Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures.

In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34++CD33,38,71(-). Bone marrow and umbilical cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34(++)CD33,38,71(-) subset. In comparison to this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the overall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.

Serial bone marrow sampling for long-term follow up of human hematopoiesis in NOD/SCID mice.

The study of long-term human hematopoiesis in immunodeficient mice is greatly facilitated by sequential bone marrow (BM) sampling in individual animals. Until now, however, the only way to obtain these samples was by sacrificing the mice. In this paper we describe a novel technique for obtaining BM cells by aspiration from the femur of living mice. The technique is simple and efficient and does not disable the animals. On average 1.6+/-1x10(6) nucleated cells can be collected from one femur at a time, which is sufficient for flow cytometry analysis, cytospin preparations, and polymerase chain reaction assays. The cellular composition of the samples obtained by puncture is identical to that of BM harvested by flushing the femur after sacrificing the animals. We present the results of 81 punctures of the femur in Hu-NOD/SCID chimeras engrafted with Ficoll-separated or CD34bright purified cells from human umbilical cord blood.

T lymphocytes determine the development of xeno GVHD and of human hemopoiesis in NOD/SCID mice following human umbilical cord blood transplantation.

Over the past decade the human-immunodeficient mouse chimera has become a well-established in vivo model for studying the human immune system and/or hemopoiesis. Under certain experimental conditions and depending on the composition of the human cell graft, the recipient mice may develop a fatal disease, designated as discordant xenogenic graft-versus-host-disease (GVHD), which differs in target tissues and histopathology from allogenic GVHD. Experimental evidence is presented that immunodeficient mice are equally susceptible to allogenic GVHD as normal immunocompetent mice. Whole human cord blood and distinct cellular subpopulations from a single cord blood harvest were transplanted in NOD/severe combined immunodeficient mice and the repopulation of human cells was monitored over time. Depending on the ratio of lymphocytes to hemopoietic stem cells, proliferation of human T cells, hemopoiesis or a combination of the two is observed in widely varying proportions. When the graft contains a preponderance of lymphocytes, fatal protracted discordant xenogenic GVHD develops. Mice receiving purified CD34 cells survived up to 207 days in good health with more than 95% human cells in the bone marrow. In those mice all lineages (B and T lymphocytes, monocytes, granulocytes, erythrocytes and thrombocytes) were demonstrated in the bone marrow and peripheral blood.