Dr. Zaret began by observing that current strategies to use stem cells for regenerative therapies for the liver and pancreas are directed toward generation of hepatocytes and beta-cells, although future approaches will hopefully lead to the generation of tissue sections. New cells are needed to replace those damaged by acute liver failure, genetic liver disease, and type 1 diabetes. Potential sources of these cells include cadaveric tissue, duct-associated progenitors that are activated after damage to hepatocytes or beta-cells, fetal liver or pancreatic cells, cells that are trans-differentiated from other cell types, ES cells, and iPS cells. To use these cell types, cells must be directed from a progenitor state to an advanced state. While the developmental programming pathway from blastomere to hepatocyte or beta-cell may appear straightforward on paper, each step in the pathway represents an alternative cell fate choice and a concomitant activation or suppression of cellular programs. To arrive at the desired product cell, emphasis must be placed on understanding the signaling and effector molecules that govern the pathways.
Hepatocytes secrete serum proteins and bile and metabolize nutrients and toxins using cytochromes p450. Beta-cells must secrete insulin in response to elevated glucose concentration. Despite the complete potential of hES cells to differentiate into each of these cell types, incomplete programming is often observed in experiments. For example, researchers have sequentially differentiated hES cells to express insulin, yet the cells failed to respond to glucose (D’Amour KA, et.al Nat Biotechnol 2006;24:1392-1401). Other experiments have shown that non-insulin-expressing, glucose-responsive cells can be generated that respond to glucose long after transplantation (Kroon E, et.al. Nat Biotechnol 2008;26:443-452). These cells, however, produced occasional teratomas. In general, more fully differentiated cells lower the potential for teratomas, indicating that basic research is needed to identify additional factors that induce differentiation. Furthermore, experiments have indicated that different hES cell lines give varying results even when similar culture methods are used.
In a recent experiment, Jiang, et.al. (Stem Cells 2007;25:1940-1953) created non-adherent clusters of insulin-expressing, glucose responsive cells. This discovery suggests the principle of co-differentiation—reciprocal signaling occurs between cells as a tissue matures, and this signaling may direct multipotent progenitor cells toward a particular fate (Matsumoto K, et.al. Science 2001;294:559-563; Lammert E, et.al. Science 2001;294:564-567; Yoshitomi H and Zaret KS. Development 2004;131:807-817). For example, the selection for endodermal and liver markers in mouse ES cells also produced endothelial cells (Gouon-Evans V, et.al. Nat Biotechnol 2006;24:1402-1411). Thus, different cells within an emerging tissue undergo mutually inductive interactions. Therefore, it may prove easier to develop differentiated tissues than single cell populations in vivo. However, these experiments suggest a need for improved animal models and strategies to enhance and ensure transplant survival.
Dr. Zaret noted that there are numerous ways to program stem cells to become specific tissue types in normal mammalian development (Zaret KS. Nat Rev Genet 2008;9:329-340). Different liver domains are modulated by unique signaling pathways; lateral and medial liver progenitors are affected by different timing of hepatogenetic signaling (Jung J, et.al. Science 1999;284:1998-2003; Rossi JM, et.al. Genes Dev 2001;15:1998-2009; Calmont A, et.al. Dev Cell 2006;11:339-348). But can developmental competence be predicted? Chromatin occupancy states and forkhead box (FoxA) transcription factors offer possible clues to the differentiation of progenitor cells. Pioneer transcription factors can enter chromatin and mark it for specific activity, thereby allowing other factors to enter. Thus, transcription factors help to endow developmental competence. Transcription factors can therefore also be thought of as epigenetic marks that initiate the restoration of gene expression programs after mitosis. To utilize these tools, additional small-molecule agonists/antagonists of chromatin-modifying enzymes are needed to control cell programming prospectively.
Early progenitor cells are highly proliferative and expand the progenitor pool. As cells differentiate, they become less proliferative. However, early progenitors have less cross-control and exist in metastable states (e.g., they lose their commitment and can be transdifferentiated to different phenotypes) (Bort R, et.al. Development 2004;131:797-806; Zaret KS. Nat Rev Genet 2008;9:329-340). Specified progenitors in the proliferative state are most prone to trans-differentiation. To expand the progenitor stage at will, cell cycle control and the genetic and epigenetic control of conversion to stable states must be better understood.
Dr. Zaret concluded by noting that patient-specific cell therapy could be made possible in the future. In the near term, however, the study of the onset of genetic diseases and cancer in vitro will inform the development of tailored drugs and thus likely have the greatest impact.
One attendee asked about differences seen between zebrafish development and the patterns described in this presentation. Dr. Zaret noted that zebrafish endoderm develops from a rod that sits above the yolk sac, whereas mammals develop endodermal tissues from a tube. The substantial differences in developmental patterns confound a direct comparison between zebrafish and mammals.