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Tissue and Cell 45 (2013) 295– 305

Contents lists available at SciVerse ScienceDirect

Tissue and Cell

jou rn al hom epage: www.elsev ier .com/ locate / t i ce

ew methods for inducing the differentiation of amniotic-derived mesenchymaltem cells into motor neuron precursor cells

u Weia,b,2, Guan Fang-xiac,1,3, Li Yuanb,4, Tang You-jiaa,5, Yang Fenga,6, Yang Bob,∗

Department of Neurosurgery, The First People’s Hospital of Jiujiang, Jiujiang, Jiangxi Province, People’s Republic of ChinaDepartment of Neurosurgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, People’s Republic of ChinaDepartment of Bioengineering, Zhengzhou University, Zhengzhou, Henan Province, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 6 September 2012eceived in revised form 23 February 2013ccepted 13 March 2013vailable online 24 June 2013

eywords:ifferentiationxtracellular matrixN precursor cells

mniotic-derived mesenchymal stem cells

a b s t r a c t

Our study investigates the differentiation of amniotic-derived mesenchymal stem cells (ADMSCs) intomotor neuron (MN) precursor cells induced by a combination of extracellular matrix (ECM) and multi-cellfactors. Membrane-like ECM was made by an enzymatic and chemical extraction method and exhibitedgood biological compatibility. Cells in the experimental group (EG) were treated with ECM and multi-cell factors in a multi-step induction process, while the control group (CG) was treated similarly, exceptwithout ECM. In the EG, after induction, the cells formed processes that connected with neighboring cellsto form a net that had directionality. In these cells, neuron-specific enolase (NSE) and synaptophysin(SYN) expression levels increased and glial fibrillary acidic protein (GFAP) expression decreased. TheSYN expression in the EG cells was higher compared with those in the CG. In the CG, NSE expressionincreased, while the expression of Nestin and SYN did not change. These were several changes in thelevels of other genes: ADMSCs at passage 1 expressed Nanog, SOX2, octamer-binding transcription factor4 (OCT4) and Nestin. In the EG, at the beginning of induction, the expression of Nanog decreased and thatof SOX2 and Nestin increased. After 2 days, the cells expressed Nestin, OCT4 and SYNIII, and after 3 days,

they expressed Olig2, OCT4, Nestin, SYNII and Islet1 (ISL1). Finally, at day 6, the cells expressed Nestin,SYNI, SYNIII, ISL-1, homeobox 9 (Hb9) and oligodendrocyte lineage transcription factor 2 (Olig2). In theCG, the cells never expressed SYNI, SYNII or Hb9. Our studies therefore demonstrate that the extracted

otinral p

ECM was capable of promcell subsets, including neu

human ADMSCs to differentiat

Abbreviations: AChE, acetylcholine esterase; ASCs, adult stem cells; ADMSCs,mnion mesenchymal stem cells; BDNF, brain derived neurophic factor; bFGF,asic fibroblast growth factor; ChAT, choline acetyltransferase; CG, control group;MSO, dimethyl sulfoxide; ESCs, embryonic stem cells; EG, experimental group;CS, fetal calf serum; GFAP, glial fibrillary acidic protein; H&E, hematoxylin andosin; Hb9, homeobox 9; MN, motor neuron; NTs, neurotrophic factor; MAPCs,ulti-potent adult progenitor cells; NSE, neuronspecific enolase; NF, neurofilament

rotein; OCT4, octamer-binding transcription factor4; Olig2, oligodendrocyte lin-age transcription factor 2; PBS, phosphate buffer saline; RA, retinoic acid; SYN,ynaptophysin-1.∗ Corresponding author at: Department of Neurosurgery, The First Affiliated Hos-ital of Zhengzhou University, No. 1 Jianshe-East Road, Zhengzhou 450052, People’sepublic of China. Tel.: +86 0371 65163795.

E-mail addresses: zzdxhuw@163.com (W. Hu), guanfangxia@126.comF.-x. Guan), liyuans889@gmail.com (Y. Li), yjtang0808@163.com (Y.-j. Tang),fwlsy@163.com (F. Yang), yangbo96@126.com (B. Yang).1 Equal contribution to this paper.2 Tel.: +86 13755280372; fax: +86 0792 8582312.3 Tel.: +86 13613813972.4 Tel.: +86 15937140683.5 Tel.: +86 13907928809.6 Tel.: +86 13970218665.

040-8166/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Ltd. All rittp://dx.doi.org/10.1016/j.tice.2013.03.002

g the maturation of synapses. Human ADMSCs are composed of multiplerogenitor cells. The multi-step induction method used in this study causese into MN precursor cells.

Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Due to the inability of the central nervous system to com-pletely self-repair following injury, great hope has been placedon stem cells. Specifically, the transplantation of stem cells as acure for central nervous system injury has become a major focus ofcurrent neuroscience research (Srivastava et al., 2009; Kulbatskiet al., 2008). Stem cells include embryonic stem cells (ESCs),adult stem cells (ASCs) and trophectoderm stem cells. ASCs areadvantageous for clinical applications because they are capable ofself-duplication, can be obtained easily, do not cause immunologi-cal rejection and avoid some ethical concerns. Therefore, a carefulexamination of the differentiation of ASCs, especially with respectto the regulation of neural differentiation (Munoz et al., 2005), is

critical. Currently, ASCs are grafted directly into the nervous sys-tem with the potential risk that cell types that are not beneficialfor tissue repair will be generated in the damaged tissue microen-vironment. Therefore, more studies examining the differentiation

ghts reserved.

296 W. Hu et al. / Tissue and Cell 45 (2013) 295– 305

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ig. 1. Morphologic change of acellular matrix. (A) Epithelial cells connected tightumble, which has three-dimensional structure (inverted microscope, ×400). (C) Thn fresh amnion (H&E staining, ×400). (D) The acellular membranous matrix: plumtaining, ×400). (For interpretation of the references to color in this figure legend, t

echanisms of ASCs and finding new methods for differentiatingSCs into the functional cells needed for tissue repair are required

o develop clinical treatments.Currently, various factors, including growth factors, such as

rain derived neurophic factor (BDNF) and basic fibroblast growthactor (bFGF), antioxidants, such as �-mercaptoethanol, dimethylulfoxide (DMSO) and retinoic acid (RA), and the cellular microen-ironment, such as a peripheral neural cell suspension and ancellular nervous scaffold, have been used for neural induction.owever, the positive ratios of neuron-like cells utilized in manyrevious studies were low. Therefore, in this study, we aimed toiscover a more reliable condition for ASC differentiation that wille more suitable for clinical applications.

There has not yet been a side-by-side comparison of ASCs dif-erentiated into nervous system cells using extracellular matrixECM) along with cell factors, chemical agents and biological fac-ors (Woodbury et al., 2000). Here, we have examined the processf in vitro differentiation of amnion-derived mesenchymal stemells (ADMSCs) into motor neuron (MN)-like cells after a four-stepnduction with membranous ECM and cell factors.

. Results

.1. Acellular matrix

.1.1. Morphologic changesUnder microscope, epithelial cells on fresh amnion con-

ected tightly and look like paving stones. Acellular membranousatrix was humble, semi-transparent with good flexibility, easy

ig. 2. Cellular compatibility. (A) Cells adhere to the material surface at 12th hour after huells unfolded and look like spindles with multiple protrusions at 24th hour. (inverted mth day after seeded (inverted microscope, ×400).

fresh amnion (inverted microscope, ×400). (B) Acellular membranous matrix was had round-like appearance with large nucleus, abundant and blue-stain kytoplasmagen was showed, without blue-stain nuclear, cellular structures and debris (H&Eder is referred to the web version of the article.)

rehydration and turn to transparent, which has three-dimensionalstructure (see Fig. 1).

2.1.2. Morphological featuresUnder the microscope, epithelial cells plated on fresh amnion

appeared tightly connected and resembled paving stones. Thethree-dimensional acellular membranous matrix was malleableand semi-transparent, had good flexibility, was easily rehydratedand turned transparent upon put in the culture media (Fig. 1).

2.1.3. Cellular compatibilityTwelve hours after the 3rd passage, human ADMSCs were

seeded onto the acellular matrix, and the cells were allowedto adhere. Changes were observed with an inverted microscope(Fig. 2). These results indicated that human ADMSCs were capableof normal growth and proliferation when grown on the membra-nous matrix.

2.1.4. Effects on cell proliferation

Human ADMSCs proliferated quickly on the membranous

matrix. In fact, the cells on the membranous matrix proliferatedsignificantly more than cells grown in 24-well plates (P = 0.003), asmeasured on the 4th day after induction (Fig. 3 and Table 1).

man ADMSCs seeded on the acellular matrix (inverted microscope, ×400). (B) Someicroscope, ×400). (C) The number of cells adhered to the material was increased at

W. Hu et al. / Tissue and Cell 45 (2013) 295– 305 297

0

2

4

6

8

10

12

24h 48 h 72 h

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lls (1

*105 )

�me of see d

matrix

plates

Fig. 3. Cell proliferation.

Table 1Effects of membranous matrix on cell proliferation.

24 h 48 h 72 h 96 h

Matrix 0.53 ± 0.09 0.79 ± 0.16 3.18 ± 0.61 9.27 ± 0.62*

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Table 3Nestin, NSE, SYN and GFAP expression of 2nd to 4th step cells in CG and EG (%, n = 10).

G Index d 1 d 3 d 6

CG

Nestin 86.87 ± 4.86 80.92 ± 6.58 87.72 ± 5.34*

NSE 45.98 ± 8.74 87.36 ± 11.83 83.82 ± 8.33�

SYN 5.09 ± 1.03 8.14 ± 0.83 10.36 ± 1.26*

GFAP 9.05 ± 1.03 6.22 ± 0.25 7.43 ± 0.71*

EG

Nestin 80.69 ± 16.18 73.05 ± 10.12 70.87 ± 14.31*,�

NSE 57.63 ± 5.98 83.18 ± 5.90 88.26 ± 10.85� , **

SYN 4.35 ± 1.37 33.03 ± 5.58 65.71 ± 6.97� ,�

GFAP 7.76 ± 1.40 3.11 ± 0.75 3.06 ± 0.91� ,�

* Ver d1: P > 0.05.

24-Well plates 0.45 ± 0.06 0.82 ± 0.14 2.59 ± 0.35 6.30 ± 0.99

* P = 0.003 (compare between groups, t-test).

.2. Differentiation

.2.1. Morphological changesThe 3rd passage human ADMSCs had a fibrocyte-like appearance

ith long, slim cell bodies, spindle shapes and large nuclei, andrew in small swirls or clusters of cells. In the experimental groupEG), the morphological changes were observed under an inverted

icroscope (Fig. 4A–I).In the control group (CG), the morphological changes were quite

ifferent from those in the EG (Fig. 5A–C).

.2.2. Results of immunohistochemical stainingThe Nestin, NSE, SYN and GFAP expression levels in human

DMSCs in the 1st to 3rd passages and cells in the 2nd through 4thteps of induction (four step methods was seen in phase “Experi-ental groups and induced differentiation”) were determined by

he SP method. Furthermore, the levels of NSE and SYN in cells onhe 6th day of differentiation were determined by the immunofluo-escence double staining method.

The Nestin and NSE expression of the 3rd passage human ADM-Cs was significantly higher than that of the 1st passage after therst step (Table 2, P < 0.01, Fig. 6), and the SYN and GFAP expres-ion had no significant changes (Table 2, P > 0.05). Then after threeteps, compared d 6 with d 1, the Nestin expression of cells in EG

egraded but had no statistical significance (Table 3, P < 0.01); theestin, SYN and GFAP expression in CG had no significant differ-nce (Table 3, P > 0.05), and the NSE expression up-regulation with

able 2estin, SYN, NSE and GFAP expression of the 1st to 3rd passage cells (%, n = 10).

Index P 1 P 2 P 3

Nestin 35.13 ± 6.85 67.11 ± 7.22 86.52 ± 2.20*

NSE 9.69 ± 1.29 39.27 ± 2.75 47.10 ± 10.06*

SYN 3.01 ± 0.52 3.53 ± 0.73 3.09 ± 0.52#

GFAP 7.81 ± 0.70 5.16 ± 0.82 8.67 ± 1.72#

s P1:* P < 0.01.# P > 0.05.

� Ver d1: P < 0.05.� Ver CG: P < 0.05.** Ver CG: P > 0.05.

significant difference (Table 3, P < 0.01). Indexes compared betweentwo groups at d 6, the variance of NSE expression had no statisti-cal significance (Table 3, P > 0.05), SYN expression was higher thanthat in CG and Nestin, GFAP expression were lower than that in CGwith statistical significance both (Table 3, P < 0.01), the SYN expres-sion after induced differentiation was as high as 65.71% in EG and10.36% in CG (Table 3 and Fig. 7).

2.2.3. RT-PCR resultsBefore and during the four-step differentiation process, the first

passage human ADMSCs and ADMSCs on the 0th, 2nd, 3rd and 6thdays of the differentiation process were collected. The mRNA lev-els of several genes were measured, including Nanog, SOX2, OCT4,Nestin, Olig2, SYNI, SYNII, SYNIII, ISL-1 and Hb9. The results areshown in Fig. 8.

3. Discussion

Based on studies of embryonic development and cytogenetics,the differentiation of stem cells into cellular lineages involves vari-ous developmental stages of cells expressing specific sets of genes.For example, neural differentiation is a process where the expres-sion of specific genes is acquired at defined times over the course ofdevelopment through external environment changes. Several pro-posed models, including the “dynamics relevance” model (Bissel,1982), the neuron differentiation model (Sun et al., 2001) and the“stem cell niche” theory published recently (Moore and Lemischka,2006), have provided further evidence that the differentiation ofstem cells follows the rules of neurodevelopment, which are closelyrelated to the surrounding microenvironment, cell factors and ECM.An ECM with cell factors enables cells to build a microenvironmentthat contains signals for survival and differentiation; these signalscan be given both through direct contact and by indirect contact(i.e., paracrine or cell factors). Stem cells respond to specific reg-ulatory signals in these microenvironments and differentiate intovarious types of cells. Therefore, imitate the proper microenviron-ment for the neurodevelopment process is necessary to inducestem cells to differentiate into nervous system cells, and the neu-ral differentiation of ASCs can also use a multi-part method tosimulate the microenvironment during neurodevelopment forinduced differentiation.

Currently, scientists have conducted three-dimensional culti-vation of ESCs on ECM, and discovered that this cultivation canpromote ESCs to differentiate into cells of various types, includingneural cells. Furthermore, it is possible to build reasonable spatialstructures of stem cells (Levenberg et al., 2003), and many cases

of successful differentiation have been reported with multi-stepinduced differentiation of ESCs into neural cells (Wang et al., 2008;Lee et al., 2000; Li et al., 2005). However, there have not beenany reports on neural differentiation of ASCs with a multi-step

298 W. Hu et al. / Tissue and Cell 45 (2013) 295– 305

Fig. 4. Morphological changes of cells in EG. (A–C) 24th hour after inoculation, cells became round and adhered to the membranous matrix tightly with high refractivity,c Protrui s extenc n (inv

itttfv

fueiwr

Fac

ell bodies stretch out protrusion (inverted microscope; ×200, ×200, ×400). (D–F)

nduced differentiation (inverted microscope; ×200, ×200, ×400). (G–I) Protrusiononnected to form a network structure at the 6th day during induced differentiatio

nduction in conjunction with ECM and cell factors to simulatehe neurodevelopment process. Therefore, we have examinedhe differentiation of human ADMSCs into neural cells based onhe stem cell niche principle, with common chemokines and cellactors involved in neurodevelopment present on a natural ECM initro.

Basic fibrocyte growth factor (bFGF) is an important mitogenicactor and one of the multifunctional signaling proteins that reg-lates normal embryonic development and adult physiological

quilibrium. It is highly expressed at early developmental phasesn animals but only expressed at low levels once neurogenesis

as nearly complete (Raballo et al., 2000). bFGF can promote neu-al progenitors to differentiate into neural precursors (Pappas and

ig. 5. Morphological changes of cells in CG. (A) Cells adherence in CG (inverted microscodded, with poor process of proliferation (inverted microscope; ×200). (C) The protrusionsells when retinoic acid was removed (inverted microscope; ×200).

sions in most cells were extended and touch with each other at the 3rd day duringded and form the primary and secondary branches, the cells and their protrusions

erted microscope; ×200, ×200, ×200).

Parnavelas, 1998). It is an important factor for the survival andproliferation of neural precursor cells in early development stage(Okabe et al., 1996), is capable of promoting neural precursor cellproliferation and the survival and proliferation of the daughter cellsof stem cells (Alanko et al., 1996) and prolonging the differentia-tion of neural precursor cells into mature neural cells (Cavanaghet al., 1997; Vaccarino et al., 1999). If removed, bFGF can cause neu-ronal cells to dedifferentiate and become similar to neuronal stemcells (Palmer et al., 1997; O’Shea, 2001). Retinoic acid (RA) is an

important component in the microenvironment of early neurode-velopment and is a strong inducer of differentiation. Specifically, itsbiological effect is to regulate transcription and affect gene expres-sion in conjunction with nuclear RA receptors (RARs). RA is an

pe; ×200). (B) In CG, only few protrusions occurrence in cells after retinoic acid was disappeared and the cell proliferated significantly but returned to the fibrocyte-like

W. Hu et al. / Tissue and Cell 45 (2013) 295– 305 299

F ) Nes(

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ig. 6. Nestin expression of P 1–3 in human ADMSCs (SP method, DAB staining). (AC) Nestin expression of 3rd passage (×100).

mportant biologically active substance in the generation of neu-al plates (Danet et al., 2002), which enable distinct parts of therain to distribute and differentiate along the cephalocaudal axisf the neural plates in early stages of brain development. RA alsoas important effects on the development of the rhombencephalon,erebellum and mesencephalon (Deans, 2000; Eriees et al., 2000;ang et al., 2004). RA regulates the expression of several recep-ors for neurotrophic factors on neural cells, which are importantactors for the development of the nervous system. Neurotrophicactors (NTs) and their receptors are important regulatory factorsn the differentiation of NSCs. Therefore, RA may have effects (Sunt al., 2002; Wohl and Weiss, 1998) on the differentiation of NSCs byncreasing the expression of NT receptors in NSCs and by increasinghe expression of NT receptors in NSCs and by modulating the affin-ty of NT receptors for their ligands. One study has demonstratedhat RA can promote the expression (Gearhart, 1998) of proto-ncogenes, such as c-fos, c-jun and others, which control severalrowth factors and play important roles in the neurodevelopmentrocess. Finally, RA can promote survival and proliferation of multi-otent neural crest precursor cells and induce them to differentiate

nto neurons (Sanchez-Ramos, 2002).ECM is largely composed of collagens, diastins, fibronectins, glu-

oproteins, proteoglycans and laminins. These proteins performarious functions, including the following: providing structuralupport for cell adhesion and movement, maintaining tension, reg-lating the differentiation and metabolism of cells (Feltri et al.,002; Li et al., 2003a,b) and aiding in medullary sheath forma-ion (Mirsky et al., 2002), neuron protection and the regulationf synapse integration (Podratz et al., 2001; Lu et al., 2001; Harit al., 2004). These ECM components can limit the diffusion ofrowth factors (Ard et al., 1987) and promote the creation ofigand–receptor complexes to strengthen the effects of growth

actors.

Natural ECM contains the biological elements and three-imensional structure necessary for nerve regeneration without

ig. 7. NSE, SYN expression at d 6 (immunofluorescence). (A) D 6, NSE expressed in cell bell body in the same location in EG (inverted microscope; ×400).

tin expression of 1st passage (×100). (B) Nestin expression of 2nd passage (×100).

immunogenicity, thus more and more scholars who started deepresearches on it. Qi and Zhu (2007) proved that laminins were con-tribute to NSCs’ directional differentiation to neurons. Chalazonitiset al. (1997) demonstrated that � chain of laminin-1 could promotethe precursor cells from the nerve crest to develop into neuronsafter binding with plasma membrane protein.

Natural ECM contains the biological elements and three-dimensional structure necessary for nerve regeneration withoutthe risk of immunogenicity; thus, increasing numbers of scho-lars have begun studying it. Qi and Zhu (2007) demonstrated thatlaminins contributed to the differentiation of NSCs into neurons.Chalazonitis et al. (1997) reported that the � chain of laminin-1could promote precursor cells from the nerve crest to develop intoneurons after binding with after binding to an unknown receptor.

We used bFGF to pre-induce differentiation in the 1st step of ourprotocol due to the fact that bFGF is known to affect neural differen-tiation and physiology of NSCs. After this treatment, we employedimmunoassays to determine that the percentages of Nestin positivecells significantly increased from 35.13% in the 1st to 86.52% in the3rd passage. Similarly, we observed a significant increase in theamount of cells expressing NSE (9.69–47.10%) but no significantchange in SYN expression. Jiang et al. (2002) previously demon-strated that low numbers of embryonic stem cells are present inmany tissues in adults; these cells are referred to as multi-potentadult progenitor cells (MAPCs). MAPCs could be induced in vitroto differentiate into cells of various embryonic layers, such as theendoderm, mesoderm and ectoderm or into stem cells of varioustissues. Woodbury et al. (2000) demonstrated that gene expressionspecific to cells in different embryonic layers could be detectedin undifferentiated mesenchymal stem cells. Based on the liter-ature, this study suggests that some subsets of human ADMSCsmight have characteristics of neural progenitor cells and that the

subgroup with neural progenitor characteristics rapidly differenti-ated into neural precursor cells and continued to proliferate whentreated with bFGF. In conjunction with this idea, the percentage

ody in EG (inverted microscope; ×400). (B) D 6, SYN expressed in protrusions and

300 W. Hu et al. / Tissue and Cel

Fig. 8. Variation of genetic expression in EG, CG. (EP1) In first passage human ADM-SCs of EG, Nanog, SOX2, OCT4, Nestin expressed at different level, Olig2, SYNI, SYNII,SYNIII, ISL-1, Hb9 did not expressed. (ED0) Nanog expression lowered, SOX2, Nestinexpression enhanced, Olig2, SYNI, SYNII, SYNIII, ISL-1, Hb9 did not expressed. (ED2)Nanog, SOX2 expression disappeared, Nestin expression unchanged, OCT4, SYNIIIexpressions were poor, Olig2, SYNI, SYNII, ISL-1, Hb9 did not expressed. (ED3)Nanog, SOX2 did not expressed, Olig2, OCT4 expressions were poor, Nestin, SYNII,ISL-1 expressed obviously, Hb9, SYNI did not expressed. (ED6) Nanog, OCT4, SOX2did not expressed, Nestin expression was low, SYNI, SYNIII, ISL-1, Hb9 expressedobviously, Olig2 expressed poorly. Vs EG, SYNI, SYNII, Hb9 had not expression inCG. (N) P1 = human ADMSCs of passage 1; D0 = after preinduction, before seededoa

odpontt

scoEititerf3cicdo

Consequently, we added RA and NGF in this step to promote the dif-

f human ADMSCs; D2 = 2 days after seeded; D3 = 3 days after seeded; D6 = 6 daysfter seeded; E = experimental group; C = control group.

f cells expressing Nestin increased swiftly after bFGF-inducedifferentiation, and this step was equivalent to the proliferationhase of neural progenitor cells that occurs in development. Inur experiment, some cells also expressed NSE but not SYN, whileo morphological changes typical of neurons occurred. Together,hese results indicate that bFGF treatment alone was not sufficiento differentiate human ADMSCs into mature neural precursor cells.

Next, we replaced the serum in the media with B27 in the secondtep in an attempt to recreate the no-serum microenvironment thatells would be exposed to during neural developmental. Through-ut this process, the heparin sulfate proteoglycan present in theCM could bind to bFGF to prevent its degradation and maintaints biological activity for long periods of time (Li et al., 2000). Fur-hermore, bFGF increased the expression of retinoic acid receptorn neural precursor cells (Shiotsugu et al., 2004) in preparation forhe next step of induced differentiation. The Nestin, SYN and GFAPxpression levels in cells in the EG were 80.69%, 4.35% and 7.76%,espectively, on the first day after inoculation, although these dif-erences were not significant compared with the first step (86.52%,.09% and 8.67%, respectively). The NSE expression (57.63%) of theells in the EG was significantly increased compared with thosen the first step (47.10%) and the CG (45.98%). This result indi-

ated that the ECM provided cues to the neural precursor cells toifferentiation into neurons, which is corroborated by the resultsf Chalazonitis et al. (1997). Cells in this step were cultivated on

l 45 (2013) 295– 305

the ECM for two days, after which, a decrease in proliferation wasobserved, possibly because the serum was removed. On the 1st dayafter inoculation, the expression of some neural marker proteinsincreased, and the cells appeared round or mostly round, with largesaturated nuclei and only a small number of prominences. How-ever, up to the end of the 2nd day, no interconnected mesh-likestructure had formed among the cells.

We removed bFGF and added RA and nerve growth factor (NGF)in 3rd step. RA was capable to increase the activity of cholineacetyltransferase (ChAT) and acetylcholine esterase (AChE) of thecells and differentiate them into cholinergic neurons (Matsuokaet al., 1989; Hill and Robertson, 1998). Furthermore, RA increasedexpression of various neurotrophic factor’s receptor on cell surface,neurotrophic factors and their receptors were important regulatedfactors for stem cell differentiation (Wohl and Weiss, 1998), theycould accelerate cells differentiate into mature one (Takahashi et al.,1999). It was well known that NGF played prominent role on main-taining telotism of central cholinergic neurons. Inject NGF slowlyinto the brains of adult mice and aged mice could increase phe-notype expression of cholinergic neurons as well as synthesis andrelease of Ach (Rylett and Williams, 1994). RA could increase NGFreceptor-TrkA expression of the neural stem cell and strengthenthe effect of NGF, thus RA and NGF could collaborate to promotethe stem cells differentiate into cholinergic neurons (Berse andBlusztajn, 1995). Consequently, we added RA and NGF in this step topromote differentiation of human ADMSCs. The protein expressionof two groups was analyzed statistically in the 3rd day after inocu-lation, the Nestin and GFAP expressions of the EG were 73.05% and3.11% respectively, which was significantly lower (P > 0.05) thanthose at d 0 and d 1 (86.52%, 8.67%; 80.69%, 7.76%). The NSE andSYN expressions were 83.18% and 33.03% respectively, which wassignificantly higher (P < 0.05) than those of d 0 and d 1 (47.10%,3.09%; 57.63%, 4.35%). SYN expression was significantly increased(33.03% versus 8.14%; P < 0.05) and the GFAP expression was sig-nificantly lowered (3.11% versus 6.22%; P < 0.05) compared EG withCG. Compared expression of different time points in CG, NSE, SYNexpressions in d 3 (87.36%, 8.14%) was significantly higher thanthe expressions in d 0 and d 1 (47.10%, 3.09%; 45.98%, 5.09%). Thedata above showed that collaborated application of RA and NGFcould promote stem cells differentiate to neurons and inhibit themdifferentiate to glial cells. The collaboration of RA, NGF and ECMwas more significant, obvious expression of SYN which hinted thegrowth of axons, part cells extended prominences this moment andinterconnected, but had not formed the mesh shape.

We removed bFGF and added RA and nerve growth factor (NGF)in the 3rd step of differentiation. RA is known to increase the activ-ity of choline acetyltransferase (ChAT) and acetylcholine esterase(AChE) in the cells and cause them to differentiate into cholin-ergic neurons (Matsuoka et al., 1989; Hill and Robertson, 1998).Furthermore, RA increased the expression of receptors for variousneurotrophic factors on the cell surface. These neurotrophic factorsand their receptors are important regulatory factors for stem celldifferentiation (Wohl and Weiss, 1998), and can cause cells to dif-ferentiate into mature cell types (Takahashi et al., 1999). It is wellknown that NGF plays a prominent role in maintaining telotism ofcentral cholinergic neurons. Slow injection of NGF into the brains ofadult mice and aged mice increased the expression of proteins asso-ciated with cholinergic neurons and the synthesis and release ofAch (Rylett and Williams, 1994). RA increases the expression of theNGF receptor TrkA in neural stem cells and strengthens the effect ofNGF; thus RA and NGF may synergize to promote the differentiationof stem cells into cholinergic neurons (Berse and Blusztajn, 1995).

ferentiation of human ADMSCs. The protein expression in the EGand CG was analyzed on the 3rd day after inoculation. The percent-ages of cells positive for Nestin and GFAP expression in the EG were

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W. Hu et al. / Tissue a

3.05% and 3.11%, respectively, both of which were significantlyower (P > 0.05) than those of cells at d 0 and d 1 (Nestin: 86.52%nd 80.69%, respectively; GFAP: 8.67% and 7.76%, respectively). Theercentages of cells with NSE and SYN expression were 83.18%nd 33.03%, respectively, both of which were significantly higherP < 0.05) than those of cells at d 0 and d 1 (NSE: 47.10% and 57.63%,espectively; SYN: 3.09% and 4.35%, respectively). The percentagef cells expressing SYN was significantly increased (33.03% versus.14%; P < 0.05), and the GFAP expression was significantly lowered3.11% versus 6.22%; P < 0.05) in cells in the EG compared with thosen the CG. The percentage of cells that were positive for NSE and SYNxpression in the CG on d 3 (87.36% and 8.14%, respectively) wereignificantly higher than those on d 0 and d 1 (NSE: 47.10% and5.98%, respectively; SYN: 3.09% and 5.09%, respectively). Theseata demonstrate that the combined application of RA and NGFaused stem cells to differentiate into neurons and blocked theirifferentiation into glial cells. The synergy between RA, NGF andCM was more significant, as evidenced by the clear expression ofYN, which usually correlates with the growth of axons, and the facthat some cells extended protrusions and became interconnected,ven though an extensive network of cells had not yet formed.

We removed RA and added bFGF in the 4th step of the differ-ntiation protocol. The immunohistochemical analysis on the 6thay showed that in cells in the EG compared with those in the CG,SE expression (88.26% versus 83.82%) was not significantly dif-

erent (P > 0.05), SYN expression (65.71% versus 10.36%) increasedignificantly (P < 0.05) and the GFAP expression decreased signifi-antly (3.06% versus 7.43%, P > 0.05). At this stage of differentiation,he cell proliferation significantly increased, compared with ear-ier stages, when bFGF was added. When it grown to 80% bottomrea, an integrated body was formed and was made up of cells, cellembrane receptors, membrane proteins, ECM and growth fac-

ors. The contact points between cells were increased, and the cellsxtended secondary branches to form a mesh-like structure witheighboring cells that was significantly different from the mor-hological changes that occurred in the 3rd step. These changes

ndicated that contact between the ECM and the cells was requiredn addition to other differentiation-inducing factors in the forma-ion of cell synapses, which is similar to what occurs in endogenouseurodevelopment. The cells at this step were further differenti-ted toward mature neuronal cells, and a significant number ofells expressed SYN and formed a mesh-like structure. Based on ourbservations of the morphological appearance, the control groupad not formed the neural network-like structures.

The neural cells of nervous system are derived from neuralrogenitor cells, which come from a specific region of the neuralctoderm. There are roughly three steps that a cell must undergoo develop from a cell of the neural ectoderm into a mature neu-on: (1) acquisition of a neural qualification, i.e., a cell determineshat it will not differentiate into an epithelial cell; at this time, theell is called to a neural progenitor cell; (2) specialization of theeural pedigree, i.e., further differentiation toward the neural lin-age; the differentiation of neurons and spongiocytes starts aroundhis time. At this point, a neural epithelial cell first acquires a head-ail axis, which is determined by extracellular signaling molecules,ncluding bFGF, Wnt and retinoic acid excreted from the paraxial

esoderm (Wichterle et al., 2002; Hébert et al., 2002; Shimogorit al., 2004); (3) Determination of neuron characteristics, includ-ng neuronal-specific morphology, excretion ability and electronxcitation (Chen and Lu, 2002). A long, complicated and preciselyegulated process is necessary for CNS development.

A block in the signaling pathway downstream of the fibroblast

rowth factor inhibits the expression of the RA receptor, and over-xpression of the RA receptor can rescue this defect in fibroblastrowth factor, which indicates that the RA and fibroblast growthactor pathways are located upstream and downstream of the

l 45 (2013) 295– 305 301

neural differentiation signal chain, and that the RA receptor is adirect downstream target (Shiotsugu et al., 2004) of the fibroblastgrowth factor pathway. bFGF can induce cultured human embry-onic neural stem cells to form cholinergic neurons independently(Wu et al., 2002), which indicates that bFGF is an effective factor inthe differentiation to MN in vitro. The joint effect of RA and bFGFcan cause differentiation into MNs during spinal cord development(Mizuseki et al., 2003; Wichterle et al., 2002). Thus, we hypothe-sized that human ADMSCs may differentiate and acquire an MNphenotype under the combined effects of bFGF, RA and NGF.

Therefore, in addition to observing morphological and immuno-histochemical changes during our multi-step induction, weperformed gene profiling at the transcriptional level of the humanADMSCs during the induction. Expression of Nanog, SOX2, OCT4and Nestin were observed at different levels in 1st passage humanADMSCs, but they did not express Olig2, SYNI, SYNII, SYNIII, ISL-1 orHb9. Nanog and OCT4 are multi-potent markers of ESCs (Chamberset al., 2003; Mitsui et al., 2003; Young and Black, 2004), and SOX2is expressed in neural progenitor cells (Miyagi et al., 2006), whichindicates that human ADMSCs are composed of multiple subsets,some of which have markers for ESCs and some for neural progen-itor cells. Next, the multi-step differentiation was carried out. Thefirst step was to pre-induce the cells with bFGF for 3 days and exam-ine the levels of various gene transcripts before inoculation. A thistime point, the expression levels of Nanog and OCT4 were slightlydecreased, while the levels of SOX2 and Nestin were increased.Olig2, SYNI, SYNII, SYNIII, ISL-1 and Hb9 were not expressed. Itcould be inferred from the perspective of neurodevelopment thatthe human ADMSCs at this time were in a phase where endogenousneural progenitor cells proliferate and differentiate into neural pre-cursor cells. bFGF is known to play a critical role at this stage.During this phase, after three passages, the neural progenitor cellsubgroups within the human ADMSCs population differentiatedand expanded, and a large quantity of neural precursor cells wasacquired that could be used to establish a foundation for furtherdifferentiation. The second step was to inoculate the cells on themembranous ECM, and thereby provide a co-induction with bFGFand ECM. The cells were inspected on the 2nd day after inoculation.There were many transcriptional changes: Nanog and SOX2 werenot expressed, Nestin expression remained unchanged, OCT4 andSYNIII expression levels were low, and Olig2, SYNI, SYNII, ISL-1 andHb9 were not expressed. These results indicate that, at this time, theneural precursor cells were a more pure population, and the SYNIIIexpression increase indicated the formation of cell growth cones.In the third step, after the culture media was replaced and differ-entiation was induced by the ECM and retinoic acid together for24 h, Olig2 and OCT4 were expressed at low levels, while Nanog andSOX2 were not expressed, Nestin, SYNII and ISL-1 were expressed atsignificantly increased levels and Hb9 and SYNI were not expressed.At this time, the neural precursor cells were further differentiatedby RA, which is equivalent to the phase where the head–tail axisusually established, the expression of ESCs disappears, SYNII beginsto be expressed, cell processes extend and some cells become inter-connected. These changes were consistent with a previous report(Ferreira et al., 1994). The combined effect of RA and NGF wasable to promote stem cell differentiation into cholinergic neurons(Hébert et al., 2002), and the ISL-1 expression supports this hypoth-esis. At this stage, the neural precursor cells had the foundation tofurther differentiate into MNs (Hutchinson and Eisen, 2006). TheOlig2 expression at this point indicated that the cells also had theability to differentiate into oligodendrocytes. In the 4th step, thecells further proliferated due to the presence of bFGF and became

interconnected. As for the transcriptional changes, Nanog, OCT4and SOX2 were not expressed, Nestin and Olig2 were expressed atlow levels and the levels of SYNI, SYNIII, ISL-1 and Hb9 were signifi-cantly increased. Hb9 is a necessary gene (Arber et al., 1999) for MN

3 nd Cell 45 (2013) 295– 305

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4

1

2

3

5

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Table 4Meaning of the indexes in protein level.

Index Distribution Function

Nestin CNS Identify neural progenitorsNSE Neurons, neuroendocrine

cellsIdentify neurons

SYN Axon terminal; cell body(during development)

Neurotransmitter release,synapses occurrence and

02 W. Hu et al. / Tissue a

ifferentiation, and thus the high expression of Hb9 illustrated thathe human ADMSCs had differentiated into MN precursor cells. Theuman ADMSCs in this period were equivalent to neuraxis ventralomite cells: after SYNII expression enabled the neurons to gener-te membrane protrusions and extend gradually, SYN I and SYNIIIxpression allowed the cells synapses to grow and mature fully,hereby forming the primary and secondary branches (Chin et al.,995).

The POU transcription factor Oct4, which is encoded by Pou5flay be necessary for neurogenesis (Shimozaki et al., 2003). Our RT-

CR result indicated that Oct4 expression remained constant longerhan other ESC differentiation markers (i.e., Nanog) and decreasedlowly during the neural differentiated process. This result was con-istent with previous reports (Gerrard et al., 2005; Nat et al., 2007;errier et al., 2004), which indicates that constant expression ofct4 for a short period may be essential for the differentiation of

tem cells into neural cells in vitro.We discovered in CG that SYNI and SYN II expression lacked, and

SL-1 and Hb9 expressions were significantly lower than that in EG.nd RT-PCR resultS was shown in Fig. 7.

We discovered that there was no SYNI or SYNII expression in theG, and ISL-1 and Hb9 levels were significantly lower than those inhe EG. The RT-PCR results are shown in Fig. 7.

Studies on the directional differentiation of ASCs into neuronsre sparser than those on ESC differentiation. Many mature andunctional neurons have been successfully differentiated from ESCs,uch as dopamine neurons (Perrier et al., 2004) and MNs (Wichterlet al., 2002). The successful differentiation of ASCs into functionaleurons is significant because they could replace ESCs in futurelinical treatments, which would avoid difficulties regarding acqui-ition, immunological rejection and ethics. This experiment utilizedhe ESC induction method as a reference, and the induction programnvolves providing multiple factors that are required for completeell differentiation. Upon exposure to a step-by-step induction pro-ocol including a matrix and other differentiation factors (RA, bFGFnd ECM), ADMSCs differentiated and expressed mature synapto-hysins and markers for MN precursor cells. Furthermore, theyenerated a neural network-like structure. These results indicatehat a step-wise differentiation is able to cause human ADMSCso differentiate into MNs under a model that simulates embryoniceurodevelopment.

. Conclusion

. The ECM extracted with the method used in this paper hadgood biological compatibility and was capable of promoting theformation of mature synapses during the neural differentiationprocess of human ADMSCs.

. Human ADMSCs are composed of a group of phenotypically dif-ferent cell subsets, including neural progenitor cells.

. The step-by-step induction method allows human ADMSCs todifferentiate into MN precursor cells.

. Materials and methods

.1. Samples and antibodies

Amnion samples from healthy women who underwent cesareanections under strict aseptic conditions were collected from theaternity Department of No. 1 Affiliated Hospital of Zhengzhouniversity.

Antibodies against the following proteins were obtainedrom Chemicon (Temecula, CA): nestin, NSE, NF, GFAP and SYN.econdary antibodies (FITC goat anti-mouse IgG and Guani-inium Isothiocyanate goat anti-rabbit IgG) were obtained from

extensionGFAP Cytoplasm of glial cell Marker of astrocyte

Jackson ImmunoResearch (West Grove, PA). DAPI fluorchrome wasobtained from AppliChem (Beijing). An RT-PCR kit was obtainedfrom TaKaRa (DRR014A type, Japan).

5.2. Culture media

The cells were cultured in three different types of medium:Basic culture media (DMEM/F12, 10% fetal calf serum (FCS) and20 �g/L bFGF), Neural basic culture media (DMEM/F12, 20 g/L B27and 20 �g/L bFGF) and Neural induced culture media (DMEM/F12,10–3 mM/L RA, 20 g/L B27 and 20 ng/ml �-NGF).

5.3. Separation and culture of human ADMSCs

Separation: placental amnions were separated from the pla-centa, rinsed with phosphate buffered saline (PBS) and cut intosmall pieces. Pancreatin (2.5 g/L) was added, and the tissue wasdigested for 30 min at 37 ◦C. The digest was terminated by the addi-tion of FCS, and then the tissue was further digested with 1 g/Lcollagenase for 60 min. A single cell suspension was formed byfiltration through stainless steel mesh. Cells were collected aftercentrifugation for 10 min at 1000 rpm and were stained with Try-pan Blue to count live cells. Finally, they were used to inoculate a75 ml culture flask at 2 × 105 cells/ml.

Culture, Purification and Amplification: DMEM/F12 (v/v = 1:1)culture media supplemented with 10% FCS and 20 g/L bFGF wasadded to the culture flask. The cells were added to 75 ml cultureflasks separately and grown at 37 ◦C, 5% CO2 in an incubator withsaturated humidity. After approximately 48–72 h, the medium wasreplaced to discard nonadherent cells and the media was subse-quently replaced once every 3–4 days. When the cells reached80–90% confluency, they were collected by trypsinization and inoc-ulated for subculturing.

5.4. Preparation of the membranous matrix

The amnion was sheared into 3 cm × 3 cm pieces, soaked in glyc-erine at 4 ◦C for 24 h and washed with saline repeatedly. It wassubsequently passed over a Millipore filter and soaked in a hypo-tonic solution (100 ml normal saline in 100 ml distilled water) for3 min. Next, the tissue was trypsinized at 4 ◦C for 10 h, disrupted ona stirring incubator in saline for 1 h and finally an acellular mem-branous matrix was formed.

5.5. Experimental groups and induced differentiation

Cells were separated into the EG and CG. Eight independentrepeats were performed for each group.

The EG was differentiated by a step-wise induction as follows.The 1st step: the human ADMSCs were pre-induced by cultiva-tion in basic culture media until the 3rd passage. Day (d) 0 was set

when this step was ended. The 2nd step: the 3rd passage humanADMSCs (4 × 104 cells/ml) were inoculated on cover glasses coatedwith membranous matrix in 24-well plates and cultured with basicnerve culture media for 2 days. This step occurred over d 0 to d 2.

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W. Hu et al. / Tissue a

he 3rd step: after reaching approximately 40% confluency on theembrane, the cells were washed with PBS, and nerve induction

ulture media was added. The cells were subsequently cultured forn additional 24 h on the membranous matrix, and this step wasounted d 2–3. The 4th step: the cells were washed with PBS, andasic culture media was added. The cells were then cultured fornother 3 days, which were recorded as d 4–6.

For the cells in the CG, the culture procedures were identical tohose in the EG except that there was no membranous matrix onhe cover glass.

.6. Acellular matrix

Morphological changes: small samples of membranous matrixnd fresh amnion were randomly collected and fixed for 6 h in 4%olyoxymethylene. They were then stained with hematoxylin andosin (H&E) and observed under a light microscope to determinehether there were any residual cells.

Cellular compatibility: the acellular matrix was hydrated withulture media before the addition of 1 ml (1 × 105 cells/ml) of 3rdassage human ADMSCs. The culture wells were filled with 9 ml ofMEM/F12 media, the samples were incubated in a 5% CO2 cham-er with saturated humidity. The culture media was replaced everyther day, and we observed the adhesion and growth of the cellsnder an inversed phase contrast microscope.

Effects of the membranous matrix on cell proliferation: the 3rdeneration human ADMSCs (4 × 104 cells/ml) were seeded in 24-ell plates either coated with membranous matrix or not. The cellsere cultured with basic culture media, and 6 samples were ran-omly collected every 24 h and digested with pancreatic enzyme.fter terminating the digestion, the cells were resuspended 1 ml ofulture media. Then a 50 �l samples was mixed with 50 �l Trypanlue (w/v, 0.2%), and the live cells were counted on a hemocytome-er. This process was repeated 3 times and the mean cell numberas determined for each day.

.7. Determination of morphological changes and protein and

ene level in the cells

Cells in the 1st, 2nd and 3rd passages were pre-inoculated andells at d 1, d 3 and d 6 from the EG and CG were collected. Six

able 5rimer sequences list used in the experiment.

Genes Pimers (sequences 5′–3′)

Nanog Sence: GCT′ATT′CTT′CGG′CCA′GTT

Anti-sence AAC′TGG′CCG′AAG′AAT′AGC

SOX2 Sence: ACC TTT GTA GGC TGG GAA TCAnti-sence ATC ACG GCA GAA ATC ACC AA

OCT4 Sence: GTT′CCC′AAT′TCC′TTC′CTT′A

Anti-sence TAA′GGA′AGG′AAT′TGG′GAA′C

Nestin Sence: CCC′TTC′CAG′ACT′CCA′CTC

Anti-sence ACA′CTC′CTC′TTC′TCC′CTC′C

SYN I Sence: GCC′TGG′TAT′TTG′GGC′ACT′T

Anti-sence AAG′TGC′CCA′AAT′ACC′AGG′C

SYN II Sence: CCA′CTA′AAA′CTC′ACA′GCG′AAAnti-sence GTT′CGC′TGT′GAG′TTT′AGT′GG

SYN III Sence: CAC′GCT′GGT′GAT′GTC′CTG

Anti-sence CAG′GAC′ATC′ACC′AGC′GTG

ISL-1 Sence: GAT′TAC′ACT′CCG′CAC′ATT

Anti-sence AAT′GTG′CGG′AGT′GTA′ATC

Hb9 Sence: GCT′GCG′TTT′CCA′TTT′CAT′CC

Anti-sence GGA′TGA′AAT′GGA′AAC′GCA′G

l 45 (2013) 295– 305 303

random samples were collected from each well, rinsed with PBSand fixed with 4% polyoxymethylene.

The fixed cells were then stained with H&E. Cellular morphol-ogy was observed pre-stain and post-stain under an inverted phasecontrast microscope (OLMPUS) and images were collected.

Some of the fixed cells were used to determine the levels ofNestin, NSE, GFAP, SYN and BrdU by the SP method. Images werealso collected on a fluorescence microscope. Immunofluorescencewas employed to detect the expression of SYN and NSE on the6th day by fluorescence microscope and images were taken. Theindexes of protein levels are listed in Table 4.

5.8. Assessment of immunohistochemical staining

After staining and mounting was performed, an area concen-trated with positive staining was selected from the slides under afluorescence microscope (×100 magnification). Ten photos weretaken with a high power lens (×400). The number of cells thatstained positive for Nestin, SYN, NSE and GFAP, and the total cellnumbers in the same area were calculated with the grid-countingmethod. Positive expression was calculated as the number of pos-itive cells/number of total cells × 100%. The Image-pro plus 6.0colored pathological image analysis system was adopted for imageanalysis.

5.9. Detection of mRNA expression

Cells in the 1st passage and cells at 0, 2nd, 3rd and 6th day dur-ing the differentiation process were selected, washed three timeswith PBS, then the mRNA levels of several genes were detected. Theprimer sequences used are listed in Table 5.

GAPDH was used as and internal control. The reaction prod-uct was separated by gel electrophoresis on a 2% agarose gel. Thebands were visualized and their densities were analyzed with a

gel imaging analyzer to obtain the relative levels of various genetictranscripts in the EG and CG. The primers were synthesized afterdetermining the appropriate sequences with the NCBI gene bank.The primer sequences are listed in Table 2.

bp T Accession

267 52.0 NM 024865

G 131 64 NM 005634C

167 52.6 NM 002701

197 52.6 NM 006617

188 54.2 NM 006950

C 214 52.8 NM 133625

235 53.2 AF 046873

172 50.7 NM 002202

221 60.4 AF 107457C

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A

A

A

B

B

C

C

C

C

C

D

D

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H

H

H

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04 W. Hu et al. / Tissue a

.10. Statistical methods

All experiments were repeated at least three times. Data areresented as the mean ± standard deviation (SD). Statistical analy-is was carried with SPSS software (version 16.0; SPSS, Chicago, IL,SA). The Student’s t-test was used to analyze differences between

wo groups and a one-way analysis of variance (ANOVA) was usedo compare multiple groups. The statistical significance level waset at P < 0.05.

eferences

lanko, T., Tienari, J., Lehtonen, E., Saksela, O., 1996. FGF-2 inhibits apoptosis inhuman teratocarcinoma cells during differentiation on collagen substratum.Exp. Cell Res. 228, 306–312 (PubMed: 8912724).

rber, S., Han, B., Mendelsohn, M., Smith, M., Jessell, T.M., Sockanathan, S., 1999.Requirement for the homeobox gene Hb9 in the consolidation of motor neuronidentity. Neuron 23, 659–674 (PubMed: 10482234).

rd, M.D., Bunge, R.P., Bunge, M.B., 1987. Comparison of the Sehwann cell sur-face and Sehwann cell extracellular matrix as promoters of neurite growth. J.Neurocytol. 16, 539–555 (PubMed: 3681353).

erse, B., Blusztajn, J.K., 1995. Coordinated up-regulation of choline acetyltrans-ferase and vesicular acetylcholine transporter gene expression by the retinoicacid receptor alpha, cAMP, and leukemia inhibitory factor/ciliary neurotrophicfactor signaling pathways in a murine septal cell line. J. Biol. Chem. 22,22101–22104 (PubMed: 7673184).

issel, M.J., 1982. How does the extracellular matrix direct gene expression? J. Theor.Biol. 99, 31 (PubMed: 6892044).

avanagh, J.F., Mione, M.C., Pappas, I.S., Parnavelas, J.G., 1997. Basic fibroblast growthfactor prolongs the proliferation of rat cortical progenitor cells in vitro with-out altering their cell cycle parameters. Cereb. Cortex 7, 293–302 (PubMed:9177761).

halazonitis, A., Tennyson, V.M., Kibbey, M.C., Rothman, T.P., Gershon, M.D., 1997.The alpha 1 subunit of laminin-1 promotes the development of neurons by inter-acting with LBP110 expressed by neural crest-derived cells immunoselectedfrom the fetal mouse gut. J. Neurosci. 33, 118–138 (PubMed: 9240369).

hambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., Smith, A., 2003.Functional expression cloning of Nanog, a pluripotency sustaining factors in

embryonic stem cells. Cel1 113, 643–655 (PubMed: 12787505).hen, Y., Lu, C., 2002. Decision of the Neuronal Phenotype-Neurological Develop-

ment and Molecular Biology. Hubei Science and Technology Press, Hubei, China,pp. 55.

hin, L.S., Li, L., Ferreira, A., Kosik, K.S., Greengard, P., 1995. Impairment of axonaldevelopment and of synaptogenesis in hippocampal neurons of synapsin I-deficient mice. Proc. Natl. Acad. Sci. U. S. A. 92, 9230–9234 (PubMed: 7568107).

anet, G.H., Luongo, J.L., Butler, G., Lu, M.M., Tenner, A.J., Simon, M.C., Bonnet, D.A.,2002. 1C1qRP defines a new human stem cell population with hematopoieticand hepatic potential. Proc. Natl. Acad. Sci. U. S. A. 99, 10441–10445 (PubMed:12140365).

eans, R.J., 2000. Mesenehymal stem cells: biology and potential clinical uses. Exp.Hematol. 28, 875–884 (PubMed: 10989188).

riees, A., Conget, P., Minguell, J.J., 2000. Mesenchymal progenitor cells in humanumbilical cord blood. Br. J. Haematol. 109, 242–253 (PubMed: 10848804).

eltri, M.L., Graus Porta, D., Previtali, S.C., Nodari, A., Migliavacca, B., Cassetti, A.,Littlewood-Evans, A., Reichardt, L.F., Messing, A., Quattrini, A., Mueller, U.,Wrabetz, L., 2002. Conditional disruption of beta 1 integrin in Schwann cellsimpedes interactions with axons. J. Cell Biol. 156, 199–209 (PubMed: 11777940).

erreira, A., Kosik, K.S., Greengard, P., Han, H.Q., 1994. Aberrant neurites and synapticvesicle protein deficiency in synapsin II-depleted neurons. Science 264, 977–979(PubMed: 8178158).

earhart, J., 1998. New potential for human embryonic stem cells. Science 282,1061–1062 (PubMed: 9841453).

errard, L., Rodgers, L., Cui, W., 2005. Differentiation of human embryonic stem cellsto neural lineages in adherent culture by blocking bone morphogenetic proteinsignaling. Stem Cells 23, 1234–1241 (PubMed: 16002783).

ari, A., Djohar, B., Skutella, T., Montazeri, S., 2004. Neurotrophins and extracellularmatrix molecules modulate sensory axon outgrowth. Int. J. Dev. Neurosci. 22,113–117 (PubMed: 15036386).

ébert, J.M., Mishina, Y., McConnell, S.K., 2002. BMP signaling is required locallyto pattern the dorsal telencephalic midline. Neuron 35, 1029–1041 (PubMed:12354394).

ill, D.P., Robertson, K.A., 1998. Differentiation of LA-N-5 neuroblastoma cells intocholinergic neurons: methods for differentiation, immunohistochemistry andreporter gene introduction. Brain Res. Brain Res. Protoc. 2, 183–190 (PubMed:9507116).

utchinson, S.A., Eisen, J.S., 2006. Islet1 and Islet2 have equivalent abilities topromote motoneuron formation and to specify motoneuron subtype identity.

Development 133, 2137–2147 (PubMed: 16672347).

iang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz-Gonzalez,X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low,W.C., Largaespada, D.A., Verfaillie, C.M., 2002. Pluripotency of mesenchymalstem cells derived from adult marrow. Nature 418, 41–49 (PubMed: 12077603).

l 45 (2013) 295– 305

Kulbatski, I., Mothe, A.J., Parr, A.M., Kim, H., Kang, C.E., Bozkurt, G., Tator,C.H., 2008. Glial precursor cell transplantation therapy for neurotraumaand multiple sclerosis. Prog. Histochem. Cytochem. 43, 123–176 (PubMed:18706353).

Lang, K.J., Rathjen, J., Vassilieva, S., Rathjen, P.D., 2004. Differentiation of embryonicstem cells to a neural fate: a route to re-building the nervous system? J. Neurosci.Res. 76, 184–192 (PubMed: 15048916).

Lee, S.H., Lumelsky, N., Studer, L., Auerbach, J.M., McKay, R.D., 2000. Efficient gen-eration of midbrain and hindbrain neurons from mouse embryonic stem cells.Nat. Biotechno1. 18, 675–679 (PubMed: 10835609).

Levenberg, S., Huang, N.F., Lavik, E., Rogers, A.B., Itskovitz-Eldor, J., Langer, R., 2003.Differentiation of human embryonic stem cells on three-dimensional polymerscaffolds. Proc. Natl. Acad. Sci. U. S. A. 100, 1246–1271 (PubMed: 14561891).

Li, J., Zhang, Y.P., Kirsner, R.S., 2003a. Angiogenesis in wound repair: angiogenicgrowth factors and the extracellular matrix. Microsc. Res. Technol. 60, 107–114(PubMed: 12500267).

Li, Y., Sauvé, Y., Li, D., Lund, R.D., Raisman, G., 2003b. Transplanted olfactoryensheathing cells promote regeneration of cut adult rat optic nerve axons. J.Neurosci. 23, 7783–7788 (PubMed: 12944507).

Li, X.J., Du, Z.W., Zarnowska, E.D., Pankratz, M., Hansen, L.O., Pearce, R.A., Zhang, S.C.,2005. Specification of motoneurons from human embryonic stem cells. Nat.Biotechnol. 23, 215–221 (PubMed: 15685164).

Lu, J., Feron, F., Ho, S.M., et al., 2001. Transplantation of nasal olfactory tissuepromotes partial recovery in paraplegic adult rats. Brain Res. 889, 344–357(PubMed: 11166728).

Matsuoka, I., Mizuno, N., Kurihara, K., 1989. Cholinergic differentiation of clonal ratpheochromocytoma cells (PC12) induced by retinoic acid: increase of cholineacetyltransferase activity and decrease of tyrosine hydroxylase activity. BrainRes. 502, 53–60 (PubMed: 2573410).

Mirsky, R., Jessen, K.R., Brennan, A., Parkinson, D., Dong, Z., Meier, C., Parmantier, E.,Lawson, D., 2002. Schwann cells as regulators of nerve development. J. Physiol.Paris 96, 17–24 (PubMed: 11755779).

Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama,M., Maeda, M., Yamanaka, S., 2003. The homeoprotein Nanog is required formaintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642(PubMed: 12787504).

Miyagi, S., Nishimoto, M., Saito, T., Ninomiya, M., Sawamoto, K., Okano, H., Mura-matsu, M., Oguro, H., Iwama, A., Okuda, A., 2006. The SOX2 regulatory region2 functions as a neural stem cell specific enhancer in the telencephalon. J. Biol.Chem. 281, 13374–13381 (PubMed: 16547000).

Mizuseki, K., Sakamoto, T., Watanabe, K., Muguruma, K., Ikeya, M., Nishiyama, A.,Arakawa, A., Suemori, H., Nakatsuji, N., Kawasaki, H., Murakami, F., Sasai, Y.,2003. Generation of neural crest-derived peripheral neurons and floor platecells from mouse and primate embryonic stem cells. Proc. Natl. Acad. Sci. U. S.A. 100, 5828–5833 (PubMed: 12724518).

Moore, K.A., Lemischka, I.R., 2006. Stem cells and their niches. Science 311,1880–1885 (PubMed: 16574858).

Munoz, J.R., Stoutenger, B.R., Robinson, A.P., Spees, J.L., Prockop, D.J., 2005. Humanstem/progenitor cells from bone marrow promote neurogenesis of endogenousneural stem cells in the hippocampus of mice. Proc. Natl. Acad. Sci. U. S. A. 102,18171–18176 (PubMed: 16330757).

Nat, R., Nilbratt, M., Narkilahti, S., Winblad, B., Hovatta, O., Nordberg, A., 2007. Neuro-genic neuroepithelial and radial glial cells generated from six human embryonicstem cell lines in serum-free suspension and adherent cultures. Glia 55, 385–399(PubMed: 17152062).

Okabe, S., Forsberg-Nilsson, K., Spiro, A.C., Segal, M., McKay, R.D., 1996. Developmentof neuronal precursor cells and functional postmitotic neurons from embryonicstem cells in vitro. Mech. Dev. 59, 89–102 (PubMed: 8892235).

O’Shea, K.S., 2001. Neuronal differentiation of mouse embryonic stem cells: lineageselection and forced differentiation paradigms. Blood Cells Mol. Dis. 27, 705–712(PubMed: 11482885).

Palmer, T.D., Takahashi, J., Gage, F.G., 1997. The adult rat hippocampus containprimordial neural stem cells. Mol. Cell Neurosci. 8, 389–404 (PubMed: 9143557).

Pappas, I.S., Parnavelas, J.G., 1998. Basic fibroblast growth factor promotes the gen-eration and differentiation of calretinin neurons in the rat cerebral cortex invitro. Eur. J. Neurosci. 10, 1436–1445 (PubMed: 9749798).

Perrier, A.L., Tabar, V., Barberi, T., Rubio, M.E., Bruses, J., Topf, N., Harrison, N.L., Studer,L., 2004. Derivation of midbrain dopamine neurons from human embryonicstem cells. Proc. Natl. Acad. Sci. U. S. A. 101, 12543–12548 (PubMed: 15310843).

Podratz, J.L., Rodriguez, E., Windebank, A.J., 2001. Role of the extracellu-lar matrix in myelination of peripheral nerve. Glia 35, 35–40 (PubMed:11424190).

Qi, Z., Zhu, X., 2007. Different extracellular matrix on differentiation of neural stemcells in rats. Heilongjiang Med. Pharm. 30, 1–3 (article in Chinese).

Raballo, R., Rhee, J., Lyn-Cook, R., et al., 2000. Basic fibroblast growth factor (Fgf2)is necessary for cell proliferation and neurogenesis in the developing cerebralcortex. J. Neurosci. 20, 5012–5023 (PubMed: 10864959).

Rylett, R.J., Williams, L.R., 1994. Role of neurotrophins in cholinergic-neuronefunction in the adult and aged CNS. Trends Neurosci. 17, 486–490 (PubMed:7531891).

Sanchez-Ramos, J.R., 2002. Neural cells derived from adult bone marrow and umbil-

ical cord blood. Neurosci. Res. 69, 880–8931 (PubMed: 12205681).

Shimogori, T., Banuchi, V., Ng, H.Y., Strauss, J.B., Grove, E.A., 2004. Embry-onic signaling centers expressing BMP, WNT and FGF proteins interactto pattern the cerebral cortex. Development 131, 5639–5647 (PubMed:15509764).

nd Cel

S

S

S

S

S

T

W. Hu et al. / Tissue a

himozaki, K., Nakashima, K., Niwa, H., Taga, T., 2003. Involvement of Oct3/4 in theenhancement of neuronal differentiation of ES cells in neurogenesis-inducingcultures. Development 130, 2505–2512 (PubMed: 12702663).

hiotsugu, J., Katsuyama, Y., Arima, K., Baxter, A., Koide, T., Song, J., Chandraratna,R.A., Blumberg, B., 2004. Multiple points of interaction between retinoic acid andFGF signaling during embryonic axis formation. Development 131, 2653–2667(PubMed: 15128657).

rivastava, N., Seth, K., Khanna, V.K., Ansari, R.W., Agrawal, A.K., 2009. Long-termfunctional restoration by neural progenitor cell transplantation in rat modelof cognitive dysfunction: co-transplantation with olfactory ensheathing cellsfor neurotrophic factor support. Int. J. Dev. Neurosci. 27, 103–110 (PubMed:18765279).

un, Y., Nadal-Vicens, M., Misono, S., Lin, M.Z., Zubiaga, A., Hua, X., Fan, G.,Greenberg, M.E., 2001. Neurogenin promotes neurogenesis and inhibits glialdifferentiation by independent mechanisms. Cell 104, 365–376 (PubMed:11239394).

un, Y., Shi, J., Lu, P.H., 2002. Neurotrophic factors and neural stem cells. Progr.Physiol. Sci. 33, 313–317 (PubMed: 12650066) (article in Chinese).

akahashi, J., Palmer, T.D., Gage, F.H., 1999. Retinoic acid and neurotrophins col-laborate to regulate neurogenesis in adult-derived neural stem cell cultures. J.Neurobiol. 38, 65–81 (PubMed: 10027563).

l 45 (2013) 295– 305 305

Vaccarino, F.M., Schwartz, M.L., Raballo, R., Nilsen, J., Rhee, J., Zhou, M., Doetschman,T., Coffin, J.D., Wyland, J.J., Hung, Y.T., 1999. Changes in cerebral cortex size aregoverned by fibroblast growth factor during embryogenesis. Nat. Neurosci. 2,246–253 (PubMed: 1046229).

Wang, J., Shang, X., Li, Y., 2008. Induced differentiation of human embryonicstem cells to neural stem cells in vitro. Acad. J. Second Military Med. Univ. 29,1141–1146 (article in Chinese).

Wichterle, H., Lieberam, I., Porter, J.A., Jessell, T.M., 2002. Directed differentia-tion of embryonic stem cells into motor neurons. Cell 110, 385–397 (PubMed:12176325).

Wohl, C.A., Weiss, S., 1998. Retinoic acid enhances neuronal proliferation andastroglial differentiation in cultures of CNS stem cell-derived precursors. J. Neu-robiol. 37, 281–290 (PubMed: 9805273).

Woodbury, D., Schwarz, E.J., Prockop, D.J., Black, I.B., 2000. Adult rat and human bonemarrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364–370(PubMed: 10931522).

Wu, P., Tarasenko, Y.I., Gu, Y., Huang, L.Y., Coggeshall, R.E., Yu, Y., 2002. Region-specific generation of cholinergic neurons from fetal human neural stem cellsgrafted in adult rat. Nat. Neurosci. 5, 1271–1278 (PubMed: 12426573).

Young, H.E., Black Jr., A.C., 2004. Adult stem cells. Anat. Rec. A Discov. Mol. Cell. Evol.Biol. 276, 75–102 (PubMed: 14699636).