Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior
Sareen et al. Stem Cell Research & Therapy
Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior
Niketa Sareen 0 1
Glen Lester Sequiera 0 1
Rakesh Chaudhary 1
Ejlal Abu-El-Rub 1
Subir Roy Chowdhury 3
Vikram Sharma 2
Arun Surendran 1
Meenal Moudgil 1
Paul Fernyhough 3
Amir Ravandi 1
Sanjiv Dhingra 1
0 Equal contributors
1 Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Department of Physiology and Pathophysiology, University of Manitoba , Winnipeg , Canada
2 School of Biomedical and Healthcare Sciences, Plymouth University Peninsula Schools of Medicine and Dentistry , Plymouth, England
3 Division of Neurodegenerative Disorders, St. Boniface Hospital Research Centre, Department of Pharmacology & Therapeutics, University of Manitoba , Winnipeg , Canada
Background: Bone marrow-derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged and their clinical application for regenerative medicine is under evaluation. However, data from preclinical and initial clinical trials indicate that allogeneic MSCs after transplantation provoke a host immune response and are rejected. In the current study, we evaluated the effect of an increase in passage number in cell culture on immunoprivilege of the MSCs. Since only limited numbers of MSCs can be sourced at a time from a donor, it is imperative to expand them in culture to meet the necessary numbers required for cell therapy. Presently, the most commonly used passages for transplantation include passages (P)3-7. Therefore, in this study we included clinically relevant passages, i.e., P3, P5, and P7, for evaluation. Methods: The immunoprivilege of MSCs was assessed with the mixed leukocyte reaction assay, where rat MSCs were cocultured with peripheral blood leukocytes for 72 h. Leukocyte-mediated cytotoxicity, apoptosis (Bax/Bcl-xl ratio), leukocyte proliferation, and alterations in cellular bioenergetics in MSCs were assessed after the coculture. Furthermore, the expression of various oxidized phospholipids (oxidized phosphatidylcholine (ox-PC)) was analyzed in MSCs using a lipidomic platform. To determine if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis using a liquid chromatography/mass spectrometry (LC/MS) proteomic platform. Results: Our data demonstrate that MSCs were immunoprivileged at all three passages since coculture with leukocytes did not affect the survival of MSCs at P3, P5, and P7. We also found that, with an increase in the passage number of MSCs, leukocytes did not cause any significant effect on cellular bioenergetics (basal respiration rate, spare respiratory capacity, maximal respiration, and coupling efficiency). Interestingly, in our omics data, we detected alterations in some of the ox-PCs and proteins in MSCs at different passages; however, these changes were not significant enough to affect their immunoprivilege. Conclusions: The outcome of this study demonstrates that an increase in passage number (from P3 to P7) in the cell culture does not have any significant effect on the immunoprivilege of MSCs.
Mesenchymal stem cells; Lipidomics; Proteomics; Immunoprivilege; Bioenergetics; Passage; Apoptosis
Bone marrow (BM)-derived mesenchymal stem cells
(MSCs) are attractive candidates for cell therapy since
these cells are immunoprivileged [
allogeneic (unrelated donor) MSCs can suppress the host
immune system and survive after transplantation. In fact,
the outcome of numerous preclinical studies and initial
clinical trials have demonstrated that allogeneic
BMMSCs after transplantation were able to initiate a
regenerative process [
]. However, long-term survival of
transplanted MSCs was not detected in the recipient
system. We recently reported in a rat model of
myocardial infarction that allogeneic MSCs after transplantation
into the infarcted heart lost their immunoprivilege and
were rejected by the host immune system [
]. The outcome
of our studies was confirmed by several other reports that
allogeneic MSCs after transplantation become
immunogenic and are rejected by the recipient immune system [
Therefore, for a successful bench-to-bedside translation of
MSC-based regenerative therapies, it is imperative to
understand the mechanisms of immune switch in MSCs
from the immunoprivileged to immunogenic state.
To maintain uniformity in the quality of the cell product
for any allogeneic cell-based clinical trial, MSCs derived
from a single healthy donor are used for transplantation
in multiple recipients. Therefore, it is imperative to
expand them in culture to facilitate generation of the
required number of MSCs. The safety of various passages of
MSCs in the in-vitro studies has been amply
demonstrated. A recent study reported no changes in telomeric
ends for up to 25 passages [
]. However, subjecting MSCs
to passages in cell culture, even in the short term, has
been associated with physiological changes. Madeira et al.
reported major differences in the pathways pertaining to
culture-induced senescence [
]. There are numerous
factors that may influence the overall optimum physiology of
the cells, and these may be affected by the passaging of
MSCs. The cell surface lipidome analysis of MSCs is
garnering attention of late. The passaging of MSCs was
found to alter levels of cell surface lipids and
]. The oxidized phospholipids (oxidized
phosphatidylcholine (ox-PC)), in particular, are known to
affect the immunological behavior of cells [
Furthermore, cellular bioenergetics including cellular respiration
and metabolic pathways are instrumental to stem cell
renewal, maintenance, and general cell health [
metabolic pathways lead to a difference in the functional
capabilities of stem cells [
]. It is reported that, with an
increase in passage number in the culture, cellular
bioenergetics and growth rate are affected [
Therefore, in this study, we have attempted to
investigate the effect of an increase in passage number in cell
culture on the immunological behavior of MSCs. The
most commonly used passages for cell therapy in
ongoing clinical studies include passage (P)3–7. In this
study, we included the clinically relevant passages, i.e.,
P3, P5, and P7, for evaluation. We have employed
functional evaluation as well as whole cell high-throughput
assessment. We have chosen parameters including cell
surface ox-PCs, cellular bioenergetics, global proteomic
assessment, and general immune as well as cell survival
pathways for our investigations. These characteristics
have hitherto not been studied in detail. With MSCs
poised at the cusp of clinical application, it is becoming
increasingly apparent that our knowledge is very limited
in various attempts to transfer understanding from the
in-vitro setting into in-vivo applications.
Unrelated male Sprague-Dawley (SD) rats (200–250 g)
were used for the isolation of bone marrow MSCs and
for the isolation of peripheral blood leukocytes. The
study protocol was approved by the Animal Care
Committee of the University of Manitoba and
conformed to the ‘Guide for the Care and Use of Laboratory
Animals’ published by the US National Institutes of
Health (NIH Publication No. 85–23, revised 1985).
MSC isolation and characterization
Bone marrow cells were flushed from the cavities of
femur and tibias of SD rats. After the connective tissue
was removed from around the bones, both ends were
cut. The bone marrow plugs were flushed with
Dulbecco’s modified Eagle’s medium supplemented with 15%
fetal bovine serum (FBS), 100 units/ml penicillin G, and
0.1 mg/ml streptomycin. Cells were plated and cultured
in the same medium followed by a media change and
the removal of nonadherent hematopoietic cells the next
day. The medium was replaced every 3 days, and the
cells were subcultured when confluency exceeded 90%.
MSCs from passages 3, 5, and 7 were used for the
studies described herein.
MSCs were characterized by flow cytometry as
described previously [
]; the cell population which was
identified as CD90.1+, CD29+, CD45−, and CD34− was
used for further experiments. To further characterize the
cells, MSCs were analyzed for their ability to differentiate
into osteogenic, adipogenic, and chondrogenic lineages
using a kit (R&D systems, catalogue number SC020). The
cells were induced to differentiate and stained using
reagents and primary antibodies provided in the kit
(osteocalcin for osteogenic differentiation; FABP4 for
adipogenic differentiation; aggrecan for chondrogenic
differentiation). The secondary antibodies used (AF488 for
osteocytes; AF647 for adipocytes and chondrocytes) were
purchased separately. The nuclei were stained with DAPI.
The images were captured using a Cytation5 at 20×
magnification. Additionally, differentiated MSCS were
also stained using Alizarin Red (osteogenic) and Oil
Red-O stain (adipogenic).
MSC population doubling
The population doubling time of MSCs at different
passages was calculated using a trypan blue cell viability
assay. The cells were plated at 100,000 cells/well in
sixwell dishes. After 96 h of culture, the MSCs were
detached using trypsin EDTA followed by staining with
trypan blue and counting of the live cell number using
an automated cell counter (BioRad). The doubling time
was calculated as follows:
Doubling time ¼ time of culture
logð2Þ= log ðfinal cell numberÞ
– log ðinitial cell numberÞ
Mixed leukocyte-mediated cytotoxicity
Leukocytes were isolated from rat spleen using
HISTOPAQUE 1083 (Sigma-Aldrich) and cocultured
with allogeneic MSCs at different passages (3, 5, and
7) at a ratio of 10:1 (leukocytes:MSCs). After 72 h of
coculture, leukocyte-mediated cytotoxicity in MSCs
was assessed by a Live/Dead viability/cytotoxicity
assay kit (Thermo Fisher Scientific, L3224).
Assessment of apoptosis
Apoptosis in MSCs at different passages (P3, P5, and P7)
was assessed after 72 h of coculture with mixed
leukocytes by measuring Bax and Bcl-xl levels using Western
blot. Briefly, total protein levels were measured by
Bradford protein assay, and 25 μg of protein was used in each
group for SDS-PAGE electrophoresis. After separation
with electrophoresis, proteins were transferred to PVDF
membranes and probed with primary antibodies for Bax
and Bcl-xl (Santa Cruz Biotechnologies Inc., CA, USA)
and secondary antibodies (Biorad Inc.). The membranes
were developed using x-ray film, and bands were
quantified using Quantity One software for densitometry.
The effect of MSCs on the proliferation of leukocytes
was analyzed using an MLR assay. Leukocytes were
cocultured with allogeneic MSCs at different passages
(P3, P5, and P7) for 72 h at a ratio of 1:10
(MSCs:leukocytes). The leukocyte proliferation was measured by flow
cytometry (BD Accuri). Briefly, after coculture, the
leukocytes in the supernatant were collected and spun at
1000 rpm for 5 min. The pellet was washed three times
using 1× phosphate-buffered saline (PBS), and
suspended in 100 μl cold PBS. The cells were then fixed
using 5 ml ice-cold 70% ethanol followed by RNase
(20 μg/ml) treatment for 30 min. The cells were then
stained with propidium iodide (PI; 5 μg/ml) for 5 min at
room temperature and analyzed using flow cytometry.
To measure leukocyte proliferation, a cell cycle analysis
was performed by counting the number of cells entering
the S phase (proliferating phase) and the G2/M phase
from the G0/G1 phase (resting cells) of the cell cycle.
Assessment of the secretion profile of leukocytes
The leukocytes were cocultured with MSCs at different
passages for 72 h at a ratio of 1:10 (MSCs:leukocytes).
The cytokine secretion profile of leukocytes was
analyzed using a multianalyte rat cytokine ELISArray kit
(Qiagen; MER 336161) following instructions from the
manufacturer. We analyzed the levels of 12 different
cytokines including interleukin (IL)-1α, IL-1β, IL-2, IL-4,
IL-6, IL-10, IL-12, IL-13, interferon (IFN)-δ, tumor
necrosis factor (TNF)-α, granulocyte-macrophage
colonystimulating factor (GM-CSF), and RANTES. The plate
was read at 450 and 570 nm using a Cytation5 analyzer
(BioTek Inc.) in plate reader mode.
Measurement of cellular bioenergetics
The cellular bioenergetics were determined using the
extracellular flux (XF24) analyzer (Seahorse Bioscience).
MSCs (4 × 104 cells/well) and leukocytes were cocultured
at a ratio of 1:10 (MSCs:leukocytes) in XF24 plates for
72 h. The mean basal respiration was determined by
recording oxygen consumption rate (OCR) measurements
before adding inhibitors or activators. ATP-linked OCR
and proton leak were determined by injecting oligomycin
(1 μM). The maximal respiration rate was determined
after adding FCCP (an uncoupler of the electron transport
chain) at a concentration of 1 μM. The difference between
the basal rate and this FCCP-stimulated rate is the reserve
capacity of the mitochondria, which is a measure of the
maximal potential respiratory capacity the cell can utilize
under conditions of stress and/or increased energetic
demands. To completely inhibit mitochondrial electron
transport, antimycin A (1 μM) and rotenone (1 μM) were
used. The OCR determined after rotenone and antimycin
A injection is attributable to nonmitochondrial oxygen
consumption. Mitochondrial basal respiration, proton
leak, and the maximal respiration were calculated after
corrections were performed for the nonmitochondrial
OCR for each assay. Under these conditions, viability was
over 90% for all cell types and remained so over the time
course of the assay. At the end of the assay period,
trypsinized cells were collected, and values were normalized to
the total cell number in each well [
Cell surface oxidized phosphatidylcholine (ox-PCs)
levels were measured in MSCs at different passages
(P3, P5, and P7) by liquid chromatography/mass
spectrometry (LC/MS) analysis. Total cellular lipids were
extracted from cell pellets using a protocol adapted from
Folch et al. [
]. Oxylipidomic analysis was performed
with reverse-phase high-performance liquid
chromatography (HPLC) using an Ascentis Express C18 column
(Supelco Analytical, Bellefonte, PA, USA). Data were
collected using analyst 1.6 software (Applied Biosystems,
Canada) and quantified using MultiQuant 2.1 (Absciex,
Ontario, Canada). The mass spectrometry data were log
transformed and autoscaled (mean-centered and divided
by standard deviation of each variable) before applying
statistical analysis. To determine the changes in ox-PCs
that were statistically significant between different
passages P3, P5, and P7, we performed a one-way analysis of
variance (ANOVA) with a p value cut-off of 0.05, followed
by Tukey’s Honestly Significant Difference (Tukey’s HSD).
E06 antibody treatment
E06 is a blocking antibody that specifically inhibits
oxidized phosphatidylcholines in cells. To assess the
involvement of ox-PCs in regulating leukocyte-mediated
cytotoxicity, apoptosis, and cellular bioenergetics in
MSCs, we cocultured MSCs and leukocytes with or
without E06 antibody (1 ng/ml) (MSC + L + Ab) for 72 h
and measured the abovementioned parameters.
Sample preparation for mass spectrometry
Whole cell proteomic analysis was performed in MSCs
at different passages (P3, P5, and P7) by the LC/MS
proteomic platform. MSCs were cultured at different
passages, and cell pellets were collected and washed in
ice-cold PBS (pH 7.2) followed by treatment with urea
lysis buffer (8 M urea in 0.1 M Tris-HCl, pH 8.5).
Protein estimation was performed by Qubit fluorescence
assay (Invitrogen). A total of 50 μg protein was digested
using the FASP procedure as described previously [
Liquid chromatography tandem mass spectrometric
analysis of tryptic peptides (500 ng) was carried out using a
Proxeon nano spray ESI source (Thermo Fisher, Hemel,
UK) and analyzed using Orbitrap Velos Pro FTMS
(Thermo Finnigan, Bremen, Germany) [
Proteomic data analysis by MaxQuant
Peptides and proteins were identified by Andromeda via
an automated database search of all tandem mass spectra
against a curated target/decoy database (using forward
and reverse versions of the Rattus norvegicus [Taxonomy
ID 10116]) and Uniprot protein sequence database
(http://www.uniprot.org; release October 2015) containing
all rat protein entries from Swiss-Prot and TrEMBL.
Cysteine carbamidomethylation was searched as a fixed
modification, whereas N-acetyl protein, deamidated NQ,
and oxidized methionine were searched as a variable
modification. The resulting Andromeda peak list-output
files were further processed using MaxQuant software.
The downstream bioinformatics data analysis was carried
out using the Perseus software suite (22.214.171.124) and the
Ingenuity Pathway Analysis software tool (Ingenuity
Systems, Qiagen, Redwood City).
Experimental values are expressed as mean ± SD. The
comparison of mean values between various groups was
performed by one-way ANOVA followed by multiple
comparisons by Tukey test using the software GraphPad
Prism. A p value < 0.05 was considered to be significant.
Differentiation of MSCs
To characterize MSCs, cells were induced to
differentiate toward the adipogenic, osteogenic, and
chondrogenic lineages. Our data demonstrate that MSCs have
the ability to differentiate toward these three lineages
(Additional file 1: Figure S1).
Population doubling of MSCs at different passages
To investigate the effect of an increase in passage
number on population doubling time of MSCs, a cell viability
assay was performed; our data demonstrate that there
was no significant difference in the population doubling
time of MSCs in the culture at P3, 5, or 7 (Fig. 1a).
MSCs show no significant changes in immunological behavior with an increase in passage number
Bone marrow-derived MSCs are reported to be
immunoprivileged and thus are able to escape the host
immune system after transplantation. To understand the
effect of an increase in passage number on the
immunoprivilege of MSCs, cells at P3, P5, and P7 were
cocultured with mixed leukocytes. The leukocyte-mediated
cytotoxicity was measured in MSCs. Our results
demonstrate that MSCs were immunoprivileged at all three
passages (P3, P5, and P7) since we found more than 80%
live cells even after 72 h of coculture with leukocytes
(Fig. 1b). Interestingly, there was no significant difference
detected in the number of live/dead MSCs at different
passages after the coculture (Fig. 1b). We also assessed
leukocyte-mediated apoptosis of MSCs and found no
significant difference in the ratio of the antiapoptotic protein
Bcl-xl and the proapoptotic protein Bax at P3, P5, and P7
after coculture with leukocytes (Fig. 1c).
Mesenchymal stem cells have the ability to suppress
immune cell proliferation and promote immune
tolerance. We analyzed the effect of an increase in passage
number of MSCs on their ability to suppress leukocyte
proliferation and found that there were no significant
differences in the level of suppression of leukocyte
proliferation by MSCs at different passages (Fig. 2a). The
data are represented as different stages of the cell
cycle including G0/G1 phase, S phase, and G2/M
phase. G1/G0 phase represents the stage where the
cells prepare for the next division cycle by synthesizing
proteins and RNA required for the division and
multiplication. In S phase, the DNA synthesis occurs allowing the
cells in G2/M phase to have double DNA which becomes
divided equally in the cells once they undergo mitotic cell
division. There were no significant differences observed at
any stage among the different passages, indicating that the
MSCs at P3, 5, and 7 affect leukocyte proliferation at the
To further assess the effect of MSCs at different
passages on the immunomodulatory effects of leukocytes,
we analyzed the levels of several proinflammatory cytokines
including IL-1α, IL-1β, IL-2, IL-6, IL-12, IFN-γ, TNF-α,
GM-CSF, and RANTES, and the anti-inflammatory
cytokines IL-4, IL-10, and IL-13 in leukocytes after coculture
with MSCs. Our data demonstrate that there was no
significant change in the levels of these soluble factors in
leukocytes after coculture with MSCs at different passages
(Fig. 2b, c). These results suggest that an increase in
the passage number from P3 to P7 does not affect
immunoprivilege and immune tolerance of MSCs.
Effect of an increase in passage number on cellular bioenergetics
It is reported that intracellular energy metabolism has a
primary influence on the presence or absence of T-cell
activation signals. Therefore, cellular bioenergetics are a
key factor for determining the response of transplanted
cells toward the host immune system. We assessed the
effect of an increase in passage number on the
intracellular energy metabolism using a SeaHorse Bioscience XF24
analyzer. We found no significant difference in basal
respiration rate and spare respiratory capacity in MSCs at P3,
P5, and P7 before and after coculture with leukocytes
(Fig. 3a, b). We also measured the maximal respiration
along with coupling efficiency of MSCs in the presence
as well as absence of leukocytes. Our data indicate that
there was no significant difference observed in any of
these parameters at different passages (Fig. 3c, d).
Oxidized phosphatidylcholine (ox-PCs) levels change in
MSCs without affecting immunoprivilege
The cellular ox-PCs have been recognized as important
mediators of immune signaling. To assess changes in the
total cell oxylipidome at different passages and their
effect on immunoprivilege of MSCs, the cells at P3, P5,
and P7 were subjected to LC/MS analysis. Our data
demonstrate that, overall, there were no significant
features identified between the passages (Fig. 4a, b).
However, some of the ox-PCs which are already reported (in
other cell types) to play a significant role in immune cell
suppression and were found to be altered with an
increase in passage number in the current study are
SOVPC, KDdiA SPC, PAPC-OOH, SAPC-keto, and
SECPC (Fig. 4b).
To explore whether changes in the levels of ox-PCs have
any effect on the immunological behavior of MSCs, we
added E06 antibody in the coculture experiments and
assessed leukocyte-mediated cytotoxicity and apoptosis in
MSCs. The E06 antibody is responsible for blocking cell
surface ox-PCs. Our data demonstrate that the presence
of the antibody did not have any significant effect on the
number of live/dead MSCs and apoptosis after coculture
with leukocytes (Fig. 1b, c). Furthermore, the presence of
E06 antibody did not cause any difference in cellular
bioenergetics. We did not see any significant changes in the
basal respiration rate, spare respiratory capacity, coupling
efficiency, or maximal respiration rate before and after the
addition of the E06 blocking antibody (Fig. 3a–d). Hence,
alterations in individual cell surface ox-PCs from P3 to P7
do not affect the immunological properties or cellular
bioenergetics of MSCs.
The stem cell proteome is largely unchanged over different passages
We performed whole-cell proteomic analysis at different
passages using LC/MS to study changes in the levels of
intracellular proteins related to cellular senescence,
immunogenicity, and bioenergetics. In total, over 800
proteins were screened (Fig. 5a). Our proteomic data
recorded some changes in the levels of proteins
associated with cellular senescence and aging (Fig. 5b) and
immunological synapse (Fig. 5c). Furthermore, some of the
proteins that have been reported to play a role in cellular
respiration pathways including glycolysis and oxidative
phosphorylation as well as tricarboxylic acid (TCA) cycle
showed changes with the increase in passage number
(Fig. 5d–f ). The proteomic analyses for mitochondrial
pathways also indicated changes in some proteins in P7
versus P3 (Additional file 2: Figure S2). However, overall,
the extent of change recorded in intracellular proteins
was not significantly different among P3 and P7, and the
changes recorded were not able to affect immunological
behavior of MSCs.
In various animal models of degenerative diseases,
transplantation of bone marrow-derived allogeneic MSCs has
triggered regenerative processes. Based on the
encouraging outcome of animal-based studies, several clinical
trials have tested the efficacy of allogeneic MSCs.
Although the outcome of allogeneic cell-based clinical
trials has been encouraging, it is not as effective as the
outcome of preclinical studies. Some of the recent
studies reported that allogeneic MSCs after transplantation
were immunogenic and were rejected by the host
immune system. Therefore, understanding the mechanisms
of the switch in the immunological behavior of MSCs
from immunoprivileged to the immunogenic state would
help in preserving the benefits of allogeneic MSCs. The
current study is the first to evaluate the possible role of
cellular bioenergetics and cell surface ox-PCs in
combination with intracellular proteomic analysis in regulating
the immunoprivilege of MSCs over different passages.
The notion that an increase in passage number in cell
culture may affect cellular physiology, morphology, and
particularly cell surface molecules is being actively
debated. It has been suggested that senescence of MSCs
starts as soon as their culturing is initiated. Furthermore,
the cells start losing the ability to differentiate, doubling
capacity, and telomere length as the passage number
increases in cell culture [
]. The outcomes of the
majority of MSC-based animal studies and clinical trials have
suggested that allogeneic MSCs after transplantation
were immunoprivileged and there was no immune
response detected in the recipient system against
transplanted cells [
]. However, some recent studies
revealed that antidonor alloantibodies were detected
against transplanted MSCs [
] and cells were rejected
by the recipient immune system. One of the important
variables in these studies was that cells employed for these
studies were from different passages. Therefore, in this
study we investigated the effect of an increase in passage
number in cell culture on the immunological behavior
of MSCs. The most commonly used passages for cell
therapy in concluded or ongoing studies include
passages 3–7. Therefore, in the current study, we included
P3, P5, and P7 for evaluation. Our investigations were
focused on hitherto untested parameters of cell surface
ox-PCs, cellular bioenergetics, global, and proteomic
Previous studies have reported an association of
cellular senescence with alterations in the
immunological behavior of MSCs [
]. Senescent cells are
reported to attract immune cells; for instance, fibroblasts
undergoing senescence secrete various cytokines as well
as chemokines which lead to activation of lymphocytes
and macrophages [
]. At the organ level, it is reported
that kidneys from old donors are more prone to
rejection by the host immune system compared with those
from young donors [
]. Additionally, in the case of
bone marrow-derived MSCs, senescence is associated
with a reduced differentiation and proliferation potential
]. Furthermore, radiation-induced senescence in MSCs
is associated with abrogation of the immunomodulatory
properties and impaired therapeutic potential in vivo in a
mouse model of sepsis [
]. However, the effect of an
increase in passage number on immunoprivilege of MSCs
has not been investigated thoroughly. Therefore, to
investigate this, in the current study we analyzed cellular
changes in immunogenicity at different passages. We
found that an increase in passage number from P3 to P7
did not have any significant effect on immunoprivilege of
MSCs since leukocyte-mediated cytotoxicity and cell
death in MSCs did not change between the different
passages. Furthermore, there was no significant difference
observed in the MSC-mediated suppression of leukocyte
proliferation with different passages of MSCs.
Several studies in the literature have reported the role
of the cellular bioenergetics profile in the regulation of
immune response. The mode of intracellular respiration
plays a key role in influencing the presence or absence
of T-cell activation signals and thus regulating
immunoprivilege of a cell [
]. The choice of fuel (glucose or
fatty acids) used for mitochondrial metabolism
regulates the interaction of the cell with the immune system
]. In dendritic cells, there is a switch in the mode
of metabolism from oxidative phosphorylation to
glycolysis that triggers their activation [
]. In another
study, the effect of blocking mitochondrial respiration
was reported to reduce the binding of TNF-α to the
cells, indicating that mitochondrial respiration might be
an important mediator of the immune responses
controlled by TNF-α [
]. To assess the effects of an
increase in passage number on the cellular respiratory
profile in MSCs and its association with the
immunoprivilege of MSCs, we performed Seahorse XF24
analysis at different passages. Our data demonstrate that
within clinically relevant passages an increase in
passage number in cell culture from P3 to P7 did not affect
basal respiration rate, spare respiratory capacity,
maximal respiration, or coupling efficiency in MSCs before
and after coculture with leukocytes.
Another major modulator for the immunogenicity of
cells is the expression of cell surface ox-PCs. Several
studies have described the role of oxidized
phosphatidylcholines in modulating the immunological behavior of
immune cells. Oxidation of phospholipids under various
conditions such as inflammation, apoptosis, and senescence
leads to the generation of proinflammatory
damageassociated molecular products (DAMPs) that leads to
recognition of the cells expressing these epitopes by the innate
immune system [
]. During atherosclerosis, ox-PCs
including phosphatidylcholines served as signals for uptake of
the cells expressing the phospholipids [
antagonistic to their role in immune cell activation, oxidized
phospholipids are also reported to be involved in
preventing the activation of T lymphocytes [
]. Adding to their
immunosuppressive role, oxidized phospholipids are also
known to prevent the activation of dendritic cells by
Tolllike receptor (TLR)3- and TLR4-mediated pathways [
These lipids can be both the mediators and the result of
cellular apoptosis in different cell types [
]. In various
models, the cells expressing the oxidized phospholipids are
recognized by macrophages for apoptosis [
the macrophages might themselves undergo apoptosis due
to activation of the TLR pathway by oxidized phospholipids
]. Therefore, ox-PCs play an important role in mediating
the cellular response to the immune system. To assess the
effect of oxidized phospholipids on the immunoprivilege of
MSCs in different passages, we performed LC/MS
lipidomic analysis. Our data revealed that some of the ox-PCs
were changing with the increase in passage number.
However, alterations in ox-PCs did not affect immunoprivilege
of MSCs. To determine if the ox-PCs were acting in
tandem with downstream intracellular protein alterations, we
performed proteome analysis using the LC/MS proteomic
platform to screen more than 800 proteins. The overall
trend recorded in intracellular proteins did not change in
MSCs with an increase in passage number. However, we
found changes in some proteins involved in cellular
senescence and metabolism, but these changes were not able to
affect the immunological behavior of MSCs.
Our study suggests that an increase in passage number
from P3 to P7 does not affect immunoprivilege of MSCs.
However, more studies are needed to delineate the
mechanisms of switch in the immunological behavior of
MSCs after transplantation.
Additional file 1: Figure S1. Differentiation of MSCs into osteocytes,
adipocytes, and chondrocytes. MSCs were induced to differentiate
toward osteocytes (a), adipocytes (b), and chondrocytes (c). MSCs
(undifferentiated cells, control group) and differentiated MSCs (D-MSC)
were stained for osteocalcin and Alizarin Red (osteocyte lineage), FABP4
and Oil Red-O stain (adipocyte lineage), and aggrecan (chondrocyte
lineage). The images were taken using Cytation5 (BioTek Instruments)
(20× magnification). (n = 6) (PPTX 2648 kb)
Additional file 2: Figure S2. Heat map showing the highly upregulated
or downregulated proteins that are known to be associated with
mitochondrial respiration and immune pathways at P3 versus P7.
(PPTX 5354 kb)
This work was supported by research grant from the Canadian Institute of
Health Research to SD. NS and GLS are funded by a fellowship from
This work was supported by a research grant from the Canadian Institute of
Health Research to SD.
Availability of data and materials
All data generated and/or analyzed during this study are included in this
published article and its Additional files.
NS, GLS, and SD conceptualized the study and designed the experiments;
NS, GLS, RC, EAER, SRC, VS, AS, and MM carried out experiments and
acquired the data; NS, GLS, AS, VS, AR, PF, and SD interpreted the data and
carried out data analysis and statistical analysis; NS, GLS, and SD drafted the
manuscript. All authors read and approved the final manuscript.
The study protocol was approved by the Animal Care Committee of the
University of Manitoba and conformed to the ‘Guide for the Care and Use
of Laboratory Animals’ published by the US National Institutes of Health
(NIH Publication No. 85–23, revised 1985).
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Faiella W , Atoui R . Immunotolerant properties of mesenchymal stem cells: updated review . Stem Cells Int . 2016 ;2016 https://doi.org/10.1155/ 2016 /1859567.
2. Fazel S , Chen L , Weisel RD , Angoulvant D , Seneviratne C , Fazel A , et al. Cell transplantation preserves cardiac function after infarction by infarct stabilization: augmentation by stem cell factor . J Thorac Cardiovasc Surg . 2005 ; 130 : 1310 .
3. Dhingra S , Li P , Huang X-P , Guo J , Wu J , Mihic A , et al. Preserving prostaglandin E2 level prevents rejection of implanted allogeneic mesenchymal stem cells and restores postinfarction ventricular function . Circulation . 2013 ; 128 ( 11 suppl 1 ): S69 - 78 .
4. Oliveira R , Linhares G , Chagastelles PC , Sesterheim P , Pranke P. In vivo immunogenic response to allogeneic mesenchymal stem cells and the role of preactivated mesenchymal stem cells cotransplanted with allogeneic islets . Stem Cells Int . 2017 ; 2017 : e9824698 .
5. Bernardo ME , Zaffaroni N , Novara F , Cometa AM , Avanzini MA , Moretta A , et al. Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms . Cancer Res . 2007 ; 67 : 9142 - 9 .
6. Madeira A , da Silva CL , dos Santos F , Camafeita E , Cabral JMS , Sá-Correia I . Human mesenchymal stem cell expression program upon extended ex-vivo cultivation, as revealed by 2-DE-based quantitative proteomics . PLoS One . 2012 ; 7 : e43523 .
7. von Bahr L , Sundberg B , Lönnies L , Sander B , Karbach H , Hägglund H , et al. Long-term complications, immunologic effects, and role of passage for outcome in mesenchymal stromal cell therapy . Biol Blood Marrow Transplant . 2012 ; 18 : 557 - 64 .
8. Bochkov VN , Oskolkova OV , Birukov KG , Levonen A-L , Binder CJ , Stöckl J . Generation and biological activities of oxidized phospholipids . Antioxid Redox Signal . 2010 ; 12 : 1009 - 59 .
9. Ito K , Suda T. Metabolic requirements for the maintenance of self-renewing stem cells . Nat Rev Mol Cell Biol . 2014 ; 15 : 243 - 56 .
10. Costello LC , Franklin RB . A review of the important central role of altered citrate metabolism during the process of stem cell differentiation . J Regen Med Tissue Eng . 2013 ;2 https://doi.org/10.7243/2050-1218-2-1.
11. Stab BR , Martinez L , Grismaldo A , Lerma A , Gutiérrez ML , Barrera LA , et al. Mitochondrial functional changes characterization in young and senescent human adipose derived MSCs . Front Aging Neurosci . 2016 ;8 https://doi.org/ 10.3389/fnagi. 2016 . 00299 .
12. Ziegler DV , Wiley CD , Velarde MC . Mitochondrial effectors of cellular senescence: beyond the free radical theory of aging . Aging Cell . 2015 ; 14 : 1 - 7 .
13. Roy Chowdhury SK , Smith DR , Saleh A , Schapansky J , Marquez A , Gomes S , et al. Impaired adenosine monophosphate-activated protein kinase signalling in dorsal root ganglia neurons is linked to mitochondrial dysfunction and peripheral neuropathy in diabetes . Brain . 2012 ; 135 : 1751 - 66 .
14. Folch J , Lees M , Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues . J Biol Chem . 1957 ; 226 : 497 - 509 .
15. Bouyer G , Reininger L , Ramdani G D Phillips L , Sharma V , Egee S , et al. Plasmodium falciparum infection induces dynamic changes in the erythrocyte phospho-proteome . Blood Cells Mol Dis 2016 ; 58 : 35 - 44 .
16. Suárez-Cortés P , Sharma V , Bertuccini L , Costa G , Bannerman N-L , Sannella AR , et al. Comparative proteomics and functional analysis reveal a role of Plasmodium falciparum osmiophilic bodies in malaria parasite transmission . Mol Cell Proteomics . 2016 ; 15 : 3243 - 55 .
17. Bonab MM , Alimoghaddam K , Talebian F , Ghaffari SH , Ghavamzadeh A , Nikbin B . Aging of mesenchymal stem cell in vitro . BMC Cell Biol . 2006 ; 7 : 14 .
18. Makkar RR , Price MJ , Lill M , Frantzen M , Takizawa K , Kleisli T , et al. Intramyocardial injection of allogenic bone marrow-derived mesenchymal stem cells without immunosuppression preserves cardiac function in a porcine model of myocardial infarction . J Cardiovasc Pharmacol Ther . 2005 ; 10 : 225 - 33 .
19. Amado LC , Saliaris AP , Schuleri KH , St John M , Xie J-S , Cattaneo S , et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction . Proc Natl Acad Sci U S A . 2005 ; 102 : 11474 - 9 .
20. Ankrum JA , Ong JF , Karp JM . Mesenchymal stem cells: immune evasive, not immune privileged . Nat Biotechnol . 2014 ; 32 : 252 - 60 .
21. Agrawal A , Tay J , Yang G-E , Agrawal S , Gupta S . Age-associated epigenetic modifications in human DNA increase its immunogenicity . Aging . 2010 ; 2 : 93 - 100 .
22. Gupta S. Role of dendritic cells in innate and adaptive immune response in human aging . Exp Gerontol . 2014 ; 54 : 47 - 52 .
23. Burton DGA , Faragher RGA . Cellular senescence: from growth arrest to immunogenic conversion . Age Dordr Neth . 2015 ; 37 : 27 .
24. Fijter JWD , Mallat MJK , Doxiadis IIN , Ringers J , Rosendaal FR , Claas FHJ , et al. Increased immunogenicity and cause of graft loss of old donor kidneys . J Am Soc Nephrol . 2001 ; 12 : 1538 - 46 .
25. Turinetto V , Vitale E , Giachino C. Senescence in human mesenchymal stem cells: functional changes and implications in stem cell-based therapy . Int J Mol Sci . 2016 ; 17 https://doi.org/10.3390/ijms17071164.
26. Carlos Sepúlveda J , Tomé M , Eugenia Fernández M , Delgado M , Campisi J , Bernad A , et al. Cell senescence abrogates the therapeutic potential of human mesenchymal stem cells in the lethal endotoxemia model . Stem Cells . 2014 ; 32 : 1865 - 77 .
27. Buck MD , O'Sullivan D , Pearce EL . T cell metabolism drives immunity . J Exp Med . 2015 ; https://doi.org/10.1084/jem.20151159.
28. MacIver NJ , Michalek RD , Rathmell JC . Metabolic regulation of T lymphocytes . Annu Rev Immunol . 2013 ; 31 : 259 - 83 .
29. Dranka BP , Benavides GA , Diers AR , Giordano S , Zelickson BR , Reily C , et al. Assessing bioenergetic function in response to oxidative stress by metabolic profiling . Free Radic Biol Med . 2011 ; 51 : 1621 - 35 .
30. van der Windt GJW , Chang C-H , Pearce EL . Measuring bioenergetics in T cells using a Seahorse Extracellular Flux Analyzer . Curr Protoc Immunol . 2016 ; 113 : 3 . 16B . 1 - 3 . 16B .14. Ed John E Coligan Al
31. Krawczyk CM , Holowka T , Sun J , Blagih J , Amiel E , DeBerardinis RJ , et al. Toll-like receptorinduced changes in glycolytic metabolism regulate dendritic cell activation . Blood . 2010 ; 115 : 4742 - 9 .
32. Sánchez-Alcázar JA , Hernández I , la Torre MPD , García I , Santiago E , MuñozYagüe MT , et al. Down-regulation of tumor necrosis factor receptors by blockade of mitochondrial respiration . J Biol Chem . 1995 ; 270 : 23944 - 50 .
33. Weismann D , Binder CJ . The innate immune response to products of phospholipid peroxidation . Biochim Biophys Acta . 1818 ; 2012 : 2465 - 75 . BBA - Biomembr
34. Berliner JA , Leitinger N , Tsimikas S. The role of oxidized phospholipids in atherosclerosis . J Lipid Res . 2009 ; 50 ( Suppl ): S207 - 12 .
35. Seyerl M , Blüml S , Kirchberger S , Bochkov VN , Oskolkova O , Majdic O , et al. Oxidized phospholipids induce anergy in human peripheral blood T cells . Eur J Immunol . 2008 ; 38 : 778 - 87 .
36. Blüml S , Kirchberger S , Bochkov VN , Krönke G , Stuhlmeier K , Majdic O , et al. Oxidized phospholipids negatively regulate dendritic cell maturation induced by TLRs and CD40 . J Immunol . 2005 ; 175 : 501 - 8 . Baltim Md 1950
37. Serbulea V , DeWeese D , Leitinger N. The effect of oxidized phospholipids on phenotypic polarization and function of macrophages . Free Radic Biol Med . 2017 ; 111 : 156 - 68 .
38. Chang M-K , Binder CJ , Miller YI , Subbanagounder G , Silverman GJ , Berliner JA , et al. Apoptotic cells with oxidation-specific epitopes are immunogenic and proinflammatory . J Exp Med . 2004 ; 200 : 1359 - 70 .
39. Seimon TA , Nadolski MJ , Liao X , Magallon J , Nguyen M , Feric NT , et al. Atherogenic lipids and lipoproteins trigger CD36-TLR2-dependent apoptosis in macrophages undergoing endoplasmic reticulum stress . Cell Metab . 2010 ; 12 : 467 - 82 .