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Neuroscience Letters xxx (2013) xxx–xxx
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 Shaoguan University, Shaoguan, Guangdong, PR China
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, Guangzhou, 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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