Our program is focused on improving methods to purify blood-forming and heart-forming stem cells so that they can be used safely and effectively for therapy. Current methods to identify and isolate blood-forming stem cells from bone marrow and blood are efficient. In addition, we found that if blood-forming stem cells are transplanted, they create in the recipient an immune system that will tolerate (i.e., not reject) organs, tissues, or other types of tissue stem cells (e.g. skin, brain, or heart) from the same donor. Many living or recently deceased donors often cannot contribute these stem cells, so we need, in the future, a single biological source of each of the different types of stem cells (e.g., blood and heart) to change the entire field of regenerative medicine. The ultimate reason to fund embryonic stem cell and other pluripotent stem cell research is to create safe banks of predefined pluripotent cells. Protocols to differentiate these cells to the appropriate blood-forming stem cells could then be used to induce tolerance of other tissue stem cells from the same embryonic stem cell line. However, existing protocols to differentiation stem cells often lead to pluripotent cells (cells that generate multiple types of tissue), which pose a risk of generating normal tissue in the wrong location, abnormal tissue, or cancers called teratomas. To address these problems, we have concentrated our efforts to devise strategies to (a) make pluripotent cells develop into desired tissue-specific stem cells, and (b) to separate these desired cells from all other cells, including teratoma-causing cells. In the first funding period of this grant, we succeeded in raising antibodies that identify and eliminate teratoma-causing cells.
In the past year, we identified surface markers of cells that can only give rise to heart tissue. First we studied the genes that were activated in these cells, further confirming that they would likely give rise to heart tissue. Using antibodies against these surface markers, we purified heart stem cells to a higher concentration than has been achieved by other purification methods. We showed that these heart stem cells can be transplanted such that they integrate into the human heart, but not mouse heart, and participate in strong and correctly timed beating.
In the embryo, a group of early stem cells in the developing heart give rise to (a) heart cells; (b) cells lining the inner walls of blood vessels; and (c) muscle cells surrounding blood vessels. We identified cell surface markers that could be used to separate each of these subsets from each other and from their common stem cell parents. Finally, we determined that a specific chemical in the body, fibroblast growth factor, increased the growth of a group of pluripotent stem cells that give rise to more specific stem cells that produce either blood cells or the lining of blood vessels. This chemical also prevented blood-forming stem cells from developing into specific blood cells.
In the very early embryo, pluripotent cells separate into three distinct categories called ‘germ layers’: the endoderm, mesoderm, and ectoderm. Each of these germ layers later gives rise to certain organs. Our studies of the precursors of mesoderm (the layer that generates the heart, blood vessels, blood, etc.) led us by exclusion to develop techniques to direct ES cell differentiation towards endoderm (the layer that gives rise to liver, pancreas, intestinal lining, etc.). A graduate student before performed most of this work before he joined in our effort to find ways to make functional blood forming stem cells from ES cells. He had identified a group of proteins that we could use to sequentially direct embryonic stem cells to develop almost exclusively into endoderm, then subsets of digestive tract cells, and finally liver stem cells. These liver stem cells derived from embryonic stem cells integrated into mouse livers and showed signs of normal liver tissue function (e.g., secretion of albumin, a major protein in the blood). Using the guidelines of the protocols that generated these liver stem cells, we have now turned our attention back to our goal of generating from mesoderm the predecessors of blood-forming stem cells, and, ultimately, blood-forming stem cells.
In summary, we have continued to discover signals that cause pluripotent stem cells (which can generate many types of tissue) to become tissue-specific stem cells that exclusively develop into only heart, blood cells, blood vessel lining cells, cells that line certain sections of the digestive tract, or liver cells. This work has also involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.