Sean V. Murphy

3D bioprinting of tissues and organs

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.

Predicting the Functional Effect of Amino Acid Substitutions and Indels

As next-generation sequencing projects generate massive genome-wide sequence variation data, bioinformatics tools are being developed to provide computational predictions on the functional effects of sequence variations and narrow down the search of casual variants for disease phenotypes. Different classes of sequence variations at the nucleotide level are involved in human diseases, including substitutions, insertions, deletions, frameshifts, and non-sense mutations. Frameshifts and non-sense mutations are likely to cause a negative effect on protein function. Existing prediction tools primarily focus on studying the deleterious effects of single amino acid substitutions through examining amino acid conservation at the position of interest among related sequences, an approach that is not directly applicable to insertions or deletions. Here, we introduce a versatile alignment-based score as a new metric to predict the damaging effects of variations not limited to single amino acid substitutions but also in-frame insertions, deletions, and multiple amino acid substitutions. This alignment-based score measures the change in sequence similarity of a query sequence to a protein sequence homolog before and after the introduction of an amino acid variation to the query sequence. Our results showed that the scoring scheme performs well in separating disease-associated variants (n = 21,662) from common polymorphisms (n = 37,022) for UniProt human protein variations, and also in separating deleterious variants (n = 15,179) from neutral variants (n = 17,891) for UniProt non-human protein variations. In our approach, the area under the receiver operating characteristic curve (AUC) for the human and non-human protein variation datasets is ∼0.85. We also observed that the alignment-based score correlates with the deleteriousness of a sequence variation. In summary, we have developed a new algorithm, PROVEAN (Protein Variation Effect Analyzer), which provides a generalized approach to predict the functional effects of protein sequence variations including single or multiple amino acid substitutions, and in-frame insertions and deletions. The PROVEAN tool is available online at http://provean.jcvi.org.

Evaluation of hydrogels for bio-printing applications.

In the United States alone, there are approximately 500,000 burn injuries that require medical treatment every year. Limitations of current treatments necessitate the development of new methods that can be applied quicker, result in faster wound regeneration, and yield skin that is cosmetically similar to undamaged skin. The development of new hydrogel biomaterials and bioprinting deposition technologies has provided a platform to address this need. Herein we evaluated characteristics of twelve hydrogels to determine their suitability for bioprinting applications. We chose hydrogels that are either commercially available, or are commonly used for research purposes. We evaluated specific hydrogel properties relevant to bioprinting applications, specifically; gelation time, swelling or contraction, stability, biocompatibility and printability. Further, we described regulatory, commercial and financial aspects of each of the hydrogels. While many of the hydrogels screened may exhibit characteristics suitable for other applications, UV-crosslinked Extracel, a hyaluronic acid-based hydrogel, had many of the desired properties for our bioprinting application. Taken together with commercial availability, shelf life, potential for regulatory approval and ease of use, these materials hold the potential to be further developed into fast and effective wound healing treatments.

Amniotic fluid and placental membranes: unexpected sources of highly multipotent cells.

Gestational tissue such as the placenta, placental membranes, and amniotic fluid are usually discarded following birth. Recently, researchers have identified gestational tissue as an untapped source of stem cells that are highly multipotent and possess potent immunosuppressive properties. Placental mesenchymal stem cells (MSCs), human amnion epithelial cells (hAECs), and amniotic fluid-derived stem cells (AFSCs) have been shown to differentiate into various cell types including adipogenic, osteogenic, myogenic, endothelial, pulmonary, neurogenic, hepatogenic, cardiac, and pancreatic lineages. Their immunomodulatory properties suggest that gestational stem cells may have an important application in the treatment of various inflammatory diseases such as graft versus host and autoimmune diseases. In clinical and preclinical studies, gestational stem cells have shown efficacy in the treatment of Crohn disease, lung disease, diabetes, repair of bone defects, heart disease, kidney disease, neural degeneration, and blood disorders. Stem cells derived from the placenta, placental membranes, and amniotic fluid are a valuable resource for the field of regenerative medicine.

Multi-tissue interactions in an integrated three-tissue organ-on-a chip platform

Many drugs have progressed through preclinical and clinical trials and have been available – for years in some cases – before being recalled by the FDA for unanticipated toxicity in humans. One reason for such poor translation from drug candidate to successful use is a lack of model systems that accurately recapitulate normal tissue function of human organs and their response to drug compounds. Moreover, tissues in the body do not exist in isolation, but reside in a highly integrated and dynamically interactive environment, in which actions in one tissue can affect other downstream tissues. Few engineered model systems, including the growing variety of organoid and organ-on-a-chip platforms, have so far reflected the interactive nature of the human body. To address this challenge, we have developed an assortment of bioengineered tissue organoids and tissue constructs that are integrated in a closed circulatory perfusion system, facilitating inter-organ responses. We describe a three-tissue organ-on-a-chip system, comprised of liver, heart, and lung, and highlight examples of inter-organ responses to drug administration. We observe drug responses that depend on inter-tissue interaction, illustrating the value of multiple tissue integration for in vitro study of both the efficacy of and side effects associated with candidate drugs.

Preterm human amnion epithelial cells have limited reparative potential

The collection and use of stem cells from the fetal membranes as cell therapy for a variety of lung diseases, including preterm lung disease, have been previously proposed. To date, only cells from term amnion have been assessed. In the setting of a future therapy for the preterm neonate, it would be ideal if autologous cells could be given. However, the reparative and anti-inflammatory actions of stem cells isolated from preterm amnions have not been evaluated. In this study, with a view to developing an autologous cell therapy for preterm lung injury, we compared the differentiation potential and efficacy of term versus preterm human amnion epithelial cells (hAECs) to protect against inflammation and fibrosis in a bleomycin mouse model of lung injury. We found that, unlike term hAECs, preterm hAECs did not differentiate into a lung lineage following culture in small airway growth media. Preterm hAECs also exerted significantly less protective effects than term hAEC following acute lung injury. Specifically, preterm hAEC did not improve Ashcroft scoring or collagen deposition in the lung despite a reduction in activated myofibroblasts. Term hAECs expressed double the levels of HLA-G compared to preterm hAECs. These findings indicate that while hAECs can be isolated from term and preterm amnions in similar numbers, they bear distinctive characteristics, which may impact upon their clinical use.

Prospect for kidney bioengineering: shortcomings of the status quo

Introduction: Dialysis and renal transplantation are the only two therapeutic options offered to patients affected by end-stage kidney disease; however, neither treatment can be considered definitive. In fact, dialysis is able to replace only the filtration function of the kidney without substituting its endocrine and metabolic roles, and dramatically impacts on patient’s quality of life. On the other hand, kidney transplantation is severely limited by the shortage of transplantable organs, the need for immunosuppressive therapies and a narrow half-life. Regenerative medicine approaches are promising tools aiming to improve this condition. Areas covered: Cell therapies, bioartificial kidney, organ bioengineering, 3D printer and kidney-on-chip represent the most appealing areas of research for the treatment of end-stage kidney failure. The scope of this review is to summarize the state of the art, limits and directions of each branch. Expert opinion: In the future, these emerging technologies could provide definitive, curative and theoretically infinite options for the treatment of end-stage kidney disease. Progress in stem cells-based therapies, decellularization techniques and the more recent scientific know-how for the use of the 3D printer and kidney-on-chip could lead to a perfect cellular-based therapy, the futuristic creation of a bioengineered kidney in the lab or to a valid bioartificial alternative.

A tunable hydrogel system for long-term release of cell-secreted cytokines and bioprinted in situ wound cell delivery.

For many cellular therapies being evaluated in preclinical and clinical trials, the mechanisms behind their therapeutic effects appear to be the secretion of growth factors and cytokines, also known as paracrine activity. Often, delivered cells are transient, and half-lives of the growth factors that they secrete are short, limiting their long-term effectiveness. The goal of this study was to optimize a hydrogel system capable of in situ cell delivery that could sequester and release growth factors secreted from those cells after the cells were no longer present. Here, we demonstrate the use of a fast photocross-linkable heparin-conjugated hyaluronic acid (HA-HP) hydrogel as a cell delivery vehicle for sustained growth factor release, which extends paracrine activity. The hydrogel could be modulated through cross-linking geometries and heparinization to support sustained release proteins and heparin-binding growth factors. To test the hydrogel in vivo, we used it to deliver amniotic fluid-derived stem (AFS) cells, which are known to secrete cytokines and growth factors, in full thickness skin wounds in a nu/nu murine model. Despite transience of the AFS cells in vivo, the HA-HP hydrogel with AFS cells improved wound closure and reepithelialization and increased vascularization and production of extracellular matrix in vivo. These results suggest that HA-HP hydrogel has the potential to prolong the paracrine activity of cells, thereby increasing their therapeutic effectiveness in wound healing.

Human Amnion Epithelial Cells Induced to Express Functional Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis, an autosomal recessive disorder caused by a mutation in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), remains a leading cause of childhood respiratory morbidity and mortality. The respiratory consequences of cystic fibrosis include the generation of thick, tenacious mucus that impairs lung clearance, predisposing the individual to repeated and persistent infections, progressive lung damage and shortened lifespan. Currently there is no cure for cystic fibrosis. With this in mind, we investigated the ability of human amnion epithelial cells (hAECs) to express functional CFTR. We found that hAECs formed 3-dimensional structures and expressed the CFTR gene and protein after culture in Small Airway Growth Medium (SAGM). We also observed a polarized CFTR distribution on the membrane of hAECs cultured in SAGM, similar to that observed in polarized airway cells in vivo. Further, hAECs induced to express CFTR possessed functional iodide/chloride (I−/Cl−) ion channels that were inhibited by the CFTR-inhibitor CFTR-172, indicating the presence of functional CFTR ion channels. These data suggest that hAECs may be a promising source for the development of a cellular therapy for cystic fibrosis.

Human Mesenchymal Stem Cells Reduce Lung Injury in Immunocompromised Mice but Not in Immunocompetent Mice

Background: The immunomodulatory and immunosuppressive capacity of human mesenchymal stem cells (hMSC) is well recognized, but efficacies of hMSC in immunocompetent and immunocompromised animals have never been directly compared. Objectives: We aimed to compare the efficacy of hMSC in preventing bleomycin-induced lung injury in immunocompromised SCID and immunocompetent C57Bl/6 mice. Methods: SCID and C57Bl/6 mice were subjected to a single bolus intranasal instillation of bleomycin to induce lung injury. One million hMSC were administered intravenously 24 h following the induction of bleomycin lung injury. Results: hMSC xenotransplantation into SCID mice resulted in transient improvements in lung weight and tidal volume and to persistent improvement in inspiratory duty cycle, inspiratory flow rate and inspiration/expiration ratio. We did not observed protective effects in C57Bl/6 mice. This correlated with histological changes, where hMSC administration reduced Ashcroft scores, collagen deposition and inflammatory influx in the lungs of SCID mice, but not in those of C57Bl/6 mice. Conclusion: The application of hMSC for the treatment of acute and chronic lung injury is significantly affected by the immune status of the recipient. Lack of hMSC-mediated repair observed in C57Bl/6 mice was likely to be due to limitations of their immune privilege and differential priming of hMSC in immunocompetent versus immunocompromised hosts.