The Haematopoietic Stem Cell Laboratory is based in the Cambridge Institute for Medical Research, and forms part of the Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute. The Cambridge Stem Cell Institute is dedicated to providing new medical treatments from its stem cell research, and supports 25 stem cell laboratories whose principal investigators use stem cells in experimental models to study development, cancer, tissue regeneration, and aging.
The Stem Cell Institute‘s Dr. Bertie Göttgens is studying how differences in expression of a network of transcription factors in heterogeneous populations of blood stem and progenitor cells contribute to the development of mature blood cells. Dr. Göttgens reasons that cell fate decisions are made by a single cell, not by bulk cells. Current methods of sorting bulk cells into distinct populations of cells, such as flow cytometry, look at cell surface markers that are often not immediately relevant to cell fate. By contrast, transcription factors are known major drivers of cell fate decision-making. It is important therefore to study transcription factor gene expression in single cells to characterize the network of transcription factors that drive cell fate. To perform high-throughput single-cell gene expression analysis, Dr. Göttgens is using the Fluidigm Biomark HD System.
“By using the Biomark HD System, we get added layers of information. In a single experiment, we [can] generate a whole range of knockdown levels that would otherwise be completely obscure.”
— Berthold Göttgens, Ph.D.
In his recent paper in Nature Cell Biology, Dr. Göttgens and his team used the Biomark HD System to see if there is a correlation between differential expression of a targeted set of 18 transcription factors and specific blood stem and progenitor cell types. The team used FACS to identify and separate five populations of blood stem and progenitor cells (haematopoietic stem cell, lymphoid-primed multipotent progenitor, common myeloid progenitor, granulocyte-monocyte progenitor, megakaryocyte-erythroid progenitor). Next, the team performed real-time PCR to measure the expression of the transcription factors in 597 single cells (~120 each from the five stem / progenitor cell populations listed above). After statistical analysis, the team could link differences in expression of the transcription factors to cell type. Their observation of consistent differences in gene expression according to cell type is consistent with the idea that cell fate decision-making in blood stem cells critically depends on controlling gene expression levels of a tight network of transcription factors. Moreover, Göttgens’s team found that three transcription factors (Gata2, Gfi1, and Gfi1b) appear to form a “regulatory triad” that may be important for deciding the type of mature blood cell into which a given stem cell will mature.
Dysregulation of transcription factor expression is linked to cancer. In the context of the triad described by the Göttgens’ team, it is noteworthy that loss of Gata2 function predisposes people to leukemia, and inhibition of Gfi1 prolongs survival in some leukemia mouse models. It is therefore tempting to speculate that dysregulation of the Gata2/Gfi1/Gfi1b regulatory circuit contributes to the aberrant cell fate decision making that occurs in leukemia, where cells proliferate without proper differentiation.
By using the Biomark HD System, “we get added layers of information,” said Dr. Göttgens. “In a single experiment, we [can] generate a whole range of knockdown levels that would otherwise be completely obscure.”
Dr. Göttgens is committed to the single-cell experiment. He plans to at least double the number of cells under study into the thousands. Future studies by Dr. Göttgens and other teams at the Cambridge Stem Cell Institute could include use of the Biomark HD System to study knockdowns of selected transcription factors with animal leukemia models. Whatever direction the Cambridge investigators take in their quest to better understand normal and malignant stem cells, Dr. Göttgens believes that single-cell analysis is so critical to his research, he intends to make single-cell analysis a key component of all his future studies.
Victoria Moignard, et al., “Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis,” Nature Cell Biology, published online 24 March 2013; DOI: 10.1038/ncb2709.