Colony forming cell assays for human hematopoietic progenitor cells.

Hematopoietic stem cells (HSCs) present in small numbers in adult bone marrow (BM), peripheral blood (PB) and umbilical cord blood (CB) produce a heterogeneous pool of progenitors that can be detected in vitro using colony forming cell (CFC) assays. Hematopoietic progenitor cells proliferate and differentiate to produce colonies of maturing cells when cultured in a semisolid methylcellulose-based medium that is supplemented with suitable growth factors and other supplements. The colonies are then classified and enumerated in situ by light microscopy or an automated imaging instrument.

Simultaneous cloning and selection of hybridomas and transfected cell lines in semisolid media.

Selection and cloning are essential but often laborious and time-consuming steps during the generation of hybridomas and genetically modified cell lines that produce monoclonal antibodies or other proteins with desired properties. Methods for the simultaneous selection and cloning of hybridomas and transfected cell lines (e.g.

Interlaboratory assessment of a novel colony-forming unit assay: a multicenter study by the cellular team of Biomedical Excellence for Safer Transfusion (BEST) collaborative.

Background: Interlaboratory scoring performances were determined using a traditional 14-day colony-forming unit (CFU) assay and a new 7-day CFU assay.

Study Design And Methods: Digital images of colonies were utilized to train personnel at each site. A central laboratory inoculated methylcellulose with progenitors and sent the samples by overnight courier to participating labs for plating.

An in vitro model for cytogenetic conversion in CML. Interferon-alpha preferentially inhibits the outgrowth of malignant stem cells preserved in long-term culture.

IFN-alpha has been shown to prolong survival in chronic myeloid leukemia patients, but its mechanism of action is still not understood. The human cobblestone area-forming cell (CAFC) assay allows for the measurement of the concentration of normal as well as malignant stem cells, while their progeny can be measured in parallel long-term culture (LTC) in flasks. Using CAFC and LTC assays, we have examined direct effects of IFN-alpha (500; 5,000 IU/ml) on the maintenance and outgrowth of CD34-enriched normal and malignant stem cells, obtained from six patients with an established major cytogenetic response to IFN-alpha and from four nonresponding patients.

Long-term leukemia-initiating capacity of a CD34-subpopulation of acute myeloid leukemia.

Acute myeloid leukemia (AML) proliferation in vivo is maintained by a small fraction of progenitor cells. These cells have been assumed to express an immature phenotype and to produce most colony-forming units (CFU-AML). For one case of AML (French-American-British [FAB] M1, normal cytogenetics), we examined the capacity of the CD34+ (25% of unseparated AML cells) and CD34- fractions to initiate leukemia in severe combined immunodeficient (SCID) mice.