A method to maintain and propagate pluripotent human ES cells

A method to maintain and propagate pluripotent human ES cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00174
Approved funds: 
$760,042
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Human embryonic stem (hES) cells are pluripotent such that they can differentiate into all three germ layers, thus potentially all different types of tissues of the body. Pluripotency is characteristic of only embryonic cells, but it can also be achieved by reprogramming differentiated cells by transferring nuclear contents into unfertilized, enucleated oocytes or by fusing with ES cells. To achieve the initial embryo-like state, it is a pre-requisite to be able to maintain and propagate these ES cells in culture conditions in vitro. Currently, such recipe exists for mouse ES cells. Surprisingly, similar media components for hES cells do not work. This very first technical barrier needs to be overcome in order to realize full clinical potential of stem cell therapy. We propose to develop a novel recipe of chemically defined culture media and culture conditions to grow and maintain pluripotency of hES cells. The media we will evaluate are combinatorial mixtures containing only recombinant proteins, chemically synthesizable reagents, or human source factors. To achieve new sets of recombinant protein reagents known to be involved in controlling differentiation and pluripotency of embryo-like cells, we will develop a novel biochemical strategy of producing a set of target protein reagents effectively in test tubes. To screen conditions using these chemically defined components and various culture conditions, we will develop a new cell line containing a reporter gene (GFP) recombined into human Oct4 gene. Human Oct4 gene is the prominent marker for stemness of the hES cells. There are three specific Aims for this proposed study. They are, 1) production of the media components biochemically, 2) development of two Oct4-reporting hES cell lines, and 3) screening of culture media and conditions for maintaining pluripotency of hES cells. These experiments will be carried out in parallel as collaboration between two laboratories {REDACTED}. Once Aims 1 and 2 are completed, we will evaluate these hES cell lines in various culture conditions systematically (Aim 3). In doing these high-throughput assays for functional characterization, we will also conduct screening of known chemical library of selected drugs and metabolites to glean into their potential ability to augment or inhibit actions of the engineered biologic reagents in controlling the growth and pluripotency of hES cells. From the screening using these two cell lines, we will establish the firm method of propagating and maintaining pluripotency of hES cells for subsequent clinical applications.
Statement of Benefit to California: 
Establishing the methods to promote and maintain pluripotency of human embryonic stem cells is a groundwork absolutely necessary to facilitate stem cell research in general and to be adopted for its immediate clinical applications. For instance, the proposed study will generate essential data to facilitate biotechnological approaches to scale up to meet the industrial demand of the much needed protein reagents to culture hES cells. These reagents we established are also critical for basic research of developmental and cell biology, structural biology, and drug discovery. So scientific and industrial benefits to California are enormous. The proposed study requires combination of extensive biochemistry and developmental biology expertise. Development of new techniques and reagents for the maintenance and proliferation of pluripotent hES cells are fundamentally essential in order to fully exploit therapeutic potentials of hES cell therapy. We believe that the prime benefit from results of the proposed study is to add substantially to the body of knowledge on growing, maintaining, and finally guiding hES cells to the differentiated states as needed to develop effective therapeutic means.
Progress Report: 

Year 1

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).

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