Katie Matatall

Type II Interferon Promotes Differentiation of Myeloid‐Biased Hematopoietic Stem Cells

Interferon gamma (IFNγ) promotes cell division of hematopoietic stem cells (HSCs) without affecting the total HSC number. We postulated that IFNγ stimulates differentiation of HSCs as part of the innate immune response. Here, we report that type II interferon signaling is required, both at baseline and during an animal model of LCMV infection, to maintain normal myeloid development. By separately evaluating myeloid‐biased and lymphoid‐biased HSC subtypes, we found that myeloid‐biased HSCs express higher levels of IFNγ receptor and are specifically activated to divide after recombinant IFNγ exposure in vivo. While both HSC subtypes show increased expression of the transcription factor C/EBPβ after infection, only the myeloid‐biased HSCs are transiently depleted from the marrow during the type II interferon‐mediated immune response to Mycobacterium avium infection, as measured both functionally and phenotypically. These findings indicate that IFNγ selectively permits differentiation of myeloid‐biased HSCs during an innate immune response to infection. This represents the first report of a context and a mechanism for discriminate utilization of the alternate HSC subtypes. Terminal differentiation, at the expense of self‐renewal, may compromise HSC populations during states of chronic inflammation. Stem Cells 2014;32:3023–3030

Loss of c-Kit and bone marrow failure upon conditional removal of the GATA-2 C-terminal zinc finger domain in adult mice.

Heterozygous mutations in the transcriptional regulator GATA‐2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA‐2 DNA binding. Deregulated hematopoiesis with GATA‐2 mutation frequently develops in adulthood, yet GATA‐2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA‐2 C‐terminal ZnF (C‐ZnF) coding sequences in adult mice. Upon Gata2 C‐ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c‐Kit expression on hematopoietic progenitors. Transplant studies indicated GATA‐2 has a cell‐autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA‐2 regulates c‐Kit expression in multilineage progenitor cells. Enforced c‐Kit expression in Gata2 C‐ZnF‐deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA‐2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c‐Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA‐2 mutation.

Detecting Hematopoietic Stem Cell Proliferation Using BrdU Incorporation

Cellular quiescence is a key component of hematopoietic stem cell (HSC) homeostasis; therefore, a reliable method to measure HSC cell division is critical in many studies. However, measuring the proliferation rate of largely quiescent and rare populations of cells can be challenging. Bromo-deoxyuridine (BrdU) incorporation into replicating DNA is a commonly used and highly reproducible method to detect cell division history. Here, we describe a protocol for BrdU incorporation analysis in hematopoietic stem and progenitor cells that can provide a sensitive measure of cell division even in rare cell populations. In combination with flow cytometry, this method can be generalized to analyze other cell populations and other tissues as identified by cell surface markers.