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Tag Archives: Gordon Keller

Friday Science Review: February 18, 2011

Mapping the Development of the Pancreatic Lineage

McEwen Centre for Regenerative Medicine ♦ Published in Development, Mar. 2011 (Epub ahead of print)

Human pluripotent stem cells (PSCs) are being investigated as a means to produce insulin-positive cells for the treatment of diabetes. The most efficient mode of producing functional cell types in vitro is to navigate the signaling pathways and temporal cues that lead to their formation during embryonic development. In the case of insulin-producing cells the key is to recreate the pivotal steps in pancreatic development including the induction of definite endoderm, specification of endoderm to the pancreatic fate, and finally the generation of mature endocrine/exocrine cells. Despite the scientific community having a grasp on pancreatic development, current differentiation protocols suffer from low efficiency and an inability to produce homogenous results across a variety of PSC lines using identical treatments. We have yet to identify the optimal signaling pathways that must be leveraged to produce insulin+ cells. Robust differentiation protocols are also hampered by variations in the characteristics of PSC lines which lead to variability in the quality of differentiation cultures. Dr. Gordon Keller and his lab team ambitiously probed this issue by mapping the pancreatic development of several different PSC lines in order to identify the optimal signaling pathways and temporal requirements essential for producing cells of the pancreatic fate.

Keller’s team found that temporally modulating activin/nodal signaling early in the differentiation protocol was crucial for the development of definite endoderm and ultimately for pancreatic differentiation. Wnt signaling and inhibition of BMP signaling at various stages was also prerequisite for the production of insulin+ cells, noting that the degree of BMP inhibition required for efficient differentiation varied extensively amongst PSC lines. By implementing this stage-specific optimization approach for different cell lines, Keller and his colleagues were able to increase insulin expression in cell cultures by a whopping 250 times; some populations contained as much as 25% C-peptide+ cells (prior to C-peptide being cleaved from the pro-insulin molecule, it acts as a linker between the A and B chains of insulin).

This is the second, recent, body of research from Gordon Keller’s lab that emphasizes the importance of identifying the crucial temporal steps that must be satisfied for highly efficient differentiation to terminal cell fates. This paper also reminds us that individual PSC lines will likely require unique treatments in culture to produce maximal results for transplantation therapy.

IL-7 Therapy: A Stimulus Package for the Immune System

Campbell Family Institute for Cancer Research ♦ Published in Cell, Feb. 18, 2011 (Epub ahead of print)

After the immune system succumbs to uncontrollable viral turnover, it eventually fails, leaving the host prone to any number of opportunistic infections. This is the case with HIV infection. One of the primary focuses of HIV research today is the modulation of immune response to encourage the clearance of chronic viral infections. It appears that a certain cytokine, interleukin-7 (IL-7), may be able to prop up the immune system allowing it to move around mechanisms that circumvent immune response during times of chronic infection. In this study led by Dr. Tak Mak, researchers hypothesized that cytokines supporting homeostatic proliferation would be promising candidates for promoting immune response. Indeed, IL-7 did just that.

After administering IL-7 to mice that were chronically infected with lymphocytic chorimeningitis virus (LCMV) variant clone 13, researchers observed an increase in size of the naive T-cell pool, and an enhanced function and cytokine output in LCMV-specific T-cells. IL-7 therapy resulted in clearance of LCMV from chronically infected mice. The cytokine also appears to serve a second function — bolstering levels of the cytoprotective cytokine IL-22. An increase in IL-22 levels has the added benefit of protecting the liver from viral infection, an organ that is particularly prone to damage under the circumstances. Researchers believe IL-7 exerts its effects by downregulating suppressor of cytokine signaling 3 (Socs3) expression in T-cells through the suppression of the FoxO transcription factors.

Friday Science Review: February 11, 2011

Cardiac Differentiation: A Customized Approach

McEwen Centre for Regenerative Medicine ♦ University of Toronto ♦ SickKids

Published in Cell Stem Cell, Feb. 4, 2011

Dr. Gordon Keller of the McEwen Centre for Regenerative Medicine has been a pioneer in the stem cell world and was the first researcher to produce functioning cardiomyocytes from embyronic stem cells. These cells form clusters in the petri-dish that beat in unison, a rather marvelous sight to behold. The efficient differentiation of embryonic stem cells to cardiac cells requires monitoring the very earliest stages of their development. Monitoring the expression of one gene, Flk-1, has been instrumental in recognizing the formation of cardiac mesoderm, an early step in the developmental path of cardiomyocytes. A problem that remains however, is that Flk-1 is expressed in different forms of mesoderm, not all of which lead to the cardiac lineage. A second gene, PdgfR-α, can be used to separate cardiac and hematopoietic lineages when monitored in conjunction with Flk-1. Fractions of differentiating cells that coexpress the two genes have greater cardiomyocyte potential. Keller’s lab group used these two genes to study the stage-specific effects that Activin/Nodal and BMP signaling have on the development of cardiomyoctyes. They found that very small changes in the amount of Activin/Nodal or BMP had profound effects on the proportion of Flk-1+/PdgfR-α+ cells that appeared early on in the differentiation protocol, and that optimization of these concentrations in cultures of human pluripotent stem cells could give rise to structures that contain more than 50% Flk-1+/PdgfR-α+ cells. A major finding by Keller’s team is that different mouse and human pluripotent stem cell lines required unique optimization to produce maximal results, stressing the importance of using differentiation protocols that are in effect customized to individual pluripotent stem cell lines.

β-Catenin Maintains Pluripotency of Stem Cells with Two Divergent Signaling Cascades

Stem Cell and Cancer Research Institute ♦ McMaster University ♦ University of Guelph

Published in Cell Stem Cell, Feb. 4, 2011

It is widely assumed that β-catenin, a key molecule in the Wnt/β-catenin signaling pathway, helps sustain pluripotency through its interaction with TCL/LEF transcription factors. However, recent research shows that β-catenin also promotes pluripotency by complexing with and stabilizing Oct-4, a key member of the transcriptional network that maintains the pluripotent nature of stem cells. Glycogen synthase kinase-3 (GSK-3) has emerged as an important regulatory of pluripotency, in part because β-catenin is one of its primary substrates. After GSK-3 phosphorylates β-catenin it is degraded, which encourages stem cells to exit the pluripotent state and differentiate to other cell types. Dr. Bradley Doble and his colleagues previously showed that mouse embryonic stem cells (mESCs) that are entirely deficient in GSK-3 express very high levels of β-catenin and exhibit a severe impairment in their capacity to differentiate into the three germ layers. In this recent work, Doble and his team hypothesized that hyperactivated β-catenin/TCF was responsible for the pluripotent “lock” that was imposed on mESCs lacking GSK-3 expression. To the surprise of researchers, GSK-3α/β double knock out mESCs still maintained pluripotency even when they stably expressed a dominant negative form of the TCF transcription factor. How were they doing this? Apparently β-catenin can maintain pluripotency independent of functioning TCF. Researchers showed that β-catenin promotes the maintenance of pluripotency by interacting with Oct-4 in a divergent signaling cascade.

Next Generation Gene Therapy for Hemophilia A: Pre-clinical Progress

Queen’s University ♦ Published in Molecular Therapy, Feb. 1, 2011

Researchers pursuing therapies for Hemophilia A have turned to gene therapy for answers but have struggled to provide convincing pre-clinical results. Patients with the disorder have vastly decreased plasma concentrations of FVIII, a clotting factor that prevents blood loss after injury. Although viral vectors can produce the protein following system injection into animal models, its efficacy is compromised by the introduction of neutralizing anti-FVIII antibodies. Researchers hypothesized that the development of neutralizing antibodies was the result of transgene expression in the antigen presenting cells of mice. The solution to this problem was to “hide” the transgene by placing it under the control of a liver-specific promoter. This approach worked in normal mice, however researchers studying mice with hemophilia B still found that an anti-FVIII immune response was mounted in the presence of the new tissue-specific promoter. As a second layer of defense against this response researchers incorporated target sequences into the transgenic construct that had perfect complementarity to hematopoietic-specific miRNA sequences. These target sequences led to suppression of the transgene specifically in hematopoietic cells, including antigen expressing cells, limiting the neutralizing response. Dr. David Lillicrap and his team at Queen’s University have now used a similar approach to produce some very promising results in a mouse model of hemophilia A. A combination of a liver-restricted promoter, a miRNA regulated FVIII transgene, and a pseudotyped viral envelope seemed to do the trick.

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