Laertis Ikonomou

Regeneration and orthotopic transplantation of a bioartificial lung

About 2,000 patients now await a donor lung in the United States. Worldwide, 50 million individuals are living with end-stage lung disease. Creation of a bioartificial lung requires engineering of viable lung architecture enabling ventilation, perfusion and gas exchange. We decellularized lungs by detergent perfusion and yielded scaffolds with acellular vasculature, airways and alveoli. To regenerate gas exchange tissue, we seeded scaffolds with epithelial and endothelial cells. To establish function, we perfused and ventilated cell-seeded constructs in a bioreactor simulating the physiologic environment of developing lung. By day 5, constructs could be perfused with blood and ventilated using physiologic pressures, and they generated gas exchange comparable to that of isolated native lungs. To show in vivo function, we transplanted regenerated lungs into orthotopic position. After transplantation, constructs were perfused by the recipients circulation and ventilated by means of the recipients airway and respiratory muscles, and they provided gas exchange in vivo for up to 6 h after extubation.

Insect cell culture for industrial production of recombinant proteins.

Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.

Efficient Derivation of Purified Lung and Thyroid Progenitors from Embryonic Stem Cells

Two populations of Nkx2-1(+) progenitors in the developing foregut endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is known about these cells because they are difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFβ and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling, can specify these cells efficiently from definitive endodermal precursors. When derived using Nkx2-1(GFP) knockin reporter ESCs, these progenitors can be purified for expansion in culture and have a transcriptome that overlaps with developing lung epithelium. Upon induction, they can express a broad repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D lung tissue scaffold. Thus, we have derived a pure population of progenitors able to recapitulate the developmental milestones of lung/thyroid development.

Human Islet‐Derived Precursor Cells Are Mesenchymal Stromal Cells That Differentiate and Mature to Hormone‐Expressing Cells In Vivo

Islet transplantation offers improved glucose homeostasis in diabetic patients, but transplantation of islets is limited by the supply of donor pancreases. Undifferentiated precursors hold promise for cell therapy because they can expand before differentiation to produce a large supply of functional insulin‐producing cells. Previously, we described proliferative populations of human islet‐derived precursor cells (hIPCs) from adult islets. To show the differentiation potential of hIPCs, which do not express insulin mRNA after at least 1,000‐fold expansion, we generated epithelial cell clusters (ECCs) during 4 days of differentiation in vitro. After transplantation into mice, 22 of 35 ECC preparations differentiated and matured into functional cells that secreted human C‐peptide in response to glucose. Transcripts for insulin, glucagon, and somatostatin in recovered ECC grafts increased with time in vivo, reaching levels approximately 1% of those in adult islets. We show that hIPCs are mesenchymal stromal cells (MSCs) that adhere to plastic, express CD73, CD90, and CD105, and can differentiate in vitro into adipocytes, chondrocytes, and osteocytes. Moreover, we find a minor population of CD105+/CD73+/CD90+ cells in adult human islets (prior to incubation in vitro) that express insulin mRNA at low levels. We conclude that hIPCs are a specific type of pancreas‐derived MSC that are capable of differentiating into hormone‐expressing cells. Their ability to mature into functional insulin‐secreting cells in vivo identifies them as an important adult precursor or stem cell population that could offer a virtually unlimited supply of human islet‐like cells for replacement therapy in type 1 diabetes.

Synergistic Interaction between Candida albicans and Commensal Oral Streptococci in a Novel In Vitro Mucosal Model

ABSTRACT Candida albicans is a commensal colonizer of the gastrointestinal tract of humans, where it coexists with highly diverse bacterial communities. It is not clear whether this interaction limits or promotes the potential of C. albicans to become an opportunistic pathogen. Here we investigate the interaction between C. albicans and three species of streptococci from the viridans group, which are ubiquitous and abundant oral commensal bacteria. The ability of C. albicans to form biofilms with Streptococcus oralis, Streptococcus sanguinis, or Streptococcus gordonii was investigated using flow cell devices that allow abiotic biofilm formation under salivary flow. In addition, we designed a novel flow cell system that allows mucosal biofilm formation under conditions that mimic the environment in the oral and esophageal mucosae. It was observed that C. albicans and streptococci formed a synergistic partnership where C. albicans promoted the ability of streptococci to form biofilms on abiotic surfaces or on the surface of an oral mucosa analogue. The increased ability of streptococci to form biofilms in the presence of C. albicans could not be explained by a growth-stimulatory effect since the streptococci were unaffected in their growth in planktonic coculture with C. albicans. Conversely, the presence of streptococci increased the ability of C. albicans to invade organotypic models of the oral and esophageal mucosae under conditions of salivary flow. Moreover, characterization of mucosal invasion by the biofilm microorganisms suggested that the esophageal mucosa is more permissive to invasion than the oral mucosa. In summary, C. albicans and commensal oral streptococci display a synergistic interaction with implications for the pathogenic potential of C. albicans in the upper gastrointestinal tract.

Design of an Efficient Medium for Insect Cell Growth and Recombinant Protein Production

SummaryWe report the development of a new serum-free medium based on the use of factorial experiments. At first, a variety of hydrolysates were screened using a fractional factorial approach with High-Five cells. From this experiment yeastolate ultrafiltrate was found to have, by far, the most important effect on cell growth. Furthermore, Primatone RL® was found to remarkably prolong the stationary phase of Sf-9 and High-Five cell cultures. The optimal concentrations for yeastolate and Primatone were determined to be 0.6 and 0.5%, respectively, on the basis of a complete factorial experiment. This new medium, called YPR, supported good growth of both Sf-9 and High-Five cells in batch cultures, with maximal densities of 5.4 and 6.1×106 cells/ml, respectively. In addition, both cell lines achieved good growth in bioreactor batch culture and had a prolonged stationary phase of 3–4 d in YPR medium compared to Insect-XPRESS medium. The ability of the new medium to support recombinant protein expression was also tested by infecting Sf-9 or High-Five cells at high density (2×106 cells/ml) with a baculovirus expressing secreted placental alkaline phosphatase (SEAP). The maximum total SEAP concentration after 7 d was about 43 IU/ml (58 mg/L) and 28 IU/ml (39 mg/L) for High-Five and Sf-9 cells, respectively.

Regeneration of Thyroid Function by Transplantation of Differentiated Pluripotent Stem Cells

Differentiation of functional thyroid epithelia from pluripotent stem cells (PSCs) holds the potential for application in regenerative medicine. However, progress toward this goal is hampered by incomplete understanding of the signaling pathways needed for directed differentiation without forced overexpression of exogenous transgenes. Here we use mouse PSCs to identify key conserved roles for BMP and FGF signaling in regulating thyroid lineage specification from foregut endoderm in mouse and Xenopus. Thyroid progenitors derived from mouse PSCs can be matured into thyroid follicular organoids that provide functional secretion of thyroid hormones in vivo and rescue hypothyroid mice after transplantation. Moreover, by stimulating the same pathways, we were also able to derive human thyroid progenitors from normal and disease-specific iPSCs generated from patients with hypothyroidism resulting from NKX2-1 haploinsufficiency. Our studies have therefore uncovered the regulatory mechanisms that underlie early thyroid organogenesis and provide a significant step toward cell-based regenerative therapy for hypothyroidism.

Microcarrier Culture of Lepidopteran Cell Lines: Implications for Growth and Recombinant Protein Production

Several microcarrier systems were screened with Sf‐9 and High‐Five cell lines as to their ability to support cell growth and recombinant (β‐galactosidase) protein production. Growth of both cell lines on compact microcarriers, such as Cytodex‐1 and glass beads, was minimal, as cells detached easily from the microcarrier surface and grew as single cells in the medium. Cell growth was also problematic on Cytopore‐1 and ‐2 porous microcarriers. Cells remained attached for several days inside the microcarrier pores, but no cell division and proliferation were observed. On the contrary, insect cells grew well in the interior of Fibra‐Cel disks mainly as aggregates at points of fiber intersection, reaching final (plateau) densities of about 4 × 106 (Sf‐9) and 2.7 × 106 (High‐Five) cells mL‐1 (8 × 106 and 5.5 × 106 cells per cm2 of projected disk area, respectively). Their growth was described well by the logistic equation, which takes into account possible inhibition effects. β‐Galactosidase (β‐gal) production of Sf‐9 cells on Fibra‐Cel disks (infected at 3.3 × 106 cells mL‐1) was prolonged (192 h), and specific protein production was similar to that of high‐density free cell infection. Cultispher‐S microcarriers were found to be a very efficient system for the growth of High‐Five cells, whereas no growth of Sf‐9 cells took place for the same system. Concentrations of about 9 × 106 cells mL‐1 were reached within 120 h, with cell growth in both microcarriers and aggregates, appearance of cellular bridges between microcarriers and aggregates, and eventual formation of macroaggregates incorporating several microcarriers. Specific protein productions after β‐gal baculovirus infection at increasing cell concentrations were almost constant, thus leading to elevated volumetric protein production: final β‐gal titers of 946, 1728, and 1484 U mL‐1 were obtained for infection densities of 3.4, 7.2, and 8.9 × 106 cells mL‐1, respectively.

Retinoschisin (RS1) interacts with negatively charged lipid bilayers in the presence of Ca2+: an atomic force microscopy study.

Retinoschisin (RS1) is a retina-specific secreted protein encoding a conserved discoidin domain sequence. As an adhesion molecule, RS1 preserves the retinal cell architecture and promotes visual signal transduction. In young males, loss-of-function mutations in the X-linked retinoschisis gene (RS1) cause X-linked retinoschisis, a form of progressive blindness. Neither the structure of RS1 nor the nature of its anchoring and organization on the plasma membranes is fully understood. The discoidin C2 domains of coagulation factors V and VIII are known to interact with extracellular phosphatidylserine (PS). In this study we have used atomic force microscopy (AFM) to study the interactions of murine retinoschisin (Rs1) with supported anionic lipid bilayers in the presence of Ca(2+). The bilayers consisting of a single lipid, PS, and mixtures of lipids with or without PS were used. Consistent with previous X-ray diffraction studies, AFM imaging showed two distinct domains in pure PS bilayers when Ca(2+) was present. Upon Rs1 adsorption, these PS and PS-containing mixed bilayers underwent fast and extensive reorganization. Protein localization was ascertained by immunolabeling. AFM imaging showed the Rs1 antibody bound exclusively to the calcium-rich ordered phase of the bilayers pointing to the sequestration of Rs1 within those domains. This was further supported by the increased mechanical strength of these domains after Rs1 binding. Besides, changes in bilayer thickness suggested that Rs1 was partially embedded into the bilayer. These findings support a model whereby the Rs1 protein binds to PS in the retinal cell plasma membranes in a calcium-dependent manner.

The Global Emergence of Unregulated Stem Cell Treatments for Respiratory Diseases. Professional Societies Need to Act.

Laertis Ikonomou, Robert J. Freishtat, Darcy E. Wagner, Angela Panoskaltsis-Mortari, and Daniel J. Weiss The Pulmonary Center, Boston University School of Medicine and Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, Massachusetts; George Washington University School of Medicine and Children’s National Medical Center, Washington, DC; Comprehensive Pneumology Center, Ludwig-Maximilians-Universität, Munich, Germany; Departments of Pediatrics and Medicine, University of Minnesota, Minneapolis, Minnesota; and University of Vermont College of Medicine, Burlington, Vermont