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Isolation and trans-differentiation of mesenchymal stromal cells into smooth muscle cells: Utility and applicability for cell-sheet engineering
Abstract
Background:
Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) have shown potential to differentiate into various cell types, including smooth muscle cells (SMCs). The extracellular matrix (ECM) represents an appealing and readily available source of SMCs for use in tissue engineering. In this study, we hypothesized that the ECM could be used to induce MSC differentiation to SMCs for engineered cell-sheet construction.
Methods:
Primary MSCs were isolated from the BM of Wistar rats, transferred and cultured on dishes coated with 3 different types of ECM: collagen type IV (Col IV), fibronectin (FN), and laminin (LM). Primary MSCs were also included as a control. The proportions of SMC (a smooth muscle actin [aSMA] and SM22a) and MSC markers were examined with flow cytometry and Western blotting, and cell proliferation rates were also quantified.
Results:
Both FN and LM groups were able to induce differentiation of MSCs toward smooth muscle-like cell types, as evidenced by an increase in the proportion of SMC markers (aSMA; Col IV 42.3 ± 6.9%, FN 65.1 ± 6.5%, LM 59.3 ± 7.0%, Control 39.9 ± 3.1%; P = 0.02, SM22; Col IV 56.0 ± 7.7%, FN 74.2 ± 6.7%, LM 60.4 ± 8.7%, Control 44.9 ± 3.6%) and a decrease in that of MSC markers (CD105: Col IV 64.0 ± 5.2%, FN 57.6 ± 4.0%, LM 60.3 ± 7.0%, Control 85.3 ± 4.2%; P = 0.03). The LM group showed a decrease in overall cell proliferation, whereas FN and Col IV groups remained similar to control MSCs (Col IV, 9.0 ± 2.3%; FN, 9.8 ± 2.5%; LM, 4.3 ± 1.3%; Control, 9.8 ± 2.8%).
Conclusions:
Our findings indicate that ECM selection can guide differentiation of MSCs into the SMC lineage. Fibronectin preserved cellular proliferative capacity while yielding the highest proportion of differentiated SMCs, suggesting that FN-coated materials may be facilitate smooth muscle tissue engineering.
... Delivery of a tissue-engineered construct that maintains important interactions between EPCs and SMCs enhances mature neovascularization within the border zone myocardium, minimizes post-infarction adverse remodeling, strengthens ventricular function in a rodent model of ischemic cardiomyopathy, and is a highly optimal therapeutic cell delivery platform [5,6]. We have recently identi ed a modi ed method for SMC transdifferentiation from bone marrow-derived mesenchymal stem cells (MSCs) [7]. We are currently working on developing preclinical human-origin EPCs and SMCs from human bone marrow. ...
... ± 0.0%), SM22-α (99.9% ± 0.1%), and caldesmon (89.5% ± 2.7%) (Fig. 1E). Our protocol yielded human-derived EPCs and SMC lineages with high purity, which is consistent with previous reports [5][6][7] and is a promising cell source for translatable clinical applications. ...
... These cell sheets preserve the cell-cell junctions and the extracellular matrix (ECM) deposited on the basal surface of the cell sheet in addition to regional morphological differences between different cell types following mobilization from the UpCell dish. SEM images showed spherical cells of the SMC layer, and a thin, lm-like EPC mono-layer, consistent with the results reported previously [7]. ...
We demonstrated that joint delivery of human-derived endothelial progenitor cell (EPC) and smooth muscle cell (SMC) sheets mimics the native architecture of structurally mature blood vessels and contributes to limiting ventricular remodeling using a confluent SMC-EPC bi-level cell sheet engineered by cell-sheet technology and transplanted into an athymic rodent model of myocardial infarction. Enhanced vasculogenic potential was observed in vitro when EPCs were stimulated with SMC-conditioned culture medium, augmenting angiogenesis in vivo. Increased structurally mature vessel density, myocardial upregulation of biological adhesion, and vasculature developmental genes in the ischemic border zone myocardium showed interaction between cells and the extracellular matrix. Cell fate tracking experiments featuring xenogeneic transplantation showed transplanted EPCs and SMCs to have elements of the newly formed vasculature. Specialized magnetic resonance imaging of the cell-sheet-transplanted rodents suggested prolonged cell retention. The robust angiogenic effect of the transplanted cell sheets induced reverse ventricular remodeling of the ischemic heart. Bioinformatic analyses indicated that these cell sheets promote transcriptome-wide changes in the left ventricular response to acute ischemia, promote productive remodeling, and prevent pathological ventricular dilation. Thus, the human-derived, spatially arranged SMC-EPC bi-level cell sheet is a promising therapy for increasing myocardial viability and limiting adverse ventricular remodeling after myocardial infarction.
... We have addressed the limitations of cell dispersion, destruction, and isolation in the hostile post-infarction environment with the creation of a tissueengineered SMC-EPC bi-level cell sheet. The SMC-EPC bi-level cell sheet provides EPCs with a natural SMC biologic support environment, and significantly improves cardiac function in a rodent model of ischemic cardiomyopathy [18][19][20]. Furthermore, SMC-EPC bi-level cell sheets secrete an abundance of angiogenic cytokines, which directly contribute to the maintenance of a functional microvasculature [18,21,22]. ...
... Rat MSCs were obtained from male Wistar rats (8 weeks old, 250-300 g; Charles River, San Jose, CA), as previously described [18,19]. Briefly, bone marrow (BM) mononuclear cells were isolated from the long bones, filtered through a 40 μm cell strainer, and centrifuged at 300g for 8 min. ...
... EPCs were isolated and cultured as described previously [18][19][20]. Briefly, BM mononuclear cells were isolated from the long bones of Wistar rats and cultured on vitronectin-coated dishes (Sigma-Aldrich, St. Louis, MO) in EBM-2 supplemented with EGM-2 SingleQuot (Lonza, Walkersville, MD). ...
Background
Diabetes mellitus is a risk factor for coronary artery disease and diabetic cardiomyopathy, and adversely impacts outcomes following coronary artery bypass grafting. Current treatments focus on macro-revascularization and neglect the microvascular disease typical of diabetes mellitus-induced cardiomyopathy (DMCM). We hypothesized that engineered smooth muscle cell (SMC)-endothelial progenitor cell (EPC) bi-level cell sheets could improve ventricular dysfunction in DMCM.
Methods
Primary mesenchymal stem cells (MSCs) and EPCs were isolated from the bone marrow of Wistar rats, and MSCs were differentiated into SMCs by culturing on a fibronectin-coated dish. SMCs topped with EPCs were detached from a temperature-responsive culture dish to create an SMC-EPC bi-level cell sheet. A DMCM model was induced by intraperitoneal streptozotocin injection. Four weeks after induction, rats were randomized into 3 groups: control (no DMCM induction), untreated DMCM, and treated DMCM (cell sheet transplant covering the anterior surface of the left ventricle).
Results
SMC-EPC cell sheet therapy preserved cardiac function and halted adverse ventricular remodeling, as demonstrated by echocardiography and cardiac magnetic resonance imaging at 8 weeks after DMCM induction. Myocardial contrast echocardiography demonstrated that myocardial perfusion and microvascular function were preserved in the treatment group compared with untreated animals. Histological analysis demonstrated decreased interstitial fibrosis and increased microvascular density in the SMC-EPC cell sheet-treated group.
Conclusions
Treatment of DMCM with tissue-engineered SMC-EPC bi-level cell sheets prevented cardiac dysfunction and microvascular disease associated with DMCM. This multi-lineage cellular therapy is a novel, translatable approach to improve microvascular disease and prevent heart failure in diabetic patients.
... We then per- www.nature.com/scientificreports/ formed gene set enrichment analysis by ranking the DEGs in descending order of fold change and by analyzing this rank list to identify enriched biological processes in annotated gene ontology (GO) 18,19 . We identified 114 significantly enriched processes in the cell sheet treated group, including pathways related to embryogenic morphogenesis, biological adhesion, and vasculature development, whereas the infarct-only group showed enrichment for pathways related to mitochondrial energetics, oxidative phosphorylation, and protein translation ( Fig. 5C-E). ...
... SMC growth medium (Medium 231 with SMGS) was used as the nutrient medium and exchanged every 2 days. MSCs grown under SMC growth conditions were passaged several times for expansion and induction toward SMC lineage differentiation before biological tests (Fig. 1A) 18 . In addition, commercially available human aortic smooth muscle cells (AoSMC; Lonza Inc., NJ, USA) were cultured in the same medium condition at a density of 3.5 × 10 3 cm 2 for comparative purposes. ...
Many cell-based therapies are challenged by the poor localization of introduced cells and the use of biomaterial scaffolds with questionable biocompatibility or bio-functionality. Endothelial progenitor cells (EPCs), a popular cell type used in cell-based therapies due to their robust angiogenic potential, are limited in their therapeutic capacity to develop into mature vasculature. Here, we demonstrate a joint delivery of human-derived endothelial progenitor cells (EPC) and smooth muscle cells (SMC) as a scaffold-free, bi-level cell sheet platform to improve ventricular remodeling and function in an athymic rat model of myocardial infarction. The transplanted bi-level cell sheet on the ischemic heart provides a biomimetic microenvironment and improved cell–cell communication, enhancing cell engraftment and angiogenesis, thereby improving ventricular remodeling. Notably, the increased density of vessel-like structures and upregulation of biological adhesion and vasculature developmental genes, such as Cxcl12 and Notch3, particularly in the ischemic border zone myocardium, were observed following cell sheet transplantation. We provide compelling evidence that this SMC-EPC bi-level cell sheet construct can be a promising therapy to repair ischemic cardiomyopathy.
... Endothelial cells regulate the activity, migration, and differentiation of MSCs [70,73] to stabilize the newly formed blood vessels. In addition, the co-culture of MSCs and SMCs is conducive to the myogenic phenotype expression of MSCs [71,74,75]. ...
The esophagus is an important part of the human digestive system. Due to its limited regenerative capacity and the infeasibility of donor transplantation, esophageal replacement has become an important problem to be solved urgently in clinics. In recent years, with the rapid development of tissue engineering technology in the biomedical field, tissue engineering stent (artificial esophagus) provides a new therapeutic approach for the repair and reconstruction of esophageal defects and has made remarkable progress. Biomedical esophageal stent materials have also experienced the development process from non-absorbable materials to absorbable materials, and then to new materials with composite cells and biological factors. In this paper, the composition, functional characteristics, and limitations of non-degradable scaffolds, biodegradable scaffolds, and Decellularized Matrix (DM) scaffolds specially designed for these applications are reviewed. Non-absorbable stents are typically composed of synthetic polymers or metals that provide structural support but fail to bind to surrounding tissues over time. In contrast, biodegradable stents are designed to break down gradually in the body while promoting cell infiltration and promoting new tissue formation. DM scaffolds can alleviate autoimmune reactions, preserve natural tissue characteristics, and enable recellularization during auto-repair. In addition, the significance of various cell-loaded materials in esophageal replacement has been explored, and the inclusion of cells in scaffold design has been shown to have the potential to enhance integration with host tissue and improve postoperative functional outcomes. These advances underscore ongoing efforts to closely mimic the structure of the natural esophagus.
... Generally, our findings on laminin and collagen IV indicate that their deposition within CSs plays a minor role in their formation, as far as both were deposited intracellularly and were barely detectable without permeabilization (Figures 4 and 5). Nevertheless, their deposition may contribute to increase in MSC number, as far as collagen IV enhances the FAK-ERK signaling pathway associated with the activation of MSC proliferation [22]. ...
Cell sheet (CS) engineering using mesenchymal stromal cells (MSC) draws significant interest for regenerative medicine and this approach translates to clinical use for numerous indications. However, little is known of factors that define the timing of CS assembly from primary cultures. This aspect is important for planning CS delivery in autologous and allogeneic modes of use. We used a comparative in vitro approach with primary donors’ (n = 14) adipose-derived MSCs and evaluated the impact of healthy subject’s sex, MSC culture features (population doubling time and lag-phase), and extracellular matrix (ECM) composition along with factors related to connective tissue formations (α-SMA and FAP-α) on CS assembly duration. Using qualitative and quantitative analysis methods, we found that, in seeded MSCs, high contents of collagen I and collagen IV had a direct correlation with longer CS assembly duration. We found that short lag-phase cultures faster turned to a ready-to-use CS, while age, sex, fibronectin, laminin, α-SMA, and FAP-α failed to provide a significant correlation with the timing of assembly. In detachable CSs, FAP-α was negatively correlated with the duration of assembly, suggesting that its concentration rose over time and contributed to MSC activation, transitioning to α-SMA-positive myofibroblasts and ECM turnover. Preliminary data on cell density and collagen I deposition suggested that the TGF-β1 signaling axis is of pivotal importance for ECM composition and construct maturation.
... Generally, our findings on laminin and collagen IV indicate that their deposition within CS play a minor role in its formation as far as majority of both were deposited intracellularly and not visualized until permeabilization (Figures 4 and 5). Nevertheless, its deposition may contribute to increase of MSC number as far as collagen IV enhances FAK-ERK signaling pathway associated with activation of MSC proliferation [23]. ...
Cell sheet (CS) engineering using human MSC is of significant interest for regenerative medicine and this rapidly growing field translates to clinical use in a number of indications. Nevertheless, little is known of factors that define the timing of CS assembly from primary cultures which is important for planning treatments in both – autologous and allogeneic modes of use. We used a comparative in vitro approach with primary donors’ (n=14) adipose-derived MSC and evaluated the impact of healthy subject’s characteristics (age and sex), MSC culture features (population doubling time and lag-phase) and extracellular matrix (ECM) composition along with factors related to connective tissue formations (-SMA and FAP-) on CS assembly duration.
... MSCs from Wharton's jelly, boosted the synthesis of pro-angiogenic factors by stimulating the sonic hedgehog factor, making it a good cell source for inducing blood cell development [68]. According to studies, activating the intracellular signalling pathways of phosphatidylinositol 3-kinase/protein kinase B promotes the MSC transdifferentiation into smooth muscle cells can be enhanced by fibronectin and laminin [69]. ...
PurposeThis review focused on the effectiveness of utilising natural compounds like curcumin to induce the trans-differentiation of mesenchymal stem cells into cells resembling neurons.Methods
The literature works published in the last 10 years on the role of curcumin in inducing neuronal trans-differentiation of mesenchymal stem cells was studied using databases like PubMed, ScienceDirect, and Google Scholar. Various experiments regarding mesenchymal stem cell (MSCs) trans-differentiation, neuroprotective property of curcumin was investigated, and the data obtained from the experiments relating curcumin’s efficiency in targeting Wnt signalling pathway which plays a major role in the trans-differentiation of mesenchymal stem cells into neuronal like cells have been concised into a review article to provide a clear and better understanding of the associated mechanism. A workflow depicting the review process is mentioned in Fig. 1.
ResultsThe results of many experimental studies have concluded that curcumin aids in neuronal trans-differentiation through its role as a GSK-3β inhibitor to activate Wnt/β-catenin signalling pathway and the neuronal like cells obtained through this process could act as a therapeutic tool in treating neurodegenerative disorders involving the damage of neurons.Conclusion
Curcumin’s numerous beneficial properties have been used in the treatment of diseases, particularly it has given rise to a possibility of treating neurodegenerative disorders. Curcumin’s role in the induction of neuronal trans-differentiation has gained popularity in the scientific world although there are drawbacks involving the efficiency of trans-differentiation. Future studies need to focus on the molecular biology level of the trans-differentiated neuronal like cells and the precise role of curcumin in the process to emerge as a potential therapeutic agent.Lay SummaryCurcumin, obtained from Curcuma longa, has vast therapeutic qualities that could be used to treat various disorders. Mesenchymal stem cell’s advantage over embryonic stem cells in ethical restraints, combined with immunosuppressive nature, has made them the preferred cellular transplantation mode for neurodegenerative illnesses. There are several works where neuronal trans-differentiation of MSCs triggered by curcumin and resveratrol has been shown to be efficient. In this review, the neuronal trans-differentiating potential of mesenchymal stem cells and curcumin’s role in modulating the associated signalling pathways have been discussed based on the reported literatures available till date. It also highlights the therapeutical efficacy of curcumin in neurodegenerative disease management.Description of Future WorksRecent advances have proved curcumin’s importance in the neuronal trans-differentiation process. However, further research on the efficiency of trans-differentiation and molecular characterisation studies on the neuronal resemblance of the transdifferentiated cells in order to develop into fully functioning neurons is required to strengthen its role as a novel therapy for neurodegenerative disorders.
... MSCs can differentiate into osteoblast 9,60 , chondroblast 9,60 , cardiomyocytes 2,60,61 , smooth muscle cells 62,63 , endothelial cells 64 among the others 9,13,60,61 . Differentiation of MSC strongly depend on the tissue microenvironment 60,65,66 . ...
The voltage-gated proton channel Hv1 is widely expressed, among others, in immune and cancer cells, it provides an efficient cytosolic H⁺extrusion mechanism and regulates vital functions such as oxidative burst, migration and proliferation. Here we demonstrate the presence of human Hv1 (hHv1) in the placenta/chorion-derived mesenchymal stem cells (cMSCs) using RT-PCR. The voltage- and pH-dependent gating of the current is similar to that of hHv1 expressed in cell lines and that the current is blocked by 5-chloro-2-guanidinobenzimidazole (ClGBI) and activated by arachidonic acid (AA). Inhibition of hHv1 by ClGBI significantly decreases mineral matrix production of cMSCs induced by conditions mimicking physiological or pathological (inorganic phosphate, Pi) induction of osteogenesis. Wound healing assay and single cell motility analysis show that ClGBI significantly inhibits the migration of cMSCs. Thus, seminal functions of cMSCs are modulated by hHv1 which makes this channel as an attractive target for controlling advantages/disadvantages of MSCs therapy.
... Endothelial cells regulate the activity [27], migration [28], and differentiation [29] of MSCs for the stabilization of newly formed vasculature. Additionally, co-culture of MSCs with SMCs contributed to myogenic phenotype expression of MSCs [30][31][32][33], and the use of muscle cells was associated with a decreased inflammatory reaction and enhanced muscle regeneration [4]. To evaluate which combination of cell types was best for artificial esophagus formation and transplantation, the structures were analyzed using tensile test and immunohistochemistry. ...
Various strategies have been attempted to replace esophageal defects with natural or artificial substitutes using tissue engineering. However, these methods have not yet reached clinical application because of the high risks related to their immunogenicity or insufficient biocompatibility. In this study, we developed a scaffold-free structure with a mixture of cell types using bio-three-dimensional (3D) printing technology and assessed its characteristics in vitro and in vivo after transplantation into rats. Normal human dermal fibroblasts, human esophageal smooth muscle cells, human bone marrow-derived mesenchymal stem cells, and human umbilical vein endothelial cells were purchased and used as a cell source. After the preparation of multicellular spheroids, esophageal-like tube structures were prepared by bio-3D printing. The structures were matured in a bioreactor and transplanted into 10-12-week-old F344 male rats as esophageal grafts under general anesthesia. Mechanical and histochemical assessment of the structures were performed. Among 4 types of structures evaluated, those with the larger proportion of mesenchymal stem cells tended to show greater strength and expansion on mechanical testing and highly expressed α-smooth muscle actin and vascular endothelial growth factor on immunohistochemistry. Therefore, the structure with the larger proportion of mesenchymal stem cells was selected for transplantation. The scaffold-free structures had sufficient strength for transplantation between the esophagus and stomach using silicon stents. The structures were maintained in vivo for 30 days after transplantation. Smooth muscle cells were maintained, and flat epithelium extended and covered the inner surface of the lumen. Food had also passed through the structure. These results suggested that the esophagus-like scaffold-free tubular structures created using bio-3D printing could hold promise as a substitute for the repair of esophageal defects.
... 16 In addition, our laboratory described a protocol to transdifferentiate mesenchymal stem cells, which can be acquired from peripheral blood, into smooth muscle cells. 29 Another step toward clinical application will be the upscaling of the EVC to the size of a human cardiovascular bypass graft. This process will involve the use of an Angiocath needle with an outer diameter of at least 3 mm and construction with more cell layers. ...
Background:
Cardiovascular bypass grafting is an essential treatment for complex cases of atherosclerotic disease. Because the availability of autologous arterial and venous conduits is patient-limited, self-assembled cell-only grafts have been developed to serve as functional conduits with off-the-shelf availability. The unacceptably long production time required to generate these conduits, however, currently limits their clinical utility. Here, we introduce a novel technique to significantly accelerate the production process of self-assembled engineered vascular conduits.
Methods:
Human aortic smooth muscle cells and skin fibroblasts were used to construct bilevel cell sheets. Cell sheets were wrapped around a 22.5-gauge Angiocath needle to form tubular vessel constructs. A thin, flexible membrane of clinically approved biodegradable tissue glue (Dermabond Advanced) served as a temporary, external scaffold, allowing immediate perfusion and endothelialization of the vessel construct in a bioreactor. Subsequently, the matured vascular conduits were used as femoral artery interposition grafts in rats (n=20). Burst pressure, vasoreactivity, flow dynamics, perfusion, graft patency, and histological structure were assessed.
Results:
Compared with engineered vascular conduits formed without external stabilization, glue membrane-stabilized conduits reached maturity in the bioreactor in one-fifth the time. After only 2 weeks of perfusion, the matured conduits exhibited flow dynamics similar to that of control arteries, as well as physiological responses to vasoconstricting and vasodilating drugs. The matured conduits had burst pressures exceeding 500 mm Hg and had sufficient mechanical stability for surgical anastomoses. The patency rate of implanted conduits at 8 weeks was 100%, with flow rate and hind-limb perfusion similar to those of sham controls. Grafts explanted after 8 weeks showed a histological structure resembling that of typical arteries, including intima, media, adventitia, and internal and external elastic membrane layers.
Conclusions:
Our technique reduces the production time of self-assembled, cell sheet-derived engineered vascular conduits to 2 weeks, thereby permitting their use as bypass grafts within the clinical time window for elective cardiovascular surgery. Furthermore, our method uses only clinically approved materials and can be adapted to various cell sources, simplifying the path toward future clinical translation.
In vitro assessment of small‐diameter synthetic vascular grafts usually uses standard cell culture conditions with early‐passage cells. However, these conduits are mainly implanted in elderly patients and are subject to complex cellular interactions influenced by age and inflammation. Understanding these factors is central to the development of vascular grafts tailored to the specific needs of patients. In this study, the effects of aged endothelial cells subjected to pro‐ and anti‐inflammatory agents and cultivated on a newly developed biodegradable electrospun thermoplastic polyurethane/poly(urethane‐urea) blend (TPU/TPUU), on clinically available expanded polytetrafluorethylene (ePTFE), and on decellularized extracellular matrix (dECM) grafts were investigated. Young and aged endothelial cells were exposed to pro‐ and anti‐inflammatory agents and characterized by morphology, migration capacity, and gene expression. In addition, the cells were seeded onto the various graft materials and examined microscopically alongside gene expression analyses. When exposed to pro‐inflammatory cytokines, young and aged cells demonstrated signs of endothelial activation. Cells seeded on ePTFE showed reduced attachment and increased expression of pro‐inflammatory genes compared with the other materials. dECM and TPU/TPUU substrates provided better support for endothelialization with aged cells under inflammatory conditions compared with ePTFE. Moreover, TPU/TPUU showed positive effects on reducing pro‐thrombotic and pro‐inflammatory gene expression in endothelial cells. Our results thus emphasize the importance of developing new synthetic graft materials as an alternative for clinically used ePTFE.
Late lung development is a period of alveolar and microvascular formation, which is pivotal in ensuring sufficient and effective gas exchange. Defects in late lung development manifest in premature infants as a chronic lung disease named bronchopulmonary dysplasia (BPD). Numerous studies demonstrated the therapeutic properties of exogenous bone marrow and umbilical cord-derived mesenchymal stromal cells (MSCs) in experimental BPD. However, very little is known regarding the regenerative capacity of resident lung MSCs (L-MSCs) during normal development and in BPD. In this study we aimed to characterize the L-MSC population in homeostasis and upon injury. We used single-cell RNA sequencing (scRNA-seq) to profile in situ Ly6a+ L-MSCs in the lungs of normal and O2-exposed neonatal mice (a well-established model to mimic BPD) at three developmental timepoints (postnatal days 3, 7 and 14). Hyperoxia exposure increased the number, and altered the expression profile of L-MSCs, particularly by increasing the expression of multiple pro-inflammatory, pro-fibrotic, and anti-angiogenic genes. In order to identify potential changes induced in the L-MSCs transcriptome by storage and culture, we profiled 15,000 Ly6a+ L-MSCs after in vitro culture. We observed great differences in expression profiles of in situ and cultured L-MSCs, particularly those derived from healthy lungs. Additionally, we have identified the location of Ly6a+/Col14a1+ L-MSCs in the developing lung and propose Serpinf1 as a novel, culture-stable marker of L-MSCs. Finally, cell communication analysis suggests inflammatory signals from immune and endothelial cells as main drivers of hyperoxia-induced changes in L-MSCs transcriptome.
Late lung development is a period of alveolar and microvascular formation, which is pivotal in ensuring sufficient and effective gas exchange. Defects in late lung development manifest in premature infants as a chronic lung disease named bronchopulmonary dysplasia (BPD). Numerous studies demonstrated the therapeutic properties of exogenous bone marrow and umbilical cord-derived mesenchymal stromal cells (MSCs) in experimental BPD. However, very little is known regarding the regenerative capacity of resident lung MSCs (L-MSCs) during normal development and in BPD. In this study we aimed to characterize the L-MSC population in homeostasis and upon injury. We used single-cell RNA sequencing (scRNA-seq) to profile in situ Ly6a ⁺ L-MSCs in the lungs of normal and O 2 -exposed neonatal mice (a well-established model to mimic BPD) at three developmental timepoints (postnatal days 3, 7 and 14). Hyperoxia exposure increased the number, and altered the expression profile of L-MSCs, particularly by increasing the expression of multiple pro-inflammatory, pro-fibrotic, and anti-angiogenic genes. In order to identify potential changes induced in the L-MSCs transcriptome by storage and culture, we profiled 15,000 Ly6a ⁺ L-MSCs after in vitro culture. We observed great differences in expression profiles of in situ and cultured L-MSCs, particularly those derived from healthy lungs. Additionally, we have identified the location of L-MSCs in the developing lung and propose Serpinf1 as a novel, culture-stable marker of L-MSCs. Finally, cell communication analysis suggests inflammatory signals from immune and endothelial cells as main drivers of hyperoxia-induced changes in L-MSCs transcriptome.
Tissue engineering is an essential component of developing effective regenerative therapies. Here, we introduce a promising method to create scaffold-free three dimensional (3D) tissue engineered multi-layered microstructures from cultured cells using the "3D tissue fabrication system" (Regenova®, Cyfuse, Japan). This technique utilizes the adhesive nature of cells. When cells are cultured in non-adhesive wells, they tend to aggregateand form a spheroidal structure. The advantage of this approach is that cellular components can be mixed into one spheroid, thereby promoting the formation of extracellular matrices, such as collagen and elastin. This system enables one to create a pre-designed 3D structure composed of cultured cells. We found the advantages of this system to be: (1) the length, size, and shape of the structure were designable and highly reproducible because of the computer controlled robotics system, (2) the graftable structure could be created within a reasonable period (8 days), and (3) the constructed tissue did not contain any foreign material, which may avoid the potential issues ofcontamination, biotoxicity, and allergy. The utilization of this robotic system enabled thecreation of a 3D multi-layered microstructure made of cell based spheres with a satisfactory mechanical properties and abundant extracellular matrix during a short period of time. These results suggest that this new technology will represent a promising, attractive, and practical strategy in the field of tissue engineering.
This book is a comprehensive and up-to-date resource on the use of regenerative medicine for the treatment of cardiovascular disease. It provides a much-needed review of the rapid development and evolution of bio-fabrication techniques to engineer cardiovascular tissues as well as their use in clinical settings. The book incorporates recent advances in the biology, biomaterial design, and manufacturing of bioengineered cardiovascular tissue with their clinical applications to bridge the basic sciences to current and future cardiovascular treatment.
The book begins with an examination of state-of-the-art cellular, biomaterial, and macromolecular technologies for the repair and regeneration of diseased heart tissue. It discusses advances in nanotechnology and bioengineering of cardiac microtissues using acoustic assembly. Subsequent chapters explore the clinical applications and translational potential of current technologies such as cardiac patch-based treatments, cell-based regenerative therapies, and injectable hydrogels. The book examines how these methodologies are used to treat a variety of cardiovascular diseases including myocardial infarction, congenital heart disease, and ischemic heart injuries. Finally, the volume concludes with a summary of the most prominent challenges and perspectives on the field of cardiovascular tissue engineering and clinical cardiovascular regenerative medicine.
Cardiovascular Regenerative Medicine is an essential resource for physicians, residents, fellows, and medical students in cardiology and cardiovascular regeneration as well as clinical and basic researchers in bioengineering, nanomaterial and technology, and cardiovascular biology.
Cardiovascular disease remains the leading cause of morbidity and mortality in the United States and worldwide. While improvements in prevention strategies and secondary interventions have led to enhanced survival, many patients still succumb to heart failure. This necessitates the development of novel strategies to target the microvascular dysfunction and malperfusion which lead to progressive functional deterioration. Cardiovascular tissue engineering strategies represent an important class of therapeutics which aim to address the insufficiencies of current management of heart failure and related sequelae. Here, we review the various tissue engineering strategies which address the limitations of current therapies and represent promising adjunct treatments to the current gold standard.
Objective:
The osteogenesis effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) and osteogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs) has been generally acknowledged. However, human bone formation effect of rhBMP-2 loaded calcium phosphate cememts (CPC) combined with allogenic BMSCs is still undefined. This study aimed to investigate how composite osteogenic bone marrow mesenchymal stem cells (BMSCs) and osteoinductive bone morphogenetic protein-2 (BMP-2) influenced the process of bone formation.
Methods:
We chose calcium phosphate cements (cancellous bone granules; cancellous bone granules contained rhBMP-2) to facilitate osteogenesis. The composites of CPC+BMSC and CPC/rhBMP-2+BMSC were implanted respectively into nude mice via subcutaneous implantation and the tooth socket after maxillary canine teeth extraction. MAR analysis, Micro-CT analysis and immunohistochemistry assay were performed in this study to observe and compare by the schedule.
Results:
It was showed that combined with CPC+BMSC or CPC/rhBMP-2+BMSC induced stronger ability of the proliferation of allogenic BMSCs observed by scanning electron microscopy (SEM). Picric acid-fuchsin staining and immunohistochemical analysis illustrated the stronger new bone forming ability of CPC+BMSC group and CPC/rhBMP-2+BMSC group than those of controlled group.
Conclusion:
The combinations of rhBMP2-loaded CPC with allogeneic BMSCs promote new bone tissue formation.
Objective
The angiogenic potential of endothelial progenitor cells (EPCs) may be limited by the absence of their natural biologic foundation, namely smooth muscle pericytes. We hypothesized that joint delivery of EPCs and smooth muscle cells (SMCs) in a novel, totally bone marrow-derived cell sheet will mimic the native architecture of a mature blood vessel and act as an angiogenic construct to limit post infarction ventricular remodeling.
Methods
Primary EPCs and mesenchymal stem cells (MSCs) were isolated from bone marrow of Wistar rats. MSCs were transdifferentiated into SMCs by culture on fibronectin-coated culture dishes. Confluent SMCs topped with confluent EPCs were detached from an Upcell dish to create a SMC-EPC bi-level cell sheet. A rodent model of ischemic cardiomyopathy was then created by ligating the left anterior descending artery. Rats were randomized into three groups: cell sheet transplantation (n=9), no treatment (n=12), or sham surgery control (n=7).
Results
Four weeks post infarction mature vessel density tended to increase in cell sheet-treated animals compared with controls. Cell sheet therapy significantly attenuated the extent of cardiac fibrosis compared with that of the untreated group (untreated vs. cell sheet, 198 degrees (IQR, 151 degrees – 246 degrees) vs. 103 degrees (IQR, 92 degrees -113 degrees) , p=0.04). Furthermore, EPC-SMC cell sheet transplantation attenuated myocardial dysfunction, as evidenced by an increase in LV ejection fraction (untreated vs. cell sheet vs sham, 33.5% (IQR, 27.8%-35.7%) vs. 45.9% (IQR, 43.6%-48.4%) vs. 59.3% (IQR, 58.8%-63.5%), p=0.001) and decreases in LV dimensions.
Conclusions
The bone marrow-derived, spatially arranged SMC-EPC bi-level cell sheet is a novel, multi-lineage cellular therapy obtained from a translationally practical source. Interactions between SMCs and EPCs augment mature neovascularization, limit adverse remodeling, and improve ventricular function after myocardial infarction.
Cell-based therapies are a promising alternative to grafts and organ transplantation for treating tissue loss or damage due to trauma, malfunction, or disease. Over the past two decades, mesenchymal stem cells (MSCs) have attracted much attention as a potential cell population for use in regenerative medicine. While the proliferative capacity and multilineage potential of MSCs provide an opportunity to generate clinically relevant numbers of transplantable cells, their use in tissue regenerative applications has met with relatively limited success to date apart from secreting paracrine-acting factors to modulate the defect microenvironment. Presently, there is significant effort to engineer the biophysical properties of biomaterials to direct MSC differentiation and further expand on the potential of MSCs in tissue engineering, regeneration, and repair. Biomaterials can dictate MSC differentiation by modulating features of the substrate including composition, mechanical properties, porosity, and topography. The purpose of this review is to highlight recent approaches for guiding MSC fate using biomaterials and provide a description of the underlying characteristics that promote differentiation toward a desired phenotype.
A family of shear-thinning hydrogels for injectable encapsulation and long-term delivery (SHIELD) has been designed and synthesized with controlled in situ stiffening properties to regulate the stem cell secretome. The authors demonstrate that SHIELD with an intermediate stiffness (200-400 Pa) could significantly promote the angiogenic potential of human adipose-derived stem cells.
Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.
Objectives:
The longevity of homografts is determined by the activation of the recipients' immune system resulting from allogenic antigen exposition. Fresh decellularized pulmonary homografts (DPH) have shown promising early results in pulmonary valve replacement in children and young adults and could potentially avoid significant activation of the immune system, as more than 99% of the donor DNA is removed during the decellularization process. While the humoral immune response to decellularized allografts has been studied, detailed information on the more significant cellular immune response is currently lacking.
Methods and results:
Peripheral blood samples were obtained from patients undergoing pulmonary valve replacement with DPH before, after, and for approximately 3 years after implantation. Absolute counts and percentages of mature T- (CD3(+)), B- (CD19(+)), and natural killer- (CD16(+)/CD56(+)) cells, as well as T helper- (CD4(+)) and cytotoxic T-cell- (CD8(+)) subsets, were determined by fluorescence-activated cell sorting (FACS). Between May 2009 and September 2013, 199 blood samples taken from 47 patients with a mean age at DPH implantation of 16.6±10.8 years were analyzed. The hemodynamic performance of DPH was excellent in all but one patient, and no valve-related deaths or conduit explantations were observed. The short-term follow up revealed a significant postoperative decrease in cell counts of most subtypes with reconstitution after 3 months. Continued assessment did not show any significant deviations in cell counts from their baseline values.
Conclusion:
The absence of cellular immune response in patients receiving DPH supports the concept that decellularization can provide a basis for autologous regeneration.
Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.
Bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into various types of cell, and the extracellular matrix (ECM) is acknowledged to be important for the regulation of cell functions. In this study, we demonstrated the effects of ECMs on the differentiation of human bone marrow-derived MSCs into a smooth muscle cell (SMC) lineage.
Human MSCs (hMSCs) were cultured on dishes coated with 3 types of ECM including laminin (LM), collagen type IV (Col-IV) and fibronectin for 7 days, and simultaneously cultured on a noncoated dish as a control. Cell numbers of these cultured hMSCs were counted, and their expression of SMC-specific genes and proteins was evaluated. hMSCs were then seeded on LM-coated biodegradable sheets and implanted into rat subcutaneous space. After 2 weeks of implantation, these tissues were evaluated.
The number of hMSCs was significantly increased by culture on Col-IV-coated dishes. The expression of SMC-specific genes and proteins (alpha-smooth muscle actin, ASMA; h1-calponin, CALP) in hMSC was significantly upregulated from culture on LM-coated dishes. LM-coated sheets showed a significantly increased expression of ASMA and CALP protein in vivo. Moreover, a fully differentiated marker (SM2) was expressed in the in vivo implanted hMSCs in the course of 2 weeks on the LM-coated sheet.
These results suggest that the signal transduction of the cell-matrix interaction for the differentiation of hMSCs into SMCs was activated when cultured with LM. LM-coated materials may thus be useful for cardiovascular tissue engineering.
Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as
undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage,
fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated
from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro.
These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages.
Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.
Integrin-initiated extracellular signal-regulated kinase (ERK) activation by matrix adhesion may require focal adhesion kinase (FAK) or be FAK-independent via caveolin and Shc. This remains controversial for fibroblast and endothelial cell adhesion to fibronectin and is less understood for other matrix proteins and cells. We investigated Caco-2 intestinal epithelial cell ERK activation by collagen I and IV, laminin, and fibronectin. Collagens or laminin, but not fibronectin, stimulated tyrosine phosphorylation of FAK, paxillin, and p130(cas) and activated ERK1/2. Shc, tyrosine-phosphorylated by matrix adhesion in many cells, was not phosphorylated in Caco-2 cells in response to any matrix. Caveolin expression did not affect Caco-2 Shc phosphorylation in response to fibronectin. FAK, ERK, and p130(cas) tyrosine phosphorylation were activated after 10-min adhesion to collagen IV. FAK activity increased for 45 min after collagen IV adhesion and persisted for 2 h, while p130(cas) phosphorylation increased only slightly after 10 min. ERK activity peaked at 10 min, declined after 30 min, and returned to base line after 1 h. Transfection with FAK-related nonkinase, but not substrate domain deleted p130(cas), strongly inhibited ERK2 activation in response to collagen IV, indicating Caco-2 ERK activation is at least partly regulated by FAK.
Organ shortage and suboptimal prosthetic or biological materials for repair or replacement of diseased or destroyed human organs and tissues are the main motivation for increasing research in the emerging field of tissue engineering. No organ or tissue is excluded from this multidisciplinary research field, which aims to provide vital tissues with the abilities to function, grow, repair, and remodel. There are several approaches to tissue engineering, including the use of cells, scaffolds, and the combination of the two. The most common approach is biodegradable or resorbable scaffolds configured to the shape of the new tissue (e.g. a heart valve). This scaffold is seeded with cells, potentially derived from either biopsies or stem cells. The seeded cells proliferate, organize, and produce cellular and extracellular matrix. During this matrix formation, the starter matrix is degraded, resorbed, or metabolized. First clinical trials using skin or cartilage substitutes are currently under way. Both the current state of the field and future prospects are discussed.
Cell adhesion to the extracellular matrix inhibits apoptosis, but the molecular mechanisms underlying the signals transduced by different matrix components are not well understood. Here, we examined integrin-mediated antiapoptotic signals from laminin-10/11 in comparison with those from fibronectin, the best characterized extracellular adhesive ligand. We found that the activation of protein kinase B/Akt in cells adhering to laminin-10/11 can rescue cell apoptosis induced by serum removal. Consistent with this, wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, or ectopic expression of a dominant-negative mutant of Akt selectively accelerated cell death upon serum removal. In contrast to laminin-10/11, fibronectin rescued cells from serum depletion-induced apoptosis mainly through the extracellular signal-regulated kinase pathway. Cell survival on fibronectin but not laminin was significantly reduced by treatment with PD98059, a specific inhibitor of mitogen- or extracellular signal-regulated kinase kinase-1 (MEK1) and by expression of a dominant-negative mutant of MEK1. Laminin-10/11 was more potent than fibronectin in preventing apoptosis induced by serum depletion. These results, taken together, demonstrate laminin-10/11 potency as a survival factor and demonstrate that different extracellular matrix components can transduce distinct survival signals through preferential activation of subsets of multiple integrin-mediated signaling pathways.
We report here that cells co-purifying with mesenchymal stem cells--termed here multipotent adult progenitor cells or MAPCs--differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. When injected into an early blastocyst, single MAPCs contribute to most, if not all, somatic cell types. On transplantation into a non-irradiated host, MAPCs engraft and differentiate to the haematopoietic lineage, in addition to the epithelium of liver, lung and gut. Engraftment in the haematopoietic system as well as the gastrointestinal tract is increased when MAPCs are transplanted in a minimally irradiated host. As MAPCs proliferate extensively without obvious senescence or loss of differentiation potential, they may be an ideal cell source for therapy of inherited or degenerative diseases.
Ocular trauma or disease may lead to severe corneal opacification and, consequently, severe loss of vision as a result of complete loss of corneal epithelial stem cells. Transplantation of autologous corneal stem-cell sources is an alternative to allograft transplantation and does not require immunosuppression, but it is not possible in many cases in which bilateral disease produces total corneal stem-cell deficiency in both eyes. We studied the use of autologous oral mucosal epithelial cells as a source of cells for the reconstruction of the corneal surface.
We harvested 3-by-3-mm specimens of oral mucosal tissue from four patients with bilateral total corneal stem-cell deficiencies. Tissue-engineered epithelial-cell sheets were fabricated ex vivo by culturing harvested cells for two weeks on temperature-responsive cell-culture surfaces with 3T3 feeder cells that had been treated with mitomycin C. After conjunctival fibrovascular tissue had been surgically removed from the ocular surface, sheets of cultured autologous cells that had been harvested with a simple reduced-temperature treatment were transplanted directly to the denuded corneal surfaces (one eye of each patient) without sutures.
Complete reepithelialization of the corneal surfaces occurred within one week in all four treated eyes. Corneal transparency was restored and postoperative visual acuity improved remarkably in all four eyes. During a mean follow-up period of 14 months, all corneal surfaces remained transparent. There were no complications.
Sutureless transplantation of carrier-free cell sheets composed of autologous oral mucosal epithelial cells may be used to reconstruct corneal surfaces and can restore vision in patients with bilateral severe disorders of the ocular surface.
Background:
Neuregulin-1β (NRG) is a member of the epidermal growth factor family possessing a critical role in cardiomyocyte development and proliferation. Systemic administration of NRG demonstrated efficacy in cardiomyopathy animal models, leading to clinical trials using daily NRG infusions. This approach is hindered by requiring daily infusions and off-target exposure. Therefore, this study aimed to encapsulate NRG in a hydrogel to be directly delivered to the myocardium, accomplishing sustained localized NRG delivery.
Methods and results:
NRG was encapsulated in hydrogel, and release over 14 days was confirmed by ELISA in vitro. Sprague-Dawley rats were used for cardiomyocyte isolation. Cells were stimulated by PBS, NRG, hydrogel, or NRG-hydrogel (NRG-HG) and evaluated for proliferation. Cardiomyocytes demonstrated EdU (5-ethynyl-2'-deoxyuridine) and phosphorylated histone H3 positivity in the NRG-HG group only. For in vivo studies, 2-month-old mice (n=60) underwent left anterior descending coronary artery ligation and were randomized to the 4 treatment groups mentioned. Only NRG-HG-treated mice demonstrated phosphorylated histone H3 and Ki67 positivity along with decreased caspase-3 activity compared with all controls. NRG was detected in myocardium 6 days after injection without evidence of off-target exposure in NRG-HG animals. At 2 weeks, the NRG-HG group exhibited enhanced left ventricular ejection fraction, decreased left ventricular area, and augmented borderzone thickness.
Conclusions:
Targeted and sustained delivery of NRG directly to the myocardial borderzone augments cardiomyocyte mitotic activity, decreases apoptosis, and greatly enhances left ventricular function in a model of ischemic cardiomyopathy. This novel approach to NRG administration avoids off-target exposure and represents a clinically translatable strategy in myocardial regenerative therapeutics.
Introduction:
Functional skeletal myoblasts (SMBs) are transplanted into the heart effectively and safely as cell sheets, which induce functional recovery in myocardial infarction (MI) patients without lethal arrhythmia. However, their therapeutic effect is limited by ischemia. Mesenchymal stem cells (MSCs) have prosurvival/proliferation and antiapoptotic effects on co-cultured cells in vitro. We hypothesized that adding MSCs to the SMB cell sheets might enhance SMB survival post-transplantation and improve their therapeutic effects.
Methods and results:
Cell sheets of primary SMBs of male Lewis rats (r-SMBs), primary MSCs of human female fat tissues (h-MSCs), and their co-cultures were generated using temperature-responsive dishes. The levels of candidate paracrine factors, rat hepatocyte growth factor and vascular endothelial growth factor, in vitro were significantly greater in the h-MSC/r-SMB co-cultures than in those containing r-SMBs only, by real-time PCR and enzyme-linked immunosorbent assay (ELISA). MI was generated by left-coronary artery occlusion in female athymic nude rats. Two weeks later, co-cultured r-SMB or h-MSC cell sheets were implanted or no treatment was performed (n=10 each). Eight weeks later, systolic and diastolic function parameters were improved in all three treatment groups compared to no treatment, with the greatest improvement in the co-cultured cell sheet transplantation group. Consistent results were found for capillary density, collagen accumulation, myocyte hypertrophy, Akt-signaling, STAT3 signaling, and survival of transplanted cells of rat origin, and were related to poly (ADP-ribose) polymerase-dependent signal transduction.
Conclusions:
Adding MSCs to SMB cell sheets enhanced the sheets' angiogenesis-related paracrine mechanics and, consequently, functional recovery in a rat MI model, suggesting a possible strategy for clinical applications.
Cell-mediated angiogenic therapy for ischemic heart disease has had disappointing results. The lack of clinical translatability may be secondary to cell death and systemic dispersion with cell injection. We propose a novel tissue-engineered therapy, whereby extracellular matrix scaffold seeded with endothelial progenitor cells (EPCs) can overcome these limitations using an environment in which the cells can thrive, enabling an insult-free myocardial cell delivery to normalize myocardial biomechanics.
EPCs were isolated from the long bones of Wistar rat bone marrow. The cells were cultured for 7 days in media or seeded at a density of 5×10(6) cells/cm(2) on a collagen/vitronectin matrix. Seeded EPCs underwent ex vivo modification with stromal cell-derived factor-1α (100 ng/mL) to potentiate angiogenic properties and enhance paracrine qualities before construct formation. Scanning electron microscopy and confocal imaging confirmed EPC-matrix adhesion. In vitro vasculogenic potential was assessed by quantifying EPC cell migration and vascular differentiation. There was a marked increase in vasculogenesis in vitro as measured by angiogenesis assay (8 versus 0 vessels/hpf; P=0.004). The construct was then implanted onto ischemic myocardium in a rat model of acute myocardial infarction. Confocal microscopy demonstrated a significant migration of EPCs from the construct to the myocardium, suggesting a direct angiogenic effect. Myocardial biomechanical properties were uniaxially quantified by elastic modulus at 5% to 20% strain. Myocardial elasticity normalized after implant of our tissue-engineered construct (239 kPa versus normal=193, P=0.1; versus infarct=304 kPa, P=0.01).
We demonstrate restoration and normalization of post-myocardial infarction ventricular biomechanics after therapy with an angiogenic tissue-engineered EPC construct.
Endothelial progenitor cells (EPCs) possess robust therapeutic angiogenic potential, yet may be limited in the capacity to develop into fully mature vasculature. This problem might be exacerbated by the absence of a neovascular foundation, namely pericytes, with simple EPC injection. We hypothesized that coculturing EPCs with smooth muscle cells (SMCs), components of the surrounding vascular wall, in a cell sheet will mimic the native spatial orientation and interaction between EPCs and SMCs to create a supratherapeutic angiogenic construct in a model of ischemic cardiomyopathy.
Primary EPCs and SMCs were isolated from Wistar rats. Confluent SMCs topped with confluent EPCs were spontaneously detached from the Upcell dish to create an SMC-EPC bi-level cell sheet. A rodent ischemic cardiomyopathy model was created by ligating the left anterior descending coronary artery. Rats were then immediately divided into 3 groups: cell-sheet transplantation (n=14), cell injection (n=12), and no treatment (n=13). Cocultured EPCs and SMCs stimulated an abundant release of multiple cytokines in vitro. Increased capillary density and improved blood perfusion in the borderzone elucidated the significant in vivo angiogenic potential of this technology. Most interestingly, however, cell fate-tracking experiments demonstrated that the cell-sheet EPCs and SMCs directly migrated into the myocardium and differentiated into elements of newly formed functional vasculature. The robust angiogenic effect of this cell sheet translated to enhanced ventricular function as demonstrated by echocardiography.
Spatially arranged EPC-SMC bi-level cell-sheet technology facilitated the natural interaction between EPCs and SMCs, thereby creating structurally mature, functional microvasculature in a rodent ischemic cardiomyopathy model, leading to improved myocardial function.
Background:
The implantation of skeletal myoblast (SMB) cell-sheets over the damaged area of a myocardial infarction (MI) has been shown to improve global left ventricular (LV) function through a paracrine effect. However, the regeneration process has not been fully evaluated. We hypothesized that the use of tissue Doppler strain M-mode imaging to assess myocardial layer-specific strain might enable detailed visual evaluation of the regenerative ability of SMBs.
Methods and results:
SMBs were cultured on temperature-responsive culture dishes to generate cell-sheets. At 4 weeks after inducing anterior MI, the animals were divided into 2 groups: SMB cell-sheet implantation and sham operation (n=6 in each). A total of 30 cell-sheets (1.5×10(7) cells/sheet) were placed on the epicardium, covering the infarct and border regions. Subendocardial and subepicardial strain values were measured in the infarct, border, and remote regions by tissue Doppler strain analysis. SMB cell-sheet implantation produced the following major effects: progression of LV remodeling was prevented and global LV ejection fraction increased; the subendocardial strain was significantly greater than the subepicardial strain in the treated border region; vascular density in the subendocardium was significantly higher than in the subepicardium in the treated region; the expression of vascular endothelial growth factor was significantly increased.
Conclusions:
Tissue Doppler strain analysis allows precise evaluation of the effect of cell-sheet implantation on layer-specific myocardial function.
We studied the feasibility of creating new tissue engineered tendons, using bovine tendon fibroblasts (tenocytes) attached to synthetic biodegradable polymer scaffolds in athymic mice. Calffore- and hind-limbs were obtained from a local slaughterhouse within 6 hours of sacrifice. Tenocytes were isolated from the calf tendons. Cells were seeded onto an array of fibers composed of polymer (PGA) configured either as a random mesh of fibers, or as an array of parallel fibers. Fifty cell-polymer constructs were implanted subcutaneously in athymic mice and harvested at 3, 6, 8, 10 and 12 weeks. Grossly, all excised specimens resembled the tendons from which the cells had been isolated. Histologic sections stained with hematoxylin and eosin (H&E) and Masson's trichrome showed cells arranged longitudinally within parallel collagen fibers in the periphery. Centrally, collagen fibers were more randomly arranged, although they seemed to attain a parallel arrangement of cells and fibers over time. By 10 weeks, specimens showed very similar histologic characteristics to normal tendon. Histologically, 12-week samples were virtually identical to normal tendon. When longitudinal polymer fibers seeded with cell had been implanted, the collagen fibers seen in the neo-tendons became organized at an earlier interval of time. Biomechanical tests demonstrated linear increase in tensile strength of the neo-tendons over time. Eight-week specimens showed 30% the tensile strength of normal tendon samples of similar size. By 12 weeks, tensile strength was already 57% that of normal bovine tendon.
The use of esophageal endoscopic submucosal dissection (ESD) to remove superficial esophageal neoplasms is gradually becoming more common in Japan. However, large-scale esophageal ESD often requires subsequent multiple balloon dilations to prevent postoperative esophageal stricture. We investigated the safety and efficacy of endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in preventing formation of strictures after ESD.
We performed an open-label, single-arm, single-institute study. We collected specimens of oral mucosal tissue from 9 patients with superficial esophageal neoplasms. Epithelial cell sheets were fabricated ex vivo by culturing isolated cells for 16 days on temperature-responsive cell culture surfaces. After a reduction in temperature, these sheets were endoscopically transplanted directly to the ulcer surfaces of patients who had just undergone ESD. All patients were monitored by endoscopy once a week until epithelialization was complete.
Autologous cell sheets were successfully transplanted to ulcer surfaces using an endoscope. Complete re-epithelialization occurred within a median time of 3.5 weeks. No patients experienced dysphagia, stricture, or other complications following the procedure, except for one patient who had a full circumferential ulceration that expanded to the esophagogastric junction.
Sutureless, endoscopic transplantation of carrier-free cell sheets composed of autologous oral mucosal epithelial cells safely and effectively promotes re-epithelialization of the esophagus after ESD. Patients in this study did not experience any serious complications. This procedure might be used to prevent stricture formation following ESD and improve patients' quality of life. Further study will be needed to show that stricture formation can be prevented.
Dilated cardiomyopathy (DCM) is a heart muscle disease characterized by progressive heart failure, and is a leading cause of mortality and morbidity. Recently, cellular therapy for end-stage heart failure has been emerging. We herein report a 56-year-old male who received a transplant of autologous myoblast sheets manufactured in temperature-responsive culture dishes. His clinical condition improved markedly, leaving him without any arrhythmia and able to discontinue using a left ventricular assist system and avoid cardiac transplantation. These findings suggest that cellular therapy using myoblast sheets is a promising new strategy for treating patients with end-stage DCM. This method might be an effective alternative to heart transplantation in the near future.
Poly(N-isopropyl acrylamide) (PIPAAm) demonstrated a fully expanded chain conformation below 32 degrees C and a collapsed, compact conformation at high temperatures. This unique temperature responsive polymer was grafted onto surfaces of commercial polystyrene dishes and used as temperature switches for creating hydrophilic surfaces below 32 degrees C and hydrophobic surfaces above 32 degrees C. Cell attachment and the growth of bovine endothelial cells and rat hepatocytes on PIPAAm-grafted surfaces at 37 degrees C demonstrated similar behavior to the commercialized culture dishes. Both cell types were observed to detach from the PIPAAm-grafted surface simply by reducing the temperature below the polymer transition temperature (collapse). Cells recovered by this method maintained substrate adhesivity, growth, and secretion activities nearly identical to those found in primary cultured cells in contrast to the compromised function found in cultured cells damaged by trypsinization. These results provide strong evidence that PIPAAm-grafted surfaces, as thermal switches are very effective for reversing cell attachment and detachment without cell damage. Properties of cell culture surfaces can be readily transformed by this technique reversibly into hydrophilic and hydrophobic coatings of PIPAAm-grafted polymers.
The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in human health care.
A new field, tissue engineering, applies the principles of biology and engineering to the development of functional substitutes
for damaged tissue. This article discusses the foundations and challenges of this interdisciplinary field and its attempts
to provide solutions to tissue creation and repair.
Marrow stromal cells can be isolated from other cells in marrow by their tendency to adhere to tissue culture plastic. The
cells have many of the characteristics of stem cells for tissues that can roughly be defined as mesenchymal, because they
can be differentiated in culture into osteoblasts, chondrocytes, adipocytes, and even myoblasts. Therefore, marrow stromal
cells present an intriguing model for examining the differentiation of stem cells. Also, they have several characteristics
that make them potentially useful for cell and gene therapy.
The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.
- Je Cohen
- Bp Purcell
- Jw Macarthur
- Jr
- A Mu
- Y Shudo
- Jb Patel
Cohen JE, Purcell BP, MacArthur JW Jr, Mu A, Shudo Y,
Patel JB, et al. Circ Heart Fail 2014;7:619–26.
Metre-long cell-laden microfibers exhibit tissue morphologies and functions
- H Onoe
- T Okitsu
- A Itoh
- M Kato-Negishi
- R Gojo
- D Kiriya
Onoe H, Okitsu T, Itoh A, Kato-Negishi M, Gojo R, Kiriya
D, et al. Metre-long cell-laden microfibers exhibit tissue
morphologies and functions. Nat Mater 2013;12:584–90.
- J E Cohen
- B P Purcell
- J W Macarthur
- A Mu
- Y Shudo
- J B Patel
Cohen JE, Purcell BP, MacArthur JW Jr, Mu A, Shudo Y,
Patel JB, et al. Circ Heart Fail 2014;7:619-26.