The field of regenerative medicine revolves around the capacity of a subset of cells, called stem cells, to become the mature tissues of the adult human body. By studying stem cells, we hope to develop methods and reagents for treating disease. For instance, we hope to develop methods for making stem cells become cardiovascular cells in the lab which could then be used to rapidly screen large numbers drugs that may be used to treat cardiovascular disease. In another example, if we are able to create bone in the lab from stem cells, we may be able to help treat people with catastrophic skeletal injuries such as wounded soldiers. Until recently, the most flexible type of stem cell known was the embryonic stem cell. Embryonic stem cells are pluripotent, meaning they can give rise to all of the adult tissues. In contrast, stem cells found in the adult are considered only multipotent, in that they can only become a limited number of mature cells. For example, bone marrow stem cells can give rise to all of the components of the blood, but cannot make nerves for a spinal chord. Breakthroughs in the past couple of months have indicated that it is possible to "reprogram" adult skin cells and make them become pluripotent, like stem cells from an embryo. These new kind of cells ares called "induced pluripotent cells" or iPS cells for short. This has lead to great excitement within the scientific community because it raises the possibility that we may use this technology to rapidly create pluripotent stem cells from a large host of human diseases using skin from affected individuals. However, whether the new iPS cells made from skin cells and embryonic stem cells are functionally the same in all applications remains to be seen. Our lab is in the unique position to test this hypothesis. We have derived several normal embryonic stem cell lines and are in the process of deriving iPS cells from normal skin. Furthermore, we are fortunate enough to have begun deriving a new embryonic stem cell line harboring an inherited mutation that results in severe cardiovascular and bone disease that affects more than 7,500 Californians. What's more, one of our collaborators has over the past ten years assembled a cell bank of more that 50 unique adult skin cell lines with the same inherited disease. Therefore, for our proposal, we will make new normal and disease specific iPS and embryonic stem cell lines. We will use these new stem cell lines to test whether the iPS and embryonic stem cells are truly functionally the same, by comparing them after we make them become cardiovascular and bone cells. This work will allow us to advance the field of regenerative medicine on two fronts. 1. We will perform an important comparison of iPS and embryonic stem cell lines. 2. We will compare the disease specific cells with normal cells which will help us better understand cardiovascular and bone disease and pave the way for the development of new therapies.
Our proposal compares normal and disease specific pluripotent stem cells derived from embryonic and adult skin sources. This proposal will benefit the state of California and its citizens in several specific ways. First, the specific inherited disease we are studying affects approximately one in every 5,000 people worldwide. That translates into over 7,500 Californians and over 60,000 men, women and children of every race and ethnic group in the United States. By examining the characteristics of the disease specific lines, we hope to better understand the mechanisms of the disease and create assays for screening new drugs that can be used to treat people with the disease. Second, this disease is one of a broad class of cardiovascular disease, called thoracic aortic disease. An estimated 3,700 Californians are treated for thoracic aortic disease every year. Our findings may provide insight into the mechanisms underlying these diseases and other cardiovascular diseases. Third, this disease also results in skeletal defects. By studying the mechanisms of the skeletal defects, we will better understand the mechanisms of bone development, which will lead to improved applications of stem cell therapies for individuals with bone injury and disease. Finally, by providing detailed comparisons of iPS and embryonic stem cells, our work will have important ramifications for the future direction of the entire field of stem cell research and regenerative medicine.
This proposal aims at deriving pluripotent stem cell lines carrying mutations that cause Marfan Syndrome (MFS) and a related disease, Loeys-Dietz Syndrome (LDS). MFS patients suffer from connective tissue defects in the skeletal, ocular, and cardiovascular compartments. The applicant will derive disease-specific pluripotent cell lines, both induced pluripotent stem cell (iPSC) lines and human embryonic stem cell (hESC) lines, and intends to test whether they are similar in their capacities for prolonged undifferentiated euploid growth and differentiation potential. The focus is on differentiation of cardiovascular and osteogenic cell types from the pluripotent stem cells.
The proposed iPSC and hESC lines may provide new approaches to study the disease progression and pathology of MFS and LDS, and to develop drug screens relevant to thoracic aortic disease, a clinical manifestation of MFS and an important clinical target. The PI thus provides an excellent rationale for the project, which was considered to be of high significance. During the discussion, reviewers considered whether the generation of iPSC and hESC lines was necessary to study the mesenchymal derivatives as proposed in this application, rather than studying patient-derived fibroblasts directly. However, it is important to note (as one reviewer did) that the disease phenotypes may be initiated early during differentiation, so that only pluripotent cells could provide insight into early defects that may be responsible for the ultimate disease phenotype.
This is a very well written, focused proposal that provides the right balance of experimental detail, so that reviewers had reasonable expectations for success. The principal investigator (PI) has a very good, relevant track record of peer-reviewed publications, and the assembled team seems ideally suited to carry out these derivations. Although some of the proposed cell line characterization studies are technically challenging, the approach is well described and the PI has access to appropriate and outstanding resources. Although not essential, it is hoped that hESC can be generated from donor embryos, and iPSC from the same donor couple, such that the same mutation could be studied in both contexts (yielding extra information about differences between iPSC and primary hESC). The direct comparison between iPSC and hESC was considered a major strength of this proposal. Overall, the proposed work has a high likelihood of success in generating novel human pluripotent stem cell lines carrying clinically-relevant disease genes.
This is a project to derive iPSC and hESC lines representing Marfan Syndrome (MFS) or the related Loeys-Dietz Syndrome (LDS).
Reviewer One Comments
This proposal aims at deriving pluripotent cells from two related diseases [Marfan Syndrome (MFS) and Loeys-Dietz (LDS)] to test whether iPS and hES cells are similar in their capacities for prolonged undifferentiated euploid growth and differentiation potential. This proposal enjoys a high significance.
This proposal has three specific aims. The first is to derive iPSC and hESC lines with MFS and LDS. The second aim is to compare iPSC and hESC lines during spontaneous in vivo differentiation. The third specific aim is the comparison of iPSC and hESC lines following in vitro directed differentiation towards osteogenic and cardiovascular fates and stimulation with TGF-beta.
Dr. Longaker (PI, 10% effort) is an expert in TGFbeta signaling with a very good track record of peer-reviewed publications. Dr. Eric Chiao (Co-PI, 25% effort)
Collaborators include Dr. Uta Francke (2% effort, has 50 MFS fibroblast lines, Stanford); Dr. Wing Wong (2% effort, Stanford); Dr. Renee Reijo Pera (effort as needed to assist with iPSC derivation, Stanford); and Dr. David Liang (effort as needed).
Stanford University is an excellent setting for these studies, including the Stanford University Center for Marfan Syndrome and Related Aortic Disorders directed by Dr. Liang and the Human Embryonic Core Facility directed by Dr. Chiao.
Responsiveness to RFA:
This proposal is adequately responsive to the RFA, including the derivation of new hESC lines that can differentiate into all three germ layers, as well as a suitable means of sharing these lines and data with colleagues.
Reviewer Two Comments
Marfan is an autosomal dominant negative syndrome caused by mutations in the fibrillin gene, which is an ECM component important for elastin fiber structure, and associated with binding TGF-beta. Patients therefore have defects in connective tissue in the skeletal, ocular, and CV compartments that are associated with excessive TGFb signaling. In addition to skeletal abnormalities, serious complications include aortic dilatation, aneurysm, and valve defects. LDS is caused by mutations in TGFb1 or 2 receptors. The iPSC lines may provide new approaches to study the disease progression and pathology and to develop drug screens relevant to thoracic aortic disease. This is an excellent rationale and the project is of high significance.
This is a very well written proposal that provides the right measure of detail and reasonable expectations for success. The goal is to compare iPS and ES cells carrying MFS and related mutations, and then to test if they can be used to model thoracic aortic disease. There are 3 Aims. The first is to generate at least 7 new human cell lines: 1 MFS from an IVF blastocyst (this is already underway), 3 iPS from MFS fibroblasts, and 3 iPS from LDS fibroblasts. The team seems ideally suited to carry out these derivations. The co-investigator Chiao has experience in hES derivation, the PI has access to over 50 MFS fibroblast lines through the collaborator Dr. Francke, and Dr. Reijo-Pera is recruited to assist with the iPS derivation. Although not essential, it is hoped that ES cells can be generated from donor embryos, and iPS from the donor couple, such that the exact same mutation could be studied in both contexts.
Aim 2 will carry out experiments to compare the lines by generating teratomas. Those lines that can generate tissues representing all 3 germ layers will be studied further. The main idea is to use laser capture microdissection to isolate samples of representative smooth muscle or chondrocytes, and to use HT sequencing to compare the transcriptomes. Although technically challenging, the approach is well described and the PI has the facilities in principle to carry out the comparison.
Aim 3 is to compare phenotypic differences among the cell lines in terms of directed differentiation in vitro. The focus is on generation of CV fates: endothelium and smooth muscle, and on osteogenic fates via derivation of mesenchymal stem cells. The latter plays to the expertise of the PI. Again, although ambitious and still unclear how efficient it will be, the PI describes the protocols well, with a good sense of the challenges and without excessive ambition (ie with good focus).
Responsiveness to RFA:
This proposal has a high likelihood of success in generation of novel human pluripotent cell lines carrying relevant disease genes. MFS is ideal for this purpose since it is dominant autosomal and the PI is well situated to recruit donors via the Stanford Center for Marfan Studies. Dr. Longaker is an expert in TGFb and FGF signaling relevant to osteoblast biology (2 R01s, 1 T32, 1 CIRM TG) with a strong focus on regenerative medicine. Therefore, the proposal is well focused within his expertise. In addition, he has recruited outstanding experts to complement, and can take advantage of outstanding resources.