An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

Abstract

We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma- dependent bone marrow culture assay in vitro. The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics. The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas. CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony- forming units (CFU-S), or vice versa. The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were studied. Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC. The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.

The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitor cells

“…The profound cellular quiescence induced by constitutive WT1 expression in this stem cell-enriched population was further supported by reduced activity in short-term colony-forming (CFU) assays and in long-term culture with primary bone marrow stroma (Ploemacher et al, 1989;Sutherland et al, 1990). Sorted GFP(+)CD34(+) cells expressing WT1(±KTS) cells showed a 10-fold decrease in clonogenic ef®ciency compared with control vector and WT1(+KTS) (P = 0.027; Figure 6B), and very low or undectable proliferative activity at weeks 1±5 following plating onto stroma, despite preserved viability (data not shown).…”

A Highly Efficient Aorta-Gonad-Mesonephros-Like Definitive Hemogenic Endothelium From Human Pluripotent Stem Cells

“…HPSCs as defined by positive for CD34, CD45, CD90 and negative for CD38 and a lineage cocktail described above were sorted into a 96-well plate containing human methylcellulose in a limiting dilution of 20, 10, 5, 2, or 1 cell. The 96-well plates were placed in a tissue culture incubator at 37 degrees for 14-21 days and CFC frequency was calculated based on established methods 35 .…”