Strategies for moving embryonic stem cells toward pancreatic or blood cells
Embryonic stem cells are, for most scientists, a means to an end. The cells themselves matter less than the cells they can produce, but making them differentiate into the desired quantities of particular cell types is easier said than done. That is especially true for tissues of the two inner germ layers, the endoderm (which forms most glands) and the mesoderm (which forms the muscles and cardiovascular system). Now, two new protocols promise more efficient ways to generate these tissues from embryonic stem cells.
Pancreatic cells come from the endoderm, but turning embryonic stem (ES) cells into endoderm cells has been easier than getting endoderm to move into the pancreatic lineage. Researchers led by Douglas Melton of Harvard University in Cambridge, Massachusetts, first used existing protocols to differentiate human ES (hES) cells into endoderm and then into heterogeneous cultures in which at least a few cells expressed Pdx-1, an early marker of pancreatic development. Next, they screened these cells against a library of 5,000 compounds, using antibody staining to see if any of the compounds could increase the number or percentage of Pdx-1-expressing cells.
This process yielded a molecule called indolactam V (ILV). Subsequent tests showed that ILV functions at a particular stage of differentiation. It did not work by causing undifferentiated ES cells to shift to endoderm or by causing the proliferation of Pdx-1 cells, but rather by inducing early endoderm to differentiate into Pdx-1-expressing cells1. The effects were more pronounced when combined with fibroblast growth factor 10 (FGF10). In fact, when combinations where tested on hES cells, about 27% expressed Pdx-1 when treated with ILV alone, compared with 46% when treated with ILV and FGF10. When treated with FGF10 alone, the rate was about 6%, and when treated with neither, less than about 2.5% of the cells expressed Pdx-1. Additionally, when the Pdx-1-expressing cells were transplanted under mouse kidney capsules, a small number went on to produce insulin-expressing cells.
Though the molecules are not chemically similar, ILV seems to work via the same mechanism of retinoic acid, a molecule that activates protein kinase C and is involved in pancreatic development. However, ILV seems to be more potent than retinoic acid. The next steps, write the authors, are finding additional small molecules that guide various stages of differentiation and figuring out just how far along the pancreatic lineage the cells would need to be for them to help patients with diabetes.
In unrelated work, Mickie Bhatia of McMaster University in Ontario, Canada, found a surprising way to make mesoderm, the germ layer that gives rise to blood. The finding involves the Wnt signaling pathway. The 18 Wnt proteins are soluble molecules implicated in a variety of ways throughout development, with effects that vary from species to species. The researchers tested two forms of Wnt — Wnt3a and Wnt11 — on hES cells that had been allowed to differentiate into embryoid bodies and found that each protein encouraged the development of haematopoietic precursors in different ways2. Wnt3a causes cells already committed to the haematopoietic lineage to proliferate, whereas Wnt11 acts at a much earlier stage, apparently by disrupting a cell's pluripotent state and pushing it toward becoming mesoderm. In this instance, the Wnt protein acts via a 'noncanonical' mechanism (that is, without its usual signaling partner beta-catenin) previously unappreciated in making mesoderm. This knowledge could lead, in turn, to more efficient generation of blood and other mesodermal cells.''