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UCSD Team Wins $1.2M NIH Grant for Stem-Cell Study Using Cellular Screening Array

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By Justin Petrone

The National Institutes of Health has awarded $1.2 million to a group of researchers at the University of California, San Diego, that will use an internally developed cellular microarray platform to study stem-cell differentiation. The ultimate goal of the study is to produce cells that can be used in vivo to treat patients with diabetes.

As part of the project, the UCSD researchers will use the cellular arrays to develop ways of deriving glucose-responsive, insulin-producing cells from human embryonic stem cells or patient-derived induced pluripotent stem cells.

The grant for the study, entitled "Epigenetic strategies for the in vitro generation of replacement beta cells," began Sept. 17 and ends June 30, 2013. The funding amount for the first year, which will run to June 30, 2011, is $1,158,534.

According to the grant's abstract, the researchers will use cues from pancreatic development to directly differentiate beta cells from stem or progenitor cells that can be used in cell-replacement therapies for diabetes.

To do this, Maike Sander, the principal investigator on the project and an associate professor of pediatrics and cellular and molecular medicine at UCSD, and colleagues will perform genome-wide mapping of key histone modifications in a variety of primary embryonic and adult human cells and tissues.

This knowledge will be used to improve existing protocols for differentiating human embryonic stem cells into pancreatic progenitors and eventually glucose-responsive insulin-producing beta-cells.

A "key" component of the study is UCSD's cellular microarray technology, which allows for "combinatorial screening of extracellular matrix components, factors, and molecular pathways for their ability to support the efficient generation of each intermediary precursor along the step-wise differentiation path from human embryonic stem cell to mature beta cell."

The epigenetic signatures will be used as endpoints to assess how closely the in vitro-generated, human embryonic stem cell-derived cells resemble in vivo pancreatic counterparts, the grant states. Human embryonic stem cell-derived beta cells will eventually be tested for their ability to correct elevated blood glucose levels upon transplantation into diabetic mouse models.

"There is huge heterogeneity in how cells respond to different protocols," Sander told BioArray News this week. "When you think about applying these cells clinically, we can't do that with the current protocol."

Sander said her team will use the array platform to try to "identify robust conditions to make cell lines more responsive. Hopefully, using bioinformatics, we can extract a signature indicative of a particular progenitor stage, a set of marker modifications that can be used to see if you are on track to producing true pancreatic cells."

As part of the project, the team will work with Viacyte, a San Diego-based stem cell-engineering firm. ViaCyte, formerly known as Novocell, is focused specifically on developing cell therapies to treat diabetes. It is led by former Solexa and Illumina official John West.

Sander said her team and Viacyte plan to isolate hESC-derived beta cells from transplanted animals and compare their transcriptional profiles and epigenomes to cadaver human beta cells. They will also use the array platform to compare epigenetic and transcriptional profiles of progenitor stages with human fetal pancreatic progenitors.

"We are trying to generate diagnostic tools that can be used by scientists to quickly assess whether stem-cell derived cells are on the right path to become pancreas or beta cells," said Sander. "To date, this is usually done by checking for the expression of two or three markers, but this is clearly not enough."

UCSD's Platform

UCSD's cellular microarray platform has been in existence for several years, but this is the first time that it will be used toward the goal of differentiating pancreatic progenitor cells, according to Karl Willert, an assistant professor of cellular and molecular medicine at UCSD who is working with Sander on the project. That will pose a challenge for the project.

"There is no generalizing involved," he told BioArray News this week. "Virtually every cell line can be productive, but you have to do the work up front to get the right conditions."

UCSD's platform was created to replace culture-based methods of differentiating cells in a more uniform, high-throughput manner. As described in a recent paper in Biomaterials, the platform consists of glass substrates functionalized with a polyacrylamide gel layer. The polymers, which are printed on the arrays, create microenvironments capable of monitoring up to 1,280 unique biomedical conditions.

The researchers then seed the cultured cells onto the array and allow them to settle on the printed polymer spots. The final steps include using an automated confocal microscope to view the slides and to quantify the images with GenePix software, available from MDS Analytical Technologies, now part of Danaher.

UCSD has licensed its cellular microarray technology to a year-old startup called Microstem, based in nearby Coronado, which uses it as part of a general strategy to "facilitate the growth, expansion, and differentiation of human stem cells in vitro for partners in the biomedical arena," according to its website.

While Willert has used the platform in several previous studies, he said Sander's project has several hurdles to overcome: The group has never worked on pancreatic progenitor cells before; it can be difficult to maintain the cells over time; and marker lines are not widely available.

"The cellular microarray technology needs to be refined and optimized for the cell type of interest," Willert said, referring to working with pancreatic progenitor cells. "For example, seeding density is critical: Cells that grow fast will outgrow the spot size of 150 microns more rapidly than slow-growing cells."

Timing is also a factor. "We have maintained cells on the arrays for up to one week and monitored their properties," Willert said. "However, longer culture periods, especially with proliferating cells, will outgrow the spot and dislodge."

Additionally, the timing issue becomes "critical" when differentiation protocols are long. "If it takes two weeks for a human embryonic stem cell to differentiate into the cell type of interest then we have to perform serial array experiments, each starting with an intermediate stage on the road toward the final state."

Finally, marker lines for this particular study are limited. "To perform live cell imaging it is helpful, and even critical, to have cells that are expressing a fluorescent protein under control of a cell or tissue-specific promoter," said Willert. "Such marker lines are not common and there are virtually none available for the pancreatic lineage."

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