CFU-S assay: a historical single-cell assay that offers modern insight into clonal hematopoiesis

Abstract

Hematopoietic stem cells (HSCs) have been studied extensively since their initial functional description in 1961 when Dr. James Till and Dr. Ernest McCulloch developed the first in vivo clonal strategy, termed the spleen colony-forming unit (CFU-S) assay, to assess the functional capacity of bone marrow-derived hematopoietic progenitors at the single-cell level. Through transplantation of bone marrow cells and analysis of the resulting cellular nodules in the spleen, the CFU-S assay revealed both the self-renewal and clonal differentiation capacity of hematopoietic progenitors. Further development and use of this assay have identified highly proliferative, self-renewing, and differentiating HSCs that possess clonal, multilineage differentiation. The CFU-S strategy has also been adapted to interrogating single purified hematopoietic stem and progenitor cell populations, advancing our knowledge of the hematopoietic hierarchy. In this review, we explore the major discoveries made with the CFU-S assay, consider its modern use and recent improvements, and compare it with commonly used long-term transplantation assays to determine the continued value of the CFU-S assay for understanding HSC biology and hematopoiesis.

Figure 1

Schematic of CFU-S assays. (A) In the original CFU-S assay developed by Till and McCulloch in 1961 [2], bone marrow cells were harvested from donor mice and irradiated prior to intravenous injection into irradiated recipient mice. Ten days post transplant, spleens were counted, harvested, and sectioned for histologic analysis. For serial transplantations to determine self-renewal of CFU-S cells within spleen colonies [15], individual colonies were dissected and transplanted into secondary recipients as single-cell suspensions. Spleen colonies formed after secondary transplantation were analyzed similarly to those from primary transplantations [15]. (B) In the updated CFU-S assay with high throughput, quantitative analysis, HSCs (or other hematopoietic progenitors) were FACS-purified and transplanted into irradiated recipient mice. At 13.5 days post transplan, individual spleen colonies were counted, harvested, and dissected under a fluorescent microscope [22]. Single-cell suspensions from each colony were then analyzed by flow cytometry to assess colony composition and cell fluorescence. Four lineages (erythroid, megakaryocytic, granulocytic, and B cell) were identified using the markers listed in the table [22,41]. BM=bone marrow; CFU-S=spleen colony-forming unit; HSCs=hematopoietic stem cells.