on 96 % of analyzed cells d approximately 85 % of cells analyzed were CD90-positive. On bone marrow-derived MSCs at 7 days post-
induction: e 18 % of cells were positive for CD34. f approximately 18 % were CD45-positive. g Approximately 99 % were positive for CD44 h analysis of the CD90 cell surface marker showed that 95 % of cells analyzed contained this marker
Table 1 MSCs available for subsequent clinical treatment and the time (in days) to reach listed cell count ðx sÞ
Table 2 The number of NSCs available for subsequent clinical treatment and the time (in days) to reach listed cell count ðx sÞ
Groups
Time (days)
Cell count (107 cells/mL)
Groups
Time (d)
A
6.3 ± 1
2.45
A
12.1 ± 1
2.81
B C
6.3 ± 2 6.6 ± 1
4.304 3.26m4
B C
10.0 ± 14 12.3 ± 1m
5.384 3.834m
D
6.7 ± 1
2.13m*
D
12.3 ± 1m
2.334m*
MSC mesenchymal stem cell
NSC neural stem cell
4
P \ 0.05, compared with group A
4
P \ 0.05, compared with group A
m
P \ 0.05, compared with group B
m
P \ 0.05, compared with group B
Cell count (107 cells/mL)
* P \ 0.05, compared with group C
* P \ 0.05, compared with group C
cultures gradually formed a tapered, triangular, and irregular shape. The cell soma on day 7 was similar to the dendrite and axon-like structures of astrocytes. Compared with cytokine-induced cells, those that were induced with CSF were quicker to differentiate and which also occurred in higher proportions. This has resolved numerous problems associated with the use of culture media and stimulating factors. In conclusion, CSF contains sufficient components to induce the differentiation of MSCs into NSCs. CSF also
provides a better microenvironment for the differentiation of MSCs into NSCs. The resulting neural-like cells are also produced in sufficient numbers and meet the minimum requirements for clinical treatment. However, the number of cells produced and the time taken to reach the end-point of differentiation differed between our study groups. Our results mirror those of Li-Dong et al. [10], who showed that the induction effects of CSF from different patients were inconsistent. Further research is needed to determine what factor(s) in the CSF is/are responsible for these effects.
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