Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse

Abstract

We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal “feeders” depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay, a series of unsorted and physically sorted bone marrow cells were simultaneously assayed in vivo and overlaid on the feeders in a range of concentrations, while frequencies of cells forming hematopoietic clones (cobblestone area forming cells, CAFC) were calculated by means of Poisson statistics. Linear regression analysis of the data showed high correlations between the frequency of CFU-S day 12 and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10. This correlation study validates the CAFC system as a clonal assay facilitation both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierarchy and the study of single long-term repopulating cells in vitro.

Efficient mobilization of haematopoietic progenitors after a single injection of pegylated recombinant human granulocyte colony‐stimulating factor in mouse strains with distinct marrow‐cell pool sizes

“…Figure 2 shows that, after administration of G-CSF, very few CAFC subsets were detected. In sharp contrast, administration of SD/01 resulted in a massive increase of all CAFC subsets, including large numbers of late-appearing colonies that have been associated with cells with long-term repopulating ability (Ploemacher et al, 1991).…”

“…For each of the six cell dilutions used, ten replicate wells were tested. Early appearing CAFCs (d 7) corresponded to relatively committed progenitor cells, whereas late-appearing CAFCs (d 35) reflected more primitive cell subsets (Ploemacher et al, 1991).…”

Haematopoietic stem cells depend on Gαs-mediated signalling to engraft bone marrow

“…No significant differences were observed between the genotypes in colony number ( Fig.2b ), morphology or size. Similarly, analysis of the more primitive cell fraction using the long-term culture initiating cell (LTC-IC) assay 10 , demonstrated no significant differences between the WT and KO cells ( Fig.2c ). Taken together, these data demonstrate that deletion of the G s α gene does not alter the differentiation potential of primitive BM MNCs in vitro .…”

Cobblestone-Area Forming Cells Derived from Patients with Mantle Cell Lymphoma Are Enriched for CD133+ Tumor-Initiating Cells

“…More specifically, during long-term co-culture we observed clusters of phase-dark cells beneath the stromal layer. These clusters are morphologically similar to CAFC that form under stromal cell monolayer when normal HSC are co-cultured with BM stromal cells [25] , [44] , [46] . In this study, immunophenotypic characterization of MCL associated CAFCs reveal that these clusters are composed of cells with a unique and primitive phenotype (CD45+CD19−CD133+) compared to cells that remain in suspension and the bulk population (CD45+CD19+CD133−).…”