ORIGINAL RESEARCH

Induction of Human Bone Marrow Mesenchymal Stem CellsDifferentiation into Neural-Like Cells Using Cerebrospinal Fluid

Ying Ye • Yin-Ming Zeng • Mei-Rong Wan •

Xian-Fu Lu

Published online: 6 January 2011

� Springer Science+Business Media, LLC 2011

Abstract To optimize a technique that induces bone

marrow mesenchymal stem cells (BMSCs) to differentia-

tion into neural-like cells, using cerebrospinal fluid (CSF)

from the patient. In vitro, CSF (Group A) and the cell

growth factors EGF and bFGF (Group B) were used to

induce BMSCs to differentiate into neural-like cells. Post-

induction, presence of neural-like cells was confirmed

through the use of light and immunofluorescence micros-

copy. BMSCs can be induced to differentiate into neural-

like cells. The presence of neural-like cells was confirmed

via morphological characteristics, phenotype, and biologi-

cal properties. Induction using CSF can shorten the pro-

duction time of neural-like cells and the quantity is

significantly higher than that obtained by induction with

growth factor (P \ 0.01). The two induction methods can

induce BMSCs to differentiate into neural-like cells. Using

CSF induction, 30 ml bone marrow can produce a

sufficient number of neural-like cells that totally meet the

requirements for clinical treatment.

Keywords Cerebrospinal fluid � Bone marrow �Mesenchymal stem cells � Induced differentiation � Neural-

like cells

Abbreviations

BMSCs Bone marrow mesenchymal stem cells

CSF Cerebrospinal fluid

auto-CSF Autologous cerebrospinal fluid

CNS The central nervous system

IMDM Iscove’s modified Dulbecco’s medium

PBS Phosphate-buffered saline

FBS Fetal bovine serum

Introduction

Recent research indicates that stem cells can be used for

tissue restoration. Stem cell transplantation is a new strat-

egy for treating refractory diseases of the central nervous

system (CNS). The clinical application of bone marrow

mesenchymal stem cell transplantation is expanding [1]

and, based on the results of pre-clinical investigations [2–

6], has entered phase I clinical trials [7, 8]. The possible

uses for this type of treatment for nervous system diseases

include degenerative and hereditary diseases [9–11], cere-

bral vascular diseases [12], and brain and spinal cord

injuries and diseases [13–16.]. However, there have been

large differences in treatment results and, to date, the

effectiveness of the treatment has not been incontrovertibly

established. One reason that results have been varied could

Y. Ye � Y.-M. Zeng � X.-F. Lu

Department of Anesthesiology, the first Affiliated Hospital,

China Medical University, Shenyang 110001, China

Y. Ye � Y.-M. Zeng � M.-R. Wan � X.-F. Lu

Jiangsu Institute of Anesthesiology, Jiangsu Key Laboratory

of Anesthesiology, Xuzhou Medical College,

Xuzhou 221002, China

Y. Ye

Emergency Center, Affiliated Hospital of Xuzhou Medical

College, Xuzhou 221002, China

Y. Ye (&) � Y.-M. Zeng (&)

Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou

Medical College, Xuzhou, Jiangsu 221002, China

e-mail: xzmcyy@163.com

Y.-M. Zeng

e-mail: zym_xzmc@163.com

123

Cell Biochem Biophys (2011) 59:179–184

DOI 10.1007/s12013-010-9130-z

be the heterogeneous response of BMSCs to culture and

expansion, which may result in induction and differentia-

tion into undesirable cell types. The study we report is

focused on establishing an efficient method of growing and

expanding BMSCs in culture. Thus, we attempted to

establish a means of inducing BMSCs to differentiate into

neural-like cells while maintaining their stem cell proper-

ties of the cells.

Materials and Methods

Specimen Sources

Bone marrow and cerebrosprinal fluid (CSF) samples were

taken from healthy adult volunteers. The hospital medical

ethics committee approved the study.

Separation and Cultivation of BMSCs

Under aseptic conditions, bone marrow (30 ml) obtained

from seven healthy volunteers. The aliquots were mixed

with an anticoagulant heparin solution (10,000 U/l) and an

equal volume (30 ml) of phosphate-buffered saline (PBS),

centrifuged at 1,5009g for 15 min and the fat and super-

natant were removed. An equal volume of Percoll in PBS

was added and the cells were resuspended and then spun at

1,2009g for 25 min. Cells were washed three times by

resuspension in PBS (10 ml) and resuspended in Iscove’s

Modified Dulbecco’s medium (IMDM) supplemented with

10% fetal bovine serum (FBS, HyClone, USA). After

counting, cell suspension was seeded in uncoated T25

culture flasks (BD FalconTM, Becton-Dickinson, No.

353018) at a concentration of 1 9 106 cells/ml, Cultures

were maintained at 37�C in a humidified atmosphere con-

taining 5% CO2. These cells were marked as the primary

generation (Passage 0, P0). After 3 days, half of the culture

media was changed, and then, after 5 days, all of the

medium was changed. One to three weeks later, when

fibroblast-like cells at the base of the flask reached con-

fluence, they were harvested with 0.25% trypsin (1 ml,

HyClone,USA) and passaged at 1:3 dilution as passage 1.

Then, all of the medium was changed with IMDM con-

taining 10% FBS every 3 day. Five to nine days later,

when fibroblast-like cells at the base of the flask reached

confluence, each P1 flask was split in a similar manner into

P2 and P3.

BMSC Induction Cultivation

For differentiation into neural-like cells, BMSCs from

seven healthy volunteers at each generation (P1, P2, and

P3) were, respectively, plated at 2 9 106 cells/well

(0.1 ml) on poly-L-lysine-coated (100 mg/ml, Sigma) cov-

erslips in six-well plate. When the cells grew to 70% con-

fluence, every six-well plate were divided into two groups

designated A and B. For Group A, after 72 h, 10 ll of auto-

CSF (collected from seven healthy volunteers under sterile

conditions of gathering by lumbar puncture) was added to

the culture medium every day for 7 days. The same protocol

was followed for Group B. After 72 h, 10 ll of IMDM

supplemented with 10% FBS, 10 lmol/l EGF and

bFGF(Sigma) was added to the culture medium every day

for 7 days.

Observation of Cell Growth and Cell Morphology

Cell growth and morphological features of primary BMSCs

and those from the passages were observed and photo-

graphed at room temperature (25�) using an inverted phase

contrast microscope (moticam 3000, motic group Co., Ltd.)

BMSC Differentiation

Cell differentiation was induced for 48, 72, and 96 h. The

expression of each antigen was examined in separate

experiments at least three times as previously described

[17], with some modifications. Briefly, Cells on the Cov-

erslips were fixed for 30 min with 4% paraformaldehyde

plus 0.3% glutaraldehyde and washed three times with

PBS. The cells were incubated with mouse anti-human

b-tubulin monoclonal antibody (NO BM1453, 1:500 dilu-

tion, Wuhan Boster Biological Technology, LTD) or rabbit

anti-human GFAP polyclonal antibody BA0056, 1:1000

dilution, Wuhan Boster Biological Technology, LTD) at 4�overnight. They were then washed three times in PBS.

These cells were incubated with FITC-Goat anti- Rabbit

IgG (NO BA1107, 1:500 diluton, Wuhan Boster Biological

Technology, LTD) or TRITC-goat Anti-rabbit IgG (NO

BA1090, 1:500 dilution, Wuhan Boster Biological Tech-

nology, LTD) for 30 min at 25� and then washed three

times with PBS. The cells were observed and photographed

with a light microscope or fluorescence microscope (Laser

Scanning Confocal Microscope LEICA TCS-SP2, Germany

LEICA Inc.). For the primary control, PBS replaced incu-

bation with the primary antibody.

Cells Counting

The cells of P1, P2, and P3 passage from each culture

bottles used trypsin to digest, washed with 0.01 mol/l PBS,

and centrifuged three times. A total of cells were resus-

pended in 100 ml PBS, resuspended cells are counted by

hand held automated cell counter (Millipore Corporate,

US).

180 Cell Biochem Biophys (2011) 59:179–184

123

Statistical Analysis

SPSS 12.0 software was adopted for statistical processing.

The data were expressed as the mean value ‘‘standard

deviation (x ± s). For comparison among many groups,

analysis of variance and q examination were employed

while the T-test was used to determine statistical signifi-

cance (P \ 0.05) of the difference between the means of

two groups.

Results

Cytomorphology and Growth

At 24 h after inoculation, the BMSCs were flat in shape.

After 48 h, they became adherent and a ‘‘budding phe-

nomenon’’ was visible. After 72 h, the formation of pro-

cesses was evident (Fig. 1a). The cells gradually became

spindle shaped after the addition of fresh media. Between

15 and 50 cells begin to fuse after 1 week, gradually

forming a large colony (Fig. 1b). After 10 days of culture,

the cells were arranged unidirectionally in a swirling pat-

tern (Fig. 1c).

Morphological Changes Observed in BMSCs

Post-Induction

After 24 h of induction with auto-CSF, cells from Group A

exhibited a significant morphological change including

soma retraction and transparency (Fig. 2a). After 3 days, a

number of neurites were formed (Fig. 2b). The soma of

4 day cultures gradually formed a tapered, triangular, and

irregular shape. The soma of 7 day cultures was similar to

the dendrite and axon-like structure of astrocytes (Fig. 2c).

In Group B, the neurites of the soma of cells cultured for

5 days were further elongated, filamentous, and reticular

(Fig. 2d). The somas were transparent. Some neurites were

formed under the retraction balls formed after 7 days of

culture. The somas of 12 day cultures gradually formed a

tapered, triangular, and irregular shape with dendrites, and

axon-like structures similar to those of astrocytes (Fig. 2f).

Immunohistochemistry and Immunofluorescence

Identification of BMSC Differentiation

After 96 h of induction, (Fig. 3), the cells exhibited both

normal GFAP and of b-tubulin immunohistochemical

staining and immunofluorescence. In contrast, the control

group displayed very slight unspecific staining, which was

similar to uninduced normal MSCs.

Cell Number

The number of cells in Group A were significantly higher

than in Group B. Furthermore, in group A, the cells of

P2 [(6.70 ± 0.45) 9 107] and P3 [(6.93 ± 0.32) 9 107]

were significantly higher than P1 [(4.90 ± 0.31) 9 107,

P [ 0.01] (Table 1).

Discussion

It is difficult to control BMSC differentiation when the

cells are directly transplanted into the CNS because they

tend to differentiate into glial cell when placed in a dam-

aged environment [18]. Regarding repair of CNS damage,

controlling the differentiation process of transplanted cells

is a key question that needs to be answered. One study

found that if the transplanted cells were pre-cultured, the

survival rate of transplanted cells was high and the devel-

opment of mature neurons was significantly increased [19].

Another study suggested that the induction of stem cells to

differentiate into neural precursor cells before transplan-

tation may help to control the differentiation of trans-

planted cells in a damaged environment [20]. Research in

the directional differentiation of MSCs is still in an

exploratory stage. At present, there are several methods for

Fig. 1 Adherent growth of primary BMSCs culture. a 3 day culture. The beginning of the budding phenomenon is visible (arrows). b 7-day

culture. Between 15 and 50 cells begin to fuse and c 10-day culture. Cells are arranged in a swirling pattern

Cell Biochem Biophys (2011) 59:179–184 181

123

the induction of differentiation of MSCs into neural cells:

cytokine exposure [1]: NGF, EGF, and bFGF [21]; chem-

ical induction with b-mercaptoethanol, DMSO, and butyl-

ated hydroxyanisole [7, 22]; a combination of cytokines

and chemical inducers [8, 23, 24]; other methods including

[2]: exposure to traumatic brain homogenate [25], co-cul-

ture in or exposure to culture medium/Chinese medicines

such as Astragalus mongholicus [26] and Salvia mitiorrh-

iza [27]. Cytokines are commonly used as inducing agents

and play a role in nutritive status of neurons [28]. They can

also act as free-radical scavengers, reduce calcium over-

load and suppress the expression of nitrogen monoxide

synthase. EGF and bFGF are polypeptide factors that

promote cell growth and important mitogens that promote

neural stem cells to proliferation and differentiate into

neurocytes [29]. CSF contains a variety of electrolytes,

proteins, sugars, and other factors, such as bFGF, brain-

derived neurotrophi factor (BDNF) and GDNF, glial cell-

derived neurotrophic factor [30, 31]. Among these, bFGF

promotes proliferation and differentiation of neural stem

cells into neurocytes via binding to its corresponding cell

surface receptor. BDNF plays a role in the nutrition to

MSCs and promotes proliferation and, to a certain extent,

differentiation [32]. The results of our study show that:

cell morphology changed significantly after 24 h induction

by auto-CSF. After 3 days in culture, the soma retracts

and gradually forms a tapered, asteriform, triangular, and

irregular shape, which is similar to the dendritic and axon-

like structures of the astrocyte. The number of astrocytes in

4 and 5 day cultures increased gradually and formed con-

nections with neural-like morphology. These cultures

exhibited only a small amount of cell death (\1%). The

result of microscopical examination showed that early

neural markers (such as b-tubulin) were expressed after

induction. In addition, mature neuronal markers (such as

GFAP) also were expressed. Compared with induction by

cytokines, ASF induction can accelerate the growth of

nerve-like cells and these cells grow well. Therefore, it is

Fig. 2 Morphological changes

after MSCs induction (9100).

a Morphology after 1 day of

culture with CSF: Cells

displayed a significant

morphological change including

soma retraction and

transparency. b 3 days after

induction with CSF: Neurites

start to form (arrows). c 7 days

after induction with CSF: The

soma exhibits dendrite and

axon-like structure similar to

those of astrocytes (arrows).

d Morphology after 5 days of

induction with growth factors:

The soma are elongated,

filamentous and reticular.

e Morphology after 7 days of

induction with growth factors:

Neurite formation is evident

under the retraction ball

(arrows). f Morphology after

12 days of induction with

growth factors: The soma

gradually form tapered

triangular and irregular shapes

(arrows)

182 Cell Biochem Biophys (2011) 59:179–184

123

considered that CSF contains sufficient material and

nutrients to induce differentiation of BMSCs and provides

a better micro-environment for their differentiation into

neural-like cells.

One week after the separation of BMSCs, auto-

cerebrospinal fluid is taken to induce the P1 generation of

BMSCs; after the first cell transplantation, 5 ml auto-

cerebrospinal fluid is taken to induce the P2 generation of

BMSCs; after the second cell transplantation, 5 ml auto-

cerebrospinal fluid is taken to induce the P3 generation of

BMSCs. During the induction periods, transplanted cells

need to number more than 4 9 107 to completely meet the

requirement of clinical treatment.

In conclusion, CSF can provide nutrition for nerve cells

and it can also be a substitute for other stimulating factors,

thereby resolving many problems caused by other culture

media and stimulating factors.

During this study, CSF caused apoptosis after being in

culture for 54 days and the apoptotic time was more of an

advantage than the induction by cytokines. It was theorized

that CSF contain one or more factors that inhibit BMSC

differentiation into mature neural-like cell after a finite

period in culture. Further research is needed to determine

what factor or factors in the CSF is/are responsible for this

effect.

Our laboratory has applied for a patent for our method of

induction (Application Number: 2008100200943).

Acknowledgments This work was supported in part by a grant of

the National Natural Science Foundation of China (NSFC30972834 to

Dr. Lu, China and the Natural Science Foundation of Xuzhou City

(XM09B119 to Dr. Ye, China).

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