Comparison of endometrial regenerative cells and bone marrow stromal cells
Journal of Translational Medicine
Comparison of endometrial regenerative cells and bone marrow stromal cells
Huan Wang 1
Ping Jin 1
Marianna Sabatino 1
Jiaqiang Ren 1
Sara Civini 1
Thomas E Ichim
David F Stroncek 0 1
0 Department of Transfusion Medicine, Cell Processing Section , 10 Center Drive-MSC-1288, Building 10, Room 3C720, Bethesda, MD 20892-1288 , USA
1 Department of Transfusion Medicine, Clinical Center, National Institutes of Health , Bethesda, MD , USA
Background: Endometrial regenerative cells (ERC) and bone marrow stromal cells (BMSC) are being used in clinical trials. While they have been reported to have similar characteristics, they have not been directly compared. Methods: We compared micro RNA (miRNA) and gene expression profiles, soluble cytokine and growth factor levels and ability to inhibit ongoing mixed leukocyte reaction (MLR) of ERC and BMSC each derived from 6 healthy subjects. Results: ERC and BMSC miRNA and gene expression profiles were similar, but not identical; more differences were noted in the expression of genes than in miRNAs. Genes overexpressed in ERCs were more likely to be in immune and inflammation pathways and those overexpressed in BMSCs were more likely to be in stem cell and cancer signaling pathways. In addition, the levels of IL-8 and ICAM-1 were greater in ERC supernatants while the levels of HGF, VEGF, IL-6, CXCL12, TGFB1 and TGFB2 were greater in BMSC supernatants. Additionally, ERC demonstrated greater inhibition of the proliferation of mixed leukocyte cultures. Conclusions: These results suggest that the in vivo effects of ERC and BMSC may differ. Multiple properties of stromal cells are responsible for their in vivo effectiveness and ERC may be more effective for some of the clinical applications and BMSC for others. Studies in animal models or clinical trials will be required to more fully characterize the differences between ERC and BMSC.
Endometrial regenerative cells; Bone marrow stromal cells; Mesenchymal stromal cells; Mesenchymal stem cells; Regenerative medicine
Mesenchymal stem cells (MSC) have been the subject of
numerous studies for their ability to differentiate into various
specialized cells and their great therapeutic potential,
particularly in tissue regeneration. These cells have been isolated
from many different tissues. One of the most commonly
investigated MSC are those derived from bone marrow,
which are known as bone marrow stromal cells (BMSC).
These are fibroblast-like, plastic adherent cells from the bone
marrow  that express CD73, CD90 and CD105 [2,3].
These multipotent cells have the ability to differentiate into
osteoblast, chondrocyte, and adipocyte colonies [4,5] and
have been shown to be capable of bone regeneration [6,7],
effective in treating acute graft-versus-host-disease (GVHD)
[8-10] and have been investigated to treat other diseases such
as cirrhosis , ischemic heart disease , Crohns Disease
 and autoimmune disorders.
Meng and collaborators  have isolated a type of
stromal cell from menstrual blood, Endometrial
Regenerative Cells (ERC), which also have promising clinical
potential. These cells are not specifically classified as
MSC, though they do express most MSC markers such
as CD9, CD29, CD41a, CD44, CD59, CD73, CD90, and
CD105. ERC are distinct from previously characterized
endometrial stromal cells, or endometrial mesenchymal
stem cells, in that they do not express the BMSC marker
STRO-1 [14,15]. They can differentiate into tissue types
beyond bone, fat, and cartilage and therefore may be
pluripotent to some degree [14,15]. Studies in animal
models also suggest that ERC can be used to treat
critical limb ischemia via stimulation of angiogenesis ,
inhibit glioma growth and reduce tumor
neovascularization and possess immunosuppressive properties [15,16].
Furthermore, unlike embryonic stem cells, ERC do not
appear to pose the risk of teratoma development in vivo
, and they are easily collected and expanded. A phase
I clinical study performed on four multiple sclerosis
patients showed that ERC could be used allogenically
and safely, and the treatment seemed to have prevented
disease progression . In addition, the FDA has
granted the approval for ERC to be used in additional
human clinical trials. Although early evidence shows a
promising future for the allogeneic use of ERC in human
patients, it is nevertheless necessary to confirm and
expand our current understanding.
We compared ERC and BMSC by examining their
morphology, cytokine production, inhibition of mixed
leukocyte reactions (MLRs), micro RNA (miRNA)
expression and global gene expression. We hypothesized
that the two types of cells would exhibit different
properties which could result in distinct functional and
Materials and methods
Six different ERC samples and six different BMSC samples
from individual donors were chosen and culture for 6
7 days. Since the media normally used to support the
growth of ERC and BMSC were different, we grew the
ERC in both media to determine if any differences
between ERC and BMSC were due to culture medium type.
We compared ERC and BMSC by analyzing the global
gene expression and miRNA profiling, cytokine
production, morphology and inhibition of MLR. Hematopoietic
stem cell (HSC), embryonic stem cell (ESC) and fibroblast
lines were utilized as controls in miRNA and global gene
expression comparisons. This study was approved by the
NHLBI Institutional Review Board.
The ERC were provided by MediStem (MediStem Inc.,
San Diego, CA, USA), BMSC were obtained from marrow
aspirates of healthy subjects (Cell Processing Section,
Department of Transfusion Medicine, Clinical Center, NIH
Bethesda, MD, USA), the fibroblasts were CRL2429,
CRL2352 (ATCC, Manassas, VA, USA) and NuFF1(Global
Stem Inc, Rockville, MD, USA), the ESC were H9 (WiCell
Research Institute, Madison, WI, USA), I6
(Technion-Israel Institute of Technology, Haifa, Israel) and BG01v
(ATCC), the HSC were CD34+ cells were isolated with
monoclonal antibodies and paramagnetic beads from
GCSF-mobilized peripheral blood stem cell concentrates
(Cell Processing Section, NIH).
Culture of ERC
Six frozen human ERC lines were thawed from liquid
nitrogen, washed, and manually counted. Passage 1 cells
were first expanded to Passage 2 in Modified Eagle
Medium with Nutrient Mixture F-12 with 10% fetal
bovine serum and 10 g gentamicin (ERC medium).
Passage 2 cells were then plated on two 75 cm2 culture
flasks at a density of approximately 3x103cells/cm2 in
two types of media: ERC Medium and alpha Minimum
Essential Medium with 20% fetal bovine serum and
10 g gentamicin (BMSC medium). Cells were observed
for adherence after 24 h and culture medium was
changed after 3 days. Once cells reached >80% confluence
(67 days), they were harvested for analysis. Two to
three million cells were lysed in 1 ml Trizol and 3 ml of
supernatant was collected for proteomic analysis. Cells
cultured in ERC media were designated ERC-E and in
BMSC media ERC-B.
Culture of BMSC
Frozen human BMSC isolated from bone marrow aspirates
were thawed from liquid nitrogen, washed, and manually
counted. Passage 2 BMSC were plated on 75 cm2 flasks at a
density of 3x103cells/cm2 in BMSC medium and observed
for adherence after 24 h. Culture medium was changed
after 3 days. Passage 3 cells were harvested at >80%
confluence after 67 days for further analysis. Two to three
million cells were lysed in 1 ml Trizol and 3 ml of supernatant
were collected for proteomic analysis.
Cell imaging of embryonic stem cell marker analysis
BMSC and ERC at passage 2 were thawed and cultured
in BMSC medium at a density of 1x105 cells per well in
flat bottom six-well plates. Cell medium was changed
after 3 days and light microscopy images were taken for
both cell types. Cell medium was aspirated after 6 days
of culture; cells were washed with 1X PBS and fixed with
4% paraformaldehyde. Cells were permeabilized with 1%
saponin in PBS and were stained for the following
embryonic stem cell markers: Oct-4, Nanog, SSEA-4,
SSEA-1, Klf-4, and TRA-1-81 at room temperature
overnight. Cells were rinsed with 1% saponin and incubated
with anti-tubulin for 1 hour, washed with 1% saponin,
and rinsed with 1X PBS before observation by
immunofluorescence microscopy (Zeiss Axio Observer Inverted
Microscope Carl Ziess, Gottingen, Germany).
Measuring cytokine and growth factor levels with
proteome profiler antibody arrays
Supernatants from 3 ERC samples (ERC-B) and 3 BMSC
samples cultured in BMSC medium and 3 BMSC samples
cultured in the same medium were analyzed for soluble
proteins and cytokines related to immunomodulation
using the Proteome Profiler Human Cytokine Array
KitPanel A (R&D Systems, Minneapolis, MN, USA), which
detects most common immunomodulatory cytokines.
Each supernatant sample was incubated with a cytokine
panel membrane according to manufacturers protocol
with modifications. Incubation times were doubled to
optimize signal detection. The membranes were scanned
(ChemiDocsTM XRS + plus ImageLab) and analyzed with
Bio-Rad Image Lab software (Bio-Rad Laboratories,
Hercules, CA, USA). Semi-quantitative analysis was
performed by measuring the average pixel intensity of each
array, normalizing the values to the intensity of the
positive control, and subtracting the background. The sample
intensity to control ratio was the average of the 3 ERC
samples and 3 BMSC samples, respectively.
Supernatant cytokine and growth factor analysis using
searchlight protein array
Three ERC-B and 3 BMSC supernatant samples were
evaluated for cytokine concentrations using SearchLight Protein
Array Analysis (Aushon Biosystems, Billerica, MA, USA).
The cytokines and growth factors measured were: human
fibroblast growth factor beta (FGFb), hepatocyte growth
factor (HGF), platelet-derived growth factor-BB (PDGFBB),
Vascular endothelial growth factor (VEGF), Interleukin 10
(IL-10), Interleukin 6 (IL-6), chemokine (C-X-C) ligand 12
(CXCL12), Transforming growth factor beta 1 (TGFB1), and
Transforming growth factor beta 2 (TGFB2).
Mixed leukocyte reaction inhibition
The immunosuppressive properties of BMSC and ERC
were compared using an MLR assay (SAIC-Frederic,
Frederic, MD). Ficoll-separated peripheral blood
mononuclear cells (PBMC) were plated in 96-well plates at
1x105 responders per well. Responders were co-cultured
with 2500 cGy irradiated stimulator PBMC at a
concentration of 1x105 cells/well. BMSC and ERC were added
at the following concentrations, 104 and 105 cells/well.
Culture plates were incubated for 6 days in a humidified
5% CO2 incubator at 37C. On the day of harvest,
0.5 Ci of 3 H-thymidine (3 H-TdR) was added to each
well for 4 hours with lymphocyte proliferation measured
using a liquid scintillation counter. The effect of BMSC
on MLR was calculated as the percentage of the
suppression compared with the proliferative response of the
positive control without BMSC, where the positive
control was set to 0% suppression. The experiments were
performed three times for each variable described.
Gene expression analysis
Total RNA from ERC, BMSC, fibroblast, ESC, and HSC
samples were extracted using miRNA Easy Kit (Qiagen,
Valencia, CA, USA). RNA concentration was measured
using ND-8000 spectrophotometer (Nano Drop
Technologies, Wilmington, DE, USA).
Microarray expression experiments were performed
on 4 44 K Whole Human Genome Microarray
(Agilent technologies, Santa Clara, CA, USA), according to
the manufacturer's instructions. The Universal Human
Reference RNA (Stratagene, Santa Clara, CA, USA)
was co-hybridized with each sample. Images of the
arrays were acquired using a microarray scanner
G2505B (Agilent technologies) and image analysis was
performed using feature extraction software version 9.5
(Agilent Technologies). The Agilent GE2-v5_95
protocol was applied using default settings. The resulting
data files were either uploaded to the mAdb database
(http://nciarray.nci.nih.gov/) and further analyzed using
BRBArrayTools  developed by the Biometric
Research Branch, National Cancer Institute (http://linus.
nci.nih.gov/BRB-ArrayTools.html) or imported and
analyzed using Partek Genomics Suite 6.5 (Partek, Inc,
St Louis, MO). Hierarchical cluster analysis and
TreeView software were used to visualize the data, and
Ingenuity Pathway Analysis (IPA, Ingenuity Systems,
Redwood City, CA, USA) was used to perform gene
Quantitative reverse transcription real time PCR (qRT-PCR)
Quantitative RT-PCR was performed to confirm the
expression of selected genes differentially expressed
between ERC and BMSC as determined by microarray
studies. One g of total RNA from 6 BMSC, 6 ERC-B, 6
ERC-E, 3 Fibroblast, 2 ESC, and 2 HSC samples were
reverse transcripted (RT) with random hexamer primer
and the RT product was further diluted for TaqMan
Gene Expression Assays (Applied Biosystems, Carlsbad,
CA, USA) using standard settings. The genes selected
were tumor necrosis factor superfamily 4 (TNFSF-4,),
interleukin 8 (IL-8), intercellular adhesion molecule 1
(ICAM-1), vascular cell adhesion molecule 1 (VCAM-1),
integrin alpha 10 (ITGA-10), prostaglandin-endoperoxide
synthase 2 (PTGS2), and matrix metallopeptidase 3
(MMP3). Gene expressions were quantified with TaqMan
Gene Expression Assay of each above listed gene (Assay
IDs: Hs00182411; Hs99999034_m1; Hs00164932_m1;
Hs01003372_m1; Hs00174623_m1; Hs00153133_m1;
Hs00968305 respectively, Applied BioSystems, Life
Technologies, Santa Clara, CA, USA). All reactions were run in
duplicate. Real-time PCR was performed using ABI
7900HT Real-time PCR system. The data was acquired
using ABI SDS v2.3 software determining the threshold
cycle (Ct) by normalizing to the endogenous control 18 s
RNA (Hs99999901_s1, Applied Biosystems). The fold
change of each gene against the calibrator was calculated
using the equation 2-Ct.
miRNA expression analysis
MiRNA profiling was performed on Agilents 8x15K
human miRNA microarray chips. Total RNA from 4
ERC-B, 3 BMSC, 3 fibroblast, 3 ESC, and 3 HSC samples
were labeled and hybridized according to manufacturers
instruction. Slides were scanned using Agilent
Microarray Scanner Version C, and data were extracted using
Feature Extraction software version 11.0 (Agilent
Technology). Data analysis was performed by using Partek
Genomics Suite 6.5 (Partek, Inc, MO).
Cell morphology, growth and stem cell marker expression
ERC were cultured in both BMSC medium (ERC-B) and a
modified ERC medium based on the formulation used by
Medistem, Inc (ERC-E). We hypothesized that the growth
rate and morphology of ERC would not be affected by the
difference in tissue culture medium. Population doubling
time (PDT) was calculated revealing that ERC-B had a
slightly shorter average PDT when compared with ERC-E
(Table 1); however, no morphological differences were
observed for ERC cultured in different media (Figure 1A,
B). When compared with BMSC, ERC had a longer PDT
(Table 1), larger cytoplasmic area and were less spindly
(Figure 1 A-C). Immunofluorescence imaging revealed
that both ERC (Figure 2A,B) and BMSC (Figure 2C,D)
were positive for Oct-4 while both types of cells were
found to be negative for SSEA1, SSEA4, Nanog,
TRA-181, and Klf-4 (data not shown).
Soluble protein production
Soluble cytokines and growth factors expressed by ERC
were examined and compared to those expressed by
BMSC. ERC supernatant was taken from ERC-B to
minimize the differences due to culture medium. ERC
and BMSC produced many of the same cytokines albeit
at varying levels (Table 2). Among the proteins with high
signal intensities IL-8 and soluble ICAM-1 (CD54) were
notably higher in ERC supernatant than that of BMSC
(Table 2). We also measured the concentration of
proteins which were reported to provide some of the
beneficial effects of BMSCs  using a multiplex ELISA
platform (Searchlight) and found that BMSC
supernatants had higher concentrations of HGF, VEGF, IL-6,
CXCL12, TGFB1 and TGFB2 than ERC supernatants
Table 1 Population doubling time of ERC and BMSC
PDT = population doubling time.
(Table 3). bFGF, PDGFBB and IL-10 levels were too low
to be detected in either group.
Mixed leukocyte reaction inhibition
The immunosuppressive capabilities of both ERC and
BMSC were investigated by MLR inhibition. When
responder T cells were stimulated in the presence of
ERCB and BMSC at a concentration of 10,000 cells per well,
the BMSC were generally more immunosuppressive.
However, a two-sample equal variable T-test revealed
that this difference was not statistically significant
(Figure 3). When responder T cells were stimulated in
the presence of ERC-B and BMSC at a concentration of
100,000 cells per well, ERC-B were significantly more
immunosuppressive (P < 0.0002).
Gene expression, class comparison and pathway analysis
Global gene expression analysis was used to profile the
ERC-E, ERC-B, BMSC, HSC, fibroblasts, and ESC.
Principal Component Analysis (PCA) performed on the
entire dataset of 34,127 genes revealed that the samples
formed four distinct clusters (Figure 4A). The HSC were
in one cluster and ESC in a second; both clusters were
located far from each other and from the rest of the
samples. Another cluster was made up of fibroblasts
which was closer to the fourth cluster made up of the
ERC-E, ERC-B and BMSC. This suggested that ERC and
BMSC were similar to each other and they were more
similar to fibroblasts than to ESC or HSC from a global
perspective. There were, however, some differences
between ERC and BMSC. A PCA analysis of fibroblast,
ERC-E, ERC-B, and BMSC samples with HSC and ESC
removed revealed that the fibroblasts, ERC and BMSC
formed three distinct groups, but the analysis did not
separate ERC-E and ERC-B (Figure 4B). Unsupervised
hierarchical clustering analysis also revealed the same
cluster pattern according to cell type and there was no
difference between ERC-B and ERC-E (Figure 4C).
Class comparison analysis was performed for the six
cell types using BRB Array Tools (Ver 3.4.0), considering
a p-value less than 0.001 as significant. Comparison of
ERC-B and ERC-E showed 82 differentially expressed
genes and a small fold change differences (data not
shown). Due to small differences in gene expression
between ERC culture under the two conditions, class
comparisons and pathway analyses were subsequently
conducted by comparing only ERC-B with the other
groups. Almost three thousand genes (2974) were
differentially expressed between ERC-B and fibroblasts,
whereas 1030 genes were differentially expressed
between BMSC and fibroblasts.
Between ERC-B and BMSC, 1923 genes were
differentially expressed. Some of the most up-regulated genes in
ERC-B when compared with BMSC included somatostatin
Figure 1 Comparison of ERC and BMSC morphology in various culture conditions. A: Early passage (P3) ERC cultured in ERC medium (10 %
FBS in DMEM F-12). B: Early passage (P3) ERC from the same donor cultured in BMSC medium (20 % FBS in MEM). C: Early passage (P3) BMSC
cultured in BMSC medium.
receptor 1 (SSTR1), TNFSF4, coagulation factor 3 (F3),
and MMP3 (Table 4) while the most down-regulated
genes in ERC-B included prostaglandin I2 synthase
(PTGIS), prostaglandin-endoperoxide synthase 2 (PTGS2),
vascular cell adhesion molecule 1 (VCAM1), and integrin
alpha 10 (ITGA10) (Table 4).
Ingenuity Pathway Analysis (IPA) was performed to
annotate the differentially expressed genes between ERC
and BMSC. This analysis revealed that the up-regulated
genes in ERC-B were involved in many of the top
canonical pathways, including pro-inflammatory pathways
such as IL-1 and IL-8 signaling and immune response
pathways such as interferon signaling, CD27 signaling in
lymphocytes, B cell receptor signaling, and NF-B
activation by viruses, IL17a signaling, CD40 signaling and
antigen presentation (Figure 5). Pathways over
represented among genes down-regulated in ERC-B were
Hepatic fibrosis/hepatic stellate cell activation, TGF-B
signaling, atherosclerosis signaling, mTor signaling,
human embryonic stem cell pluripotency and hedgehog
signaling and the cancer signaling pathways, ovarian
cancer signaling, basal cell carcinoma and HER-2
signaling in breast cancer (Figure 6).
Biologically relevant genes preferentially expressed in
ERC vs BMSC
Analysis of genes preferentially expressed in ERC vs
BMSC based on biological significance was performed
manually. Several genes of interest were identified.
FoxL2 transcript, a transcription factor essential for
Figure 2 Immunofluorescence staining of ERC and BMSC. A-B: ERC were labeled for tubulin (A) and the embryonic stem cell marker Oct-4
(B), 10X magnification. C-D: Immunofluorescence staining of BMSC for tubulin (C) and Oct-4 (D), 10X magnification.
Table 2 Mean pixel intensity values from cytokine array panels of BMSC and ERC-B supernatants
*Three BMSC and 3 ERC different donors were analyzed.
ovary development  was expressed 80.5-fold higher
in ERC compared to BMSC (Table 5). Mindin (SPON2)
transcript, an innate immunity receptor involved in
bacterial recognition was expressed 53.5-fold higher as
compared to BMSC. The stem cell potency marker,
aldehyde dehydrogenase  was expressed at transcript
level 39.5-fold higher. Immune modulatory proteins
pregnancy associated glycoprotein 1  and GM-CSF
 were expressed 30.5- and 5.0-fold higher.
Angiogenesis associated proteins Angiopoietin-1  and PDGF
 were expressed 13.8- and 26.9-fold higher. The
matrix metalloprotease 3 transcript was 29.2-fold
higher expressed as compared to BMSC. Interestingly, a
comparison of MSC to ERC by Meng et al. revealed that
ERC possessing substantially higher protein production
of Angiopoietin, PDGF, GM-CSF and MMP3 as
compared to MSC .
Quantitative RT-PCR results
Quantitative RT-PCR analysis was performed to confirm
the expression of differentially expressed genes between
ERC-B and BMSC. The raw data was calculated based
on an 18sRNA endogenous control, and
log2transformed fold change against the BMSC was
calculated based on the Ct values. Fold changes between
ERC-B and ERC-E for most genes were less than 2-fold
with the exception of IL-8 and PTGS2, which had
2.5fold and 1.3-fold differences, respectively confirming
that there was little difference in ERC grown in the
different media (Figure 7). In addition, the expression of
TNFSF4, MMP3, IL-8, and ICAM-1 was several fold
greater in ERC with respect to BMSC, while the
expression VCAM-1, PTGS2, ITGA-10 was several fold lower
in ERC than in BMSC. These data were in good
accordance with our microarray finding.
miRNA microarray analysis
A miRNA profiling was performed on ERC-B, BMSC,
fibroblasts, ESC, and HSC. PCA analysis showed three
distinct clusters. The BMSC, ERC, and fibroblasts were
again closely grouped together, and they were also
clustered close to the ESC samples (Figure 8-A). HSC were
in a third distinct group of their own. The hierarchical
clustering analysis of the samples also showed that
BMSC, ERC, and fibroblasts formed a mixed cluster,
instead HSC and ESC samples formed two separate
clusters (Figure 8-B).
The increased interest in MSC for therapeutic applications
in recent years has led to many new discoveries of
regenerative cells from different origins. In addition to ERC and
Table 3 Detected levels of cytokines in BMSC and ERC-B supernatants (pg/ml)
*Three BMSC and 3 ERC donors were analyzed.
BMSC CM = Bone marrow stromal cell culture media.
Figure 3 Immunosuppression of T cell responses in mixed lymphocyte reactions (MLRs) by ERC-B and BMSC. The bars indicate responder
T cell proliferation when incubated with irradiated T cell stimulator cells and ERC-B or BMSC. Two doses of ERC-B and BMSC were tested: 10,000
and 100,000 cells. The measures were performed in triplicate and converted to percent immunosuppression by normalizing to the proliferation of
T cells without BMSC co-incubation.
BMSC, other MSC have been used clinically or considered
for clinical use and the group includes those derived from
adipose tissue and umbilical cord blood. There have been
some comparisons of the different types of stromal cells
but this is the first comparison between ERC and BMSC
both of which are being tested in clinical trials.
BMSC have been compared with other types of
stromal cells including adipose tissue, lung tissue and
umbilical cord blood derived MSC [28-32]. These studies have
found that stromal cells produced from these various
cell and tissue types have a similar phenotype [28-32].
All express CD44, CD73, CD90 and CD105, but do not
express hematopoietic cell markers. They all
demonstrate some degree of osteoblastic, chondrogenic and
adipogenic differentiation ability, but there were some
differences in differentiation ability among them [30,31],
particularly when expression of differentiation markers
were compared [29,32,33]. In addition the proliferation
of stromal cells derived from diverse sources sometimes
differ [28,31]. Stromal cells derived from different tissues
can modulate immune function, but the degree of
immune modulation can vary. For example, BMSC and
adipose tissue derived-MSC differ in their ability to
modulated the production of immunoglobulin by
mitogen-stimulated B cells  and the proliferation of
allogeneic T cells . Some studies have also found
differences in the expression of genes encoding cytokines
and growth factors among rat adipose tissue MSC, rat
cartilage MSC and rat BMSC . None of these studies
have compared global gene or miRNA expression among
different types of stromal cells. The results of our study
were similar to previous reports in that BMSC and ERC
had similar morphology and proliferation, but we found
some differences in gene expression in addition to the
differences in cytokine and growth factor production.
We noted morphological differences between ERC and
BMSC, but both cells resemble fibroblasts and
morphology differences have also been noted between other
types of MSC . The PDTs of ERC and BMSC were
similar, with BMSC having a slightly shorter PDT. Our
PDT findings for ERC differed from those found from
earlier literature, which was reported to be around
19 hours. This difference may be due to slight variations
to the culture media formula and culture conditions.
Immunofluorescence staining showed that both ERC and
BMSC were positive for the pluripotency marker Oct-4,
which is consistent with findings from earlier studies
. Comparison of miRNA expression signatures also
found that ERC were very similar to BMSC.
Gene expression analysis revealed considerable
similarities between ERC and BMSC, but there were also
distinct differences between these two types of cells.
Many immunity and inflammation related pathways
were overrepresented among genes differentially
expressed between ERC and BMSC suggesting that they
may have different immune modulatory and
antiinflammatory properties. This is supported by qRT-PCR
analysis which showed that the expression of TNFSF4,
MMP3, IL-8 and ICAM-1 were greater in ERC, and
that of VCAM-1, PTGS2 and ITGA10 were greater in
BMSC. Comparison of the levels of cytokines and
growth factors in ERC and BMSC supernatants also
Figure 4 Principal Component Analysis (PCA) and hierarchical clustering analysis of differentially expressed genes among ERC-E,
ERCB, BMSC, HSC, ERC, and fibroblast (Fb) samples. A: Principal component analysis of all six cell types based on differentially expressed genes. B:
PCA analysis comparing ERC-E, ERC-B, BMSC and fibroblast (Fb) samples using the differentially expressed genes. C: Unsupervised clustering of all
samples based on the differentially expressed genes.
found difference among ERC and BMSC. The levels of
IL-8 and ICAM-1 were greater in ERC supernatants
and the levels of HGF, VEGF, TGFB2 and IL-6 were
greater in BMSC supernatants. We found slight
differences in the inhibition of MLRs between ERC and
BMSC; the ERC being slightly more inhibitory.
Interesting, adipose derived MSC have been found to be more
immunomodulatory than BMSC[30,34]. In addition, the
expression of two factors important in angiogenesis
differed among these two types of stromal cells; MMP-3
was greater in ERC at the transcript level and VEGF
was greater in BMSC supernatants at the protein level.
This suggests the ability of ERC and BMSC to support
angiogenesis may also differ. Specific analysis of
biologically relevant genes that were overexpressed in ERC
revealed substantially higher expression of genes
associated with angiogenesis, including PDGF, and
Angiopoietin-1. This is of interest since a previous
publication by Meng et al., reported higher protein
expression of MMP-3, angiopoietin, and PDGF .
Global gene and miRNA expression analysis found
that fibroblasts were similar to ERC and BMSC. This is
consistent with the fact that, like ERC and BMSC,,
fibroblasts express CD73 and CD105 and lack
hematopoietic markers CD14, CD34 and CD45 and
they demonstrate osteogenic, chondrogenic and
adipogenic differentiation [35,36]. They also have
immunosuppressive properties. They can inhibit mitogen and
allogeneic stimulated T cell proliferation and
interferon production [35,37].
The results of this study show that although ERC and
BMSC are currently being tested in clinical trials for
similar indications, they may not have identical clinical
effects. While our studies found a number of differences
in gene expression, protein production and in vitro
function which suggest that these two cell types may differ in
Chromosome 3 open
eading frame 72
Coagulation factor III
Tumor necrosis factor
superfamily, member 4
Forkhead box L2
similarity 105, member A
transcript variant 1
Solute carrier organic anion
transporter family, member 2A1
Spondin 2, extracellular
matrix protein, transcript var. 3
domain family, member 16,
transcript var. 2
Vesicle amine transport
protein 1 homolog-like
Nbla00301 non-coding RNA
type II, transcript variant 5
1 family, member A1
(serotonin) receptor 2B
Caspase recruitment domain
family member 17
Zinc finger and BTB
domain containing 46
transcript variant 1
Chromosome 13 open
reading frame 15
Oxidized low density
lipoprotein receptor 1,
transcript var. 2
Matrix metallopeptidase 3
Table 4 Genes differentially expressed between ERC and BMSC
family 1, subfamily B
Cytochrome P450, family 1,
subfamily B, clone
Iroquois homeobox 3
Solute carrier family 14,
member 1, transcript variant 3
Paired-like homeodomain 2
Brain & acute leukemia,
cytoplasmic, transcript var. 2
Prostaglandin I2 synthase
Iroquois homeobox 5
PR domain containing 16, transcript var. 2,
Distal-less homeobox 5
synthase 2 (prostaglandin G/H
synthase and cyclooxygenase)
Collectin sub-family member 12
Keratin associated protein 1-1
PDZ and LIM domain 3,
transcript variant 1
Growth associated protein 43,
transcript var. 2
Vascular cell adhesion molecule 1,
Integrin, alpha 10
Leucine rich repeat containing 15,
transcript variant 2
Solute carrier family 14 (urea transporter),
member 1, transcript variant 4
Distal-less homeobox 6
Chemokine-like receptor 1,
transcript variant 3
Cholesterol 25-hydroxylase (CH25H), mRNA.
Placenta-specific 9 (PLAC9), mRNA.
P <0.0001 for all genes.
*The genes most up- and down-regulated according to fold-change are listed.
Procollagen C-endopeptidase enhancer 2
Figure 5 Ingenuity pathway analysis of up-regulated genes in ERC-B samples compared with BMSC. The 30 canonical pathways most
significantly overrepresented with ERC-B up-regulated genes are shown (P < 0.05). The orange bar shows the ratio of the number of genes in
each pathway divided by the total number of genes that make up that pathway. The yellow line indicated the significance of the ERC
upregulated genes in each pathway and is expressed as minus-log P-value.
their immunomodulatory and anti-inflammatory effects,
we cannot tell from these studies how they will perform
in vivo or which cell type will have greater
immunomodulatory or anti-inflammatory effects. We suspect that
multiple properties of stromal cells are responsible for
their in vivo effectiveness and ERC may be more
effective for some of the clinical applications and BMSC for
others. Studies in animal models or clinical trials will be
required to more fully characterize the differences
between ERC and BMSC.
Figure 6 Ingenuity pathway analysis of down-regulated genes in ERC-B samples when compared with BMSC. The 30 canonical pathways
most significantly overrepresented with ERC-B down-regulated genes are shown (P < 0.05). The orange bar shows the ratio of the number of
genes in each pathway divided by the total number of genes that make up that pathway. The yellow line indicated the significance of the ERC-B
down-regulated genes in each pathway and is expressed as minus-log P-value.
Expression in ERC
Matrix Metalloprotease 3
differentiation transcription factor
Bacterial recognition receptor
Associated with stem cell
potency in hematopoietic stem cells,
angiogenic stem cells and cancer stem cells
Anti-inflammatory product that
induces IL-10, protects fetus from
maternal immune system
Tissue remodeling, angiogenesis
endothelial survival, vessel maturity
Table 5 Biologically-Relevant mRNA Expression Compared Between ERC and BMSC
Figure 7 Measurement of the expression of selected genes among 6 ERC-B, 6 ERC-E, 3 HSC (CD34), 3 ESC, and 3 fibroblast samples
using q RT-PCR. The expression levels of TNFSF4, MMP3, IL-8 and ICAM-1 were up-regulated in ERC compared to BMSC and the expression of
VCAM-1, PTGS2 and ITGA-10 were down-regulated in ERC. The fold change values shown were normalized against BMSC and calculated as the
Figure 8 Principal component and hierarchical clustering analysis of miRNA differentially expressed among BMSC, ERC-B, HSC, ESC,
and fibroblast (fb) samples. A: Principal component analysis of all five cell types based on differential expressed miRNA. B: Hierarchical
clustering analysis of all five cell types based on differentially expressed miRNA.
While the miRNA and gene expression signatures of
ERC and BMSC are very similar, we found some
differences in a number of immune and inflammatory
pathways at the transcriptome and protein levels. The ability
of ERC and BMSC to inhibit MLR also differed slightly.
This suggests that the in vivo effects of these two types
of MSC may also differ.
HW helped design the study, performed experiments, analyzed data and
wrote the manuscript. PJ assisted with the study design, preformed
experiments, analyzed data analysis and helped write the manuscript. SC
performed experiments and analyzed the data. MS helped design the study,
analyzed the data and wrote the manuscript. JR provided the BMSC and
preformed experiments and analyzed the data. DFS, TEI and VB conceived
and helped design the studies. TEI and VB provided the ERC. DFS and TEI
reviewed that data and helped write the manuscript. All of the authors read
and approved the final manuscript.
We thank that the NIH Bone Marrow Stromal Cell Transplant Center for
providing the BMSC used in this study. This research was supported by the
Intramural Research Program of the NIH, Clinical Center.
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