Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1993-10-12
1995-04-25
Wax, Robert A.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
4352402, 43524021, 43524025, C12N 500, C12N 502, C12N 508
Patent
active
054098254
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a system and process for supporting human stem cells and more particularly the present invention relates to supporting hematopoietic stem cells for use in bone marrow transplant patients.
Mammalian hematopoiesis has been studied in vitro through the use of various long-term marrow culture systems (3, 10-12). Dezter and co-workers (3) described a murine system from which CFU-S an CFU-GM could be assayed for several months, with erythroid and megakaryocytic precursors appearing for a more limited time. Maintenance of these cultures was dependent on the formation of an adherent stromal cell layer composed of endothelial cells, adipocytes, reticular cells, an macrophages. These methods were soon adapted for the study of human bone marrow. Human long-term culture systems were reported to generate assayable hematopoietic progenitor cells for 8 or 9 weeks (10, 11) and, later, for up to 20 weeks (12, 13). Such cultures are again relying on the pre-establishment of a stromal cell layer which is frequently reinoculated with a large, heterogeneous population of marrow cells. Hematopoietic stem cells have been shown to home and adhere to this adherent cell multilayer before generating and releasing more committed progenitor cells (1, 14, 15). Stromal cells are thought to provide not only a physical matrix on which stem cells reside, but also to produce membrane-contact signals and/or hematopoietic growth factors necessary for stem cell proliferation and differentiation (4, 5, 16, 17). This heterogeneous mixture of cells comprising the adherent cell layer presents an inherently complex system from which the isolation of discrete variables affecting stem cell growth has proven difficult.
Recently, a study was conducted by McNiece and Langley which examined the stimulatory effect of recombinant human stem cell factor (MGF) on human bone marrow cells alone and in combination with recombinant human colony stimulating factors, GM-CSF, IL-3 and erythropoietin. The results showed that MGF stimulation of low density non-adherent, antibody depleted CD34.sup.+ cells suggests that MGF directly stimulates progenitor cells capable of myeloid and erythroid differentiation (18).
In accordance with an aspect of the present invention there is provided a process for supporting mammalian bone marrow cells wherein such cells are maintained in a culture medium essentially free of stromal cells and which includes at least one cytokine effective for supporting such cells.
Preferred embodiments of this aspect of the present invention provide a process for supporting bone marrow cells which are hematopoietic stem cells, a process for supporting bone marrow cells which are hematopoietic progenitor cells and a process for supporting bone marrow cells which are CD34.sup.+ DR.sup.- CD15.sup.- cells.
In addition, this invention provides that at least one cytokine be selected from the following cytokines: Interleukin (IL)-1, IL-3, IL-6, granulocyte/macrophate-colony stimulating factor (GM-CSF), human or murine stem cell factor, sometimes referred to as human or murine mast cell growth factor (MGF) and a fusion protein of GM-CSF/IL-3 (FP). Further, this invention provides particularly preferred embodiments wherein the cytokine MGF is included as the sole cytokine or in combination with at least one other cytokine.
In accordance with another aspect of the present invention there is provided a process for supporting mammalian bone marrow cells wherein such cells are maintained in a culture medium containing a combination of cytokines effective for supporting such cells. Preferably, the bone marrow will be supported in a culture medium which is essentially free of stromal cells.
Another aspect of the present invention provides for a process of supporting mammalian bone marrow cells wherein such cells are maintained in a culture medium which is essentially free of serum and of stromal cells. This system allows for preferred expansion of progenitor cell numbers and enables the identification of which cytokines specifically affect
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Brandt John
Hoffman Ronald
Moore William W.
Rowland Bertram I.
Sherwood Pamela J.
Wax Robert A.
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