Download PDF - Circulation Research

SHORT COMMUNICATION
Fabrication of Synthetic Mesenchymal Stem Cells for the Treatment of Acute Myocardial
Infarction in Mice
Lan Luo1, Junnan Tang3,4,5, Kodai Nishi2, Chen Yan1, Phuong-Uyen Dinh4,5, Jhon Cores4,5, Takashi Kudo2,
Jinying Zhang3, Tao-Sheng Li1, Ke Cheng4,5,6,
1
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Department of Stem Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4
Sakamoto, Nagasaki 852-8523, Japan; 2Department of Radioisotope Medicine, Atomic Bomb Disease
Institute Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; 3Department of Cardiology,
The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China; 4Department of
Molecular Biomedical Sciences and Comparative Medicine Institute, North Carolina State University,
Raleigh, North Carolina 27607, USA; 5Department of Biomedical Engineering, University of North
Carolina at Chapel Hill & North Carolina State University, Raleigh, North Carolina 27607, USA, and;
6
Molecular Pharmaceutics Division, Eshelman School of Pharmacy, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Running title: Therapeutic Potential of Synthetic Stem Cells.
L.L. and J.T. contributed equally to the work.
Subject Terms:
Stem Cells
Myocardial Infarction
Myocardial Regeneration
Address correspondence to:
Dr. Ke Cheng
North Carolina State University
1060 William Moore Drive
Raleigh, NC 27607
USA
Tel: 919 513 6157
Fax: 919 513 7301
[email protected]/ [email protected]
Dr. Tao-Sheng Li
Nagasaki University Graduate
Biomedical Sciences
1-12-4 Sakamoto
Nagasaki 852-8523
Japan
Tel: +81-95-819-7098
Fax: +81-95-819-7100
[email protected]
School
of
In February 2016, the average time from submission to first decision for all original research papers
submitted to Circulation Research was 15.4 days.
DOI: 10.1161/CIRCRESAHA.116.310374
1
ABSTRACT
Rationale: Stem cell therapy faces a number of challenges. It is difficult to grow, preserve, and transport
stem cells before they are administered to the patient. Synthetic analogs for stem cells represent a new
approach to overcome these hurdles and hold the potential to revolutionize regenerative medicine.
Objective: We aim to fabricate synthetic analogs of stem cells and test their therapeutic potential for
treatment of acute myocardial infarction in mice.
Methods and Results: We packaged secreted factors from human bone marrow-derived mesenchymal
stem cells (MSC) into Poly(lactic-co-glycolic acid) PLGA microparticles and then coated them with MSC
membranes. We named these therapeutic particles “synthetic MSC” (or synMSC). synMSC exhibited a
factor release profile and surface antigens similar to those of genuine MSC. synMSC promoted
cardiomyocyte functions and displayed cryopreservation and lyophilization stability in vitro and in vivo.
In a mouse model of acute myocardial infarction, direct injection of synMSC promoted angiogenesis and
mitigated left ventricle remodeling.
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Conclusions: We successfully fabricated a synMSC therapeutic particle and demonstrated its regenerative
potential in mice with acute myocardial infarction. The synMSC strategy may provide novel insight into
tissue engineering for treating multiple diseases.
Keywords:
Mesenchymal stem cells, paracrine factors, myocardial infarction, synthetic cells, regeneration, stem cell,
tissue engineering.
Nonstandard Abbreviations and Acronyms:
MSC
synMSC
PLGA
VEGF
IGF-1
CCNPs
MP
SEM
SDF-1
NRCM
MI
CT
18
F-FDG
PET-CT
SPECT-CT
mesenchymal stem cells
synthetic mesenchymal stem cells
Poly(lactic-co-glycolic acid)
vascular endothelial growth factor
insulin-like growth factor 1
cancer cell membrane-coated nanoparticles
microparticles
scanning electron microscopy
stromal cell-derived factor-1
neonatal rat cardiomyocytes
myocardial infarction
computed tomography
18
F-fluorodeoxglucose
positron emission tomography/computed tomography
single photon emission computed tomography/computed
tomography
DOI: 10.1161/CIRCRESAHA.116.310374
2
INTRODUCTION
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
A growing body of studies have demonstrated the therapeutic potential of different stem cells types
such as skeletal myoblasts, bone marrow-derived mesenchymal stem cells, embryonic stem cells, and
endogenous cardiac stem cells in cardiovascular diseases1. Among the cell types under investigation,
mesenchymal stem cells (MSC) have attracted great attention owing to their ability to differentiate into
mesoderm and non-mesoderm tissues, their immunomodulatory properties, and their broad spectrum
release of trophic factors2. Preclinical and clinical studies on MSC have shown promise for repair and
regeneration of cardiac tissues3. In an effort to understand the mechanisms responsible for the therapeutic
effect of MSC, scientists investigated their retention rates in the myocardium after transplantation. As a
result of the low retention rates observed4, they postulated other mechanisms of action promoting the
recovery in cardiac function and structure other than the stem cells’ in situ differentiation. Soon, they
realized that the broad spectrum release of soluble factors by MSC may be the primary mechanism for
their therapeutic effects5. More recently, they found that MSC-secreted exosomes exhibited functions
similar to MSC for repairing heart injury6. Inspired by this, scientists are considering alternative strategies
to stem cell transplantation, namely the direct delivery of MSC secretome to repair injured tissues. Indeed,
in vivo and in vitro studies have demonstrated the therapeutic effect of MSC-conditioned media for
treatment of cardiovascular diseases7, 8. Moreover, the single delivery of cytokines such as vascular
endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) have also been tested for their
cardiac therapeutic effects in clinical trials9. Unfortunately, neither has met our expectations. The reasons
may be the short half-life of cytokines in vitro, the uncertainty of effective/safe dosages, and the
possibility that multiple administration of cytokines may be necessary to act synergistically to achieve
therapeutic effect10. It is noteworthy that exosomes could circumvent a number of these challenges. The
bi-lipid membrane of exosomes could protect their contents from degradative enzymes or chemicals and
the membrane bound molecules might home the exosomes to a specific tissue or microenvironment11. In
addition, exosomes contain proteins and RNAs that may have adequate potential for cardiac repair12-16.
However, exosome-based therapeutics also face challenges such as the lack of a standard isolation
protocol, rapid clearance and wash-away due to their extremely small sizes. Poly(lactic-co-glycolic acid)
(PLGA), a biodegradable and biocompatible polymer, is emerging as a prominent element in drug
delivery system due to its capability of protecting cytokines from degradation while allowing for the
sustained release of factors that target in specific organs or cells17, 18. Further, Fang et al. reported
cancer-cell-membrane coated nanoparticles (CCNPs) formed by coating cancer cell membranes onto
PLGA-loaded immunological particles. The membrane-bound tumor-associated antigens permit CCNPs
to be efficiently delivered to antigen presenting cells to promote anticancer immune response19.
In the present study, based on the polymer encapsulation and membrane cloaking approaches, we
fabricated a therapeutic particle, namely synthetic MSC (synMSC), by coating MSC cell membranes onto
MSC-secretome-loaded PLGA particles. We then characterized its physiochemical and biological
properties in vitro, and tested its regenerative potential in mice with acute myocardial infarction. The
scientific premise of our study is that the synMSC idea overcomes several major challenges of the status
quo of cell therapy practice, namely cryopreservation stability, standardization, and “off the shelf”
feasibility. In addition, because of the MSC membrane coating, synMSC will likely avoid the
tumorigenicity and immunogenicity risks associated with stem cell transplantation. Although the present
study targets the heart, the synMSC technology represents a platform technology that is generalizable to
other stem cell types.
DOI: 10.1161/CIRCRESAHA.116.310374
3
METHODS
A detailed methods section is provided in the Online Data Supplement.
RESULTS
synMSC fabrication and biological properties.
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
The schematic design of synMSC fabrication was summarized in Figure 1A. In brief, MSC conditioned
media was incorporated in PLGA to form microparticles (MP), then the MP were coated with MSC cell
membrane to form synthetic mesenchymal stem cells (synMSC). Scanning electron microscopy and
fluorescent imaging (Figure 1B) confirmed the successful MSC cell membrane coating on MP. synMSC
had a size around 20 μm, similar to those of MP and real MSC (Figure 1C). Flow cytometry analysis
showed that synMSC exhibited similar expressions of CD105, CD90, CD45, CD31, and CD34 compared
to MSC, while MP didn’t (Figure 1D). Furthermore, synMSC could sustain the release of growth factors
like vascular endothelial growth factor (VEGF) (Figure 1E), stromal cell-derived factor-1 (SDF-1)
(Figure 1F), and insulin-like growth factor-1 (IGF-1) (Figure 1G). These results demonstrated that
synMSC and MSC were comparable in terms of secretome and surface antigen expressions.
synMSC promotes cardiomyocyte functions in vitro.
To test the cardiomyocyte protective capability of synMSC in vitro, neonatal rat cardiomyocytes
(NRCM, stained by alpha-sarcomeric actin, green in Figure 2A) were co-cultured with MP, synMSC and
MSC (red in Figure 2A). Solitary NRCM culture was included as negative control. synMSC significantly
increased NRCM number (Figure 2B) and promoted NRCM contractility (Figure 2C). Such beneficial
effects were comparable to those from MSC. The promotion of NRCM number and contractility of
synMSC might be due to its significantly higher number existed in NRCM (Figure 2D), although the
same amount of particles was originally applied to NRCM. These results demonstrated that the MSC
membrane on synMSC allow them to bind and interact with cardiomyocytes.
Cryopreservation and lyophilization stability of synMSC.
Cryopreservation stability is one of the major challenges of cell therapy products. Here, we tested the
stability of synMSC after rapid freezing and thawing. Fluorescent and white light microscopy images
revealed freeze/thaw treatment didn’t alter the structure (Figure 2E) or size (Figure 2F) of synMSC. Flow
cytometry analysis showed no significant difference on the surface antigen expressions of synMSC pre
and post freeze (Figure 2G). Further, we tested the lyophilization stability of synMSC, and found that the
lyophilization process didn’t alter the structure, size, surface antigen expressions, or sustained VEGF
release of synMSC (Online Figure II). MSC, however, could not undergo the harsh freeze/thaw process
without inducing cell death. After injecting freeze/thawed synMSC or MSC into a mouse heart, MSC
were targeted by macrophages while synMSC were not (Figure 2H, 2I). These results demonstrated the
cryopreservation and lyophilization stability and advantages of synMSC over real MSC.
synMSC injection mitigates left ventricle remodeling of infarcted heart.
To test the therapeutic effect of synMSC, we made an acute myocardial infarction (MI) model in mice
by left anterior descending artery ligation, and then synMSC were immediately injected intramyocardially.
Negative control mice received no treatment after MI. 18F-fluorodeoxglucose positron emission
tomography/computed tomography (18F-FDG PET/CT) was performed at 1 (baseline) and 14 days
DOI: 10.1161/CIRCRESAHA.116.310374
4
(endpoint) after infarction to measure the infarct area (Figure 3A). 99mTc-tetrofosmin single photon
emission computed tomography/computed tomography (SPECT/CT) was performed at 2 (baseline) and
15 days (endpoint) after infarction to measure left ventricular volume (Figure 3A). synMSC injection
showed a significant reduction of infarct area (Figure 3B). The left ventricular volume changes were
indistinguishable between the two groups (Figure 3B). Left ventricle morphometry imaged by Masson’s
trichrome staining revealed the protective effects of synMSC and MSC treatment on heart morphology
(Figure 3C). The infarct wall thickness was increased (Figure 3D) and infarct size was reduced (Figure
3E) both in synMSC and MSC treated mice as compared to the control group.
synMSC injection promotes endogenous repair in the infarcted heart.
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
To reveal the mechanisms underlying the therapeutic benefits of synMSC, we investigated whether
synMSC injection could recruit more c-kit-positive stem cells, promote angiogenesis, and improve cell
proliferation in the infarcted heart. Immunostaining analyses with c-kit (Figure 4A), CD34 (Figure 4B),
and ki67 (Figure 4C) were performed in the infarcted hearts of control, synMSC, and MSC treated mice.
Compared to control, synMSC and MSC treatments increased the c-kit positive stem cell recruitment
(Figure 4D) and vessel density (Figure 4E) of the infarcted heart. Compared to control, the proliferated
cells were slightly increased in the infarcted heart of synMSC treated mice, but significantly increased in
the infarcted heart of MSC treated mice (Figure 4F). These results suggested that the therapeutic effects
of synMSC may be through activation of c-kit-positive stem cells and promotion of angiogenesis.
DISCUSSION
In this study, we fabricated a particle named synMSC by coating MSC cell membranes onto PLGA
particles loaded with MSC secretome. This novel particle exhibited similar secretome and surface antigen
profiles as compared to real MSC. synMSC promoted cardiomyocyte function, and displayed
cryopreservation and lyophilization stability in vitro. Intramyocardial injection of synMSC mitigated left
ventricle remodeling in a mouse model of acute myocardial infarction at a level comparable to genuine
MSC.
Emerging lines of evidences indicate that adult stem cells exert their therapeutic effects mainly through
paracrine effects rather than direct differentiation. To that end, scientists have begun to consider the direct
delivery of stem cell-released soluble factors as an alternative approach to stem cell transplantation.
However, the progress is hindered by the short lived effect of injected soluble factors. The cardiac
contraction can quickly “wash away” the injected factors. Approaches that allow controlled release of
soluble factors are paramount and urgently needed for the clinical implementation of stem-cell-derived
factors for therapeutic heart regeneration. Although, exosomes show great potential in cardiac repair and
may overcome the shortcomings associated with cell transplantation, the lack of standardized protocol for
exosome isolation and the quick washout of exosomes after injection remain challenges for clinical
application. We designed synMSC, which combined the secretome (containing both soluble factors and
exosomes) and membranes of MSC. synMSC can release soluble factors such as VEGF, SDF-1, and
IGF-1, binding to cardiomyocytes in vitro. Additionally, the expression of MHC class I molecules, but not
MHC class II molecules or costimulatory molecules in MSC cell membranes allow it to escape
allorecognition by the immune system and may modulate the host immune response20. The MSC
membrane coating on PLGA particles could effectively protect synMSC from being attacked by host
immune and inflammatory cells.
A great number of cardiomyocytes die after the induction of MI. The restoration of cardiomyocyte
numbers is one important target for cell-based therapy. By co-culturing the synMSC with NRCM, we
DOI: 10.1161/CIRCRESAHA.116.310374
5
observed a significant increase in NRCM number and contractility at a level comparable to MSC, which
may be associated with the growth factors released by synMSC. The superiority of synMSC over MP
could be due to several reasons. First, the MSC membrane on synMSC allow them to closely attach to
cardiomocytes by cell-cell interactions. Second, it has been reported that the stem cell membranes are not
null in the regeneration process: direct contact may trigger downstream signaling in cardiomyocytes to
favor survival and function augmentation21.
One major challenges of stem cell based therapy is the cryopreservation stability of cells. Here we
found that snap freezing in -80 ºC and rapid thawing did not alter the structure, size, or surface antigen
expressions of synMSC. Furthermore, lyophilization did not alter the traits of synMSC. Importantly, when
the freeze/thawed MSC (with dead MSC caused by harsh freezing/thawing) were injected into a mouse
heart, they were targeted by macrophages (initiating the phagocytosis of dead MSC) while synMSC were
not. This suggested the superior cryopreservation stability of synMSC over MSC.
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Currently, as computed tomography (CT) can provide great detail in anatomic structure, hybrid
imaging of PET and SPECT with CT have been adopted in clinical and small animal cardiovascular
disease diagnosis22, 23. PET utilizing glucose tracer analog 18F- FDG allows the detection of cells with
different metabolic activity24, and gated SPECT utilizing 99mTc-tetrofosmin makes accurate assessment of
ventricular volumes25. So we evaluated the myocardial viability and left ventricle volume of mice heart by
18
F-FDG PET/CT and 99mTc-tetrofosmin SPECT/CT. synMSC significantly mitigated left ventricle
remodeling, as indicated by a significant reduction of infarct area, confirming the therapeutic potential of
synMSC. Further, the left ventricle morphometry evaluation by Masson’s trichrome staining revealed
synMSC exhibited protection of heart morphometry at a level that was comparable to MSC.
Previous reports have demonstrated that MSC provide cardio-protection by paracrine actions that
activate cardiac stem cells26, angiogenesis and cell proliferation8. Consistent with these findings, a
significant increase of c-kit-positive stem cells was found in synMSC treated mice (similar to MSC
treatment), although it is hard to distinguish the origination of these c-kit-positive stem cells
(cardiac-derived or bone marrow-derived). In addition, a larger number of vessels were found in synMSC
treated mice which would provide sufficient oxygen and nutrients to the surrounded cardiomyoytes.
Conclusions.
Taken together, we here successfully fabricate synMSC and demonstrate their prominent therapeutic
effects in an acute myocardial infarction mouse model, suggesting the feasibility of this approach in
regenerative medicine. Moreover, this synthetic stem cell approach provides novel insight into tissue
engineering for treating multiple diseases. All after all, our results suggest synthetic stem cells offer an
alternative option to stem cell-mediated regenerative therapies. Future studies should focus on
streamlining the handling and manipulations of synthetic stem cells to facilitate clinical translation.
DOI: 10.1161/CIRCRESAHA.116.310374
6
AUTHOR CONTRIBUTIONS
K. Cheng and T. Li designed the overall experiments. L. Luo, J. Tang, K. Nishi, C. Yan, P.U. Dinh, J.
Cores, T. Kudo, and J. Zhang performed the experiments and analyzed the data. L. Luo, K. Cheng, and T.
Li wrote the article. All authors read and approved the final article. All authors have provided the
corresponding author with written permission to be named in the article.
SOURCES OF FUNDING
This study was sponsored by the National Institute of Health R01 HL123920, a Grant-in-Aid from the
Ministry of Education, Science, Sports, Culture and Technology, of Japan, and a UNC General Assembly
Research Opportunities Initiative award. The funders had no role in study design, data collection, and
interpretation, or the decision to submit the work for publication.
DISCLOSURES
None.
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Segers VF, Lee RT. Stem-cell therapy for cardiac disease. Nature. 2008;451:937-942
Karantalis V, Hare JM. Use of mesenchymal stem cells for therapy of cardiac disease. Circ Res.
2015;116:1413-1430
Squillaro T, Peluso G, Galderisi U. Clinical trials with mesenchymal stem cells: An update. Cell
Transplant. 2016;25:829-848
Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells
differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation. 2002;105:93-98
Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and
therapy. Circ Res. 2008;103:1204-1219
Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS, Salto-Tellez M, Timmers L, Lee CN, El
Oakley RM, Pasterkamp G, de Kleijn DP, Lim SK. Exosome secreted by msc reduces myocardial
ischemia/reperfusion injury. Stem Cell Res. 2010;4:214-222
Angoulvant D, Ivanes F, Ferrera R, Matthews PG, Nataf S, Ovize M. Mesenchymal stem cell
conditioned media attenuates in vitro and ex vivo myocardial reperfusion injury. J Heart Lung
Transplant. 2011;30:95-102
Boomsma RA, Geenen DL. Mesenchymal stem cells secrete multiple cytokines that promote
angiogenesis and have contrasting effects on chemotaxis and apoptosis. PLoS One.
2012;7:e35685
Beohar N, Rapp J, Pandya S, Losordo DW. Rebuilding the damaged heart: The potential of
cytokines and growth factors in the treatment of ischemic heart disease. J Am Coll Cardiol.
2010;56:1287-1297
Baraniak PR, McDevitt TC. Stem cell paracrine actions and tissue regeneration. Regen Med.
2010;5:121-143
Lai RC, Chen TS, Lim SK. Mesenchymal stem cell exosome: A novel stem cell-based therapy for
cardiovascular disease. Regen Med. 2011;6:481-492
Ibrahim AG, Cheng K, Marban E. Exosomes as critical agents of cardiac regeneration triggered
by cell therapy. Stem Cell Reports. 2014;2:606-619
Vandergriff AC, de Andrade JB, Tang J, Hensley MT, Piedrahita JA, Caranasos TG, Cheng K.
Intravenous cardiac stem cell-derived exosomes ameliorate cardiac dysfunction in doxorubicin
induced dilated cardiomyopathy. Stem Cells Int. 2015;2015:960926
Gray WD, French KM, Ghosh-Choudhary S, Maxwell JT, Brown ME, Platt MO, Searles CD,
Davis ME. Identification of therapeutic covariant microrna clusters in hypoxia-treated cardiac
DOI: 10.1161/CIRCRESAHA.116.310374
7
15.
16.
17.
18.
19.
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
20.
21.
22.
23.
24.
25.
26.
progenitor cell exosomes using systems biology. Circ Res. 2015;116:255-263
Khan M, Nickoloff E, Abramova T, Johnson J, Verma SK, Krishnamurthy P, Mackie AR,
Vaughan E, Garikipati VN, Benedict C, Ramirez V, Lambers E, Ito A, Gao E, Misener S, Luongo
T, Elrod J, Qin G, Houser SR, Koch WJ, Kishore R. Embryonic stem cell-derived exosomes
promote endogenous repair mechanisms and enhance cardiac function following myocardial
infarction. Circ Res. 2015;117:52-64
Emanueli C, Shearn AI, Angelini GD, Sahoo S. Exosomes and exosomal mirnas in cardiovascular
protection and repair. Vascul Pharmacol. 2015;71:24-30
Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Preat V. Plga-based nanoparticles: An
overview of biomedical applications. J Control Release. 2012;161:505-522
Tang J, Shen D, Caranasos TG, Wang Z, Vandergriff AC, Allen TA, Hensley MT, Dinh PU, Cores
J, Li TS, Zhang J, Kan Q, Cheng K. Therapeutic microparticles functionalized with biomimetic
cardiac stem cell membranes and secretome. Nat Commun. 2017;8:13724
Fang RH, Hu CM, Luk BT, Gao W, Copp JA, Tai Y, O'Connor DE, Zhang L. Cancer cell
membrane-coated nanoparticles for anticancer vaccination and drug delivery. Nano Lett.
2014;14:2181-2188
Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood.
2007;110:3499-3506
Xie Y, Ibrahim A, Cheng K, Wu Z, Liang W, Malliaras K, Sun B, Liu W, Shen D, Cheol Cho H,
Li T, Lu L, Lu G, Marban E. Importance of cell-cell contact in the therapeutic benefits of
cardiosphere-derived cells. Stem Cells. 2014;32:2397-2406
Jaffer FA, Libby P, Weissleder R. Molecular imaging of cardiovascular disease. Circulation.
2007;116:1052-1061
Tsui BM, Kraitchman DL. Recent advances in small-animal cardiovascular imaging. J Nucl Med.
2009;50:667-670
Ghesani M, Depuey EG, Rozanski A. Role of f-18 fdg positron emission tomography (pet) in the
assessment of myocardial viability. Echocardiography. 2005;22:165-177
Abidov A, Germano G, Hachamovitch R, Slomka P, Berman DS. Gated spect in assessment of
regional and global left ventricular function: An update. J Nucl Cardiol. 2013;20:1118-1143; quiz
1144-1116
Tang JM, Wang JN, Zhang L, Zheng F, Yang JY, Kong X, Guo LY, Chen L, Huang YZ, Wan Y,
Chen SY. Vegf/sdf-1 promotes cardiac stem cell mobilization and myocardial repair in the
infarcted heart. Cardiovasc Res. 2011;91:402-411
DOI: 10.1161/CIRCRESAHA.116.310374
8
FIGURE LEGENDS
Figure 1. Fabrication and characterizaiton of synMSC. (A) Schematic illustration of the fabrication
process of synMSC. Microparticles (MP) were fabricated by treating mesenchymal stem cells
conditioned-media with Poly (lactic-co-glycolic acid) (PLGA). Synthetic mesenchymal stem cells
(synMSC) were formed by coating the MP with MSC membranes. After that, we tested the therapeutic
effects of synMSC injection in mice with acute myocardial infarction. (B) Scanning electron microscopy
images (left) and fluorescent images (right) on the structure of MP and synMSC. MP was labeled with
Texas red succinimidyl ester (red), and synMSC as cell membranes labeled with green fluorescent DiO
(red particle with green coat). Scale bar: 10 μm. (C, D) Quantitative analyses on the diameter and
expressions of MSC markers in the MP, synMSC, and MSC. (E, F and G) Quantitative analyses on the
release of vascular endothelia growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), and
insulin-like growth factor-1 (IGF-1) from synMSC. n=3 for each group. All data are mean ± SD.
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Figure 2. Potency and stability of synMSC in vitro. (A) Fluorescent images of neonatal rat
cardiomyocytes (NRCM) stained with alpha sarcomeric actin (green) and co-cultured with MP, synMSC,
and MSC (red). Scale bar: 100 μm. (B, C) Quantitative analyses of NRCM numbers and contractility
when co-cultured with MP (blue bars), synMSC (red bars), and MSC (green bars). (D) Quantitative
analyses on the number of MP and synMSC binding to NRCM. (E) Fluorescent images (above) and white
light microscopy images (below) on synMSC morphology and aggregation before and after freeze/thaw.
Scale bar: above, 10 μm; below, 100 μm. (F, G) Quantitative analyses on the size and surface antigen
expressions of synMSC pre and post freeze/thaw. (H) Representative fluorescent images and illustration
showing macrophage (green) attraction after the injection of freeze/thawed MSC and synMSC (red) into a
mouse heart. Scale bar: 100μm. (I) Quantitative analyses of the CD68+ macrophages in freeze/thawed
MSC- or synMSC- injected mouse heart. n=4 for each group. All data are mean ± SD. (B, C)* P < 0.05
when compared to control, (D)* P < 0.05 when compared to MP, (I)* P < 0.05 when compared to
synMSC.
Figure 3. Benefits of synMSC injection in mice with myocardial infarction. (A) Representative
PET/CT images and SPECT/CT images obtained at baseline and endpoint of mice after MI with or
without synMSC treatment. (B) Quantitative analyses on the percentage of altered infarct area and left
ventricular volume (endpoint vs baseline) in control and synMSC treated mice. (C) Masson’s trichrome
staining images from the base, mid-papillary and apical regions of the infarcted heart two weeks after MI
of control, synMSC and MSC treated mice. Quantitative analyses of infarct wall thickness (D) and infarct
size (E) of left ventricle in control, synMSC and MSC treated mice. n=8 for each group. All data are
mean ± SD. * P < 0.05 when compared to control.
Figure 4. Injection of synMSC promoted endogenous repair in the infarcted heart. (A, B, C)
Representative fluorescent images showing c-kit-positive, CD34-positive, and ki67-positive cells in the
infarcted heart after control, synMSC, or MSC treatement. Arrows indicate the positively stained cells.
Scale bar: (A), (C): 20 μm; (B): 50 μm. (D, E, F) Quantitative analyses on c-kit-positive cells,
CD34-positive cells, and ki67-positive cells in the infarcted heart after control, synMSC, or MSC
treatment. n=6 for each group. All data are mean ± SD. * P < 0.05 when compared to control.
DOI: 10.1161/CIRCRESAHA.116.310374
9
NOVELTY AND SIGNIFICANCE
What Is Known?
 Stem cell transplantation for heart repair has shown some benefits in animal studies and clinical trials,
but it is difficult to expand, preserve, and transport stem cells before they are administered to the patient.
 Benefits from stem cell therapy, including the injection of mesenchymal stem cells (MSCs), are
presumably from the secretion of regenerative factors rather than from direct tissue replacement.
 The stem cell membrane plays an important role in anchoring the injected stem cells to the host tissue
and mediating the repair process through cell-cell communication.
What New Information Does This Article Contribute?
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
 We describe a process to fabricate synthetic MSCs (synMSCs) by encapsulating MSC-secreted factors
in biodegradable polymer particles and then coating the particles with MSC-derived cell membranes.
 Unlike authentic, living MSCs, synMSCs can undergo harsh cryopreservation and lyophilization
processes without changing their properties. In vitro, synMSCs release various growth factors and
promote cardiomyocyte functions.
 In a murine model of myocardial infarction, injection of synMSCs leads to reduction of scar and
mitigation of ventricular remodeling without triggering inflammatory responses. Such therapeutic benefits
are similar to those from MSC therapy.
We employed a core/shell polymer particle design to fabricate synthetic stem cells designed to emulate
authentic stem cells. The new product, named as synMSCs, contained the secreted factors and surface
antigens similar to genuine MSCs. synMSCs exhibited superior cryo-stability and lyo-stability compared
to MSCs while preserving regenerative abilities of MSCs in treating mice with ischemic myocardial
injury. The synMSC technology would offer a more uniform treatment strategy from patient to patient,
rather than an inherently variable autologous or allogeneic cell-based strategy. The cell-free nature of our
synthetic approach is readily translatable to the clinic, with a potentially similar safety profile compared
to living MSCs.
DOI: 10.1161/CIRCRESAHA.116.310374
10
irc
ul
di at
st io
rib n
ut Re
e. se
D ar
es ch
tr P
oy e
af er R
te e
r u vi
se ew
. .D
rC
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Fo
o
t
no
FIGURE 1
irc
ul
di at
st io
rib n
ut Re
e. se
D ar
es ch
tr P
oy e
af er R
te e
r u vi
se ew
. .D
rC
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Fo
o
t
no
FIGURE 2
irc
ul
di at
st io
rib n
ut Re
e. se
D ar
es ch
tr P
oy e
af er R
te e
r u vi
se ew
. .D
rC
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Fo
o
t
no
FIGURE 3
irc
ul
di at
st io
rib n
ut Re
e. se
D ar
es ch
tr P
oy e
af er R
te e
r u vi
se ew
. .D
rC
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Fo
o
t
no
FIGURE 4
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Fabrication of Synthetic Mesenchymal Stem Cells for the Treatment of Acute Myocardial
Infarction in Mice
Lan Luo, Junnan Tang, Kodai Nishi, Chen Yan, Phuong-Uyen Dinh, Jhon Cores, Takashi Kudo,
Jinying zhang, Tao-Sheng Li and Ke Cheng
Circ Res. published online March 15, 2017;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2017 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/early/2017/03/14/CIRCRESAHA.116.310374
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2017/03/14/CIRCRESAHA.116.310374.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located, click
Request Permissions in the middle column of the Web page under Services. Further information about this process is
available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/
Supplemental Material
Materials and Methods
Fabrication of PLGA microparticles and synMSC
Human bone marrow-derived mesenchymal stem cells (MSC) were directly obtained from
Lonza. The cells were cultured per vendor’s instructions. To harvest conditioned media, the MSC
were cultured in serum-free media for 3 days and after that the supernatant was collected.
Conditioned media was concentrated by lyophilization and reconstitution. Briefly, conditioned
media was collected and filtered through a 0.22uM filter into a sterile 15 or 50 mL conical, as
appropriate. The filtered conditioned media was then stored at -80 ºC for at least 24 hours or until
solid and then lyophilized by a LABCONCO FreeZone 2.5 Liter Freeze Dry System. MSC
conditioned medium-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles (MP) were
fabricated by a water/oil/water (w/o/w) emulsion technique. Briefly, human MSC conditioned
media as the internal aqueous phase with polyvinyal alcohol (PVA) (0.1% w/v) was mixed in
methylene chloride (DCM) containing PLGA as the oil phase. The mixture was then sonicated on
ice for 30 s using a sonicator (Misonix, XL2020, Farmingdale, NY, USA). Subsequently, the
primary emulsion was immediately introduced into water with PVA (0.7% w/v) to produce a
w1/o/w2 emulsion. The secondary emulsion was emulsified for 5 min on a high-speed
homogenizer. The w1/o/w2 emulsion was continuously stirred overnight at room temperature to
promote solvent evaporation. The solidified microparticles, were then centrifuged, washed three
times with water, lyophilized and stored at -80 ºC. To prepare synMSC, DiO (Invitrogen)-labeled
MSC went through three freeze/thaw cycles. After which, the disrupted MSC were sonicated for
approximately 5 minutes at room temperature along with the MP. After that, the particles were
washed three times in PBS by centrifugation. Successful membrane coating was confirmed using
fluorescent microscopy.
Scanning electron microscopy
Scanning electron microscopy (SEM, Philips XL30 scanning microscope, Philips, The
Netherlands) were used for study of microparticles and synMSC morphology. Freeze-dried
microparticles were mounted on aluminum stubs with double-sided tape and coated with gold
particles. The coated specimen was then scanned and photographed at an acceleration voltage of
15 kV.
Flow cytometry
To characterize the surface antigen expressions of MP, synMSC and MSC, flow cytometry was
performed using a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA) and analyzed using
FCS Express software (De Novo Software, Los Angeles, CA). In brief, MP, synMSC, and MSC
were incubated with FITC, PE, or APC-conjugated antibodies against CD105 (3µl per 100 µl of
sample, R&D Systems), CD90 (4µl per 100 µl of sample, BD Biosciences), CD45 (5µl per 100
µl of sample, BD Biosciences), CD34 (5µl per 100 µl of sample, BD Biosciences), and CD31
(5µl per 100 µl of sample, BD Biosciences) for 60 min. Isotype-identical antibodies from BD
Company served as negative controls (3µl per 100 µl of sample).
Growth factors release study
Growth factor releases from synMSC were determined using the following method.
Approximately 1mg/ml microparticles in PBS buffer (pH 7.4) were sonicated for on ice for 2mins
using a sonicator (Misonix, XL2020, Farmingdale, NY, USA) with power set at 23 W. Total
protein and growth factor amount were determined. After that, microparticles were incubated in
PBS at 37 ºC. Supernatant was collected at various time points and the concentrations of growth
factors were determined by commercially available ELISA kits (R & D Systems, Minneapolis,
MN, USA) and expressed as cumulative release % of the total amount encapsulated.
Immunocytochemistry
MP, synMSC, and MSC were pre-labeled with red-fluorescent Texas red succinimidyl ester (1
mg/ml [Invitrogen, Carlsbad, California]). NRCM or NRCM co-cultured with pre-labeled MP,
synMSC, and MSC were plated onto fibronectin-coated chamber slides (BD Biosciences) for 3
days and subsequently fixed with 4% paraformaldehyde (PFA) before immunocytochemistry
(ICC) staining. Slides were stained with the antibodies against α-SA (1:100, a7811, Sigma) or
ki67 (1:100, ab15580, Abcam) and detected by FITC- or Texas Red-conjugated secondary
antibodies (1:100). Nuclei were stained with 4, 6-diamidino-2-phenylin-dole (DAPI). Images
were taken with an epi-fluorescent microscope (Olympus IX81).
Myocardial infarction model
All animal experiments were performed in accordance to the guidelines from Directive
2010/63/EU of the European Parliament on the protection of animals used for scientific purposes
or the NIH guidelines. Acute myocardial infarction (AMI) model was created in 9-11-weeks-old
male C57BL/6 mice (CLEA Japan, Inc.) based on our previous studies1. Briefly, after general
anesthesia with intraperitoneal injection of 160 mg/kg pentobarbital and endotracheal intubation,
mice were artificially ventilated with room air. A left thoracotomy was performed through the
fourth intercostal space, and left anterior descending artery was ligated with 8-0 prolene under
microscopy. Immediately after AMI induction, the heart was randomized to receive one of the
following treatments: 1) Control group: closing chest cavity with no treatment; 2) synMSC group:
intramyocardial injection of 1x105 synMSC particles in 50 μl PBS into the heart immediately after
AMI; 3) MSC group: intramyocardial injection of 1x105 cells in 50 μl PBS into the heart
immediately after AMI.
PET-CT and SPECT-CT evaluation
Control and synMSC treated mice were underwent PET-CT at day 1 (baseline) and day 14
(endpoint), and SPECT-CT at day 2 (baseline) and day 15 (endpoint) under anesthesia with 2.0%
to 2.5% isoflurane based on our previous study2. For PET-CT, mice were intravenously injected
with 10 MBq of 18F-FDG (Nihon Medi-physics Co., Kurume, Japan) via tail vein. Approximately
30 min later, the mice were scanned for 15 min. For SPECT-CT, mice were intravenously injected
with 80 MBq of
99m
Tc-tetrofosmin (Nihon Medi-physics Co., Chiba, Japan) via tail vein.
Approximately 5 min later, the mice were scanned for 21 min using imaging parameters as
follows; 20 sec/projection, 64 projection/360° and radius of rotation 40 mm. All acquisitions were
performed at Triumph LabPET4/SPECT4/CT (TriFoil, Imaging Inc., Chatsworth, CA, USA), a
small animal PET/SPECT/CT scanner. Single pinhole collimator (pinhole diameter 1mm, focallength 90mm) was attached on the SPECT detectors for SPECT imaging. For PET imaging data,
3D-Maximum-Likelihood Expectation Maximization (3D-MLEM) algorithm was applied using
30 iterations. For SPECT imaging data, 3D-MLEM algorithm was applied using 50 iterations.
The acquired PET and SPECT data were analyzed using OsiriX MD (FDA cleared, Pixmeo),
Dr.View (Infocom Co., Tokyo, Japan).
Histological analyses
At 15 days after MI, all mice were euthanized by severing the aorta under general anesthesia
with intraperitoneal injection of 160 mg/kg pentobarbital. The heart was injected with 5 ml PBS
to remove the blood cells and then embedded in OCT compound for histological analysis. The
heart tissue was sectioned at 7 μm thickness from the base to the apex level with 100 μm intervals
after the internal radionuclide decayed. Masson’s trichrome staining was performed according to
the manufacturer’s protocol (Sigma-Aldrich) and then mounted and imaged by Keyence BZ-9000
fluorescence microscope. Morphometric analyses were performed using the NIH ImageJ software.
Infarct size measurement was obtained at the base, mid-papillary and apical regions of the
infarcted heart by mid-length measurement3. Left ventricle infarct wall thickness measurement
was obtained from the infarcted heart.
Immunohistochemistry
Heart cryosections were fixed with 4% paraformaldehyde, permeablized and blocked with
protein block serum-free solution (Dako), and then incubated with the following antibodies at
room temperature: goat polyclonal anti-mouse c-kit antibody (10 μg/ml, R&D Systems), rat antimouse Ki-67 monoclonal antibody (1:400, Dako) and anti-mouse CD34 FITC (5 μg/ml,
eBioscience). Donkey anti-Goat IgG(H+L) secondary antibody, Alexa Flour 546 conjugate (1:400,
Thermo scientific) and Donkey anti-Rat IgG(H+L) secondary antibody, Alexa Flour 488
conjugate (1:800, Thermo scientific) were used for the conjunction with these primary antibodies.
Nuclei were then stained with DAPI. Images were taken by a fluorescent microscopy (Olympus
IX83, Olympus).
Statistical analysis
All data were presented as the mean ± SD. Comparisons between two groups were performed
with unpaired two-tailed Student’s t-test. Comparisons among more than two groups were
performed using one-way ANOVA followed by post-hoc Bonferroni test. Differences were
considered statistically significant when P < 0.05.
References
1.
Andrade JN, Tang J, Hensley MT, Vandergriff A, Cores J, Henry E, Allen TA, Caranasos
TG, Wang Z, Zhang T, Zhang J, Cheng K. Rapid and efficient production of coronary
artery ligation and myocardial infarction in mice using surgical clips. PLoS One.
2015;10:e0143221
2.
Luo L, Nishi K, Urata Y, Yan C, Hasan AS, Goto S, Kudo T, Li ZL, Li TS. Ionizing
radiation impairs endogenous regeneration of infarcted heart: An in vivo 18f-fdg pet/ct
and 99mtc-tetrofosmin spect/ct study in mice. Radiat Res. 2017;187:89-97
3.
Takagawa J, Zhang Y, Wong ML, Sievers RE, Kapasi NK, Wang Y, Yeghiazarians Y, Lee
RJ, Grossman W, Springer ML. Myocardial infarct size measurement in the mouse
chronic infarction model: Comparison of area- and length-based approaches. J Appl
Physiol (1985). 2007;102:2104-2111
Online Figure I. Characterization of mesenchymal stem cells (MSC)
(A) Representative images of the MSC morphology. (B, C, D, E, F) Flow cytometry analysis on
the expressions of CD105, CD90, CD45, CD31 and CD34 on MSCs.
Online Figure II. Lyophilization stability of synthetic mesenchymal stem cells (synMSCs)
(A) Schematic of the lyophilization process on synMSC. (B) Representative white light
microscopy images of synMSCs before and after lyophilization. Scale bar: 100μm. (C)
Quantitative analyses on the sizes of synMSCs before and after lyophilization. (D) Flow
cytometry analysis on the surface antigen expressions. (E) Quantitative analyses on the release of
VEGF from synMSC before lyophilization and after lyophilization.