The acute respiratory distress syndrome (ARDS) is a common lung disorder in adult intensive care units, resulting in death in approximately 40% of patients affected. Idiopathic pulmonary fibrosis (IPF) is a chronic, less common respiratory condition with a dismal prognosis, contributing to the death of nearly every one of its victims within 5 years of diagnosis from progressive lung failure. Both of these lung disorders share something in common - to date there is no specific pharmacologic therapy that greatly improves survival. These and other lung diseases, characterized by lung injury followed by inadequate or abnormal repair processes, may be amenable to treatment with cell-based therapies using stem cells. Stem cells, particularly human embryonic stem (hES) cells, have the capacity to differentiate into cells of a variety of tissues and may be useful in augmenting repair of damaged or diseased lungs. Unfortunately, previous attempts to treat experimental lung injury by administering undifferentiated adult bone marrow-derived stem cells have been largely unsuccessful. The goal of this proposal is to learn what conditions hES cells require before and after administration to promote repair of injured lung tissue. We have established an animal model of lung injury in our laboratory which uses a cancer chemotherapeutic agent, bleomycin, that causes acute lung injury (similar to human ARDS) early after administration, and chronic injury (similar to human IPF) later in the course. Bleomycin will be given to immunodeficient mice, who will not reject hES administered to them. To promote the repair of damaged lung tissue, hES cells may need to recapitulate the early events of lung development in the culture dish in order to establish the biological “memory” for developing new lung tissue. We propose to direct the differentiation of hES in culture before administering the cells to mice following bleomycin-induced lung injury. In addition, it may be necessary to provide an environment in the injured lung that enhances the ability of the administered hES cells to promote repair. We will determine the usefulness of this approach by first, determining whether the hES cells actually can lodge in the injured lung and change into lung cells, and second, by learning whether the lung injury had been ameliorated by treatment with hES cells. These studies should assist in the development of novel methods of treatment for these life-threatening lung diseases.
Statement of Benefit to California:
Chronic respiratory disease is the fourth leading cause of death among Californians and causes an untold amount of suffering and disability. Influenza and pneumonia are acute lung diseases and, as the sixth leading cause of death, are responsible for the demise of over 8,000 Californians each year. Many of these serious diseases are characterized by an initial acute lung injury followed by inadequate or abnormal repair leading to chronic disability. Pharmacologic therapies alone are often not available to treat these disorders, so there is a need to develop novel approaches, such as cell-based therapy in order to augment lung repair. Unfortunately, despite great promise, attempts to treat experimental lung injury by administering adult bone marrow-derived stem cells have thus far been largely disappointing and have not been shown to confer functional benefit. Human embryonic stem cells (hESC), by demonstrating far greater plasticity than adult-derived stem cells, offer a unique opportunity for novel therapies. It is unknown whether the few existing federally-approved hESC lines will be adequate to achieve the goals of cell-based therapy for lung diseases, so there will be a need to explore the use and differentiation potential of non-approved hESC lines as well in order to maximize the chances for success. Research involving non-federally approved hESC will not be funded by the U.S. federal government, potentially slowing down research and development of new strategies for the treatment of lung diseases with hESC. The ability to use appropriately-derived hESC lines beyond the few permitted by the U.S. National Institutes of Health, puts California in the forefront in the development of novel treatments of lung diseases with cell-based therapies. This could have a clear benefit to California, by attracting both researchers and biomedical investors to California. Should effective treatments for advanced lung diseases be developed as a result of this research, productivity and longevity of Californians should be positively impacted by this work.
SYNOPSIS: This project aims to generate alveolar epithelial cells (AEC) from hESCs for the treatment of lung injury and repair of gas exchange. Studies will 1) test different methods of coaxing hESCs towards an AEC phenotype, 2) test different routes of administration, 3) test whether changes in the microenvironment of the diseased lung tissue can be modulated to enhance AEC engraftment and 4) test whether lung injury is ameliorated by ESC-derived AEC. This proposal suggests the use of a murine model of lung injury; both approved and non-approved hESC lines (the investigators will use Wicell and Melton lab ESCs); experiments with induction of endoderm characteristics and avidin-low serum differentiation to definitive/lung endoderm; and monitoring for endodermal markers. Once an appropriate differentiation method is achieved, dependence of functional engraftment on the stage of differentiation and route of administration will be investigated using immune deficient mice subjected to the bleomycin lung injury model. The results will be analyzed at various levels (histology, analysis of inflammation, effect of growth factors, microCT scanning and functional studies) to determine whether the transplantation of human cells has had a positive clinical effect. SIGNIFICANCE AND INNOVATION: Acute lung-injury is a devastating disease. The clinical aspects of damage to or destruction of the alveolar epithelium are well known as well as catastrophic, and no good therapies exist. Loss of epithelium brings about loss of gas exchange area and eventual respiratory failure and death. Stem cell therapy with adult bone marrow stem cells was explored with some enthusiasm, which proved to be at least partially unwarranted, as some studies disclosed that engrafted cells were not epithelial but fibroblastic and that the beneficial effects might be due to cytokine effects. Effective hESC therapy is thus still considered the most likely to provide possible tissue repair as the generation of AEC from hESC may provide a source of cells that could be used for repair. In addition, insights into the differentiation process may provide insights into factors that govern human progenitors to differentiate into AEC. Thus far few studies have addressed this issue, making the proposal innovative. STRENGTHS: There is obviously high clinical and biological interest in this work. The investigators have extensive experience in acute lung injury models including in immunodeficient animals, and the involvement of Dr. C. Lutzko as a collaborator, an expert on hESC and head of the CHLA Embryonic Stem Cell Core Facility, is a key strength. The PI’s Laboratory is well funded by NIH and there are two large grants pending. Also, the availability of the bleomycin model, which the PI and her group has developed, is a plus. WEAKNESSES: While the approach is scholarly and systematic, the authors do not seem to have much specific hope that hESC will provide them with a straightforward solution. For example, Aim 1 presents a number of studies to be done in an attempt to induce the differentiation of hESC into AEC. It is however not clear that most of these will indeed result in AEC differentiation, and a step-wise approach where definitive endoderm, pulmonary epithelial progenitors and the AEC are generated should be attempted. The investigators consider the use of lung mesenchyme as an inducer of hESC differentiation the most hopeful, but they concede that much more work will be necessary even if there is significant progress along the path they propose to follow. The experiments are described but there is some pessimism in the authors’ conclusions on feasibility: “… minimal experimental evidence that hESC, although able to be stimulated to express endodermal markers, are able to progress toward functional lung epithelium.” Little attention is given to the degree of differentiation that will be obtained, or how to purify the differentiated progeny to minimize the chance for teratoma formation from the grafted cells. Reviewers recommend using the paradigm put forward in Aim1 C to start. Aim 2 and Aim 3 are within the technical capabilities of the investigators and reviewers have no doubt that these could be accomplished when Aim 1 is successful. However, the combination of all these studies and the necessary success Aim 1 make the proposal very ambitious, and the investigators should spend more effort on Aim 1 prior to embarking on complicated transplant studies. DISCUSSION: There was no further discussion following the reviewers' comments.