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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