Darrell N. Kotton

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.

Telomere dysfunction induces metabolic and mitochondrial compromise

Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1β, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1β promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere–p53–PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.

Induced Pluripotent Stem Cell Generation Using a Single Lentiviral Stem Cell Cassette

Induced pluripotent stem (iPS) cells can be generated using retroviral vectors expressing Oct4, Klf4, Sox2, and cMyc. Most prior studies have required multiple retroviral vectors for reprogramming, resulting in high numbers of genomic integrations in iPS cells and limiting their use for therapeutic applications. Here we describe the use of a single lentiviral vector expressing a “stem cell cassette” composed of the four transcription factors and a combination of 2A peptide and internal ribosome entry site technology, generating iPS cells from postnatal fibroblasts. iPS cells generated in this manner display embryonic stem cell‐like morphology, express stem cell markers, and exhibit in vivo pluripotency, as evidenced by their ability to differentiate in teratoma assays and their robust contribution to mouse chimeras. Combining all factors into a single transcript achieves the most efficient reprogramming system to date and allows derivation of iPS cells with a single viral integration. The use of a single lentiviral vector for reprogramming represents a powerful laboratory tool and a significant step toward the application of iPS technology for clinical purposes. STEM CELLS 2009;27:543–549

Generation of Transgene-Free Lung Disease-Specific Human Induced Pluripotent Stem Cells Using a Single Excisable Lentiviral Stem Cell Cassette

The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step toward the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. Although several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease‐specific iPSC from humans with inherited or degenerative diseases. Here, we report the use of a humanized version of a single lentiviral “stem cell cassette” vector to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either three or four reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration that on removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease‐specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, α‐1 antitrypsin deficiency‐related emphysema, scleroderma, and sickle‐cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia. STEM CELLS 2010;28:1728–1740

Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector.

The residual presence of integrated transgenes following the derivation of induced pluripotent stem (iPS) cells is highly undesirable. Here we demonstrate efficient derivation of iPS cells free of exogenous reprogramming transgenes using an excisable polycistronic lentiviral vector. A novel version of this vector containing a reporter fluorochrome allows direct visualization of vector excision in living iPS cells in real time. We find that removal of the reprogramming vector markedly improves the developmental potential of iPS cells and significantly augments their capacity to undergo directed differentiation in vitro. We further propose that methods to efficiently excise reprogramming transgenes with minimal culture passaging, such as those demonstrated here, are critical since we find that iPS cells may acquire chromosomal abnormalities, such as trisomy of chromosome 8, similar to embryonic stem cells after expansion in culture. Our findings illustrate an efficient method for the generation of transgene‐free iPS cells and emphasize the potential beneficial effects that may result from elimination of integrated reprogramming factors. In addition, our results underscore the consequences of long‐term culture that will need to be taken into account for the clinical application of iPS cells. STEM CELLS 2010;28:64–74

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.

Self-Renewing Endodermal Progenitor Lines Generated from Human Pluripotent Stem Cells

The use of human pluripotent stem cells for laboratory studies and cell-based therapies is hampered by their tumor-forming potential and limited ability to generate pure populations of differentiated cell types in vitro. To address these issues, we established endodermal progenitor (EP) cell lines from human embryonic and induced pluripotent stem cells. Optimized growth conditions were established that allow near unlimited (>10(16)) EP cell self-renewal in which they display a morphology and gene expression pattern characteristic of definitive endoderm. Upon manipulation of their culture conditions in vitro or transplantation into mice, clonally derived EP cells differentiate into numerous endodermal lineages, including monohormonal glucose-responsive pancreatic β-cells, hepatocytes, and intestinal epithelia. Importantly, EP cells are nontumorigenic in vivo. Thus, EP cells represent a powerful tool to study endoderm specification and offer a potentially safe source of endodermal-derived tissues for transplantation therapies.

Lung regeneration: mechanisms, applications and emerging stem cell populations

Recent studies have shown that the respiratory system has an extensive ability to respond to injury and regenerate lost or damaged cells. The unperturbed adult lung is remarkably quiescent, but after insult or injury progenitor populations can be activated or remaining cells can re-enter the cell cycle. Techniques including cell-lineage tracing and transcriptome analysis have provided novel and exciting insights into how the lungs and trachea regenerate in response to injury and have allowed the identification of pathways important in lung development and regeneration. These studies are now informing approaches for modulating the pathways that may promote endogenous regeneration as well as the generation of exogenous lung cell lineages from pluripotent stem cells. The emerging advances, highlighted in this Review, are providing new techniques and assays for basic mechanistic studies as well as generating new model systems for human disease and strategies for cell replacement.

Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics

Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na+ channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na+ channel current (INaL) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of INaL such that increasing the pacing rate markedly reduces INaL and, in addition, increases its inhibition by the Na+ channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband’s iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens.

The Prolonged Life-Span of Alveolar Macrophages

To further examine the half-life of alveolar macrophages, chimeric CD 45.2 mice were generated through bone marrow transplantation of donor CD 45.1 cells. Before administration of donor cells, recipient mice were divided into two cohorts: the first cohort received total body irradiation; the second cohort also received irradiation-however, the thorax, head, and upper extremities were shielded with lead. Flow cytometric analysis was then performed on blood, peritoneal, and bronchoalveolar lavage cells over time to quantify engraftment. The data generated for the unshielded cohort of mice revealed a macrophage half-life of 30 days. In the shielded cohort, however, we found that by 8 months there was negligible replacement of recipient alveolar macrophages by donor cells, despite reconstitution of the blood and peritoneum by donor bone marrow. Consistent with these findings, the mean fluorescent intensity of alveolar macrophages remained stable over a 4-week period after in vivo PKH26 dye loading. Together, these data show that previous alveolar macrophage half-life studies were confounded by the fact that they did not account for the toxic effects of irradiation conditioning regimens, and demonstrate that the bone marrow does not significantly contribute to the alveolar macrophage compartment during steady-state conditions.