Pharmacological characterization of human stem cells and their differentiation via the GPCR transcriptome

Funding Type: 
SEED Grant
Grant Number: 
RS1-00174
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
In envisioning how stem cells could be used to replace lacking or deficient cells in the central nervous system (CNS), we recognize that one of the best way would be to induce stem cells present in the brain to differentiate into the lacking or deficient cells. When considering Parkinson's disease, one of the best way to develop a therapy would be for mature neurons that can replace lacking dopamine-expressing cells. The issue is how to drive stem cells into becoming full-fledged neurons. This project proposes a new way at reaching this goal. Every cell in the organism expresses a battery of G protein-coupled receptors (GPCRs). The GPCRs bind all the small molecules that direct intercellular interactions. Importantly these include all the neurotransmitters and neuropeptides., making the GPCRs the prominent regulators of brain function. Binding of the GPCR ligands initiate a cascade of intracellular reactions that change the transcriptional activity of the recipient cell. Most notably GPCR activation can initiate differentiation, i.e. they may be able to induce a stem cell to become a neuron. Each cell in the organism, expresses a few dozen different GPCRs. Although not determined thus far, stem cells too will harbor several GPCRs, probably at least as many as the adult cells. If we can find the stem cells GPCRs we may be able to use them to transformed these cells into neurons. The first issue is to find all the stem cells GPCRs. This will be done using a well developed technique, RT-PCR. Using it we will catalog all the GPCRs express in different stem cells. We will then activate the GPCRs that we have found on stem cells to determine whether they can induce the stem cells to differentiate into neurons. There are already data that a few GPCRs have this ability. Our aim will be to discover how many can do it and whether there is some commonalities between the ones that have this ability. We will also determine whether the exhibit differences in the neuronal phenotypes and thus define the cascade of events that leads to this phenotype. Knowing which GPCRs can induce neuronal phenotypes and at which step in the differentiation process a particular GPCR acts will have direct impact on therapies. GPCRs are the most sought targets in drug development and many drugs acting on GPCRs already exist. Our research may point at some of these drugs as potential mediators of neuronal regeneration and possibly helpers in therapies aiming at replacing lacking neurons.
Statement of Benefit to California: 
The success of this proposal would have a positive outcome for California. It approaches the use of stem cells from a pharmacological stand point. In doing so it leads to therapies that do not require surgical intervention. Instead it may point at existing drugs as potential agents for neuronal regeneration. This would result in immediate applications. It may also point at new targets that should be pursued pharmaceutically.
Progress Report: 
  • Full clinical potential of human ES (hES) cell therapy can be achieved when one can grow hES cells effectively while maintaining full pluripotency. We have focused on developing stem cell culture media by which we can maintain pluripotency of human ES (hES) cells. It is critical to determine and develop a chemically defined media that are animal product-free and feeder cell-free conditions so that the media can be standardized throughout stem cell research and in clinical situations.
  • One major recombinant protein component we will use in developing chemically-defined media is a set of TGF-beta signaling ligands, receptor domains, and ligand-specific antagonists. We have established a new method of generating a diverse array of these ligands, including BMPs, Activins, inhibin, and their heteromeric ligands of the BMP/Activin class ligands. Some of these heteromeric ligands possess their signaling properties unlike their homodimeric counterparts. These reagents include Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, and their derivatives. These reagents have been engineered by chimeric recombination. They were also further modified by site-specific mutagenesis, and by combinatorial heterodimeric assembly to create and modify protein-specific binding affinity to their binding counterparts. Several of these reagents are now available as recombinant protein in sufficient quantity for large-scale screening for media composition.
  • To establish the functional characteristics and optimal culture combinations using these new reagents, we have used an established hES cell, H9. We have cultured H9 cells in various compositions of culture media containing some of the engineered reagent and followed expression of several differentiation markers to monitor for pluripotency of hES cells, and also for their differentiation-guiding and pluripotency-maintaining abilities. We have first examined effect of aforementioned reagents: Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, BMP3 S28A mutant, in our standard culture media mTeSR condition, which does contain bFGF, for proliferation and differentiation of hES cells. In these assays, hES cell line H9 was cultured and reagents were added at varying concentration (1-100 ng per ml) over 1-5 days culture period. Reagents were added in new media during the course of cell culture. We have used morphological change and the presence of markers as a means to follow the differentiation. Ectoderm markers are Nestin, Cdx2; Mesoderm by Brachyury, HBZ; Endoderm markers by CXCR4, Sox17, Gata4, HBF4 alpha, Gata6, AFP. Two BMPs had pronounced effects in inducing cells to endoderm. We have followed up by analyzing the efficiency using FACS. Up to 60% of cells have undergone to endoderm-marked cells. With the availability of a cell sorter, we evaluated pluripotency by means of proliferation rate, morphology, fluorescent signal in the reporter lines by visual inspection and FACS, then we further characterized the factors by real-time PCR for stem cell markers and karyotyping.
  • It is known that high concentration of FGF can suppress the action of BMPs, so we planned to repeat the experiments in mTeSR media with lowered levels of FGF to re-evaluate the effects of BMPs on cell differentiation abilities. After these tests were completed, we established a protocol performing these assays in high-throughput manner. We are currently in the process of writing this work for publication (Valera et al., in preparation).
  • Towards the development of chemically defined culture media to maintain pluripotency, we have then tested various newly-engineered reagent to replace a protein component in TeSR media. We have established a combination of protein factors known to maintain established hES cells without using nonhuman products except human albumin, which include basic fibroblast growth factors (bFGF), and a bone morphogenetic protein derivative known as AB2008. We have termed this new media as CAV media. We are currently in the process of writing this work for publication (Valera et al., in preparation).

© 2013 California Institute for Regenerative Medicine