Human iPS-derived pre-epicardial cells direct cardiomyocyte aggregation expansion and organization in vitro.

Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro.

Ex Vivo Modeling of Perioperative Air Leaks in Porcine Lungs.

OBJECTIVE: A novel ex vivo model is described to advance the understanding of prolonged air leaks, one of the most common postoperative complications following thoracic resection procedures. METHODS: As an alternative to in vivo testing, an ex vivo model simulating the various physiologic environments experienced by an isolated lung during the perioperative period was designed and built. Isolated porcine lungs were perfused and ventilated during open chest and closed chest simulations, mimicking intra and postoperative ventilation conditions. To assess and validate system capabilities, nine porcine lungs were tested by creating a standardized injury to create an approximately 250 cc/min air leak. Air leak rates, physiologic ventilation, and perfusion parameters were continuously monitored, while gas transfer analysis was performed on selected lungs. Segmental ventilation was monitored using electrical impedance tomography. RESULTS: The evaluated lungs produced flow-volume and pressure-volume loops that approximated standard clinical representations under positive (mechanical) and negative (physiological) pressure ventilation modalities. Leak rate was averaged across the ventilation phases, and sharp increases in leak rate were observed between positive and negative pressure phases, suggesting that differences or changes in ventilation mechanics may strongly influence leak development. CONCLUSION: The successful design and validation of a novel ex vivo lung model was achieved. Model output paralleled clinical observations. Pressure modality may also play a significant role in air leak severity. SIGNIFICANCE: This work provides a foundation for future studies aimed at increasing the understanding of air leaks to better inform means of mitigating the risk of air leaks under clinically relevant conditions.

Development of a large-volume human-derived adipose acellular allogenic flap by perfusion decellularization.

In reconstructive surgery, transfer of patients' tissue (autologous flaps) is routinely used to repair large soft tissue defects caused by surgery, trauma, chronic diseases, or malformations; unfortunately, this strategy is not always possible and often creates a secondary defect in the donor site of the tissue. Tissue-engineered synthetic flaps are currently unable to repair clinically-relevant, large-volume defects; allogenic flaps from cadaveric donors could provide a ready-to-use biological alternative if treated with methods to avoid the immune-rejection of the donor's cells. Here, we describe the successful decellularization of a large (> 800 cc) human-derived adipose flap through a perfusion apparatus; we demonstrate the complete removal of the immunogenic cellular components of the flap with the retention of its structural components and vascular network. Our aim is to obtain a universally compatible, off-the-shelf acellular allogenic flap that could be recellularized with cells from recipient patients to provide a tissue-engineered allogenic/autologous alternative for reconstruction of large-volume soft-tissue defects.

Creation of a Bioengineered Skin Flap Scaffold with a Perfusable Vascular Pedicle.

Full-thickness skin loss is a challenging problem due to limited reconstructive options, demanding 75 million surgical procedures annually in the United States. Autologous skin grafting is the gold standard treatment, but results in donor-site morbidity and poor aesthetics. Numerous skin substitutes are available on the market to date, however, none truly functions as full-thickness skin due to lack of a vascular network. The creation of an autologous full-thickness skin analogue with a vascular pedicle would result in a paradigm shift in the management of wounds and in reconstruction of full-thickness skin defects. To create a clinically relevant foundation, we generated an acellular skin flap scaffold (SFS) with a perfusable vascular pedicle of clinically relevant size by perfusion decellularization of porcine fasciocutaneous flaps. We then analyzed the yielded SFS for mechanical properties, biocompatibility, and regenerative potential in vitro and in vivo. Furthermore, we assessed the immunological response using an in vivo model. Finally, we recellularized the vascular compartment of an SFS and reconnected it to a recipient's blood supply to test for perfusability. Perfusion decellularization removed all cellular components with preservation of native extracellular matrix composition and architecture. Biaxial testing revealed preserved mechanical properties. Immunologic response and biocompatibility assessed via implantation and compared with native xenogenic skin and commercially available dermal substitutes revealed rapid neovascularization and complete tissue integration. Composition of infiltrating immune cells showed no evidence of allorejection and resembled the inflammatory phase of wound healing. Implantation into full-thickness skin defects demonstrated good tissue integration and skin regeneration without cicatrization. We have developed a protocol for the generation of an SFS of clinically relevant size, containing a vascular pedicle, which can be utilized for perfusion decellularization and, ultimately, anastomosis to the recipient vascular system after precellularization. The observed favorable immunological response and good tissue integration indicate the substantial regenerative potential of this platform.

Delivering stem cells to the healthy heart on biological sutures: effects on regional mechanical function.

Current cardiac cell therapies cannot effectively target and retain cells in a specific area of the heart. Cell-seeded biological sutures were previously developed to overcome this limitation, demonstrating targeted delivery with > 60% cell retention. In this study, both cell-seeded and non-seeded fibrin-based biological sutures were implanted into normal functioning rat hearts to determine the effects on mechanical function and fibrotic response. Human mesenchymal stem cells (hMSCs) were used based on previous work and established cardioprotective effects. Non-seeded or hMSC-seeded sutures were implanted into healthy athymic rat hearts. Before cell seeding, hMSCs were passively loaded with quantum dot nanoparticles. One week after implantation, regional stroke work index and systolic area of contraction (SAC) were evaluated on the epicardial surface above the suture. Cell delivery and retention were confirmed by quantum dot tracking, and the fibrotic tissue area was evaluated. Non-seeded biological sutures decreased SAC near the suture from 0.20 ± 0.01 measured in sham hearts to 0.08 ± 0.02, whereas hMSC-seeded biological sutures dampened the decrease in SAC (0.15 ± 0.02). Non-seeded sutures also displayed a small amount of fibrosis around the sutures (1.0 ± 0.1 mm2 ). Sutures seeded with hMSCs displayed a significant reduction in fibrosis (0.5 ± 0.1 mm2 , p < 0.001), with quantum dot-labelled hMSCs found along the suture track. These results show that the addition of hMSCs attenuates the fibrotic response observed with non-seeded sutures, leading to improved regional mechanics of the implantation region. Copyright © 2014 John Wiley & Sons, Ltd.

Functional Effects of Delivering Human Mesenchymal Stem Cell-Seeded Biological Sutures to an Infarcted Heart.

Stem cell therapy has the potential to improve cardiac function after myocardial infarction (MI); however, existing methods to deliver cells to the myocardium, including intramyocardial injection, suffer from low engraftment rates. In this study, we used a rat model of acute MI to assess the effects of human mesenchymal stem cell (hMSC)-seeded fibrin biological sutures on cardiac function at 1 week after implant. Biological sutures were seeded with quantum dot (Qdot)-loaded hMSCs for 24 h before implantation. At 1 week postinfarct, the heart was imaged to assess mechanical function in the infarct region. Regional parameters assessed were regional stroke work (RSW) and systolic area of contraction (SAC) and global parameters derived from the pressure waveform. MI (n = 6) significantly decreased RSW (0.026 ± 0.011) and SAC (0.022 ± 0.015) when compared with sham operation (RSW: 0.141 ± 0.009; SAC: 0.166 ± 0.005, n = 6) (p < 0.05). The delivery of unseeded biological sutures to the infarcted hearts did not change regional mechanical function compared with the infarcted hearts (RSW: 0.032 ± 0.004, SAC: 0.037 ± 0.008, n = 6). The delivery of hMSC-seeded sutures exerted a trend toward increase of regional mechanical function compared with the infarcted heart (RSW: 0.057 ± 0.011; SAC: 0.051 ± 0.014, n = 6). Global function showed no significant differences between any group (p > 0.05); however, there was a trend toward improved function with the addition of either unseeded or seeded biological suture. Histology demonstrated that Qdot-loaded hMSCs remained present in the infarcted myocardium after 1 week. Analysis of serial sections of Masson's trichrome staining revealed that the greatest infarct size was in the infarct group (7.0% ± 2.2%), where unseeded (3.8% ± 0.6%) and hMSC-seeded (3.7% ± 0.8%) suture groups maintained similar infarct sizes. Furthermore, the remaining suture area was significantly decreased in the unseeded group compared with that in the hMSC-seeded group (p < 0.05). This study demonstrated that hMSC-seeded biological sutures are a method to deliver cells to the infarcted myocardium and have treatment potential.

Bioengineering Human Myocardium on Native Extracellular Matrix.

RATIONALE: More than 25 million individuals have heart failure worldwide, with ≈4000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only ≈2500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. OBJECTIVE: The objective of this study is to translate previous work to human scale and clinically relevant cells for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human induced pluripotent stem cell-derived cardiomyocytes. METHODS AND RESULTS: To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiomyocytes derived from nontransgenic human induced pluripotent stem cells and generated tissues of increasing 3-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole-heart scaffolds with human induced pluripotent stem cell-derived cardiomyocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue and showed electrical conductivity, left ventricular pressure development, and metabolic function. CONCLUSIONS: Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human induced pluripotent stem cell-derived cardiomyocytes and enable the bioengineering of functional human myocardial-like tissue of multiple complexities.

Engineered composite tissue as a bioartificial limb graft.

The loss of an extremity is a disastrous injury with tremendous impact on a patient's life. Current mechanical prostheses are technically highly sophisticated, but only partially replace physiologic function and aesthetic appearance. As a biologic alternative, approximately 70 patients have undergone allogeneic hand transplantation to date worldwide. While outcomes are favorable, risks and side effects of transplantation and long-term immunosuppression pose a significant ethical dilemma. An autologous, bio-artificial graft based on native extracellular matrix and patient derived cells could be produced on demand and would not require immunosuppression after transplantation. To create such a graft, we decellularized rat and primate forearms by detergent perfusion and yielded acellular scaffolds with preserved composite architecture. We then repopulated muscle and vasculature with cells of appropriate phenotypes, and matured the composite tissue in a perfusion bioreactor under electrical stimulation in vitro. After confirmation of composite tissue formation, we transplanted the resulting bio-composite grafts to confirm perfusion in vivo.

Enhanced lung epithelial specification of human induced pluripotent stem cells on decellularized lung matrix.

BACKGROUND: Whole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs). METHODS: Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm, anteriorized to a foregut fate, and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs, which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position. RESULTS: Activin-A treatment, followed by transforming growth factor-ß inhibition, induced differentiation of human iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2), SRY (Sex Determining Region Y)-Box 17 (SOX17), and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after 5 days. Cells expressing Nkx2.1 were identified at 40% to 60% efficiency. Within whole-lung scaffolds and under perfusion culture, cells further upregulated Nkx2.1 expression. After orthotopic transplantation, grafts were perfused and ventilated by host vasculature and airways. CONCLUSIONS: Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further in vivo graft maturation.

Perfusion decellularization of whole organs.

The native extracellular matrix (ECM) outlines the architecture of organs and tissues. It provides a unique niche of composition and form, which serves as a foundational scaffold that supports organ-specific cell types and enables normal organ function. Here we describe a standard process for pressure-controlled perfusion decellularization of whole organs for generating acellular 3D scaffolds with preserved ECM protein content, architecture and perfusable vascular conduits. By applying antegrade perfusion of detergents and subsequent washes to arterial vasculature at low physiological pressures, successful decellularization of complex organs (i.e., hearts, lungs and kidneys) can be performed. By using appropriate modifications, pressure-controlled perfusion decellularization can be achieved in small-animal experimental models (rat organs, 4-5 d) and scaled to clinically relevant models (porcine and human organs, 12-14 d). Combining the unique structural and biochemical properties of native acellular scaffolds with subsequent recellularization techniques offers a novel platform for organ engineering and regeneration, for experimentation ex vivo and potential clinical application in vivo.

Perfusion decellularization of human and porcine lungs: bringing the matrix to clinical scale.

BACKGROUND: Organ engineering is a theoretical alternative to allotransplantation for end-stage organ failure. Whole-organ scaffolds can be created by detergent perfusion via the native vasculature, generating an acellular matrix suitable for recellularization with selected cell types. We aimed to up-scale this process, generating biocompatible scaffolds of a clinically relevant scale. METHODS: Rat, porcine, and human lungs were decellularized by detergent perfusion at constant pressures. Collagen, elastin, and glycosaminoglycan content of scaffolds were quantified by colorimetric assays. Proteomic analysis was performed by microcapillary liquid chromatography tandem mass spectrometry. Extracellular matrix (ECM) slices were cultured with human umbilical vein endothelial cells (HUVEC), small airway epithelial cells (SAEC), or pulmonary alveolar epithelial cells (PAECs) and evaluated by time-lapse live cell microscopy and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Whole-organ culture was maintained under constant-pressure media perfusion after seeding with PAECs. RESULTS: Rat lungs were decellularized using: (1) sodium dodecyl sulfate (SDS), (2) sodium deoxycholate (SDC), or (3) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Resulting scaffolds showed comparable loss of DNA but greatest preservation of ECM components in SDS-decellularized lungs. Porcine (n = 10) and human (n = 7) lungs required increased SDS concentration, perfusion pressures, and time to achieve decellularization as determined by loss of DNA, with preservation of intact matrix composition and lung architecture. Proteomic analysis of human decellularized lungs further confirmed ECM preservation. Recellularization experiments confirmed scaffold biocompatibility when cultured with mature cell phenotypes and scaffold integrity for the duration of biomimetic culture. CONCLUSIONS: SDS-based perfusion decellularization can be applied to whole porcine and human lungs to generate biocompatible organ scaffolds with preserved ECM composition and architecture.

Regeneration and experimental orthotopic transplantation of a bioengineered kidney.

Approximately 100,000 individuals in the United States currently await kidney transplantation, and 400,000 individuals live with end-stage kidney disease requiring hemodialysis. The creation of a transplantable graft to permanently replace kidney function would address donor organ shortage and the morbidity associated with immunosuppression. Such a bioengineered graft must have the kidney's architecture and function and permit perfusion, filtration, secretion, absorption and drainage of urine. We decellularized rat, porcine and human kidneys by detergent perfusion, yielding acellular scaffolds with vascular, cortical and medullary architecture, a collecting system and ureters. To regenerate functional tissue, we seeded rat kidney scaffolds with epithelial and endothelial cells and perfused these cell-seeded constructs in a whole-organ bioreactor. The resulting grafts produced rudimentary urine in vitro when perfused through their intrinsic vascular bed. When transplanted in an orthotopic position in rat, the grafts were perfused by the recipient's circulation and produced urine through the ureteral conduit in vivo.

A novel suture-based method for efficient transplantation of stem cells.

Advances in regenerative medicine have improved the potential of using cellular therapy for treating several diseases. However, the effectiveness of new cellular therapies is largely limited by low cell engraftment and inadequate localization. To improve on these limitations, we developed a novel delivery mechanism using cell-seeded biological sutures. We demonstrate the ability of cell-seeded biological sutures to efficiently implant human mesenchymal stem cells (hMSCs) to specific regions within the beating heart; a tissue known to have low cell retention and engraftment shortly after delivery. Cell-seeded biological sutures were developed by bundling discrete microthreads extruded from extracellular matrix proteins, attaching a surgical needle to the bundle and seeding the bundle with hMSCs. During cell preparation, hMSCs were loaded with quantum dot nanoparticles for cell tracking within the myocardium. Each biological suture contained an average of 5903 ± 1966 hMSCs/cm suture length. Delivery efficiency was evaluated by comparing cell-seeded biological suture implantation with intramyocardial (IM) cell injections (10,000 hMSCs in 35 µL) into the left ventricle of normal, noninfarcted rat hearts after 1 h. Delivery efficiency of hMSCs by biological sutures (63.6 ± 10.6%) was significantly higher than IM injection (11.8 ± 6.2%; p < 0.05). Cell-tracking analysis indicated suture-delivered hMSCs were found throughout the thickness of the ventricular myocardium: along the entire length of the biological suture track, localizing closely with native myocardium. These results suggest cell-seeded biological sutures can deliver cells to the heart more efficiently than conventional methods, demonstrating an effective delivery method for implanting cells in soft tissue.

Fibrin microthreads support mesenchymal stem cell growth while maintaining differentiation potential.

We developed a method to produce discrete fibrin microthreads, which can be seeded with human mesenchymal stem cells (hMSCs) and used as a suture to enhance the efficiency and localization of cell delivery. To assess the efficacy of fibrin microthreads to support hMSC attachment, proliferation, and survival, microthreads (100 µm diameter per microthread) were bundled together, seeded with 50,000 hMSCs for 2 h, and cultured for 5 days. Cell density on microthread bundles increased over time in culture to a maximum average density of 731 ± 101 cells/mm(2) after 5 days. A LIVE/DEAD assay confirmed that the cells were viable, and Ki-67 staining verified hMSC proliferation. In addition, functional differentiation assays demonstrated that hMSCs cultured on microthreads retained their ability to differentiate into adipocytes and osteocytes. The results of this study demonstrate that fibrin microthreads support hMSC viability and proliferation, while maintaining their multipotency. We anticipate that these cell-seeded fibrin microthreads will serve as a platform technology to improve localized delivery and engraftment of viable cells to damaged tissue.

Strategies for regeneration of heart muscle.

Regenerative medicine has emerged to the forefront of cardiac research, marrying discoveries in both basic science and engineering to develop viable therapeutic approaches for treating the diseased heart. Signifi cant advancements in gene therapy, stem cell biology, and cardiomyoplasty provide new optimism for regenerating damaged myocardium. Exciting new strategies for endogenous and exogenous regeneration have been proposed. However, questions remain as to whether these approaches can provide enough new myocyte mass to sufficiently restore mechanical function to the heart. In this article, we consider the mechanisms of endogenous cardiomyocyte regeneration and exogenous cell differentiation (with respect to myoblasts, stem cells, and induced pluripotent cells being researched for cell therapies). We begin by reviewing some of the cues that are being harnessed in strategies of gene/cell therapy for regenerating myocardium. We also consider some of the technical challenges that remain in determining new myocyte generation, tracking delivered cells in vivo, and correlating new myocyte contractility with cardiac function. Strategies for regenerating the heart are being realized as both animal and clinical trials suggest that these new approaches provide short-term improvement of cardiac function. However, a more complete understanding of the underlying mechanisms and applications is necessary to sustain longer-term therapeutic success.