New reporter proteins, imaging, and biochemical tools for measuring signaling activities in stem cells have potential to improve drug studies.

Typically, as warmer weather replaces the cold and snow, and new life is created, one is reminded of the self-renewal process that is ever-present in organisms; that is, the self-renewal process by which new cells are created, while the present ones are recycled. Stem cells take care of the business of self-renewal that occurs in the human body by differentiating from a less mature, unspecialized cell type to a more mature, specialized type.

Stem cell differentiation is dependent on cell-signaling activities, which can be targeted for drug discovery. For example, a drug researcher may be interested in determining if a specific compound triggers differentiation in a particular stem cell line. “There are several signaling pathways that regulate the survival, proliferation, differentiation, and self-renewal of both embryonic and adult tissue stem cells, with the Notch, Wnt, and Hedgehog pathways being among the best characterized,” says Stephen Szilvassy, PhD, principal scientist in Hematology/Oncology Research, Amgen Corporation, Thousand Oaks, Calif. “Some of these key regulators are over-expressed or dysregulated in tumors, and possibly also in putative cancer ‘stem’ cells, and therefore might be useful therapeutic targets.” For the last several years, Amgen has been developing both antibody- and small-molecule-based therapeutics that modulate some of the key components of these stem cell signaling pathways; these therapeutics are currently in preclinical studies.

Obtaining the cells
In order to develop therapeutics based on stem cell signaling pathways, researchers must first obtain sufficient quantities of the cell type they wish to target. Says Ralph Snodgrass, PhD, chief executive officer of VistaGen, South San Francisco, Calif.: “You can express reporters to look at a specific biochemical pathway or protein-based pathway in these cells, but first, you have to generate the cells.” Fortunately, in 2007, Takahashi et al. (Cell. 2007;131(5):861-72) reported that they had created a pluripotent stem cell line from human adult somatic cells—they termed it induced pluripotent stem (iPS) cell line. With that discovery, drug researchers gained greater control over their studies of stem cell differentiation, a fact that would greatly enhance drug studies in stem cells, such as screening for compounds that are capable of either inducing or inhibiting differentiation.

VistaGen harnesses the power of iPS cell lines. The company is developing stem cell-based systems to determine drug response, and provide information for safety assessment. The cells used in these experiments include human cardiomyocytes; human liver cells; and the insulin-producing, human beta-islet cells, all of which are derived from either embryonic stem cells or from adult iPS cells. One example of VistaGen’s efforts to utilize knowledge generated from studies of stem signaling to develop therapeutics is their work in beta-islet cells. By studying pathways that regulate insulin expression, processing, and secretion in beta-islet cells, VistaGen aims to develop compounds that stimulate insulin production in diabetics. Insulin production is directly measured in these cells by gene-reporter assay.

Reporting on stem cell activity
Using a fluorescent protein reporter, a researcher can “label” a single stem cell signaling protein to track its translocation between cellular compartments or monitor cellular differentiation by tracking specific labeled proteins. Current fluorescent protein technology allows these measurements to be performed at the single-cell level.

Clontech Laboratories, (Mountain View, Calif.) a wholly-owned subsidiary of Takara Bio Inc., offers 18 different fluorescent protein options through its Living Colors products. Living Colors fluorescent proteins span the color spectrum from far-red to cyan. For example, one of many red fluorescent proteins produced by Clontech, DsRed-Express2, was developed and validated specifically for use in stem cells. Other choices include the extremely bright red fluorescent protein tdTomato, as well as monomeric fluorescent proteins such as mCherry and AcGFP1. An improved orange fluorescent protein, mOrange2, is scheduled to become commercially available in summer 2009.

“Our reporters are provided in a format that can be tailored for use in any pathway of choice,” says Suvarna Gandlur, PhD, product manager for reporters, cell signaling, and stem cell and epigenetic products, Clontech Laboratories, “We provide easy cloning solutions by which each researcher can clone their preferred target elements into the reporter vector.”

To avoid the over-differentiation of a stem cell culture into an undesired terminal cell type, it is important for a researcher to pinpoint the exact timing of stem cell differentiation. Activation or inactivation of specific gene promoters dictates this timing. And, of course, the activity status of a stem cell-specific promoter can be monitored using a reporter gene. The most recent method for monitoring stem cell status and differentiation is to use an On-Demand Fluorescent Reporter to precisely determine the timing of stem cell-specific promoter activation and inactivation.

Imaging is another major approach for monitoring stem cell growth and differentiation. PerkinElmer, Inc. (Waltham, Mass.) produces a range of imaging systems applicable to the detection and quantitative analysis of signaling pathways in stem cells. “We have several instruments designed for high-content screening (Opera) or high-content analysis (Operetta), which enable researchers to measure, in either live-cells or fixed-cells, the various signaling pathways,” says Richard Eglen, PhD, president of Bio-discovery at PerkinElmer, Inc. “Since confocal microscopy [images] can be detected and analyzed using our proprietary Acapella or Harmony software, both the basic academic and pharma industry life sciences researcher can measure a wide range of cellular-signaling systems.”

Antibodies for detection
Of course, one does not have to use a reporter system or imaging to track stem-cell activity— antibody-based detection in a simple, well-established biochemical assay will work just fine. As with any other cell signaling protein, stem cell signaling proteins can be detected and validated via immunoassays such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation. Case in point: Cell Signaling Technology (Danvers, Mass.) produces antibodies for stem cell research. “Our antibodies help facilitate research from the initial generation and maintenance of iPS lines to measurement of changes in protein expression or modification in response to perturbations in the iPS cell system,” says Susan Kane, PhD, a development scientist at the company. “We are constantly developing new antibodies that can track pluripotency of ES/ iPS cells, track differentiation along lineage pathways, and measure signaling events in response to drug treatment.”

GenScript (Piscataway, N.J.) also provides specific reagents that are crucial in stem cell signaling, such as c-Myc ORF clones, peptides, and antibodies, and Sox2 ORF clones. According to Nick Yan, PhD, vice president of marketing at GenScript, the company has developed a series of specificity assay platforms, integrating biochemical and cell-based methods, to cover a wide range of drug targets including G-protein-coupled receptors, ion channels, proteases, and kinases, as well as other enzymes, transmembrane proteins, and adhesion molecules.

Summary
The analysis of stem-cell signaling pathways is highly dependent on biotechnology tools. Cell-culture tools are needed to cultivate the cells; antibodies are required for imaging and in vitro biochemical analysis; and reporter proteins are necessary to monitor the activity of signaling protein and promoters. But, no matter which tool is used, the objective is the same: to generate accurate biological data that can be utilized to improve drug discovery efforts.

About the Author
James Netterwald is president and CEO of BioPharmaComm LLC, a provider of writing, editing, and consulting services to the life science, pharma-biotech, and public relations industries.

This article was published in Drug Discovery & Development magazine: Vol. 12, No. 6, June, 2009, pp. 22-25.