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Contents lists available at SciVerse ScienceDirect

Neuroscience Letters

jou rnal homepage: www.elsevier.com/locate/neulet

Comparison of cell proliferation, apoptosis, cellular morphology and

ultrastructure between human umbilical cord and placenta-derived mesenchymal stem cells

a,c,1 b,1 c c d b

Shao-Fang Zhu , Zhi-Nian Zhong , Xia-Fei Fu , Dong-Xian Peng , Guo-Hui Lu , Wen-Hu Li ,

e e a a c,∗

Hong-Yan Xu , Hong-Bo Hu , Jian-Ming He , Wei-Yan Su , Yuan-Li He

a

Medical College of University, Shaoguan, , PR

b

Department of Orthopedics, First Hospital of Shaoguan, Guangdong Medical College, Shaoguan, Guangdong, PR China

c

Department of Obstetrics and Gynaecology, Zhujiang Hospital, Southern Medical University, , Guandong 510282, PR China

d

Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, PR China

e

Department of Obstetrics and Gynaecology, The Yuebei People’s Hospital of Shaoguan, Shaoguan, Guandong 512000, PR China

h i g h l i g h t s

We showed that HUCMSCs had higher self-renewal capacity than HPDMSCs.

The ability of secretion of VEGF, IGF-1, HGF from HPDMSCs was greater than HUCMSCs.

HPDMSCs had a greater number of large cuboidal or flat cells than HUCMSCs.

Myofilaments and pseudopods of HPDMSCs were more intensively organized than HUCMSCs.

Cellular characteristics should be considered when selecting an appropriate source of MSC.

a r t i c l e i n f o a b s t r a c t

Article history: Research in mesenchymal stem cells (MSCs) is mainly focused on applications for treatments of brain

Received 14 October 2012

and spinal cord injury as well as mechanisms underlying effects of MSCs. However, due to numerous

Received in revised form 26 February 2013

limitations, there is little information on selection of appropriate sources of MSCs for transplantation in

Accepted 3 March 2013

clinical applications. Therefore, in this study we compared various properties of human umbilical cord-

derived MSCs (HUCMSCs) with human placenta-derived MSCs (HPDMSCs), including cell proliferation,

Keywords:

apoptosis, cellular morphology, ultrastructure, and their ability to secrete various growth factors (i.e. vas-

Mesenchymal stem cells (MSCs)

cular endothelial growth factor, insulin-like growth factors-1, and hepatocyte growth factor), which will

Biological characteristics

Ultrastructures allow us to select appropriate MSC sources for cellular therapy. Cell culture, flow cytometry, transmission

electron microscope (TEM) and atomic force microscope (AFM) were used for assessment of HUCMSCs

and HPDMSCs. Results showed that the two types of cells appeared slightly different when they were

observed under AFM. HUCMSCs appeared more fibroblast-like, whereas HPDMSCs appeared as large

flat cells. HUCMSCs had higher proliferative rate and lower rate of apoptosis than HPDMSCs (p < 0.05).

However, HPDMSCs secreted more of the three growth factors than HUCMSCs (p < 0.05). Results of TEM

revealed that the two types of MSCs underwent active metabolism and had low degree of differentiation,

especially HUCMSCs. Results of AFM showed that HUCMSCs had stronger ability of mass transport and

cell migration than HPDMSCs. However, HPDMSCs displayed stronger adhesive properties than HUCM-

SCs. Our findings indicate that different sources of MSCs have different properties, and that care should

be taken when choosing the appropriate sources of MSCs for stem cell transplantation. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

In recent years, increasing evidence has shown that trans-

plantation of mesenchymal stem cells (MSCs) is a promising

∗ therapeutic strategy for acute brain or spinal cord injury and

Corresponding author. Tel.: +86 20 61 643 010; fax: +86 20 61 643 010.

progressive degenerative diseases of the central nervous system

E-mail addresses: [email protected], [email protected] (Y.-L. He).

1

These authors contributed equally to this work. [12], such as stroke [8], Parkinson’s disease (PD) [11], spinal cord

0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.neulet.2013.03.018

Please cite this article in press as: S.-F. Zhu, et al., Comparison of cell proliferation, apoptosis, cellular morphology and ultrastructure between

human umbilical cord and placenta-derived mesenchymal stem cells, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.03.018

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2 S.-F. Zhu et al. / Neuroscience Letters xxx (2013) xxx–xxx

injury [16], amyotrophic lateral sclerosis (ALS) [10], autoimmune 2.2.2. Primary culture of HPDMSCs

encephalomyelitis (EAE) [1], and multiple system atrophy (MSA) The tissues were collected from the chorionic plate under ster-

[5]. The therapeutic effects rely not only the differentiation of MSCs ile condition and rinsed with PBS to remove the remaining blood.

3

into cells of neural lineage to repair damaged tissues, but also on The tissues were cut into fragments (approximately 1 mm ) and

their ability to create a favorable environment for regeneration, digested with type-II collagenase solution (Sigma) in a 37 C water

such as expression of growth factors or cytokines. bath for 30 min. The resulting fluid was collected and centrifuged at

However, MSCs derived from adult tissues, such as the bone 1500 rpm for 10 min. The precipitated cells were collected, resus-

marrow, fat, liver, skin, have many disadvantages: for example, pended with DMEM-LG medium (Hyclone), and transferred into a

insufficient supply of stem cells, decreased proliferation and dif- centrifugation tube containing Ficoll–Hypaque solution, and cen-

ferentiation capacity with age, and associated risk of infection trifuged at 2000 rpm for 15 min. The white layer was aspirated,

and development of tumors after stem cell transplantation [14]. rinsed twice with PBS, and centrifuged again to separate HPDMSCs.

It has been shown that bone marrow-derived MSCs (BMSCs) can The cells were plated in DMEM-F12 medium (Hyclone) containing

cause secondary damage in patients after transplantation [15]. By 10% FBS and incubated in an atmosphere of 5% CO2 at 37 C with

contrast, MSCs derived from fetal tissues have advantages over pro- saturated humidity for 7 days (d). The incubation medium was

liferation and differentiation, however, these tissues are difficult to refreshed every 3–4 d to remove non-adherent cells. Cell growth

obtain. Moreover, embryo-derived MSCs are ethically controversial and morphology were observed daily under an inverted micro-

and they are difficult to be cultured in vitro, implying that this type scope. The cells were trypsinized and passaged after reaching a

of cells is not suitable for clinical research for large-scale appli- confluence of 80–90%.

cations. Therefore, many researchers have tried to isolate MSCs

from fetal appendages, such as the umbilical cord blood, amni-

2.3. Flow cytometric analysis

otic fluid, umbilical cord and placenta, which can be extensively

used in clinical studies because of their availability, the absence of

Secondary passages of HUCMSCs and HPDMSCs were

ethical concerns, low oncogenicity and resistance to bacterial and

trypsinized and dissociated into different single cell suspen-

viral contamination. However, the MSCs derived from the umbili-

sions. Monoclonal antibodies (Pharmingen, San Diego, USA) that

cal cord blood have unavoidable immunogenicity, and the amount

recognize CD29, CD44, CD73, CD105, CD90, CD14, CD34, CD45,

of the amniotic fluid are often limited. Therefore, human umbilical ␮ 6

CD106, CD133 (at a concentration of 20 l/10 cells) were used.

cord-derived MSCs (HUCMSCs) and human placenta-derived MSCs

HLA-DR (5 ␮L) were added into suspension of each antibody

(HPDMSCs) are the most preferable choice when compared with

solution. Mouse IgG1 (5 L) was used as a negative control. All of

the above-mentioned sources. Based on these advantages, HUCM-

the resulting suspension samples were incubated at 4 C for 30 min

SCs and HPDMSCs have been widely used in research for treatment

and then analyzed by flow cytometry (FACSCalibur, BD Bioscences,

of many diseases, such as cirrhosis [19], myocardial infarction

San Jose, CA, USA).

[6], spinal cord injury [2], diabetes [7] and tissue engineering [9].

Researchers have therefore proposed HUCMSCs and HPDMSCs to

2.4. Analyses of cell cycle distributions

be promising sources of cells for future clinical applications [17]. In

this study, we focused on comparing the biological characteristics

Appropriate volumes of above single cell suspensions were

and ultrastructures of HUCMSCs and HPDMSCs, in order to select 6

dispensed into tubes (1 × 10 cells/tube) and centrifuged. The

suitable sources of MSCs for future clinical application according to

supernatants were discarded, and the precipitated cells were

their connatural characteristics. ◦

fixed with ice-cold (−20 C) 75% ethanol. The fixed cells were re-

suspended and centrifuged. The cell pellet was treated with RNase

2. Materials and methods (Sigma) to remove RNA, followed by staining with propidium iodide

in the dark (4 C, 30 min). Cell cycle distributions were then ana-

2.1. Materials lyzed by flow cytometry.

Fresh human umbilical cords and placentae were obtained

2.5. Detection of cellular apoptosis

from healthy women ranging from 25 to 30 years old (n = 8) who

delivered healthy full-term infants by cesarean section at the

Secondary passages of HUCMSCs and HPDMSCs were rinsed

Department of Obstetrics and Gynecology of the Zhujiang Hospital

twice with 4 C PBS and resuspended in binding buffers; the cell

(Southern Medical University, Guangzhou, Guangdong, PR China) 6

concentrations were adjusted to 1 × 10 cells/mL. A small volume

(03/05/2010 to 05/21/2010). These women were healthy with no

(100 ␮L) of these cell suspensions was dispensed into a 5 mL FACS

history of infectious diseases or pregnancy complications, and were

tube, and 5 ␮L of Annexin V/FITC (Pharmingen, San Diego, USA) and

confirmed to be negative for HBV, HIV, and syphilis. Informed con-

10 ␮L of propidium iodide (20 ␮g/mL) were also added to the tube.

sent was obtained, and all procedures were approved by the Ethics

The resulting cell suspension was thoroughly mixed and incubated

Committee of our hospital.

at room temperature in the dark for 15 min. Then, 400 ␮L of PBS

(Hyclone) was added to the tube, and the levels of cellular apoptosis

were analyzed by flow cytometry.

2.2. Primary culture of MSCs

2.2.1. Primary culture of HUCMSCs 2.6. Analysis of cell proliferation

Fresh human umbilical cords were obtained after birth and

washed in phosphate-buffered solution (PBS) (Hyclone, Logan, Secondary passages HUCMSCs and HPDMSCs (1000 cells) were

USA) several times. After removal of blood vessels, the umbilical plated onto two 96-well plates with 100 ␮L per well. Over the fol-

3

cords were minced into 1 cm fragments, and subsequently homog- lowing 7 days, 10 L of CCK-8 (Beyotime Institute of Biotechnology,

3

enized into 1–2 mm pieces. Tissue cultures were maintained in Haimen, PR China) was added into 5 wells, and incubated for 1 h

DMEM medium (Hyclone) supplemented with 10% FBS (Hyclone), in CO2 incubator once daily. The absorbance module at 450 nm

and were incubated in a humidified atmosphere with 5% CO2 at was analyzed by an auto-microplate reader, and mean value was

37 ◦C. calculated.

Please cite this article in press as: S.-F. Zhu, et al., Comparison of cell proliferation, apoptosis, cellular morphology and ultrastructure between

human umbilical cord and placenta-derived mesenchymal stem cells, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.03.018

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2.7. Characterization of cell topography (Fig. 1). The cells readily attached to the bottoms of culture dishes,

grew in parallel or vortex-like patterns, and proliferated rapidly to

Secondary passages of HUCMSCs and HPDMSCs were cul- confluence after 3–4 days of culture. These cells were stably pas-

2

tured on coverslips (0.2 cm ) for 3 d. The culture mediums were saged for 20 passages. However, we found that some HPDMSCs

removed, and the cells were rinsed with PBS, followed by fixa- appeared flat when observed under the atomic force microscope,

tion with paraformaldehyde for 10 min. The surface morphologies however, such appearance was not observed under the light micro-

of cells on the slides were studied by tapping-mode atomic force scope, suggesting that HPDMSCs had different cell shape from the

microscopy (AFM) (SPM-9500J3, Shimadzu, Columbia, MD, Kyoto, HUCMSCs.

Japan).

2.8. Characterization of cellular ultrastructure

3.2. Expression of mesenchymal cell surface-specific markers on

HUCMSCs and HPDMSCs

Secondary passages of HUCMSCs and HPDMSCs were cultured to

reach confluence, rinsed with PBS, and scraped from the flasks. The

To identify whether HUCMSCs and HPDMSCs expressed spe-

cells were purified by centrifugation (1000 rpm, 10 min), processed

cific mesenchymal markers, we performed flow cytometric analysis

for transmission electron microscopy (TEM), and observed under

using mesenchymal markers (CD29, CD44, CD73, CD90, and CD105)

a transmission electron microscope (TEM, Philips CM 10; Philips,

and hematopoietic and endothelial markers (CD14, CD34, CD45,

Amsterdam, the Netherlands).

CD 106, CD 133). Results of flow cytometry revealed that cells iso-

lated from human umbilical cords and placentae expressed CD29,

2.9. Detection of secreted vascular endothelial growth factor CD44, CD73, CD90, and CD105, but not CD14, CD34, CD45, CD 106,

(VEGF), insulin-like growth factors-1 (IGF-1), and hepatocyte CD 133, or HLA-DR (MHC-II). These findings indicated that both

growth factor (HGF) types of cells we obtained only express mesenchymal-specific anti-

gens, they did not express hematopoietic- or endothelial-specific

Secondary passages of HUCMSCs and HPDMSCs were cultured in antigens (Fig. 2).

5

×

flasks (5 10 cells/flask) and were grown to confluence. The cells

were then further cultured in complete culture medium (DMEM

supplemented with 10% serum) for 24 h, and the culture medium

3.3. HUCMSCs have stronger proliferative capacity than

was collected and centrifuged. ELISA tests (ADL, USA) were per- HPDMSCs

formed on the supernatants with antibodies to detect VEGF, IGF-1,

or HGF, with culture medium serving as negative control.

We found that the rate of cell proliferation was slow during the

first 2 days (latent phase), then accelerated rapidly during days

2.10. Statistical analysis 3–6 (logarithmic phase), and slowed down thereafter (stationary

phase). The duplication time of secondary HUCMSCs in logarith-

±

Data were presented as mean ± standard deviation. A two-sided, mic phase was 2.560 0.117 days and the secondary HPDMSCs was

±

non-paired t-test was used to analyze the flow cytometry and the 2.956 0.204 days, and significant difference was found between

cumulative doubling data. Differences were considered significant HUMSCs and HPDMSCs (p < 0.05) from day 5 (Fig. 3A).

at p < 0.05. The SPSS software package (version 13.0; SPSS Inc.,

Chicago) was used for the statistical tests.

3.4. HUCMSCs had lower level of apoptosis than HPDMSCs at the

early and late stages of apoptosis

3. Results

Flow cytometry showed that secondary HUCMSCs and HPDM-

3.1. Distinct morphology of HUCMSCs and HPDMSCs in vitro

SCs had low rates of cell apoptosis, and the percentage of HUCMSCs

with apoptosis was approximately 2-fold higher than the percent-

The cells isolated from human umbilical cord and the placenta

age of HPDMSCs with apoptosis (Fig. 3B; p < 0.05). There were

exhibited an elongated spindle (fibroblast-like) morphology with

similar amount of non-apoptotic cells.

abundant cytoplasm and large nuclei under a light microscope

×

×

Fig. 1. HUCMSCs and HPDMSCs exhibited elongated spindle (fibroblast-like) morphology. (A) Morphology of HUCMSCs (200 ); (B) morphology of PDMSCs (200 ).

Please cite this article in press as: S.-F. Zhu, et al., Comparison of cell proliferation, apoptosis, cellular morphology and ultrastructure between

human umbilical cord and placenta-derived mesenchymal stem cells, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.03.018

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Fig. 2. Flow cytometric analysis showing HUCMSCs and HPDMSCs expressed CD29, CD44, CD73, CD90, HLA-1 and CD105, but not CD14, CD34, CD45, CD 106, CD 133,

and HLA-DR (MHC-II). (A) Phenotypic analysis of HUMSCs by flow cytometry; (B) phenotypic analysis of PDMSCs by flow cytometry. FITC, fluorescein isothiocyanate; PE,

phycoerythrin; PC5, phycoerythrin and cyaniding; CY5, cyanidin 5.

3.5. The percentage of HUCMSCs in the G2/M and S phases was types of MSCs had large nuclei with clear nuclear membranes and

higher than HPDMSCs distinct nucleoli.

The percentage of HUCMSCs in the G2/M and S phase (G2/M + S)

was approximately 2- and 4-folds higher than that of HPDM- 3.8. Intracellular ultrastructure of HUCMSCs and HPDMSCs

SCs, respectively (Fig. 3C). Significant differences were detected

between the HUCMSCs and HPDMSCs in the G2/M, S and G0/G1 Results of TEM analysis (Fig. 4) showed that the HPDM-

phases (p < 0.05), implying that the proliferation capacity of HUCM- SCs had more scattered microvilli-like structures than the

SCs was stronger than that of HPDMSCs (Fig. 3C; p < 0.05). HUCMSCs (Fig. 4B(4)), implying that HPDMSCs might have

stronger adhesive properties than HUCMSCs. This characteris-

3.6. HPDMSCs secrete more VEGF, IGF-1, and HGF

tic of HPDMSCs might be related to the inherent capability

of the placenta which needs to adhere to the uterus. The

The concentrations of HGF secreted from HPDMSCs were 3-fold

intracellular connections of the two types of MSCs were pri-

higher than from HUMSCs (Fig. 3D; p < 0.05), the concentrations

marily comprised of tight junctions, and gap junctions were

of IGF secreted from HPDMSCs were 1.3-fold higher than HUMSCs

also observed in some locations, which suggest that the two

(Fig. 2D; p < 0.05), and the concentrations of VEGF secreted from

types of cells involve intracellular adherence and communi-

HPDMSCs were 2-fold higher than the HUMSCs (Fig. 2D; p < 0.05).

cation. HUCMSCs and HPDMSCs contained one nucleus, and

These results suggested that the ability of secretion of the cytokines

some cells contained two nuclei. These nuclei contained exu-

in HPDMSCs were greater than that in HUMSCs.

berant nucleoplasm and single or multiple nucleoli, in which

nucleolonema were woven into spongy globules (Fig. 4A(2) and

3.7. Cell surface topography of HUCMSCs and HPDMSCs B(2)).

In addition, the rough endoplasmic reticulum membranes of the

Results of AFM analysis (Fig. 4) revealed that secondary two types of MSCs contained many ribosomes scattered throughout

HUCMSCs and HPDMSCs appeared elongated spindle-like mor- the cytoplasm (Fig. 4A(3) and B(3)) implying these cells participate

phology with microfilament bundles running parallel to their long in active synthesis of proteins, with a low degree of differenti-

axes, and HPDMSCs also appeared large and flat. Microtubules, ation. The mitochondria of HPDMSCs contained tubular cristae

microfilaments, and intermediate filaments were interwoven into (Fig. 3B(4)), which were similar to the ultrastructure of endocrine

three-dimensional networks with variable shape of cell surface cells, suggesting strong secretory capacity. In addition, HUCMSCs

and synapse-like protrusions, such structures were more obviously contained abundant secondary lysosomes (Fig. 4A(4)) indicating

observed in the HPDMSCs than those in the HUCMSCs. The two that the cells were undergoing active metabolism.

Please cite this article in press as: S.-F. Zhu, et al., Comparison of cell proliferation, apoptosis, cellular morphology and ultrastructure between

human umbilical cord and placenta-derived mesenchymal stem cells, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.03.018

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Fig. 3. HUCMSCs displayed greater proliferative capacity, shorter duplication time, lower rate of apoptosis, a greater number of cells in the mitotic phase than HPDMSCs

(p < 0.05). (A) Comparative analysis of cell proliferation between HUCMSCs and HPDMSCs; (B) comparative analysis of apoptosis between HUCMSCs and HPDMSCs; (C)

comparison of cell cycle analysis between HUCMSCs and HPDMSCs; (D) comparison of secretion of HGF, IGF, VEGF between HUCMSCs and HPDMSCs.

Fig. 4. Comparison of cell surface topography and intracellular ultrastructures between HUCMSCs and HPDMSCs. A(1) Atomic force micrographs showing surface topography

of HUCMSCs. A(2)–(4) Transmission electron micrographs showing the ultrastructure of HUMSCs. A(2) Elongated spindle-like morphology (5800×). A(3) Rough endoplas-

×

mic reticulum (11,600 ). A(4) Autophagic vacuoles (26,500×). B(1) Atomic force micrographs showing surface topography of HPDMSCs. B(2)–(4). Transmission electron

micrographs of the ultrastructures of HPDMSCs showing elongated spindle or large, flat cells morphology B(2) (1850×), intracellular connections B(3) (46,000×), microvilli

on the cell surface B(4) (9700×). NM: nuclear membrane; Nu: nucleolus; N: nucleus; RER: rough endoplasmic reticulum; Ly2: secondary lysosomes; MV: microvilli; M:

mitochondria; R: ribosomes. The arrows indicated the corresponding organelles.

Please cite this article in press as: S.-F. Zhu, et al., Comparison of cell proliferation, apoptosis, cellular morphology and ultrastructure between

human umbilical cord and placenta-derived mesenchymal stem cells, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.03.018

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4. Discussion Acknowledgements

In the present study, we showed that MSCs derived from the We thank the Neural Stem Cell Research Centre of Zhujiang Hos-

umbilical cords and placentae displayed their own unique char- pital for their technical assistance, and Xiaodan Jiang and Yingqian

acteristics. When compared with HPDMSCs, the HUCMSCs had Cai for providing valuable assistance on cell surface topography

greater proliferative capacity, shorter duplication time, lower rate analyses. This research was supported by the Natural Science Foun-

of apoptosis, greater amount of cells in the mitotic phase, and dation of China (NSFC, 81270658).

more autophagic vacuoles. It is possible that the umbilical cord

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