Cell membranes constitute one of the fundamental structural and functional elements of living organisms. These complex mixtures of lipids and proteins form the outer boundary of the cell. They distinguish between the ‘inside" and the "outside", and allow the cell to communicate with its environment and maintain its integrity. Membranes are generated when a cell is “born” and membrane lipids are continuously remodeled during the life of the cell. In this remodeling process lipids from the environment are taken up and used. All cell types in the body have a closely regulated and characteristic membrane lipid organization, and while very little is known on the lipid organization of embryonic stem cells, they are likely no exception. When stem cells are collected and grown in cell culture, it is essential to provide them with an environment that maintains their membrane integrity and allow them to proliferate and differentiate into the tissue of choice. In our proposed studies we plan to define the lipid composition and organization of stem cells in culture. We will change the cell culture conditions to modulate self-renewal and differentiation, and specifically test this in a system to where human lung epithelial cells are generated from stem cells. The biochemical, cell biology and molecular biology technology that has been established in our laboratory for cord blood derived stem cell studies will be applied for these studies on human embryonic stem cells, currently not funded by the federal government. By exploring the role of lipids in proliferation and differentiation of human embryonic stem cells, our preliminary studies will add substantially to the body of knowledge on human embryonic stem cells, and set the stage for full scale investigations to define the role of lipids in modulating the therapeutic use of embryonic stem cells.
Statement of Benefit to California:
A significant better understanding is needed to define the powerful potential of self-renewal and differentiation of human embryonic stem cell for the diagnosis, prevention and treatment of disease and injury. While lipids are essential components of all cells, including stem cells, little is known with respect to the organization of lipids in human embryonic stem cells. Moreover, we pose that lipids in the environment of these cells may modulate both self-renewal and differentiation. We anticipate that our studies will add substantially to the body of knowledge on human embryonic stem cells, and set the stage for full scale investigations to define the role of lipids in modulating the therapeutic use of embryonic stem cells. The opportunity offered by the CIRM SEED Grant program in California allows established investigators to explore studies that would otherwise be impossible due to the lack of federal funds. Our proposed studies will be part of a large effort to establish California as an important scientific center in stem cell research. It will create a scientific community with experience in embryonic stem cell research, attract scientists interested in exploring the potential of embryonic stem cells to our state, and will accelerate the development of applications of stem cells for therapeutic use.
SYNOPSIS: This application proposes to determine the nature and organization of lipids in hESC membranes, as possible indicators of SC status (apoptosis, proliferation, differentiation). It also addresses the question whether the composition of the lipids in the culture medium may affect the regulation of SC growth and induction of differentiation or apoptosis. The PI proposes to define the molecular composition of hESC phospholipids, the enzymes that regulate their turnover and the proteins that maintain phospholipid asymmetry at the cell membrane. Variations in the culture conditions that promote growth or differentiation, or that prevent apoptosis will be investigated. Addition of lipids, including synthetic peptides that mimic the LDL binding domain of ApoE, will be investigated. A second aim seeks to determine whether specific lipid composition is relevant to differentiation of stem cells into functional lung epithelial cells. The long term goal of these studies is to identify the role of specific phospholipids species on hESC differentiation and self-renewal, including the use of lipids as potential biomarkers. INNOVATION AND SIGNIFICANCE: The proposed studies are reasonable and feasible, though not hugely original or innovative. All the proposed studies can be done given time and effort. The results are likely to clarify aspects of stem cell biology and provide usful hints to guide the selection of nutrients and additives (including synthetic lipoproteins) in culture medium to prevent apoptotic changes and mitochondrial damage that reduce the efficiency of the cultures. The lipid compositon of hESCs is a timely and important question. To date lipidomic analysis is the most well developed and systematically developed form of the larger field of Metabolomics. The overall goals of this project are important and timely and the potential impact and significance of the results is high. STRENGTHS: The PI and his collaborators are currently supported by NHLBI for studies of phospholipids on red blood cell membranes in sickle cell disease and have all the necessary techniques and equipment at their disposal. Drs. Kuypers, Forte and Serikov are all experienced researchers with substantial publication records in this and related areas. Dr Kuypers has carried out studies of expansion of stem cells in cord blood. The participation of Drs. Ames and Atamna adds substance to the studies on preventing or reversing mitochondrial stress and oxidative damage to cells in culture. The environment at CHORI is excellent for these studies: several investigators in stem cell-related areas including clinical hematopoietic stem cell transplantation, will undoubtedly contribute to Dr. Kuypers work. Another strength of this application is the appreciation by the applicants that standard cell culture conditions leads to aberrant membrane lipid composition. Growing cells in 10% FBS does not provide the physiological exposure to many fatty acids and other metabolites. The studies will be conducted incorporating technical improvements to conditions such as lower O2 tension in the incubators as well as higher CO2, to reflect actual live conditions better. The applicants will actively examine the effects of various culture conditions to define the effects on membrane lipid composition. WEAKNESSES: The main weakness is the lack of proper consideration of the potential changes that can occur in cultures. The proposal even suggests that several hESC lines may have to be studied, from the same source (Harvard) or other sources. Conceivably, the studies would be detecting and characterizing artifacts of the life in culture dishes of these stem cells, including possibly the adaptation to those dishes. They might not represent the natural patterns of lipids on native hESCs. Thus, some changes may not be characteristic of the functional differentiation towards mature cells but simply further reflections of life in culture without a functional connotation. One of the deficiencies in the proposal is that the applicants are not experts in lipid mass spectrometry. Some of the lipid analysis will be subcontracted. There is no discussion of how the data will be analyzed. There is no discussion of the levels of significance, what internal standards will be used for quantitative analysis nor any of the numerous bioinformatics issues will be addressed. In short the applicants have presented an interesting question and their expertise in the relevant areas of biology are clear. However, there is no clear demonstration that they will know what to do with these massive data setsthat will be generated from the proposed studies, important in own right. DISCUSSION: Although not a particularly original approach, the question proposed by the work is fundamentally important. The lipidomic/metabolic analysis is being subcontracted; the subcontractor is not known for doing this kind of molecular species analysis. There is no explicit discussion as to how the very large resulting dataset would be handled; typically a bioinformatics core is required for these large analyses. There is a lack of discussion of appropriate controls, how many samples, how many variants scored. In short, there were methodological concerns with the application.