February 15, 2007 (Vol. 27, No. 4)

Firms Are Using Sem Cells to Better Screen Compounds for Efficacy and Toxicity

The prospect of stem cells in regenerative medicine has caught the public’s imagination. Less attention has thus been given to the use of stem cells as models for drug discovery and development. The latter, however, is perhaps a more realistic prospect, at least in the short term. Both stem cell therapy and stem cell-based drug discovery technologies will be discussed at the “European Stem Cells & Regenerative Medicine Congress” to be held in London during May.

Big pharma, major suppliers, and small companies are now developing a wide range of stem cell approaches, which reflect the interest in this emerging area. Cellerant Therapeutics (www.cellerent.com) has long had an interest in cancer stem cells, thanks to the pioneering work of its founder, Irving Weissmann, who showed that there is a cell population with stem-like properties in cancer patients. “The new thinking in cancer is that these stem cells continue to proliferate and are not the target of currently used cancer therapy,” says Bruce Cohen, president and CEO.

Cellerant is now looking at the upstream cells, which have self-renewing and tumor properties and are the cancer stem cells. These are shown to be phenotypically different and have surface markers distinct from the daughter cells, which are the target of conventional cancer therapy.

Cellerant’s expertise is in hematopoietic cells and leukemia. It has identified cancer stem cells in myeloid leukemia and shown that a small number of cancer stem cells in the hematopoietic population can create a large number of tumor cells. The same has also been found in acute myelogenous leukemia (AML). “We have developed an antibody against AML cancer stem cells that is in preclinical testing,” states Cohen. Similar stem cells have also been found in brain cancer.

Cancer Stem Cells as Discovery Tool

However, Cohen points out that in his view, “hematopoietic cancer stem cells are probably a better target for drug discovery right now.” The cancer stem cell theory could explain why cure rates in cancer have not improved, which is by no means commensurate with the research effort that has been put in, says Cohen.

In cancer, cells mutate to have stem-cell properties and so avoid apoptosis. “A series of cumulative mutations makes the cells stem-like and able to self-renew in an unregulated fashion,” explains Cohen.

“Cellerant has a cancer franchise, but there is a host of other diseases where the mutation is in an otherwise normal blood-forming stem cell—for example, in thalassemia and in sickle cell anemia, which are therefore stem-cell mediated diseases. We and others have shown that if you replace the stem cell population, you can make the disease go away.”

The same concept applies to a larger universe of diseases that have multiple-gene involvement, he adds. “These involve a predisposition to disease. As, for instance, in the autoimmune diseases where the mutation does not absolutely cause but increases the likelihood of the condition, such as type I diabetes, Crohn’s disease, and lupus.”

Pure Cell Transplants

Cellerant is developing a new approach to stem cell transplant. The conventional method is to completely eliminate the existing population.

“The more interesting way is to inject a pure cell population with no T or B cells into an animal without ablation, which is less aggressive,” states Cohen. “The resulting animal is chimeric—part self, part donor—and the donor cells confer resistance to autoimmune diseases.

“There is anecdotal evidence for this in patients with autoimmune diseases. We have shown for the first time in animal models of lupus that if you do a pure stem cell transplant without completely ablating the host immune system, you can eliminate the symptoms of the disease.

“But to take the therapy forward, there is a need to first identify the right patient population.” Cellerant has developed purified cell populations with the reduced volume needed for transplant and no B or T cells, which reduces the risk of graft versus host disease, a potentially serious complication in the recipient.

These purified cells are being applied to the support of patients receiving high-dose chemotherapy, where the blood-forming system needs to be rescued with stem cells. “We hope to show that if you give pure stem cells back to patients, it lowers the probability of giving them back their own cancer,” adds Cohen.

When it comes to doing stem cell research, Cohen sees the challenges as being more about finance than the science itself.

“There is great research being done, but it is hard to raise capital for stem cells in general, and also for cell-based therapies. We are looking to cure in a one-time procedure, which is not the classic drug model.”

Genetically Engineered for Screening

Whatever the financial situation, stem cell researchers need the tools to push their work forward. Invitrogen (www.invitrogen.com) provides reagents and services for stem cell research. These include technologies, such as cell tracking dyes, bead-based cell-separation systems, and cell banking and characterization methods that can be applied to each stage of stem cell work—isolation, characterization, expansion, and differentiation. Many of these are scalable and suited to GMP and can thus help research move toward the clinic.

As the stem cell industry matures, the company is also moving into the development of genetically engineered stem cell lines. “Invitrogen sees great potential for stem cells in many areas, particularly in the near term as platforms for drug discovery and drug screening,” says Jon Chesnut, Ph.D., R&D director for stem cells and regenerative medicine. The Invitrogen approach involves pairing promoters and reporter elements to create cell linage beacons that can be used to screen small molecules, potential regulatory proteins, or noncoding RNAs for their impact on cell differentiation.

The company is working toward proof-of-concept for this platform, which could ultimately be used to manage cell differentiation toward a specific lineage for a therapeutic application by expressing or silencing essential genes. “There is a lot to be learned about cell differentiation. This new platform is a tool that will make it easier,” says Dr. Chesnut.

The Invitrogen cell platforms could also be used in cell-based screening assays with readout geared to the pathway under study.

Applying Pluripotent Stem Cell Lines

Meanwhile, Stem Cell Innovations (SCI; www.stemcellinnovations.com) derived human pluripotent stem cells called PluriCells™ from fetal primordial germ cells. To date, 14 cell lines have been developed that can grow without feeder cells on plastic in a serum-free defined medium. “PluriCells are similar to embryonic stem cells in many of their marker proteins,” explains Helmuth van Es, Ph.D., CSO. “As they differentiate, the expression of the marker proteins disappears. The cells can differentiate into all three germ layers and are potentially capable of becoming any of the body’s 200 cells types.”

Currently, the company is looking at those differentiated cells that can most readily add value, such as in therapies for neurodegenerative disorders, including Alzheimer’s, Huntington’s, and Parkinson’s disease. “We are focusing on applications in drug discovery and preclinical development,” says Dr. van Es. “We hope to create those human biological models for discovery and molecule validation that are not currently available.”

Because of their origin, PluriCells are eligible for federal funding and so can be used for research in U.S. universities and nonprofit institutions.

SCI’s overall strategy is to make the cells widely available for research and accelerate human stem cell-based studies. Accordingly, the company recently signed an agreement with the ALS Association, under which, SCI is to develop human motor neuron cell models from the PluriCell technology. These can be used for high-throughput screening of potential ALS therapies.

Alliance partner, Galapagos(www.galapagos.com), will use the cell models to discover new ALS drug targets using its adenoviral RNAi library. SCI is also developing the motor neurons and their precursor cells as therapeutic agents.

In other collaborations, the Massachusetts Eye and Ear Institute is evaluating the PluriCells for their ability to differentiate into the cells implicated in hearing loss. In another agreement, the University of Twente in The Netherlands is looking at the ability of PluriCells to form bone and cartilage cells.

Assays to Aid Early-Stage Discovery

According to Paul Rounding, Ph.D., managing director of business development and operations, Artemis Pharmaceuticals (www.artemis-pharmaceuticals.com), there are various ways in which stem cells might be used in drug discovery. First of all, stem cells play a role in certain diseases, including cancer and neurodegenerative conditions, and it may be possible to find compounds that could modify their activity.

Moreover, stem cells differentiated into primary cells could form the basis of more predictive assays that could filter out compounds at an earlier stage of drug discovery. This could be done with either human stem cells or humanized mouse cells—the latter being an area that Artemis specializes in. Such assays are being used right now but are not yet adapted to high-throughput because of the challenges involved in handling stem cells.

Dr. Rounding believes that stem cells may also be used in single-cell, high-content screening assays using imaging and/or confocal microscopy systems, such as those developed by Cellomics (www.cellomics.com) and Evotec Technologies (www.evotec-technologies.com). These can give data on efficacy and toxicity at an earlier stage, an issue that the industry continues to struggle with.

In particular, so-called intractable toxicity of a compound must be addressed earlier, which could be done by testing on hepatocytes. Recently, the Innovative Medicines Initiative, a joint venture between the European Federation of Pharmaceutical Industries and the European Commission, met to discuss the issue of earlier prediction of intractable toxicity. “It was clear that stem cells, human or mouse, can make an important contribution to this,” says Dr. Rounding. Companies are thus beginning to use stem cells and developing protocols to differentiate them into hepatocytes for this kind of testing.

Tox-Testing with Humanized Mouse Models

Artemis uses stem cells to generate mouse models for drug testing. It can modulate the stem cells, changing a gene or genes to create knockout, knockdown, and humanized mice. “We have big programs in humanizing up to four genes in mice, and so, we are able to begin to modify whole pathways,” states Dr. Rounding.

This means that for compound testing, the mouse model begins to resemble the human one more, which can serve the increasing need in the industry for ever earlier in vivo testing. “Our belief is that the best way of doing this early-stage testing is in the mouse model with modified pathways and humanization.”

Artemis is working with the U.K. tox-testing company CXR Biosciences (www.cxrbiosciences.com)through Scotland’s ITI program to make humanized mouse models for such early testing of compounds. Several of these models will become available during 2007. “Mouse stem cell lines can be used for drug testing and also be used to generate our in vivo models,” points out Dr. Rounding. “This will allow more predictive selection of compounds for efficacy and toxicity.

“But it is not an easy challenge,” he cautions. The only way of knowing if these models work is by asking whether the data they produce is high quality and can be validated. “We have the technology to do the genetic modification and the differentiation, but we now need solid data to indicate that the models can solve actual problems.”

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