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A Larochelle

Publications and source records attributed to A Larochelle.

8 recordsLinked to original sources
PubMed1997

Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells.

Many experimental and clinical protocols are being developed that involve ex vivo culture of human hematopoietic cells on stroma or in the presence of cytokines. However, the effect of these manipulations on primitive hematopoietic cells is not known. Our severe combined immune-deficient mouse (SCID)-repopulating cell (SRC) assay detects primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of immune-deficient non-obese diabetic/SCID (NOD/SCID) mice. We have examined here the maintenance of SRC, colony-forming cells (CFC), and long-term culture-initiating cells (LTC-IC) during coculture of adult human BM or umbilical cord blood (CB) cells with allogeneic human stroma. Transplantation of cultured cells in equivalent doses as fresh cells resulted in lower levels of human cell engraftment after 1 and 2 weeks of culture for BM and CB, respectively. Similar results were obtained using CD34+-enriched CB cells. By limiting dilution analysis, the frequency of SRC in BM declined sixfold after 1 week of culture. In contrast to the loss of SRC as measured by reduced repopulating capacity, the transplanted inocula of cultured cells frequently contained equal or higher numbers of CFC and LTC-IC compared with the inocula of fresh cells. The differential maintenance of CFC/LTC-IC and SRC suggests that SRC are biologically distinct from the majority of these in vitro progenitors. This report demonstrates the importance of the SRC assay in the development of ex vivo conditions that will allow maintenance of primitive human hematopoietic cells with repopulating capacity.

ADP-ribosyl Cyclase↗
PubMed1996

Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.

The development of stem-cell gene therapy is hindered by the absence of repopulation assays for primitive human hematopoietic cells. Current methods of gene transfer rely on in vitro colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, as well as inference from other mammalian species. We have identified a novel human hematopoietic cell, the SCID-repopulating cell (SRC), a cell more primitive than most LTC-ICs and CFCs. The SRC, exclusively present in the CD4+CD8- fraction, is capable of multilineage repopulation of the bone marrow of nonobese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). SRCs were rarely transduced with retroviruses, distinguishing them from most CFCs and LTC-ICs. This observation is consistent with the low level of gene marking seen in human gene therapy trials. An SRC assay may aid in the characterization of hematopoiesis, as well as the improvement of transduction methods.

ADP-ribosyl Cyclase↗
PubMed1995

Engraftment of immune-deficient mice with primitive hematopoietic cells from beta-thalassemia and sickle cell anemia patients: implications for evaluating human gene therapy protocols.

Permanent correction of genetic deficiencies of the hematopoietic system requires gene transfer into stem cells and long-term lineage specific expression after autologous transplantation. However, progress to develop gene therapy protocols has been hampered by the absence of in vivo assays that detect genetically deficient human hematopoietic stem cells and their diseased differentiated progeny. The establishment of systems to transplant human cells into immune-deficient SCID mice provides such an assay. We report that primitive bone marrow cells from beta-thalassemia major and sickle cell anemia patients engraft immune-deficient mice, giving rise to high levels of human erythroid and myeloid cells in response to treatment with human cytokines. The bone marrow of transplanted mice contained the entire erythroid lineage from BFU-E to mature erythrocytes expressing human gamma, beta or beta s-globin. Moreover, human erythroid cells from mice transplanted with sickle cell anemia bone marrow showed characteristic sickling under reducing conditions in an in vitro assay. This model provides a powerful in vivo system that can be used to evaluate the efficiency of globin gene transfer into primitive human hematopoietic cells, lineage-specific expression in mature erythrocytes, and ultimately correction of the cellular defect found in the erythroid lineage.

Anemia, Sickle Cell↗
PubMed1994

Increased expression of a novel leukocytic factor in patients with hemolytic anemia.

Misprimed polymerase chain reaction (PCR) products were generated from the crude leukocytic DNA extract of four of five patients with hereditary hemolytic anemia, during the course of exon 6 pyruvate kinase L gene amplification. These by-products, which originated from abundant mRNA templates, were not observed in seven healthy individuals. Two markers were cloned (GenBank accession numbers M64700 and M64701). We focused further studies on one of the human markers associated with hereditary hemolytic anemia, human DNA marker B (HUMDNAMB). HUMDNAMB is a 451-bp open reading frame that has never been described previously. The nucleic acid sequence region 303-416 is 63% homologous to a coding region of the bovine interferon alpha-A gene. Matrix analysis of the amino acid sequences reveals a structural similarity between the two proteins. The HUMDNAMB protein is expected to be larger than interferons, based on the size of its 2-kb messenger RNA detected by Northern blot in human leukocytes, fetal liver, and fetal intestine.

Adult↗