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.

Genetic analysis of hemopoietic cell cycling in mice suggests its involvement in organismal life span

“…The CAFC assay, as described by Ploemacher et al (14), was slightly modified, as published earlier (15). This in vitro assay allows quantification of primitive hemopoietic cell subsets of various developmental stages.…”

Mature Accessory Cells Influence Long‐Term Growth of Human Hematopoietic Progenitors on a Murine Stromal Cell Feeder Layer

“…CAFC ( Fig. 1) frequencies per inoculated cell number were calculated using Poisson statistics [1,19,21]. The cell dilution containing one proliferating progenitor per well on average is found in the row where 37% of wells contain no CAFC, while the others show one or more.…”

Cobblestone Area‐Forming Cells in Human Cord Blood Are Heterogeneous and Differ from Long‐Term Culture‐Initiating Cells

“…The results described here refer to human CAFC seeded on human bone marrow stroma feeder layers. In the murine model initially developed for the CAFC assay by Ploemacher et al [4], there is a direct correlation between the number of nucleated cells required to produce long-term donor chimerism and the frequency of CAFC, suggesting that the false cobblestone phenomenon may not occur in this case.…”

“…Wells are scored as being positive or negative for the presence of cobblestone areas (CA, tightly knit group of phase-dark, angular cells in the stroma) and can be scored at more than one time point, e.g., after 5 and 8 weeks. In the murine model, CAFCs seen between days 28-45 of long-term culture are directly related to bone marrow repopulating ability [4].…”