Heart failure is a leading cause of mortality in California and the United States. Currently, there are no “cures” for heart failure.Other life threatening forms of heart disease include dysfunction of cardiac pacemaker cells, necessitating implantation of mechanical pacemakers. Although mechanical pacemakers can be efficacious, there are potential associated problems, including infection, limited battery half-life, and lack of responsiveness to normal biological cues.
Our research with human embryonic stem cells will be aimed at developing therapies for heart failure, and cardiac pacemaker dysfunction. In each of these disease settings, one might effect a “cure” by replacing worn out or dysfunctional cardiac cells with new ones. In the case of heart failure, the cells that need to be replaced are heart muscle cells, which do the majority of the work in the heart. In the case of pacemaker dysfunction, the cells that need to be replaced are pacemaker cells, a highly specialized type of heart muscle cell. To replace these cells, we need to find cells that can become heart muscle or cardiac pacemaker cells, understand how to generate fairly large numbers of them, and how to persuade them to become either heart muscle or cardiac pacemaker cells. Potential cardiac progenitor cells may come from a number of different sources, either from patients themselves, or from extrinsic sources. Regardless of the source,we need to define factors which will make the cells multiply and will make them become the cell type that we need for repair.
The biology of human heart cells is likely to be distinctive from that of heart cells from other animals. For example, a human heart has to function for multiple decades, unlike hearts of other animals who live in general for shorter periods of time. The size, required function, and rhythm of the human heart are also distinct from that of other animals. For these reasons, for repair of human heart, it is important to study human cardiac progenitors and to define pathways required to grow them and to differentiate them utilizing human cells as a model experimental system.
Our proposed research will utilize human embryonic stem cells as a source of cardiac progenitors. As human embryonic stem cells can turn into many different kinds of cells, we will create special lines of human embryonic stem cells that will become fluorescent when they adopt the cardiac progenitor, heart muscle, or pacemaker state. These lines will then be treated with a large number of small molecules to find small molecules which amplify cells the number of fluorescent cells in each of these states. The small molecules activate known biochemical pathways, so we can then use the small molecules themselves, or activate identified pathways to achieve the goal of obtaining sufficient numbers of specific cardiac cell types for cardiac therapy.
More Californians die each year of cardiovascular disease than from the next four leading causes of death combined. Californians continue to die or be disabled as a direct result of cardiovascular disease. Although advances in medical treatment have improved post-infarct survival, heart failure is an increasingly abundant manifestation of cardiovascular disease. A secondary complication of heart failure, and other cardiac diseases, is cardiac pacemaker dysfunction, a potentially fatal condition which is currently ameliorated by mechanical pacemakers. However, mechanical pacemakers have many associated complications,particularly for pediatric patients. For both heart failure and pacemaker dysfunction, replacement of heart muscle cells or biological pacemaker cells offers the hope of improving upon current medical practice. Our research is aimed toward developing new therapies which will allow for the replacement of these critical cell types in diseased heart.
SYNOPSIS: In this proposal, the investigators wish to generate cardiomyocytes from hESCs. This will be accomplished using cells engineerd to express fluorochromes from the Isl1 promoter alone or combined with the MLC2v or HCN4 promoter, the latter identifying ventricular myocytes vs. pacemaker cells respectively. Libraries of small molecules will be screened to identify those that support cardiomyocyte conversion and subsequently ventricular and pacemaker cell conversion.
SIGNIFICANCE AND INNOVATION: This proposal targets the differentiation of cardiac cell types from human embryonic stem cells. These cells can then be used as both a platform to understand the basics of ventricular myocyte and cardiac pacemaker cell lineage specification and alternatively as a source of cell-based cardiovascular therapy. The PI, who is a leader in her field, proposes two specific aims. The first specific aim is to develop, phenotype, and perform preliminary screens of hESC lines genetically engineered to express isl1-GFP. hESCs with GFP targeted to the isl1 locus will be established, characterized, and utilized in preliminary small molecule screens for optimal conversion to cardiovascular progenitors. The second specific aim is to genetically engineer hESC lines from parent isl1-nGFP hESC lines which co-express is isl1-nGFP and MLC2v-Red Cherry) cR) or is isl1-nGFP and HCN4-cRC, as markers specific for ventricular and pacemaker cell lineages, respectively.
Understanding the pathways required for specification, proliferation, and differentiation of hESCs into these two cardiac cell lineages are a necessary and essential component for both the basic understanding of this important differentiation event as well as the potential for clinical settings. It is thus extremely significant and of the highest priority. This grant is also innovative, as it courageously tackles genetic engineering of hESCs, which unlike the mouse system (mESCs) is either inexistent or in its infancy.
STRENGTHS: The PI has a track record of high productively in solving the molecular basis of lineage restrictions toward cardiac cell fates. Previous work from the applicant's laboratory in model systems has established the basis of our current understanding in this lineage acquisition. Additionally, generating hESCs that express stable markers of cell differentiations, in this particular case the isl-GFP line is one of the most secure ways to approach this problem if the technical problems can be overcome. The small molecule screen described on specific aim 1 is bound to identify individual compounds with the ability to turn on the isl1-GFP promoter efficiently. Finally, the generation of doubly-targeted hESCs distinguishing between ventricular and pacemaker cell lineages are inevitably going to allow linkage to specific signaling pathways as well characterization of active compounds that have the ability to induce one cell type versus the other.
WEAKNESSES: Previous attempts in characterizing small molecules with specific activities toward the induction of a particular cell type using stably marked embryonic stem cells have been efficient in characterizing compounds. However, limiting factors downstream of this characterization have been to address the specificity of these compounds toward other biological activities. So while many good candidates can be identified by this type of approach, and most are very useful in the context of basic science approaches, it is not very clear how these compounds will be useful in clinical settings.
DISCUSSION: The proposal was regarded as very well written and very well reasoned. The use of non-federally-approved lines for screening is deemed a smart choice. It was felt that the use of these cells over federally-approved lines might also allow knock-in strategies to work but there was concern that a two-step knock-in approach presents a significant challenge; the BAC "back-up" strategy was praised. A discussant voiced an alternative view that the anticipated results of this work would not be significant.