Jean A. Niles

Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation

We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.

In vitro analog of human bone marrow from 3D scaffolds with biomimetic inverted colloidal crystal geometry

In vitro replicas of bone marrow can potentially provide a continuous source of blood cells for transplantation and serve as a laboratory model to examine human immune system dysfunctions and drug toxicology. Here we report the development of an in vitro artificial bone marrow based on a 3D scaffold with inverted colloidal crystal (ICC) geometry mimicking the structural topology of actual bone marrow matrix. To facilitate adhesion of cells, scaffolds were coated with a layer of transparent nanocomposite. After seeding with hematopoietic stem cells (HSCs), ICC scaffolds were capable of supporting expansion of CD34+ HSCs with B-lymphocyte differentiation. Three-dimensional organization was shown to be critical for production of B cells and antigen-specific antibodies. Functionality of bone marrow constructs was confirmed by implantation of matrices containing human CD34+ cells onto the backs of severe combined immunodeficiency (SCID) mice with subsequent generation of human immune cells.

Human Lymphocyte Apoptosis after Exposure to Influenza A Virus

ABSTRACT Infection of humans with influenza A virus (IAV) results in a severe transient leukopenia. The goal of these studies was to analyze possible mechanisms behind this IAV-induced leukopenia with emphasis on the potential induction of apoptosis of lymphocytes by the virus. Analysis of lymphocyte subpopulations after exposure to IAV showed that a portion of CD3+, CD4+, CD8+, and CD19+ lymphocytes became apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling positive). The percentage of cells that are infected was shown to be less than the percentage of apoptotic cells, suggesting that direct effects of cell infection by the virus cannot account fully for the high level of cell death. Removal of monocytes-macrophages after IAV exposure reduced the percent of lymphocytes that were apoptotic. Treatment of virus-exposed cultures with anti-tumor necrosis factor alpha did not reduce the percentage of lymphocytes that were apoptotic. In virus-exposed cultures treated with anti-FasL antibody, recombinant soluble human Fas, Ac-DEVD-CHO (caspase-3 inhibitor), or Z-VAD-FMK (general caspase inhibitor), apoptosis and production of the active form of caspase-3 was reduced. The apoptotic cells were Fas-high-density cells while the nonapoptotic cells expressed a low density of Fas. The present studies showed that Fas-FasL signaling plays a major role in the induction of apoptosis in lymphocytes after exposure to IAV. Since the host response to influenza virus commonly results in recovery from the infection, with residual disease uncommon, lymphocyte apoptosis likely represents a part of an overall beneficial immune response but could be a possible mechanism of disease pathogenesis.

Neurogenic and neuro-protective potential of a novel subpopulation of peripheral blood-derived CD133+ ABCG2+CXCR4+ mesenchymal stem cells: Development of autologous cell-based therapeutics for traumatic brain injury

IntroductionNervous system injuries comprise a diverse group of disorders that include traumatic brain injury (TBI). The potential of mesenchymal stem cells (MSCs) to differentiate into neural cell types has aroused hope for the possible development of autologous therapies for central nervous system injury.MethodsIn this study we isolated and characterized a human peripheral blood derived (HPBD) MSC population which we examined for neural lineage potential and ability to migrate in vitro and in vivo. HPBD CD133+, ATP-binding cassette sub-family G member 2 (ABCG2)+, C-X-C chemokine receptor type 4 (CXCR4)+ MSCs were differentiated after priming with β-mercaptoethanol (β-ME) combined with trans-retinoic acid (RA) and culture in neural basal media containing basic fibroblast growth factor (FGF2) and epidermal growth factor (EGF) or co-culture with neuronal cell lines. Differentiation efficiencies in vitro were determined using flow cytometry or fluorescent microscopy of cytospins made of FACS sorted positive cells after staining for markers of immature or mature neuronal lineages. RA-primed CD133+ABCG2+CXCR4+ human MSCs were transplanted into the lateral ventricle of male Sprague-Dawley rats, 24 hours after sham or traumatic brain injury (TBI). All animals were evaluated for spatial memory performance using the Morris Water Maze (MWM) Test. Histological examination of sham or TBI brains was done to evaluate MSC survival, migration and differentiation into neural lineages. We also examined induction of apoptosis at the injury site and production of MSC neuroprotective factors.ResultsCD133+ABCG2+CXCR4+ MSCs consistently expressed markers of neural lineage induction and were positive for nestin, microtubule associated protein-1β (MAP-1β), tyrosine hydroxylase (TH), neuron specific nuclear protein (NEUN) or type III beta-tubulin (Tuj1). Animals in the primed MSC treatment group exhibited MWM latency results similar to the uninjured (sham) group with both groups showing improvements in latency. Histological examination of brains of these animals showed that in uninjured animals the majority of MSCs were found in the lateral ventricle, the site of transplantation, while in TBI rats MSCs were consistently found in locations near the injury site. We found that levels of apoptosis were less in MSC treated rats and that MSCs could be shown to produce neurotropic factors as early as 2 days following transplantation of cells. In TBI rats, at 1 and 3 months post transplantation cells were generated which expressed markers of neural lineages including immature as well as mature neurons.ConclusionsThese results suggest that PBD CD133+ABCG2+CXCR4+ MSCs have the potential for development as an autologous treatment for TBI and neurodegenerative disorders and that MSC derived cell products produced immediately after transplantation may aid in reducing the immediate cognitive defects of TBI.

Design and development of tissue engineered lung: Progress and challenges

Before we can realize our long term goal of engineering lung tissue worthy of clinical applications, advances in the identification and utilization of cell sources, development of standardized procedures for differentiation of cells, production of matrix tailored to meet the needs of the lung and design of methods or techniques of applying the engineered tissues into the injured lung environment will need to occur. Design of better biomaterials with the capacity to guide stem cell behavior and facilitate lung lineage choice as well as seamlessly integrate with living lung tissue will be achieved through advances in the development of decellularized matrices and new understandings related to the influence of extracellular matrix on cell behavior and function. We have strong hopes that recent developments in the engineering of conducting airway from decellularized trachea will lead to similar breakthroughs in the engineering of distal lung components in the future.

Modeling the lung: Design and development of tissue engineered macro- and micro-physiologic lung models for research use

Respiratory tract specific cell populations, or tissue engineered in vitro grown human lung, have the potential to be used as research tools to mimic physiology, toxicology, pathology, as well as infectious diseases responses of cells or tissues. Studies related to respiratory tract pathogenesis or drug toxicity testing in the past made use of basic systems where single cell populations were exposed to test agents followed by evaluations of simple cellular responses. Although these simple single-cell-type systems provided good basic information related to cellular responses, much more can be learned from cells grown in fabricated microenvironments which mimic in vivo conditions in specialized microfabricated chambers or by human tissue engineered three-dimensional (3D) models which allow for more natural interactions between cells. Recent advances in microengineering technology, microfluidics, and tissue engineering have provided a new approach to the development of 2D and 3D cell culture models which enable production of more robust human in vitro respiratory tract models. Complex models containing multiple cell phenotypes also provide a more reasonable approximation of what occurs in vivo without the confounding elements in the dynamic in vivo environment. The goal of engineering good 3D human models is the formation of physiologically functional respiratory tissue surrogates which can be used as pathogenesis models or in the case of 2D screening systems for drug therapy evaluation as well as human toxicity testing. We hope that this manuscript will serve as a guide for development of future respiratory tract model systems as well as a review of conventional models.

Production and utilization of acellular lung scaffolds in tissue engineering

Pulmonary disease is a worldwide public health problem that reduces the quality of life and increases the need for hospital admissions as well as the risk for premature death for those affected. For many patients, lung transplantation is the only chance for survival. Unfortunately, there is a significant shortage of lungs for transplantation and since the lung is the most likely organ to be damaged during procurement many lungs deemed unacceptable for transplantation are simply discarded. Rather than discarding these lungs they can be used to produce three‐dimensional acellular (AC) natural lung scaffolds for the generation of engineered lung tissue. AC scaffolds are lungs whose original cells have been destroyed by exposure to detergents and physical methods of removing cells and cell debris. This creates a lung scaffold from the skeleton of the lungs themselves. The scaffolds are then used to support adult, stem or progenitor cells which can be grown into functional lung tissue. Recent studies show that engineered lung tissues are capable of surviving after in vivo transplantation and support limited gas exchange. In the future engineered lung tissue has the potential to be used in clinical applications to replace lung functions lost following injury or disease. This manuscript discusses recent advances in development and use of AC scaffolds to support engineering of lung tissues. J. Cell. Biochem. 113: 2185–2192, 2012.

Giving new life to old lungs: methods to produce and assess whole human paediatric bioengineered lungs.

We report, for the first time, the development of an organ culture system and protocols to support recellularization of whole acellular (AC) human paediatric lung scaffolds. The protocol for paediatric lung recellularization was developed using human transformed or immortalized cell lines and single human AC lung scaffolds. Using these surrogate cell populations, we identified cell number requirements, cell type and order of cell installations, flow rates and bioreactor management methods necessary for bioengineering whole lungs. Following the development of appropriate cell installation protocols, paediatric AC scaffolds were recellularized using primary lung alveolar epithelial cells (AECs), vascular cells and tracheal/bronchial cells isolated from discarded human adult lungs. Bioengineered paediatric lungs were shown to contain well‐developed vascular, respiratory epithelial and lung tissue, with evidence of alveolar–capillary junction formation. Types I and II AECs were found thoughout the paediatric lungs. Furthermore, surfactant protein‐C and ‐D and collagen I were produced in the bioengineered lungs, which resulted in normal lung compliance measurements. Although this is a first step in the process of developing tissues for transplantation, this study demonstrates the feasibility of producing bioengineered lungs for clinical use. Copyright

Novel in vitro respiratory models to study lung development, physiology, pathology and toxicology

Detailed studies of lung pathology in patients during the course of development of acute lung injury or respiratory distress are limited, and in the past information related to lung-specific responses has been derived from the study of lungs from patients who died at autopsy or from animal models. Development of good in vitro human tissue models would help to bridge the gap in our current knowledge of lung responses and provide a better understanding of lung development, physiology and pathology. In vitro models of simple one-cell or two-cell culture systems as well as complex multicellular lung analogs that reproduce defined components of specific human lung responses have already been realized. A benefit of current in vitro lung models is that hypotheses generated from review of data from human or animal disease studies can be tested directly in engineered human tissue models. Results of studies done using simple in vitro lung systems or more complex three-dimensional models have already been used to examine cell-based responses, physiologic functions, pathologic changes and even drug toxicity or drug responses. In the future we will create models with specific genetic profiles to test the importance of single gene products or pathways of significance. Recent development of microfluidics-based models that support high-throughput screening will allow early-stage toxicity testing in human systems and faster development of new and innovative medical products. Model design in the future will also allow for evaluation of multiple organ systems at once, providing a more holistic or whole-body approach to understanding human physiology and responses.

Engineering Complex Synthetic Organs

At this time there is a substantial, and as yet unmet, demand for organs to replace nonfunctional tissues resulting from congenital defects, or to repair damaged or degenerated tissues. The field of regenerative medicine hopes to provide engineered replacement tissues in situations where our body’s regenerative capability or nonbiological mechanical devices cannot adequately replace lost physiological functions. This technology holds the promise to supply customized organs to overcome the severe shortages we currently face. Engineering synthetic organs is a complex process which necessitates careful (1) selection of cells or controlled proliferation of stem or progenitor cells to achieve appropriate numbers of cells for seeding onto biodegradable scaffolds to create cell-scaffold constructs, (2) design and selection of appropriate biodegradable or biomodifiable scaffold materials, and (3) design and construction of bioreactors to support generation of functional tissue replacements. To be successful, ongoing efforts to understand and engineer multicellular systems must continue, and new efforts to induce vascularization and integration of engineered tissues into the body will need to be developed. Current studies lead to improved understanding of how tissue systems can be integrated, as well as development of biomedical technologies not traditionally considered in tissue engineering, such as development of biohybrid organs or “bionic” devices.