A major area of research in regenerative medicine is the potential application of cell therapy for various disorders. Human embryonic stem (ES) cells are self-renewable and are able to differentiate into various cell types, tissues and organs if they are properly taken care. Thus, ES cells can be a great resource for therapeutic treatment of a variety of dysfunction cells, tissues, and organs. However, it these cells are not properly taken care, when they are implanted to any tissues and organs they can give rise to the tumor formation. This research application focuses on two areas, one is to see if these cells can be trained in cell culture to direct their differentiated potential to lung epithelial cell types, the other focus is to see if such a training can reduce the risk of forming unwarranted tumor in immune-deficient mice. This is based on the hypothesis that human ES cells can be trained in cell culture toward lung epithelial cell types and these newly derived cells have a full capacity as lung epithelial cells. We will then use these modified ES cells to test the feasibility in cell culture system as well as in a tracheal graft repopulation model to see if these cells mixed with isolated adult cystic fibrosis (CF) cells can correct the chloride ion transport property, which is not function properly in CF. To achieve these goals, we will develop well-defined culture medium and condition for these cells to grow and to differentiate. We intend to use NIH approved human ES cell lines, H1 and H9 for this study. There are three specific aims in this application. Aim 1 is to define the defined (optimized) medium for these human ES cells to grow and to differentiate into lung and airway epithelial cell types. Aim 2 is then to put these trained cells to transplanted tracheal graft to see if these cells form differentiated lung/airway epithelium and the potential of forming tumor in the immune-deficient mice. Aim 3 is then mixed these trained cells with isolated CF cells in culture as well as in repopulating tracheal graft to see if the mixing can correct the chloride channel defect associated with CF. Success from such a study, not only proof of principal but also provides the justification to continue this research area for future cell therapy for this disease.
Statement of Benefit to California:
A major area of research in regenerative medicine is the potential application of cell therapy for various disorders. Human embryonic stem (ES) cells are self-renewable and are able to differentiate into various cell types, tissues and organs if they are properly taken care. Thus, ES cells can be a great resource for therapeutic treatment of a variety of dysfunction cells, tissues, and organs. However, it these cells are not properly taken care, when they are implanted to any tissues and organs they can give rise to the tumor formation. In this research application, we would like to establish a proof of the principal that ES cells are indeed trainable and can be a great resource for cell therapeutic application. We focus on the possibility to use these ES-derived cells to correct the diseased phenotypes of airway/lung cells related to cystic fibrosis (CF). CF is a dreadful, genetic disease. One out of 25 Caucasians carries the defected CF gene. For California, there is a substantial population carrying the defected gene. The disease through the vicious cycles of infection and pneumonia in lung/airway resulting in many hospital visits, treatments, and care until there is no more drug can inhibit the bacteria infection. Currently, there is no cure. Attempts to use gene therapy and pharmacological agent for the treatment have met with little success. Human ES cells have a great potential to be used as the source for cell therapy, as these cells can be properly trained in vitro, as we intend to do. We think the feasibility to use this approach for cell therapy is very high since it has been reported in science literature that only 6-10% of wild-type cells are needed for CF airways. The current study used in vitro cell culture and transplant tracheal graft approach to see if these ES cells derived from cell culture manipulation can be useful for the correction of the CF defect. Thus, if this project is done successfully, the results will form the basis for future therapeutic application. Many Californian as well as CF patients will be benefited from this study.
In this proposal, Dr Reen and collaegues plan to develop pulmonary epithelial cells from human ES cells (Wisconsin lines H1 and H9) using "minimal media conditions", modeled from their studies using murine ES cells. Susequently, they will test such precomitted cells, enriched by FACS for specific subpopulations of pulmonary epithelium, for their ability to repopulate a tracheal graft, where functional assessment will be done. The hyopthesis is that as few as 10% normal cells grafted in patients with CF will result in normalization of chloride transport of the epithelium akin to normal epithelium. SIGNIFICANCE AND INNOVATION: CF is a relatively frequent genetic disorder leading to pulmonary insufficiency and death by the age of 20-40 years. The only curative therapy is lung transplantation. If replacement of cells missing the CFTR could be achieved, this would lead to correction of the pulmonary insufficiency. As ES cells could be induced to make any cell type, it should be possible to generate pulmonary epithelial cells for this purpose. The PI and two collaborators have extensive and pertinent research experience in pulmonary cell biology. The PI worked with Dr. Gordon Sato and has first-hand experience in developing serum-free media for various types of cultured cells. Although the proposed approach is not particularly innovative, its focus of the project on the pulmonary system is. This has been a neglected area of work in the hESC field until recently. STRENGTHS: This proposal is from a group of investigators with a good track record in pulmonary epitehlium characterization and differentiation, as well as genetic modification. Preliminary data using mouse ES cells show that under the conditions proposed to use for hES cells induction towards cells with some characteristics of pulomanry epithelial cells can be achieved. In addition this team has experience with the trachea repopulation model. WEAKNESSES: Reviewer one: 1. Aim 1: the authors will base studies in hESC on the prelimary data obtained with mESC, presented in the proposal. Although some induction of pulmonary markers is seen using the conditions proposed, the degree is relatively low. The histology shown is not convincing and no data as to function of the cells is shown. The likelihood that the conditions proposed will indeed result in significant numbers of cells differentiated into pulmonary epithelium is relatively slim. It would be more prudent if the investigators did studies wherein cells were taken step-wise through endoderm commitment, and then pulmonary specification, and final differentiation as has been done for pancreatic and hepatic differentiation from mESC and hESC. Little attention is also given to the degree of differentiation that will be obtained, and how to assure purity of the differentiated progeny, to minimize the chance for teratoma formation from the grafted cells 2. in the second part of the studies proposed, the investigators would select cells based on cell surface markers associated with different subpopulations of pulmonary epithelial cells and then use such cell populations to graft into a tracheal repopulation model. This is an extremely ambitious aim, in view of the fact that no evidence exists that PE cells can indeed be generated and purified. It is not clear how well the cells can be selected by FACS, and how many cells can be obtained using this approach. As an other method for asessing function, chloride currents will be tested in vitro. This appears more doable and likely more infomative at this stage of knowledge of ES-PE differentiation. Reviewer 2: The application is poorly and carelessly written. This proposal only includes 1.3 FTEs. The PI and two collaborators are committed 10% each and one technician is committed 100%. The technician is the only one on the team with any hESC experience. Her experience is not extremely extensive. This is evident in the Research Design section where it is written that the hESCs will be cultured in DMEM supplemented with 10% FBS and LIF. This is not a traditional medium formulation for hESCs, and rather is more reminiscent of mouse ESC culture medium.