Fabrication of Synthetic Mesenchymal Stem Cells for the Treatment of Acute Myocardial Infarction in MiceNovelty and Significance
Rationale: Stem cell therapy faces several 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) 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.
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.
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 (MSCs), embryonic stem cells, and endogenous cardiac stem cells in cardiovascular diseases.1 Among the cell types under investigation, MSCs have attracted great attention owing to their ability to differentiate into mesoderm and nonmesoderm tissues, their immunomodulatory properties, and their broad spectrum release of trophic factors.2 Preclinical and clinical studies on MSC have shown promise for repair and regeneration of cardiac tissues.3 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 observed,4 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 effects.5 More recently, they found that MSC-secreted exosomes exhibited functions similar to MSC for repairing heart injury.6 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 diseases.7,8 Moreover, the delivery of individual growth factors, such as vascular endothelial growth factor and insulin-like growth factor-1, has also been tested for their cardiac therapeutic effects in clinical trials.9 Unfortunately, neither has met our expectations. The reasons may be the short half-life of protein factors in vivo, the uncertainty of effective/safe dosages, and the possibility that multiple administration may be necessary to act synergistically to achieve therapeutic effect.10 It is noteworthy that exosomes could circumvent many of these challenges. The bilipid 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 microenvironment.11 In addition, exosomes contain proteins and RNAs that may have adequate potential for cardiac repair.12–16 However, exosome-based therapeutics also face challenges such as the lack of a standard isolation protocol, rapid clearance, and wash-away because of 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 because of its capability of protecting cytokines from degradation while allowing for the sustained release of factors that target in specific organs or cells.17,18 Furthermore, Fang et al19 reported cancer cell membrane–coated nanoparticles formed by coating cancer cell membranes onto PLGA-loaded immunologic particles. The membrane-bound tumor-associated antigens permit cancer cell membrane–coated nanoparticles to be efficiently delivered to antigen-presenting cells to promote anticancer immune response.
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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 (MI). 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.
A detailed Methods section is provided in the Online Data Supplement.
synMSC Fabrication and Biological Properties
The schematic design of synMSC fabrication is summarized in Figure 1A. In brief, MSC-conditioned media was incorporated in PLGA to form microparticles and then the microparticles were coated with MSC cell membrane to form synthetic MSCs (synMSC). Scanning electron microscopy and fluorescent imaging (Figure 1B) confirmed the successful MSC cell membrane coating on microparticles. synMSC had a size ≈20 μm, similar to those of microparticles and real MSC (Figure 1C). Flow cytometry analysis showed that synMSC exhibited similar expressions of CD105, CD90, CD45, CD31, and CD34 compared with MSC, whereas microparticles did not (Figure 1D). Furthermore, synMSC could sustain the release of growth factors like vascular endothelial growth factor (Figure 1E), stromal cell–derived factor-1 (Figure 1F), and insulin-like growth factor 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 α-sarcomeric actin; Figure 2A, green) were cocultured with microparticles, synMSC and MSC (Figure 2A, red). 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 because of its significantly higher number existed on 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 that freeze/thaw treatment did not 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). Furthermore, we tested the lyophilization stability of synMSC and found that the lyophilization process did not alter the structure, size, surface antigen expressions, or sustained vascular endothelial growth factor 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, MSCs were targeted by macrophages while synMSCs were not (Figure 2H and 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 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 (CT) was performed at 1 (baseline) and 14 (end point) days after infarction to measure the infarct area (Figure 3A). 99mTc-tetrofosmin single photon emission computed tomography/CT was performed at 2 (baseline) and 15 (end point) days 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 2 groups (Figure 3B). Left ventricle morphometry imaged by Masson 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 when compared with the control group.
synMSC Injection Promotes Endogenous Repair in the Infarcted Heart
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 with 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 with 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.
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 when compared with 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 MI 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 remains 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 vascular endothelial growth factor, stromal cell–derived factor-1, and insulin-like growth factor 1, binding to cardiomyocytes in vitro. In addition, the expression of MHC class I molecules, but not of MHC class II molecules or costimulatory molecules, in MSC cell membranes allows it to escape allorecognition by the immune system and may modulate the host immune response.20 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 coculturing the synMSC with NRCM, we 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 microparticles could be because of 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 augmentation.21
One major challenges of stem cell–based therapy is the cryopreservation stability of cells. Here, we found that snap freezing in −80°C 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 MSCs (with dead MSCs caused by harsh freezing/thawing) were injected into a mouse heart, they were targeted by macrophages (initiating the phagocytosis of dead MSC) whereas synMSCs were not. This suggested the superior cryopreservation stability of synMSC over MSC.
Currently, as CT can provide great detail in anatomic structure, hybrid imaging of positron emission tomography and single photon emission computed tomography with CT has been adopted in clinical and small animal cardiovascular disease diagnosis.22,23 Positron emission tomography utilizing glucose tracer analog 18F-FDG allows the detection of cells with different metabolic activities,24 and gated single photon emission computed tomography utilizing 99mTc-tetrofosmin makes accurate assessment of ventricular volumes.25 So we evaluated the myocardial viability and left ventricle volume of mice heart by 18F-fluorodeoxglucose positron emission tomography/CT and 99mTc-tetrofosmin single photon emission computed tomography/CT. synMSC significantly mitigated left ventricle remodeling, as indicated by a significant reduction of infarct area, confirming the therapeutic potential of synMSC. Furthermore, the left ventricle morphometry evaluation by Masson trichrome staining revealed synMSC exhibited protection of heart morphometry at a level that was comparable to MSC.
Previous reports have demonstrated that MSC provide cardioprotection by paracrine actions that activate cardiac stem cells,26 angiogenesis, and cell proliferation.8 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 that would provide sufficient oxygen and nutrients to the surrounded cardiomyoytes.
Taken together, we successfully fabricate synMSC and demonstrate their prominent therapeutic effects in an acute MI 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. After all, our results suggest that 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.
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 and HL137093 to K.C.), a Grant-in-Aid from the Ministry of Education, Science, Sports, Culture and Technology of Japan (to T.L.), the Network-Type Joint Usage/Research Center for Radiation Disaster Medical Science of Hiroshima University, Nagasaki University, and Fukushima Medical University (to K.C. and T.L.), and a UNC General Assembly Research Opportunities Initiative award (to K.C.). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.
In February 2016, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.4 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.310374/-/DC1.
- Nonstandard Abbreviations and Acronyms
- computed tomography
- myocardial infarction
- mesenchymal stem cells
- neonatal rat cardiomyocytes
- poly(lactic-co-glycolic acid)
- synthetic mesenchymal stem cells
- vascular endothelial growth factor
- Received November 25, 2016.
- Revision received March 9, 2017.
- Accepted March 14, 2017.
- © 2017 American Heart Association, Inc.
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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?
We describe a process to fabricate synthetic mesenchymal stem cells (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 used 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 cryostability and lyostability compared with 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 with living MSCs.