Funding opportunities

Derivation and analysis of pluripotent stem cell lines with inherited TGF-b mediated disorders from donated IVF embryos and reprogrammed adult skin fibroblasts

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00662
Principle Investigator: 
Institution: 
Funds requested: 
$1 424 412
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
Executive Summary 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. Reviewer synopsis This is a project to derive iPSC and hESC lines representing Marfan Syndrome (MFS) or the related Loeys-Dietz Syndrome (LDS). Reviewer One Comments Significance: 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. Feasibility: 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 Significance: 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. Feasibility: 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.
Conflicts: 

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