SEED: Whole Stem Cell Sorting Using an All-Electronic Microfluidic System

Funding Type: 
SEED Grant
Grant Number: 
RS1-00174
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Stem cells are typified by their ability to differentiate into a wide range of specific cell types, including nerve cells, brain cells, and other cells that do not typically regenerate in the human body. This pluripotency holds great promise for regenerative medecine, possibly enabling the repair of damaged or mis-developed tissues in adults and children. A great promise for stem cells is the production of very large quantities replacement heart muscle, skin cells, nerve cells, or other types of cells that are needed to repair these tissues. Controlling into what type of cells stem cells differentiate, and separating those that have differentiated, from those that have not differentiated, are a key elements in using stem cells as therapeutic elements. Control is presently being explored using a wide range of different chemical and physical environments, and is proving a highly complex and challenging area of research. Distinguishing and purifying the results of these control experiments, both in research and in clinical application, will clearly be a significant and important part of the eventual therapeutic use of stem cells. A multiplexed, microfluidic, all-electronic stem cell sorter, which we propose to develop under this seed grant, would enable the rapid and highly specific separation of differentiated and non-differentiated stem cells into separate groups, producing very highly purified samples of each particular type. These would then be available for further experimentation, or for direct therapeutic use. The entire microfluidic sorter is based on technology that would allow this process to be performed in a very inexpensive and disposable instrument, making the sorter readily and cheaply available to both the researcher and the biomedical industry. It is based on a very novel and possibly disruptive technology, quite distinct from the methods presently used for these applications. This all-electronic approach would provide a route to much simpler and faster sorters, and more rapid results, than present methodologies.
Statement of Benefit to California: 
The research proposed here will focus on the development of an all-electronic, microfluidic cell sorting technology, which will involve the combination of microfluidics, all-electronic biosensing, biomolecular labeling, and cell culture. The end product will be a small, hand-held unit that includes disposable components, that enables a rapid, highly specific way to sort and quantify cells in a fluid sample. Technology that will enable uniquely labeling stem cells based on surface protein expression, sorting labeled cells in a highly specific and rapid fashion, and quantifying the numbers of cells sorted into each channel, will be developed and used in creating this instrument. The benefits to California are multi-dimensional. First, the technology produced through this research will have a direct impact on stem cell research and possible clinical use, an area to which California has clearly made a strong commitment, so that efforts in these areas pursued within California would benefit directly. Second, the technology produced here will likely form the basis of a commercial enterprise to produce, market, and further develop this technology. This enterprise will most likely be based in California, and if successful would provide employment, new technology, and tax revenue to the state. Third, the participation of four researchers from quite diverse field, bringing highly distinct expertise in engineering biophysics (PI Cleland), biochemistry (co-PI Reich), molecular biology (collaborator Laird) and stem cell culture (collaborator Wesselschmidt), will provide a unique combination of backgrounds and technologies that will likely generate new ideas and more applications for this type of instrumentation. Fourth, the research efforts and education of the postdoctoral and graduate students will generate two new professionals in this cross-disciplinary area, two students that will doubtless continue to work on closely related areas of work and further develop the concepts and technology that will enable future progress.
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).

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