Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal Stromal Cells

Abstract

Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use. Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone, would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes of MSC transplantation and potentially other transient cell therapeutics.

Full text

Pharmacokinetics of Natural and Engineered Secreted
Factors Delivered by Mesenchymal Stromal Cells
Jessica S. Elman
1.
, Ryan M. Murray
1.
, Fangjing Wang
1.
, Keyue Shen
1
, Shan Gao
1
, Kevin E. Conway
4
,
Martin L. Yarmush
1,2
, Bakhos A. Tannous
4
, Ralph Weissleder
3
, Biju Parekkadan
1,5
*
1Department of Surgery, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospital for
Children, Boston, Massachusetts, United States of America, 2Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey, United States of America,
3Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 4Department of Neurology,
Experimental Therapeutics and Molecular Imaging Laboratory, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America, 5Harvard Stem Cell
Institute, Boston, Massachusetts, United States of America
Abstract
Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use.
Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-
inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis
that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone,
would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular
engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear
differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or
engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that
affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined
consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes
of MSC transplantation and potentially other transient cell therapeutics.
Citation: Elman JS, Murray RM, Wang F, Shen K, Gao S, et al. (2014) Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal
Stromal Cells. PLoS ONE 9(2): e89882. doi:10.1371/journal.pone.0089882
Editor: Eva Mezey, National Institutes of Health, United States of America
Received December 2, 2013; Accepted January 28, 2014; Published February 21, 2014
Copyright: ß2014 Elman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported of the National Institutes of Health (R01EB012521 and K01DK087770) and the Broad Medical Research Program of The Broad
Foundation (BMRP498382) and Shriners Hospitals for Children. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: biju_parekkadan@hms.harvard.edu
.These authors contributed equally to this work.
Introduction
Cell therapy is an exponentially growing field with .2,500
clinical trials in the world over the last 10 years [1]. Cell-based
therapeutic products are positioned as a billion dollar per year
industry with anticipated market growth [2,3]. The method of
using cells as drugs is particularly advantageous when a higher-
order approach to treatment is required. A fundamental issue,
particularly for transient cell therapies which are the largest of the
drug class (50%), is the lack of predictive measures of the body’s
response to the therapy and vice versa: traditionally known as a
drug’s pharmacokinetics (PK) and pharmacodynamics (PD).
Without a rigorous PK/PD model of the mechanism of action
of transient cell therapies, processes cannot be optimized to
formulate a drug and physicians will be unable to effectively
monitor and communicate the benefits and risks of a cell therapy
to a patient.
Mesenchymal stem cells (MSCs) - an adult multipotent
progenitor cell population initially derived from bone marrow
[4] – have over a decade of clinical testing in thousands of patients
worldwide. These cells have appealing manufacturing properties
such as the ease of isolation, expansion, and cryopreservation, as
well as the tolerability of allogeneic cell therapy. MSCs are widely
being evaluated for the combinatorial treatment of a variety of
diseases including myocardial infarction [5,6], bone marrow
transplantation [7], stroke [8], autoimmune disease [9], and
wound healing [10,11,12,13]. The predominant use of MSCs as
an immunomodulatory agent is substantiated by the observation
that MSCs can inhibit the activation and effector function of
numerous immune cell types [14,15,16,17]. The mechanism of
action of immune cell inhibition by MSCs is primarily due to the
release of secreted factors by cells [18]. MSCs have proven
effective in early stage clinical trials [19], yet, several Phase II and
Phase III industry-led trials either have undergone early termina-
tion or have failed to meet primary endpoints [20]. New strategies
to optimize this therapeutic are needed to help usher this
promising cell population to the clinic.
A deep understanding of MSC pharmacology can provide
insight on how to best deliver this therapeutic. A prevailing view of
MSC trafficking is that, upon intravenous administration, cell
homing, engraftment, proliferation, and/or differentiation are
critical to induce a therapeutic effect. Sensitive monitoring
techniques have demonstrated little, if any, long-term engraftment
(.1 week) of MSCs upon systemic administration with the
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majority of administered MSCs (.90%) accumulating immedi-
ately in the lungs and then cleared with a half-life of 24 h
[21,22,23]. Therefore, MSCs can be viewed conceptually as a
transient cell therapy that delivers a ‘‘payload’’ of secreted factors
to alter acute disease progression [20].
We put the concept of MSCs as a therapeutic delivery vehicle to
the test with a focus on evaluating the systemic release of secreted
factors. Investigators have studied the release of natural secreted
proteins such as TSG-6 [22] and TRAIL [24] or engineered
antibodies [25] from MSCs in the systemic circulation after
transplantation, although a thorough pharmacological analysis
was not performed nor compared to purified proteins. In this
study, conventional cell biodistribution data was combined with
serum profiles of natural or engineered secreted factors released by
the cells to understand rate-limiting processes that alter exposure
to intravenous MSC therapy, the most widely used administration
method. Based on our combined PK analysis, we observed that
molecular monitoring of serum secreted factors revealed interest-
ing phenomena regarding the delivery of natural or engineered
proteins, an active cell secretion mechanism, and the host immune
response to the graft. This study can aid in designing an optimal
cell delivery regimen to maximize MSC therapy.
Results
Comparison between the Bioavailability of Transplanted
MSCs and Secreted Molecules
Secreted factors released by MSCs after transplantation have
been a general phenomena that has been observed in several pre-
clinical therapeutic studies [22,26]. It is reported that MSCs can
secrete a wide range of cytokines and growth factors [18]. We
designed a pharmacokinetic study to monitor both the viability
and distribution of MSC transplants and overlay the serum profiles
of a specific and detectable level of interleukin (IL)-6, a pleiotropic
molecule secreted by MSCs [27]. Human MSCs were engineered
with a luciferase reporter gene, injected via intravenous (IV) route
(the convention used by the cell therapy community) and detected
after injection using whole-body, bioluminescent imaging (BLI).
By nearly 8 hours after IV cell injection the BLI signal was almost
undetectable (Figure 1A). At the same imaging time points, using
human MSCs that were not engineered, we also measured the
serum levels of human IL-6. We observed that the release of
human IL-6 by MSCs was short-lived and tracked accordingly to
the cellular BLI signal of the graft (Figure 1C). Similar
pharmacokinetics of two other protein secreted factors, monocyte
chemoattrive protein (MCP)-1 and IL-8, were observed after IV
injection in vivo further supporting our observation (Figure S2A–
B). Future experiments continued to explore and evaluate the
impact of these pharmacokinetic profiles, with a focus on
molecular delivery of IL-6 by IV administration.
Cell Delivery of IL-6 Achieves Greater Exposure than
Molecular Delivery of IL-6
Cells can be considered a ‘‘carrier’’ for their secreted factors.
We used this perspective to understand the differences of
delivering IL-6 in a purified form compared to a cell transplant.
Conditioned medium from MSCs (referred to as MSC-CM) was
generated in order to compare MSC-derived IL-6 without
concerns of post-translational modifications that take place in
other protein expression systems for recombinant forms of IL-6
that may affect bioavailability. Concentrated MSC-CM was
prepared and a volume of 400 mL (containing 40 ng IL-6) was
injected into mice. The content of soluble factors in this volume is
equivalent to the secreted levels when 3610
6
cells are cultured for
2 days in vitro and was normalized to 1610
6
cells for comparison
to cell transplants at the same cell dose. MSC-CM was
administered by IV and the serum levels of IL-6 followed a
classical bolus pharmacokinetic profile (Figure 2A). Pharmacoki-
netic parameters for serum IL-6 levels were calculated with the
assumption that the clearance of IL-6 itself was a constant 218 ml/
hr based on the literature [28]. The maximum serum IL-6
concentration was increased by ,400% when delivered by cell
transplants (Figure 2B). This amounted to a greater than 7-fold
increase in the exposure of the subject to IL-6 as measured by area
under the curve (AUC) analysis (Figure 2C). The temporal
kinetics of IL-6 was also artificially extended by way of cell
transplantation. The time to reach maximum serum concentra-
tion, the half-life, and the elimination constant of IL-6 were all
significantly modified for prolonged duration of IL-6 by the use of
cell transplantation (Figure 2D–F). These data highlight the
supplementary changes to molecular pharmacokinetic parameters
by way of cellular delivery.
MSCs Utilize an Active, Golgi-Dependent Secretion
Mechnaism to Release IL-6 In Vivo
The presence of human IL-6 in serum after cell transplantation
could be due to passive mechanisms, such as cell rupture and
cytokine release, or by active secretion pathways. Brefeldin A
(BFA), a protein-transport inhibitor, was used to block IL-6
production by inhibiting Golgi apparatus-dependent vesicle
secretion. A non-toxic concentration of 5 ug/ml BFA was chosen
by evaluating a dose response of BFA to MSCs in vitro
(Figure 3A). After incubating MSCs with BFA for one day, the
in vitro secretion of IL-6 in the supernatant was significantly
inhibited compared to the untreated controls (Figure 3B). The
secretion of IL-6 was not restored after a day of incubation with
BFA until 72 hrs later (Figure 3B). We were satisfied with
blockade of IL-6 for .60 hours in vitro and advanced this cell
formulation with impaired IL-6 secretion for in vivo PK studies.
The PK profile of MSC-derived IL-6 was dramatically different,
specifically a reduced maximal effective concentration (,56) and
AUC (,10006) compared to the cells that were not treated with
BFA (Figure 3C–D). This study highlights a powerful active
mechanism that MSCs employ to deliver IL-6 to the bloodstream
that requires the Golgi apparatus.
Exposure of MSC-derived IL-6 is a Function of Host
Immune Cells
To study the natural process of protein release from non-
engineered cells, we designed our PK model using human cell
transplants in mice. This model afforded us the ability to detect
human proteins in mouse serum with high specificity and
sensitivity and correlate that to in situ cellular production. We
were also interested in studying the immune response, albeit a
xenogenic rejection response, and its ability to alter in vivo protein
release. We transplanted human MSCs by IV administration in
mice strains that are immunocompetent (C57Bl/6), less immuno-
competent (Foxn12/2, thymic and peripheral loss of T cells), or
severely immunodeficient (NOD-SCID-IL-2rg 2/2, loss of B, T,
and NK cells). The effect of the immune response on MSC-
derived IL-6 serum kinetics was pronounced after IV administra-
tion. NOD-SCID-IL-2rg 2/2mice treated with human MSCs
had a delayed maximum effective concentration and a longer half-
life of serum IL-6 than the same data gathered from Foxn12/2,
or C57Bl/6 (Figure 4A). The exposure of the subject to MSC-
derived IL-6 was quantifiably different amongst recipients
(Figure 4B), suggesting that molecular monitoring of cell therapy
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can distinguish clearance mechanisms of the MSC transplant and/
or secreted factors, in particular a form of immune clearance.
Enhanced Systemic Exposure of Secreted Factors by
Engineered MSCs
MSC-derived IL-6 is a natural and highly expressed secreted
factor that enabled our molecular monitoring approach, however
Figure 1. Combined pharmacokinetic monitoring of MSCs and MSC-secreted IL-6. (A) Bioluminescent images of C57Bl/6 mice over a
period of three days after IV cell administration of one million luciferase-engineered human MSCs. (B) Photon flux of bioluminescent signal over time
after IV cell administration. Durable BLI signals were detected up to 24 hours in mice that were injected IV with MSCs. (C) Serum ELISA measurements
of human IL-6 released by IV cell transplants over time. Time points for serum and imaging analyses were 0.5, 8, 24, and 72 hours after cell injection.
Pooled mouse serum was serially analyzed as batches of N = 5.
doi:10.1371/journal.pone.0089882.g001
Figure 2. Enhanced delivery of IL-6 by MSC transplants compared to MSC conditioned medium. (A) Serum profiles of human IL-6 after IV
administration of concentrated conditioned medium into C57Bl/6 mice. The plot was normalized to the dose of conditioned medium that was
contributed by 1610
6
cells. Pharmacokinetic parameters (B) Cmax, (C) AUC, (D) Tmax, (E) Half-life, and (F) Elimination constant were calculated for IL-6
exposure by cell transplants compared to CM administration. Significant differences between cell transplants compared to CM whereby higher levels
of IL-6 and longer artificial duration was observed in plasma after cell transplantation. Time points for serum analyses were 0.5, 8, and 24 hours after
cell or media injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P.0.01.
doi:10.1371/journal.pone.0089882.g002
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IL-6 can be influenced by the in vivo microenvironment that MSCs
encounter. In order to understand the maximal exposure of a
subject to MSC secreted factor that was uninfluenced by host
regulation, we developed genetically engineered MSCs with
constitutive expression of the naturally secreted Gaussia luciferase
(Gluc) reporter. Gluc has a circulation half-life of 5–10 minutes in
mice, and has been used as a highly sensitive reporter (detection of
,1000 cells) for quantitative assessment of cells in vivo by
measuring its level in the blood ex vivo [29]. GLuc activity can
be easily quantified in blood by adding its substrate coelenterazine
and measuring emitted photons using a luminometer. MSCs were
first transduced with a lentivirus vector to stably express Gluc and
GFP under the control of the constitutively active CMV promoter
which yielded high transduction efficiency as monitored by
fluorescent microscopy and flow cytometry for GFP (Figure 5A–
B). Gluc expression and secretion was also readily detected in
MSCs conditioned medium (Figure 5C). When infused into
NOD-SCID mice, GLuc was detectable over a one week period
with a time to peak concentration occurring at ,8 hours post-cells
transplantation (Figure 5D). Engineering of MSCs with GLuc
revealed a longer exposure to cell therapy and suggests engineered
cell formulations for therapeutic studies may be useful for
minimizing cell dose and/or frequency to achieve durable
responses.
Discussion
Pharmacology is a powerful discipline with many available
methods to help understand and shape the characteristics of a drug
to meet a therapeutic need. Monitoring a drug’s PK is a necessary
study of a drug formulation that has been modeled extensively by
balancing the infusion, absorption, metabolism, and elimination of
a drug, assuming the body is defined by a steady-state of discrete
compartments that are permeable to a drug. Drug formulations
that contain living cells are an ever-increasing class of therapeutics
that can benefit from such pharmacological analysis. The classical
monitoring strategy for a cell therapy is to define the trafficking
and viability of a cellular formulation after introduction to the
body. In this study, we coupled this strategy with molecular
monitoring of secreted factors released by a cell transplant that
became detectable in the systemic circulation. We identified that
Figure 3. Golgi-dependent secretion mechanism of MSC-derived IL-6 in vivo. Brefeldin A pre-treatment of MSCs was used to evaluate
blockade of IL-6 release in vitro and in vivo. (A) MTT assay of MSCs treated at different concentrations of brefeldin. A non-toxic dose of 5 ug/ml was
used for functional studies. (B) Human IL-6 levels in vitro after brefeldin pre-treatment. Significant reduction in 24 hour release of IL-6 was observed
across all doses. (C) Alteration in serum IL-6 delivery by MSCs pretreated with a Golgi-apparatus inhibitor, Brefeldin A. MSCs were incubated with
5mg/ml of BFA for one day and then injected into C57Bl/6 mice and compared to untreated MSCs in terms of serum IL-6 delivery. Brefeldin treatment
of MSCs led to diminished release of human IL-6 in vitro and in vivo. (D) Area-under-curve analysis of human IL-6 after MSC pre-treatment with
brefeldin A and transplantation. Exposure to IL-6 was significantly reduced by inhibition of the Golgi apparatus. Time points for serum analyses were
0.5, 8, and 24 hours after cell injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P.0.01.
doi:10.1371/journal.pone.0089882.g003
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these two PK profiles - that is, the PK of an administered MSC
population and MSC-secreted IL-6 - were discordant. This was
dependent on the imaging modality used in this study (BLI), which
has known limitations in whole-animal reporting due to tissue
diffusion distance of light emission. BLI showed a prominent lung
expression of the MSC graft for a period of several hours, which
engineered MSC-Gluc releasing a secreted reporter could be
detected for days. Exploration of the dynamics and rate-limiting
aspects of MSC-derived secreted factors in blood serum, rather
than the cells themselves, may provide more predictive power in
their use as a therapeutic.
The PK of cell-derived molecules can be considered a lumped
model of classical drug PK with additional complexity to consider
due to cellular delivery. Cells as a delivery vehicle may be
responsible for the difference in profiles compared to isolated
molecules. Administration of a molecular drug bolus typically
follows an exponentially decaying trajectory in terms of serum
concentration of the drug. After cell administration, we observed
that serum levels of secreted IL-6 followed an exponential decay
independent of administration route. This may be attributed to
limited engraftment MSCs entrapped in the lung. The diffusion of
IL-6 from the cells would subsequently follow and be limited by
tissue transport barriers before being detected in the bloodstream.
In the systemic circulation secreted factors would likely follow their
natural clearance pathways, which, in the case of IL-6, are very
rapid (,minutes in half-life). We propose that traditional PK
models can be modified to account for an active, cellular
production term as well as the diffusion of material from an
engrafted tissue bed.
The elimination, or clearance, of a drug compound can occur
by metabolism or excretion of the drug in voided volume. In the
case of a MSC transplant, we could detect the influence that the
immune system has on the exposure of a subject to MSC secreted
factors. The immune response we observed is likely a xenogenic
rejection response that can be compartmentalized by arms of
innate and adaptive immunity. Previous studies have shown that
the immune competency of the host can have an impact on the
cellular viability after transplantation [30]. There are a number of
reports that describe the role of NK cells and other mechanisms
that affect the viability of allogeneic MSCs after transplantation
including the generation of immunological memory to the
transplanted material over time [16,31,32]. The clearance of
MSCs was substantial in IV transplanted MSCs, which were
entrapped in lung tissue. NOD-SCID-IL-2rg 2/2mice had a
much greater exposure to MSC-derived factors compared to
Foxn12/2mice or C57Bl/6 mice. The difference between the
two immunocompromised strains lies in the absence of B cells, NK
cells, and defects in cytokine signaling in NOD-SCID-IL-2rg 2/2
mice suggesting that these cell populations and cytokines may be
accountable for MSC clearance. This study, however, cannot
distinguish immunologic elimination of the human cells versus
elimination of the human secreted protein without further
evaluation.
We, and others, have observed protection of acute liver injury
by molecules derived from MSCs [33,34]. MSC molecules
collectively had a dose-dependent effect on injured hepatocytes
and stimulated a proliferation response in vitro and in vivo [33].
In this study, we used IL-6 as a model secreted factor to study the
cellular release profile of this cytokine in vivo. Although specific
mediators of therapy still remain elusive, there is considerable
evidence that IL-6, itself, may be liver-protective. [35]. IL-6 is also
shown to be important in improving acute inflammatory response
[36,37]. We envision that other cytokine and growth factors
released by MSCs will help broaden the overall PK profile of this
transient cell therapy and begin to identify associations with
relevant diseases that can be combated through the combination
of factors released by MSCs. Cognate receptors for specific
secreted factors will help clarify the pharmacodynamics of MSC
action and if dosing is enough to activate downstream signaling of
MSCs are amenable to ex vivo engineering to express therapeutic
secreted factors such as IL-2 for cancer immunotherapy [38]. In
this study, we first report the use of a non-specific GLuc reporter
Figure 4. The immune system limits the bioavailability of MSC-derived IL-6. Pharmacokinetic profile of sera IL-6 after IV cell administration.
Approximately 1610
6
MSCs were injected into C57Bl/6, Foxn12/2, or NSG mice by IV injection. At different time points after cell injection, mice were
sampled for blood plasma and serum human IL-6 levels were measured by ELISA. (B) AUC analysis of IL-6 exposure as a function of mouse strain. IV
administration was significantly affected by mouse strain, particularly in severely immunodeficient mice, which had the highest exposure of IL-6 for a
given cell mass. Data represent mean 6standard derivation of duplicate or triplicate experiments. Time points for serum analyses were 0.5, 8, and 24
hours after cell injection. Mice were serially analyzed as batches of N = 5 per study. * denotes P.0.01.
doi:10.1371/journal.pone.0089882.g004
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that was engineered into MSCs for sensitive blood detection.
MSC-Gluc revealed a longer exposure of the subject to a secreted
factor implying that MSCs were persistent in the body although
undetected by BLI. MSCs were genetically engineered ex vivo
with a self-inactivating lentivirus vector which integrates the Gluc
cDNA within the genome of the cells, leading to stable expression.
This study cannot rule out the possibility that MSCs could fuse
with a host cell (e.g. myeloid cells) after transplantation and
thereby maintain a longer serum level. These data also suggest that
constitutive secreted factors are necessary to reveal the true
bioavailability of MSCs, as IL-6 and presumably other natural
secreted factors may be regulated at the gene expression level by
the host. The initial release dynamics of IL-6 were contributed by
an active Golgi-dependent mechanism, which is presumed to be
necessary for continued secretion of other protein factors such as
GLuc. Non-protein secreted factors, such as nitric oxide or
prostaglandin E2, may not be rate-limited by Golgi-secretion
pathways but instead follow enzymatic reaction kinetics that
require stimulation to generate these mediators in vivo [14,39].
MSC-Gluc can be an extremely useful tool for cell pharmacology
studies that evaluate administration routes, initial dosage, and
dosing frequency for optimizing the exposure of a cell therapy
product.
This work serves as the first application of a combined cell
biodistribution and molecular PK modeling approach to MSC
therapeutics. By combined in silico modeling and empirical
analysis, a functional PK/PD model can now begin to be
developed to predict the nature of MSC therapy given a particular
formulation and administration route. Allometric scaling laws that
help predict the conversion of parameters from animal to human
models may be applicable to guide clinical trials using MSC
therapeutics. Although we focus on a few key mediators, a more
comprehensive view of all bioactive MSC secreted factors can lead
to second-generation models that better capture potential non-
linearity in the data. In addition, this theoretical framework may
serve as the foundation for other clinically used cell variants such
as hematopoietic and embryonic stem cells, or T cells. Such
predictive, in silico analysis of cell-based therapies may reduce
experimental costs due to a systematic minimization of required
testing, increase throughput of discovery, and ultimately lead to
more efficacious treatment regimens.
Figure 5. Blood Monitoring of Engineered Human MSCs with the Secreted Gaussia Luciferase Reporter. A lentivirus vector expressing
GLuc and GFP was transduced into human MSCs at a confluence of 70% and multiplicity of infection of 4:1 in complex with 8 ug/ml of polybrene. (A)
GFP micrograph and (B) flow cytometry showing high expression level and therefore transduction efficiency of construct. (C) The activity of GLuc was
successfully measured in MSCs conditioned medium using a luminometer. (D) Five different engineered cell lines were infused into NOD-SCID mice
and serum was individually collected at 0.5, 8, 24, 72, and 168 hours after cell injection in batches of N = 5 per study. MSCs constitutively expressing
GLuc were detected in many cases over a week in duration.
doi:10.1371/journal.pone.0089882.g005
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Materials and Methods
Mice
Athymic Foxn12/2mice (nude, male, 6–8 weeks old),
C57BL/6 mice (male, 8 weeks old), Balbc/J mice (female, 8
weeks old), and NOD.Cg-Prkdc
scid
Il2rg
tm1Wjl
/SzJ (NSG mice,
male, 8–10 weeks old) were all purchased from Jackson
Laboratories (Bar Harbor, ME) and housed at Massachusetts
General Hospital Animal Facility following approved experimen-
tal protocols by the IACUC.
Human MSC Isolation and Expansion
Human MSCs were isolated and expanded following a
previously established protocol [33,34]. Briefly, fresh human bone
marrow aspirates were purchased from Lonza. Mononuclear cells
were separated by Ficoll density gradient centrifugation (GE
Healthcare) and plated on a T-175 flask (1610
6
cells per flask).
Mononuclear cells were cultured at 37uC with 10% CO
2
in MSC
expansion medium. MSC expansion medium was composed of
15% fetal bovine serum, 2% penicillin and streptomycin, 0.2%
gentamycin, 1 ng/L fibroblast growth factor, alpha-MEM with
ribonucleosides and deoxyribonucleosides. Medium was changed
1 week later and unbound cells were washed away. The following
week, colony-forming adherent cells were re-plated into a new
flask for expansion. Medium was changed every 3–4 days. MSCs
were subcultured when they reached 70–80% confluence. Only
passage 2–5 MSCs were used for experiments. Figure S1 outlines
the immunophenotype of MSCs using antibodies purchased from
BD Biosciences.
MSC Administration and Measurement of Human IL-6,
MCP-1, and IL-8 Levels in Plasma
MSCs (1610
6
MSCs in 200 ml FBS-free medium) were injected
into mice by IV infusion. At 30 minutes, 3 hours, 8 hours, 24 hours
and 72 hours, mice were anesthetized with 60 ml ketamine, 30 ml
xylazine, and 60 ml saline per mouse and blood was withdrawn by
cardiac puncture. After centrifugation at 14,000 rpm for 10
minutes at 4uC, plasma were collected and stored at 280uC before
use. Human IL-6, MCP-1, and IL-8 levels were measured using an
ELISA kit from BD Bioscience following the supplier’s recom-
mended procedures.
Preparation of Human MSCs Expressing a Firefly
Luciferase Gene Reporter
The lentiviral vector pHR’MND-LRT containing a firefly
luciferase reporter was constructed as previously described [40].
Infectious virus was produced by triple transient co-transduction of
293T/17 cells (ATCC) with pHR’MND-LRT, pCMVDR8.91 i.e.
packaging vector, and pMD.G i.e. VSVG pseudotyping vector.
The titer of virus was determined by transduction of 293T cells
followed by flow cytometry analysis of the mRFP reporter (Ex:
594 nm/Em: 620615 nm). Cultures of 30–40% confluent human
MSCs in a T-175 flask were incubated with the virus at a
multiplicity of infection of 4 in a total of 20 ml expansion medium
containing 8 mg/ml polybrene. This transduction protocol was
repeated one more time. In each round, cells were incubated with
the viral supernatant for 8 hours and then in MSC expansion
medium for 16 hours. After the second round of infection, fresh
medium was added to each flask and cultured for 3–4 days.
Luciferase activity of transduced MSCs was confirmed with a
luciferase activity assay before in vivo use.
Bioluminescence Imaging
A total of 1610
6
luciferase-engineered MSCs were given to
C57Bl/6 mice either IM or IV. At specific time points after cell
injection, mice received an intraperitoneal injection of 4.5 mg of
luciferase substrate solution (Molecular Imaging Products) and
were imaged thereafter. The bioluminescent signal was measured
in anesthetized mice on an IVIS-100 imaging system (Caliper
LifeSciences) until a peak signal was reached. Data are expressed
as photons/second/cm
2
, encompassing a region of interest over
the implanted cells, including lung, leg and whole body.
Preparation of Concentrated Conditioned Medium
MSCs were cultured to 70–80% confluency in T-175 culture
flasks before 15 ml DMEM media consisted of 0.05% BSA and
2% penicillin and streptomycin were added. Cells were further
cultured for 1–2 days and then the supernatants were collected
and filtered. Cell number was quantified using a hemacytometer
after trypsinization. Culture media were concentrated 20–50 folds
using an Amicon filter (MWCO: 3,000 Da) by centrifuging at
3500 rpm for 2–3 hours. The human IL-6 levels were measured
by ELISA by appropriate serial dilution before injection into mice.
Concentrated conditioned medium (400 ml) were injected into
C57Bl/6 mice by IV or IM routes.
Engineering and In Vivo Monitoring of MSCs with
Secreted Gaussia Luciferase
MSCs were allowed to grow up to about 70% confluency before
viral transduction. A lentivirus vector carrying the expression
cassette for Gluc and GFP, separated by an internal ribosomal
entry site, under the control of the CMV promoter was previously
described [29] with a titer of 6.1610
7
IU/ml. Polybrene was
added to each T-175 flask diluted down to a final concentration of
16. Then 1 mL of virus was added to each flask. Cells were
allowed to grow over night and then the virus-containing media
was aspirated and replaced with fresh virus-free media. Trans-
duction efficiency was confirmed by analyzing GFP expression
using fluorescence microscopy and flow cytometery.
Fully confluent cells were trypsinized with 16Trypsin (Fisher)
and re-suspended in conditioning media at a density of 1610
6
cells
per 200 uL. 200 uL of cell suspension was injected into each
mouse via tail vein. Blood was collected in Eppendorf tubes
containing 4 mL of 20 mM EDTA via tail vein at 0.5 hour,
3 hour, 8 hour, 24 hour, 72 hour and 1 week post-MSCs
injection. 10 uL blood was mixed with 100 uL 5 ug/ml
coelenterazine substrate in a white, opaque 96-well plate and
luminescence was detected using a BioTek microplate reader.
Brefeldin A Treatment of MSCs and Proliferation Assay
In a 6-well plate, MSCs were incubated with brefeldin A
(Sigma-Aldrich) at a final concentration of 50, 10, 5, and 1 mg/ml
for 24 hours. Supernatants were collected, and then MSCs were
washed 3 times using PBS. Fresh medium was replaced, and cells
continued to culture up to 3 days. Supernatants were collected at
different time points for subsequent human IL-6 measurements.
To measure the proliferation, BFA-treated cells were reseeded in a
96-well flat bottom plate and cultured with fresh medium for 72
hours. Cell proliferation was measured using a MTT assay kit
(ATCC) following the supplier’s recommended procedures.
Statistical Analysis
In all studies batches of 3–8 mice from 2–3 independent
experiments are reported. Raw pharmacokinetic data were
analyzed using a 1-tailed Mann-Whitney U test for non-
Mesenchymal Stem Cell Pharmacokinetics
PLOS ONE | www.plosone.org 7 February 2014 | Volume 9 | Issue 2 | e89882

parametric data with the mean 6SEM shown or using a two way
ANOVA with Tukey’s multiple comparison correction where
pharmacokinetic parameters were calculated based on MATLAB
software package models.
Supporting Information
Figure S1 Immunophenotyping of MSCs. Expanded cells
were CD11b2, CD452, CD45+, and CD73+consistent with a
bone marrow MSC identity.
(TIF)
Figure S2 Pharmacokinetics of MSC-derived IL-8 and
MCP-1 after IV transplantation. ELISA measurements of
mice injected with MSCs and analyzed for human (A) MCP-1 and
(B) IL-8 over time. Kinetics follow a similar trend compared to IL-
6.
(TIF)
Acknowledgments
The authors are grateful for assistance from Ms. Jessica Sullivan and Mr.
Peter Waterman in bioluminescence imaging.
Author Contributions
Conceived and designed the experiments: FW MLY BT BP. Performed the
experiments: JE RM FW KS SG KC BP. Analyzed the data: JE RM FW
BT BP. Contributed reagents/materials/analysis tools: KC RW BT. Wrote
the paper: JE RM BT BP.
References
1. Culme-Seymour EJ, Davie NL, Brindley DA, Edwards-Parton S, Mason C
(2012) A decade of cell therapy clinical trials (2000–2010). Regen Med 7: 455–
462.
2. Mason C, Brindley DA, Culme-Seymour EJ, Davie NL (2011) Cell therapy
industry: billion dollar global business with unlimited potential. Regen Med 6:
265–272.
3. Brindley DA, Davie NL, Sahlman WA, Bonfiglio GA, Culme-Seymour EJ, et al.
(2012) Promising growth and investment in the cell therapy industry during the
first quarter of 2012. Cell Stem Cell 10: 492–496.
4. Meirelles LDS, Chagastelles PC, Nardi NB (2006) Mesenchymal stem cells
reside in virtually all post-natal organs and tissues. Journal Of Cell Science 119:
2204–2213.
5. Tang YL, Zhao Q, Zhang YC, Cheng LL, Liu MY, et al. (2004) Autologous
mesenchymal stem cell transplantation induce VEGF and neovascularization in
ischemic myocardium. Regulatory Peptides 117: 3–10.
6. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD (2002) Human
mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult
murine heart. Circulation 105: 93–98.
7. Maitra B, Szekely E, Gjini K, Laughlin MJ, Dennis J, et al. (2004) Human
mesenchymal stem cells support unrelated donor hematopoietic stem cells and
suppress T-cell activation. Bone Marrow Transplantation 33: 597–604.
8. Honma T, Honmou O, Iihoshi S, Harada K, Houkin K, et al. (2006)
Intravenous infusion of immortalized human mesenchymal stem cells protects
against injury in a cerebral ischemia model in adult rat. Experimental Neurology
199: 56–66.
9. Parekkadan B, Tilles AW, Yarmush ML (2008) Bone marrow-derived
mesenchymal stem cells ameliorate autoimmune enteropathy independently of
regulatory T cells. Stem Cells 26: 1913–1919.
10. Wu Y, Chen L, Scott PG, Tredget EE (2007) Mesenchymal stem cells enhance
wound healing through differentiation and angiogenesis. Stem Cells 25: 2648–
2659.
11. Shumakov VI, Onishchenko NA, Rasulov MF, Krasheninnikov ME, Zaidenov
VA (2003) Mesenchymal bone marrow stem cells more effectively stimulate
regeneration of deep burn wounds than embryonic fibroblasts. Bull Exp Biol
Med 136: 192–195.
12. McFarlin K, Gao X, Liu YB, Dulchavsky DS, Kwon D, et al. (2006) Bone
marrow-derived mesenchymal stromal cells accelerate wound healing in the rat.
Wound Repair Regen 14: 471–478.
13. Fu X, Li H (2009) Mesenchymal stem cells and skin wound repair and
regeneration: possibilities and questions. Cell Tissue Res 335: 317–321.
14. Ren G, Zhang L, Zhao X, Xu G, Zhang Y, et al. (2008) Mesenchymal stem cell-
mediated immunosuppression occurs via concerted action of chemokines and
nitric oxide. Cell Stem Cell 2: 141–150.
15. Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V, et al. (2006)
Human mesenchymal stem cells modulate B-cell functions. Blood 107: 367–372.
16. Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L (2006)
Mesenchymal stem cell-natural killer cell interactions: evidence that activated
NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced
NK-cell proliferation. Blood 107: 1484–1490.
17. Aggarwal S, Pittenger MF (2005) Human mesenchymal stem cells modulate
allogeneic immune cell responses. Blood 105: 1815–1822.
18. Nasef A, Ashammakhi N, Fouillard L (2008) Immunomodulatory effect of
mesenchymal stromal cells: possible mechanisms. Regen Med 3: 531–546.
19. Le Blanc K, Rasmusson I, Sundberg B, Gotherstrom C, Hassan M, et al. (2004)
Treatment of severe acute graft-versus-host disease with third party haploiden-
tical mesenchymal stem cells. Lancet 363: 1439–1441.
20. Parekkadan B, Milwid JM (2010) Mesenchymal Stem Cells as Therapeutics. In:
Yarmush ML, Duncan JS, Gray ML, editors. Annual Review of Biomedical
Engineering, Vol 12. 87–117.
21. Gao JZ, Dennis JE, Muzic RF, Lundberg M, Caplan AI (2001) The dynamic
in vivo distribution of bone marrow-derived mesenchymal stent cells after
infusion. Cells Tissues Organs 169: 12–20.
22. Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, et al. (2009) Intravenous
hMSCs improve myocardial infarction in mice because cells embolized in lung
are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 5:
54–63.
23. Schrepfer S, Deuse T, Reichenspurner H, Fischbein MP, Robbins RC, et al.
(2007) Stem cell transplantation: The lung barrier. Transplantation Proceedings
39: 573–576.
24. Sasportas LS, Kasmieh R, Wakimoto H, Hingtgen S, van de Water JA, et al.
(2009) Assessment of therapeutic efficacy and fate of engineered human
mesenchymal stem cells for cancer therapy. Proc Natl Acad Sci U S A 106:
4822–4827.
25. Balyasnikova IV, Ferguson SD, Sengupta S, Han Y, Lesniak MS Mesenchymal
stem cells modified with a single-chain antibody against EGFRvIII successfully
inhibit the growth of human xenograft malignant glioma. PLoS ONE 5: e9750.
26. Nemeth K, Keane-Myers A, Brown JM, Metcalfe DD, Gorham JD, et al. (2010)
Bone marrow stromal cells use TGF-beta to suppress allergic responses in a
mouse model of ragweed-induced asthma. Proceedings Of The National
Academy Of Sciences Of The United States Of America 107: 5652–5657.
27. Majumdar MK, Thiede MA, Haynesworth SE, Bruder SP, Gerson SL (2000)
Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic
cytokines and support long-term hematopoiesis when differentiated toward
stromal and osteogenic lineages. Journal of Hematotherapy & Stem Cell
Research 9: 841–848.
28. Castell JV, Geiger T, Gross V, Andus T, Walter E, et al. (1988) Plasm a
clearance, organ distribution and target cells of interleukin-6/hepatocyte-
stimulating factor in the rat. Eur J Biochem 177: 357–361.
29. Wurdinger T, Badr C, Pike L, de Kleine R, Weissleder R, et al. (2008) A
secreted luciferase for ex vivo monitoring of in vivo processes. Nat Methods 5:
171–173.
30. Tolar J, O’Shaughnessy M J, Panoskaltsis-Mortari A, McElmurry RT, Bell S, et
al. (2006) Host factors that impact the biodistribution and persistence of
multipotent adult progenitor cells. Blood 107: 4182–4188.
31. Sotiropoulou PA, Perez SA, Gritzapis AD, Baxevanis CN, Papamichail M
(2006) Interactions between human mesenchymal stem cells and natural killer
cells. Stem Cells 24: 74–85.
32. Zangi L, Margalit R, Reich-Zeliger S, Bachar-Lustig E, Beilhack A, et al. (2009)
Direct imaging of immune rejection and memory induction by allogeneic
mesenchymal stromal cells. Stem Cells 27: 2865–2874.
33. van Poll D, Parekkadan B, Cho CH, Berthiaume F, Nahmias Y, et al. (2008)
Mesenchymal stem cell-derived molecules directly modulate hepatocellular
death and regeneration in vitro and in vivo. Hepatology 47: 1634–1643.
34. Parekkadan B, van Poll D, Suganuma K, Carter EA, Berthiaume F, et al. (2007)
Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure.
PLoS ONE 2: e941.
35. Bouffi C, Bony C, Courties G, Jorgensen C, Noel D (2010) IL-6-dependent
PGE2 secretion by mesenchymal stem cells inhibits local inflammation in
experimental arthritis. PLoS One 5: e14247.
36. Kopf M, Baumann H, Freer G, Freudenberg M, Lamers M, et al. (1994)
IMPAIRED IMMUNE AND ACUTE-PHASE RESPONSES IN INTER-
LEUKIN-6-DEFICIENT MICE. Nature 368: 339–342.
37. Klein C, Wustefeld T, Assmus U, Roskams T, Rose-John S, et al. (2005) The IL-
6-gp130-STAT3 pathway in hepatocytes triggers liver protection in T cell-
mediated liver injury. Journal of Clinical Investigation 115: 860–869.
38. Stagg J, Lej eune L, Paquin A, Galipeau J (2004) Marrow stromal cells for
interleukin-2 delivery in cancer immunotherapy. Hum Gene Ther 15: 597–608.
39. Nemeth K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, et al. (2009)
Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent
reprogramming of host macrophages to increase their interleukin-10 production.
Nat Med 15: 42–49.
40. Love Z, Wang F, Dennis J, Awadallah A, Salem N, et al. (2007) Imaging of
mesenchymal stem cell transplant by bioluminescence and PET. Journal Of
Nuclear Medicine 48: 2011–2020.
Mesenchymal Stem Cell Pharmacokinetics
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