Hematopoietic cell culture nutrient supplement

Details Hematopoietic cell culture nutrient supplement Hematopoietic cell culture nutrient supplement

1997-01-31

2004-05-11

Falk, Anne-Marie (Department: 1632)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C424S093100, C424S093200, C435S372000, C435S383000, C435S384000, C435S386000, C435S387000, C435S388000, C435S389000, C435S404000, C435S405000, C435S406000, C435S407000

Reexamination Certificate

active

06733746

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a replacement for the serum supplementation normally required for ex vivo expansion of CD34
+
hematopoietic cells and cells of myeloid lineage.
BACKGROUND OF THE INVENTION
Blood cells in the mammal can be divided into three main categories or families: red cells, white cells of myeloid lineage, and white cells of the lymphocytic lineage. Red blood cells carry oxygen from the lungs to the tissues and cells of the body and transport CO
2
from the tissues and cells back to the lungs for elimination. White cells of myeloid lineage include: neutrophils, basophils, eosinophils, megakaryocytes, monocytes/macrophages, and dendritic cells. These cells play a role in the identification and elimination of foreign organisms (e.g. bacteria) or damaged tissue, cells and substances from the body. White cells of lymphocytic lineage are divided into two main subgroups: T lymphocytes (helper cells, killer cells, suppressor cells), which are involved in cell mediated responses to viruses, tumor cells and foreign tissue grafts; and B lymphocytes, which are involved in the production of antibodies which circulate in the blood and react in a chemically specific fashion to foreign materials (e.g. bacteria, foreign proteins).
Embryologically, blood cells are formed in the third week of development from cells of the splanchnic mesoderm (Langman,
J., Medical Embryology,
3rd ed., pp. 1-7, Williams and Wilkins Co., Baltimore, Md., 1975). In the yolk sac, hematopoiesis (i.e., blood cell formation) initially occurs in clusters or islands (“blood islands”) of splanchic mesoderm cells. The blood islands then migrate to the liver during fetal development. At full gestation and immediately after delivery, these cells migrate to the bone marrow in the shafts of long bones, ribs, and hips. Throughout the remainder of life, the bone marrow serves as the normal site of blood cell formation, releasing mature cells into the circulation (Wintrobe, M. M.,
Clinical Hematology
, pp. 1-7, Lea and Febiger, Philadelphia, Pa., 1967).
Hematopoiesis is an ordered process by which different blood cells are produced at a rate of 400 billion per day (Koller, M. R. and Palsson, B. O.,
Ex Vivo
42:909-930 (1993)). Since the early 1960s, researchers have acquired experimental evidence for the hypothesis that each of the various blood cell types are derived from a common “pluripotent” stem cell in the mouse (Till, J. E. and McCulloch, E. A.,
Radiation Res.
14:213-222 (1961); Becker, A. J. et al.,
Nature
195:452-454 (1963); Siminovitch, L. et al.,
J. Cell. Comp. Physiol.
62:327-336 (1963)). Experimental evidence also supports the existence of pluripotent stem cells in humans (Nowell, P. C. and Hungerford, D. A.,
J. Natl. Cancer Inst.
25:85-109 (1960); Tough, I. M. et al.,
Lancet
1:411-417 (1961); Barr, R. D. and Watt, I.,
Acta Haemat.
60:29-35 (1978)).
Hematopoietic cell differentiation occurs in stages (Pimentel, E., Ed.,
Handbook of Growth Factors Vol. III: Hematopoietic Growth Factors and Cytokines
, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). The first stage is represented by the hematopoietic stem cell. Development then diverges along the lymphoid and myeloid lineages as lymphoid progenitor cells and myeloid/erythroid progenitor cells are formed. Development further diverges within each of these lineages. Lymphoid progenitor cells form pre-B and pre-T precursor cells, which subsequently develop into B lymphocytes and T lymphocytes, respectively. Myeloid/erythroid progenitor cells form (1) erythroid burst-forming unit (BFU-E) cells, which eventually develop into erythrocytes; (2) megakaryocyte colony-forming unit (CFU-MEG) cells, which eventually develop into megakaryocytes and platelets; (3) granulocyte/macrophage colony-forming units (CFU-GM), which eventually develop into monocytes, macrophages, and neutrophils; (4) eosinophils; and (5) basophils.
A major focus in the field of experimental hematology continues to be the identification of the most primitive, pluripotent stem cell. One approach has been to identify cell surface markers (such as CD antigens) on the surface of progenitor cells and to correlate these markers with stages of development or differentiation by the cells' ability to form colonies of differentiated cells in methylcellulose culture systems. CD antigen expression has been shown to be modulated during cellular differentiation (Sieff, C. et al.,
Blood
60:703 (1982)). Hematopoietic stem cells are CD34
+
cells. That is, they express the CD34 surface marker. The most primitive known human progenitor cell, which has been characterized as CD34
+
/CD33
−
CD38
−
, represents only 1 to 2% of all bone marrow cells (Civin, C. I. et al.,
J. Immunol.
133:157 (1984)).
In the mid 1960's, in order to better understand the mechanisms of normal and aberrant hematopoiesis, investigators began trying to grow bone marrow cells ex vivo using both suspension and semi-solid tissue culture systems. The early studies of Bradley and Metcalf (Bradley, T. R. and Metcalf, D.,
Biol Med. Sci.
44:287-300 (1966)), as well as those of Pluznik and Sachs (Pluznik, D. H. and Sachs, L.,
Expl. Cell Res.
43:553-563 (1966)), demonstrated that serum alone was not sufficient to support the growth of myeloid progenitors in culture. Cell growth required the presence of factor(s), secreted by other cells (i.e. feeder cells) and found in the conditioned media from cultures of these cells. It is now clear that the growth of hematopoietic tissue ex vivo requires the presence of several cytokines or hematopoietic growth factors.
Several distinct factors have been identified, cloned and are now routinely manufactured as recombinant molecules for both research and/or clinical use. These include erythropoietin (Lin, F. K. et al.,
Proc. Natl. Acad. Sci. U.S.A.
82:7580-7584 (1985); Stone, W. J. et al.,
Am. J. Med. Sci
296:171-179 (1988)), interleukin-3 (IL-3) (Fung, M. C. et al.,
Nature
307(5948):233-237 (1984); Yokota, T. et al.,
Proc. Natl. Acad. Sci. U.S.A.
81:1070-1074 (1984); Ganaser, A. et al.,
Blood
76:666-676 (1990)), granulocyte macrophage-colony stimulating factor (GM-CSF) (Wong, G. G. et al.,
Science
228:810-815 (1985); Sieff, C. A. et al.,
Science
230:1171-1173 (1985)); granulocyte-colony stimulating factor (G-CSF) (Souza, L. M. et al.,
Science
232:61-65 (1986)); stem cell factor (SCF) (Copeland, N. G. et al.,
Cell.
63:175-183 (1990); Flanagan, J. G. et al.,
Cell
63:185-194 (1990); Zsebo, K. M. et al., Cell 63:195-201 (1990); Martin, F. H. et al.,
Cell
63:203-211 (1990); Szebo, K. M. et. al.,
Cell
63:213-224 (1990); Huang, E. et al.,
Cell
63:225-233 (1990)), and interleukin-11 (II-11) (Paul, W. et al.,
Proc. Natl. Acad. Sci. U.S.A
87:7512-7516 (1990)), to cite only a few.
In 1977, Dexter and his colleagues developed a long-term bone marrow culture (LTBMC) protocol (Dexter, T. M. et al.,
J. Cell Physiol.
91:335-344 (1977)). Known as “Dexter” culture, this type of cell culture system does not require the use of conditioned media and appears to establish and mimic, in vitro, the hematopoietic environment. Long term cultures have been established using human bone marrow (Grenberger, H. M. et al.,
Blood
58:724-732 (1981); Eaves, C. J. et al.,
J. Tiss. Cult. Method.
13:55-62 (1991)), as well as the marrow from other animal species (Eastment, C. E. and Ruscetti, F. W., Evolution of Hematopoiesis in Long-Term Bone Marrow Culture: Comparison of Species Differences, in:
Long
-
Term Bone Marrow Cultures, Droc Foundation Series
18, pp. 97-118, Allan R. Liss, Inc., New York, N.Y., 1984).
In long-term culture systems, growth and differentiation of stem cells and early progenitor cells appears to require either direct cell to cell contact or very close proximity between the developing hematopoietic cells and stromal cells (Dexter, T. M. et al.,
J. Cell. Physiol.
91:335-344 (1977)).
Each of the above cell culture systems (e.g., liquid static culture, semi-solid culture and long term bone marrow culture) appe

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