The researchers describe four critical stages on the way from a multi-potent precursor to a committed B-cell and suggest how combinations of regulatory proteins and signaling pathways direct maturing cells through each crossroad, guiding them down one specific developmental path, preparing them to respond to signals yet to come, and blocking off other options.
"Our findings reveal considerable complexity, but are promising from the standpoint of directing stem cell differentiation," said Harinder Singh, Ph.D., Louis Block Professor of Molecular Genetics and Cell Biology and an Investigator with the Howard Hughes Medical Institute at the University of Chicago.
"For this one cell type, about which we already knew a great deal, it's a complicated and elaborate recipe that involves multiple ingredients at each step and mixing them in a particular order. We expect that other cell types will require similarly complex regulatory networks for their generation."
"But the work is also promising," Singh added. "Once we order the components and gain insight into the design principles of such regulatory networks we may be able to make any kind of cell we want, or even produce hybrids that combine features of different cell types, such as antibody-producing skin cells."
Singh and colleagues work with hematopoietic stem cells (HSCs), which give rise to the different types of blood cells. Unlike embryonic stem cells, HSCs have already taken some steps in differentiation and are committed to producing various types of blood cells.
"This is a wonderful model system," Singh said. "We know more about the differentiation of B cells and red cells than other cell types in the blood system."
After a series of experiments that involved manipulating multiple genes encoding regulatory proteins, Singh and colleagues came up with a "hierarchical regulatory network" that orchestrates the differentiation of a stem cell into a committed B-cell precursor.
Five transcriptional regulators guide future B cells along this pathway, activating genes that move the cell to the next stage and enabling the cell to respond to specific chemical signals later on. For example, the transcription factors PU.1 and lkaros are crucial early in the process, nudging a multi-potent progenitor cell -- stage 1, which could become any type of blood cell -- toward becoming a lymphoid progenitor, stage 2. They trigger the expression of certain receptors on the cell surface, such as Flk2 followed by IL-7R, which are necessary for receiving subsequent external signals.
In the next step, the gene for a regulatory protein known as E2A cooperates with PU.1 to activate another regulatory gene called EBF. EBF and E2A act together to push the lymphoid progenitor towards stage 3, a specified pro-B cell. At this stage, many of the genes expressed in B cells have been activated and the genes that encode antibodies have begun the process of recombination.
Finally, EBF and E2A activate a regulator called Pax-5, which pushes the specified pro-B cell to stage 4, a committed pro-B cell. After this point, there is no turning back.
"This is a complicated sequence of events," Singh notes. "There's no denying it." At each stage, different markers or receptors appear on the cell surface, which helps the researchers monitor a cell's progress and enables the cell to reach the next stage.
"To make real use of stem cells we will have to assemble genetic regulatory networks such as this for each cell type we want to generate," Singh added. "This is the next challenge facing the field. Molecular biologists are used to manipulating single genes, but this may require controlling several components in an ordered manner to properly direct a stem cell through a given developmental sequence."
The research was supported by grants from The Howard Hughes Medical Institute and the Irvington Institute. Additional authors include Kay Medina, Jagan Pongubala, Karen Reddy and David Lancki of the University of Chicago; Rodney DeKoter of the University of Cincinnati; and Matthias Kieslinger and Rudolf Grosshedl of the Max-Planck-Institute of Immunobiology, Freiburg, Germany.