This issue of ASAIO Journal presents three original research articles highlighting various approaches and stages of investigation in the development of cell-based therapies. Kocyildirim et al.1 describe intratracheal delivery of bone marrow-derived stromal stem cells (B-MSC) applied in a relatively nonspecific manner to decrease the inflammatory state induced by acute respiratory distress syndrome (ARDS), and further exacerbated by venovenous extracorporeal membrane oxygenation (VV ECMO). In contrast, Pino et al.2 and Maymand et al.3 target organ-specific cell types, optimizing the design and manufacturing of an extracorporeal circuit lined with adult renal cells, and inducing differentiation of induced pluripotent stem cells (iPSC) into hepatocyte-like cells within a novel three-dimensional (3D) culture environment, respectively. These studies highlight the multitude of potential applications for cell-based therapies. Ultimately, these studies are major advancements in the field of cell-based therapies, but stop shy of a fully functional treatment for clinical use. The burden lays on the medical and scientific community to translate the new techniques and technologies presented here into actionable solutions for many challenging clinical situations.
Kocyildirim’s group tackles the dilemma of treating patients with ARDS, an entity with high morbidity and mortality and a treatment option, VV ECMO, which can effectively enhance oxygenation after failure of conventional ventilation strategies, but simultaneously worsens the systemic inflammation which causes significant collateral damage to other organ systems. In their original article, the authors explore the use of B-MSCs as an immune modulating adjunct to ECMO.1 The authors base their work on prior studies of the anti-inflammatory effects of B-MSCs in various in vivo and ex vivo models of lung injury which posit contributions from a paracrine mechanism of action, given robust responses despite limited cell engraftment.4,5 Other mechanisms include upregulation of anti-inflammatory cytokines, including IL-10, and suppression of proinflammatory cytokines IL-6, IL-1B, and IL-8. Studies have also revealed a primary protective effect of B-MSCs on endothelial integrity, thought to be mediated through keratinocyte growth factor.6
Given this evidence, B-MSCs demonstrate the potential to mitigate both the pathophysiologic derangements in ARDS, as well as the negative side effects of ECMO. The study was limited to a very acute window—the first 6 hours after acute lung injury with endotoxin exposure—and although this was insufficient time to demonstrate improvements in partial pressure of oxygen (PO2) between ECMO alone and ECMO + B-MSCs, it did confirm that B-MSC treatment may suppress the early inflammatory response based on histopathologic analysis and neutrophil response. Interestingly, despite directed intratracheal delivery of B-MSCs into the left lower lobe of sheep, bilateral lungs in the ECMO + B-MSCs group demonstrated less inflammatory and hemorrhagic change as compared with the control and ECMO alone groups, providing further evidence of paracrine and systemic immunomodulatory effects. The ECMO alone group had lower PO2 at the study end as compared with the control group, suggesting that cannulation in the acute phases of evolving lung injury may cause more harm than benefit.
Overall, this study presents many promising results and raises important questions to further elucidate the optimal management strategy for ARDS patients undergoing dual modality treatment with ECMO and B-MSCs. Longer studies are needed to fully understand the evolution of disease and recovery. In the phase I clinical trial studying B-MSC therapy in ARDS patients, the mean duration of mechanical ventilation was 11.8 days, demonstrating the prolonged time course required for restoration of pulmonary function.7 The protracted clinical course of some patients with ARDS also necessitates investigations of the durability of B-MSC effects and the utility of additional doses throughout the treatment course.
Pino et al.2 take a different approach to addressing acute, reversible end organ injury and present an ambitious effort to mass-produce a renal cell-based extracorporeal circuit for use in the treatment of acute renal injury. Much like the putative paracrine effects associated with B-MSCs, renal cell therapy is thought to replace the metabolic, endocrine, and immunologic signaling functions of the native kidneys that cannot be reproduced by traditional hemodialysis. In this way, renal cell therapy can be employed concurrently with hemodialysis as a means of modulating extrarenal organ injury.8 The bioartificial renal epithelial cell system (BREC) overcomes many of the major obstacles impeding the implementation of cell therapies for organ replacement including biocompatibility of scaffolding materials, immune-incompatibility necessitating immunosuppressive drug regimens, metabolic and circulatory integration, full mimetic functionality, and storage and distribution difficulties.
The BREC is designed to treat acute kidney injury (AKI) through supplemental renal cell functionality via an extracorporeal circuit. Due to specialized filters, the BREC is immune-isolated and thus avoids many of the issues generally associated with allotransplantation and artificial organs. The device serves as its own cell culture system within which RECs are grown on niobium-coated carbon disks. The researchers capitalize on the ability to use isolated cells and use human RECs from kidneys ineligible for transplantation due to structural defects. Of great interest, is that the device could potentially be used with any adherent cells that can be cultured in isolation or simple coculture, tolerate low-shear perfusion, and have metabolic or regulatory functions that can be harnessed within an extracorporeal unit.8 To address the remaining barriers to implementation of the BRECs device in clinical practice related to availability and production, Pino et al.2 describe the injection molding (IM) BREC device, which exhibits substantial improvements compared with previously described stereolithography (SLA) and computer numerical control (CNC) BREC devices in terms of manufacturing speed, cost of production, and freeze–thaw times.
These advancements significantly enhance the clinical applicability of the IM-BREC, reducing not only its size but also the cost of production of the bioreactor by nearly 3000%. Design improvements also dramatically increased heat transfer efficiency, resulting in uniform freezing and thawing, and reducing thaw times to as little as 10 minutes with more than 90% cell viability. In a preclinical porcine model of AKI caused by septic shock, the IM-BREC had improved therapeutic efficacy as measured by cardiovascular performance and survival time compared with acellular control devices–40% vs. 0% survival at 16 hours, respectively. Interestingly, the IM-BREC device was able to provide some degree of immunomodulation, as evidenced through alterations in systemic cytokine levels.9
Despite these advances, several questions remain. Cell viability under the prolonged metabolic and inflammatory stress present in AKI remains unclear. Clinical trials to study the impact of the IM-BREC on morbidity and mortality related to AKI are needed, and characterization of the durability and longevity of the cells within the device will define the potential for long-term renal replacement therapy. Currently, the BREC is optimized for one cell type; the ability to produce and maintain heterogeneous cell populations which mirror those found in the native kidney could further augment the therapeutic capacity of the device.
Expanding on the theme of cell-based, functional organ replacement therapies, Maymand et al.3 describe a novel method of inducing differentiation of human iPSC to hepatocyte-like cells on a collagen-coated polyethersulfone-based nanofibrous 3D scaffold (PES/COL). Cell therapies using patient-derived iPSCs are an attractive option for the treatment of end-stage liver disease (ESLD), given that currently, the only definitive treatment is liver transplantation, which is limited by donor scarcity, immune rejection, and high morbidity. Evaluating the efficiency of differentiating iPSCs to hepatocyte-like cells and maintenance of hepatocyte function with different culture techniques is critically important for the ongoing development of bioengineered hepatic replacement therapies.
Maymand et al.3 report that iPSCs can be effectively and efficiently differentiated into hepatocyte-like cells when grown within a 3D PES/COL nanofibrous scaffold, thus introducing a promising new method of liver tissue engineering. Induced pluripotent stem cells have previously been shown to differentiate into hepatocyte-like cells when grown on Matrigel matrix, a well-established ECM–scaffold complex.10,11 However, Matrigel is derived from Engelbreth-Holm-Swarm mouse sarcoma cells, with shortcomings related to batch-to-batch variability, availability, composition, and tumorigenic origin. In contrast, PES is a bioengineered, biocompatible polymer shown to support proliferation and differentiation of pluripotent cells. Increased surface area and porosity of the nanofibrous scaffold enhance the transfer of nutrients and diffusion of oxygen and other metabolites, which is especially significant given the high metabolic demand of hepatocyte-like cells.12 Traditional 2D culture of primary or iPSC-derived hepatocytes are limited by dedifferentiation and loss of hepatocyte function within days. In addition, hydrogel-embedded 3D culture is characterized by poor nutrient distribution and difficulty in cell retrieval, thus limiting its therapeutic translatability. Among 3D culture methods, PES/COL scaffold-supported 3D culture may prove to be a promising system, given its potential to promote both differentiation of iPSCs and maintenance of a hepatocyte-like phenotype.13 The authors propose the use of autologous iPSCs which obviate concerns of immunogenicity. Furthermore, the use of electrospinning allows for tunable generation of nanofibrous scaffolds with well-defined, reproducible parameters.
Future experiments to fully characterize the functionality of iPSC-derived hepatocyte-like cells are necessary to ascertain which components of liver function can effectively be replaced by cellular therapy. Evaluating glycogen production and storage, uptake of low-density lipoprotein, CYP450 metabolic activity, and urea production can more definitively confirm the functional competence of these PES/COL-supported cells. Additional studies are also needed to test the durability of hepatocyte-like cells in the hostile physiologic conditions resulting from liver failure. Characterization of phenotypes of these hepatocyte-like cells will also be revealing in understanding if a homogenous versus diverse population of cells is necessary to effectively mimic the intrinsic functions of a healthy liver.
As an alternative to whole organ allotransplantation or autologous cell transplantation, PES/COL nanofibrous scaffolds could potentially be adapted for use within a bioartificial liver construct. Over the past 20 years, at least nine different extracorporeal liver assistance devices have been in clinical trials.14 Recent publications have shown successful cryopreservation of hepatocytes in 3D culture with maintenance of viability and functionality.13 This introduces the possibility of applying PES/COL-based scaffolds seeded with primary or iPSC-derived hepatocytes as a component in the development of novel bioartificial liver designs, with a potentially great impact on patients awaiting liver transplantation.
Overall, these three original studies have the potential to help overcome numerous challenges that have hindered the field of cell-based therapy and organ bioengineering. From the data presented, one can conceive of future multimodality therapeutics that combine the systemic immune modulating effects of B-MSCs, delivered in the acute stages of shock, followed by specific organ system support via bioartificial organ platforms such as the BREC, which could potentially be modified to support renal epithelial or iPSC-derived hepatocyte-like cells. In addition, of great significance is the authors’ identification and application of abundant cell sources—from otherwise unusable adult kidneys to indefinitely proliferating iPSCs—rendering obsolete the constraints of limited supply associated with whole organ allotransplantation. It is also conceivable that the PES/COL scaffold could be optimized to support differentiation of RECs from iPSCs, thus completely eliminating reliance on human organ supply. Currently, IM-BREC devices can piggyback on hemofiltration circuits as an adjunct to conventional dialysis. Further modifications to allow connection to ECMO circuitry could leverage the bioreactor’s metabolic, regulatory, and potential paracrine functions in the support of critically ill patients. The innovations in design, delivery, cell culture, and manufacturing of cell-based therapies presented by these three pioneering groups can be synergized to advance the field of cell-based therapy and bioengineered tissues closer to high-impact, deliverable clinical therapies.
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