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

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
Approved funds: 
$1,406,636
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 
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.
Statement of Benefit to California: 
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.
Progress Report: 

Year 1

During the past year, we have used the funds from this grant to derive a new embryonic stem cell line with an inherited mutation that results in a severe cardiovascular and bone disease called Marfan syndrome that affects more than 7,500 Californians. In addition, using adult skin cell lines with the same inherited disease, we have made significant progress deriving iPS cells with Marfan syndrome. During the next year we also hope to expand our studies by recruiting patients with a disease very similar to Marfan syndrome called Loeys-Dietz syndrome, to donate skin biopsies so that we can make iPS cells to study that disease as well. Using these new stem cell lines, we are testing whether the iPS and embryonic stem cells are truly functionally the same, by comparing them after we make them become cardiovascular and bone cells. One of the biggest challenges in stem cell biology is figuring out how to make the stem cells become the adult cells we want to study and not some other random adult cells. Over the past year, we have made great strides in turning our stem cells into the cell types most severely affected in people with Marfan syndrome, namely bone and cardiovascular cells. What is most exciting to us is that even with these preliminary studies, it looks like we might be seeing differences between the stem cells with Marfan syndrome and normal stem cells after they are coaxed into become the bone and cardiovascular cells. These results are still very preliminary though, and we need to take great care during the next year to rigorously repeat our experiments before we can be certain of those results. If we can reproduce the differences, these differences may be the basis for screening for new drugs to treat people with Marfan syndrome or lead to a better understanding as to what exactly is the sequence of cellular events that leads to the patient’s symptoms. What’s more, by studying how to efficiently make bone and cardiovascular cells from human embryonic stem cells and iPS cells in the dish, we hope to provide important data that could be beneficial in a wide variety of applications such as tissue engineering or cellular replacement therapies using bone or blood vessels.

Year 2

Marfan Syndrome (MFS) is a genetic disorder that affects more than 7,500 Californians. Patients develop severe complications, affecting several parts of the body (eyes, limbs, aorta). During the last two years, we have used the funds from this grant to develop new cell lines aimed at studying MFS in a dish. These cell lines, are called pluripotent stem cells, and have been generated from: (i) an embryo that was donated for research and was known to have inherited the MFS disease (these cell lines are named human embryonic stem cells (hESCs)); and (ii) from skin biopsies of adult patients (these cell lines are named induced pluripotent stem cells (iPSCs)). These stem cell lines allow us to study MFS by differentiating the cells to adult cells (mainly bone and cardiovascular cells) and not other random adult cells. Using these new stem cell lines, we can test whether hESCs and iPSCs are functionally the same, by comparing them after we make them become cardiovascular and bone cells. We have observed that when the cells form bone or muscle cells, the stem cells with MFS are different and do not behave the same as those made with normal stem cells. We also started to use reagents that can force MFS cells to resemble and behave like normal bone cells. This is called “rescuing the disease phenotype”. For the first time, we are close to describing a stem cell-based technology not only to understand the mechanism(s) of the MFS but also to develop a screen for new drugs to treat people with MFS. However, we still need to confirm our results by repeating the experiments. Our results are very promising for understanding the bone issues in MFS, but continued efforts are also required to understand the cardiovascular issue. It is important to point out that the most important health risk associated with the disease is an aortic aneurysm that, if untreated, leads to death around 35 years old. In conclusion, we are continuing to generate data that will provide the foundation for improving our knowledge of the disease, and also will potentially assist us in developing new therapies for improving MFS patient lives.

Year 3

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 for treating a wide variety of diseases. 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 of drugs that may be used to treat cardiovascular disease. We are also trying to create skeletal tissue from stem cells so that 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 cell types in the body. In contrast, stem cells found in the adult are considered only multipotent, in that they can only become a limited number of mature cells. Breakthroughs in the past five years have indicated that it is possible to "reprogram" adult skin cells and make them become pluripotent, like stem cells from an embryo. These new kinds of cells are called "induced pluripotent cells" or iPS cells. 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 easy to obtain tissue like skin and fat from affected individuals. Our laboratory is in the unique position to test this hypothesis. We have derived several normal embryonic stem cell lines and iPS cells from normal skin. Furthermore, we have derived a new embryonic stem cell line and induced pluripotent stem cells from fibroblasts harboring an inherited mutation that results in severe cardiovascular and bone disease that affects more than 7,500 Californians, called Marfan's Syndrome. We have created stem cells lines, both embryonic and induced pluripotent stem cells from cells having this disease. We have compared these cells to normal embryonic and induced pluripotent stem cells to examine exactly what makes these diseased cells behave in a way to have impaired bone formation. In addition, we have completed the differentiation, banking and full characterization of vascular cells derived from Marfan's Syndrome embryonic stem cells and Marfan’s syndrome induced pluripotent stem cells. We have seen that the cells with Marfan’s syndrome have a particular signaling pathway that has functional disregulation compared to normal, healthy cells. We have been able to explore how this disease process manipulates this pathway to cause this specific disease. Through this kind of modeling, we can use these cells to screen for treatment as well as model the disease in a way to manipulate the specific pathways this disease impacts to hopefully bring clinical treatments to patients who suffer from this disease.

© 2013 California Institute for Regenerative Medicine