Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of culturing cells in suspension
Reexamination Certificate
2000-06-23
2001-07-17
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of culturing cells in suspension
C435S377000, C435S384000, C435S002000
Reexamination Certificate
active
06261841
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates generally to the field of maintaining and expanding populations of hematopoietic cells ex vivo.
Blood cells of all types derive from hematopoietic progenitor cells, which are multipotential (i.e., capable of differentiating into any of a variety of types of blood cells) at early stages of development. At later stages of development, a hematopoietic progenitor cell can become one of only certain types of cells, depending on the developmental path the cell has undergone. By way of example, a hematopoietic stem cell can differentiate to become either a myelo-erythroid progenitor cell or a lymphoid stem cell. If the cell becomes a myelo-crythroid progenitor cell, it can become an erythroid progenitor (and subsequently an erythrocyte) or a myeloid progenitor cell. A myeloid progenitor cell, in turn, can differentiate to become a megakaryocyte (MK), or one of several other types of blood cells. Platelets are derived from MKs. Thus, MKs and the physiological processes by which hematopoietic progenitor cells differentiate into MKs are involved in disorders associated with aberrant formation and activation of platelets.
Many pregnancy-associated diseases (e.g., pregnancy-induced hypertension, pre-eclampsia, and diabetes) result from aberrant modulation of maternal physiology. For example, although the platelet count has been observed to decrease slightly during human pregnancy in some studies (Fay et al., 1983, Obst. Gynecol. 61:238-240), the rate of platelet production apparently increases to compensate for the dramatic increase in blood volume during pregnancy (Davison et al., 1989, Baillieres Clin. Endocrinol. Metab. 3:451-472). Aberrantly high rates of platelet activation in plasma have been clinically associated with pre-eclampsia, and anti-platelet treatment is widely used to treat pregnant women afflicted with this disorder (Beaufils et al., 1985, Lancet 1:840-842; Steyn et al., 1997, Lancet 350:1267-1271; Konijnenberg et al., 1997, Am. J. Obst. Gynecol. 176:461-469). Disorders associated with aberrantly low rates of platelet production include thrombocytopenia (e.g., that associated with leukemia and alcohol-induced thrombocytopenia).
Several cytokines, including thrombopoietin (TPO), interleukin-6 (IL-6), IL-11, leukemia inhibitory factor, and kit ligand, have been demonstrated to enhance MK maturation under normal physiological conditions (Baatout, 1998, Anticancer Res. 18:1871-1882; Ellis et al., 1995, Blood Rev. 9:1-6).
It is not understood, which factor or combination of factors are responsible for pregnancy-associated thrombopoietic activity. Thus, efficacious treatment of pregnancy-associated thrombopoietic disorders has been hampered. A significant need remains for methods which can be used to treat such disorders.
Ex vivo expansion of hematopoietic progenitor cells and transplantation of those cells have several important clinical uses, including stem cell rescue following myeloablative therapy and gene therapy. However, there is presently no clinically approved method to preserve and expand hematopoietic progenitor cells, particularly at their earliest, most multi-potential stages. The most common approach to ex vivo multi-potential hematopoietic cell expansion is to culture purified progenitor cells (i.e., those expressing the CD34 marker) in the presence of early-acting cytokines such as interleukin-3. Unfortunately, using prior art cell expansion methods, there is generally an inverse relationship between cell proliferation and the percentage of primitive stem cells which are maintained in a relatively non-differentiated stage of development. Thus, using prior art methods, when hematopoietic progenitor cells are induced to proliferate or differentiate, early progenitor cells are lost. Because these early cells can be the most useful for inducing hematopoietic engraftment (i.e., in that survival of these cells in vivo can provide a source for many or all blood cell types), differentiation associated with prior art cell expansion methods represents a significant shortcoming of those methods.
Recent work has shown that inclusion, in a nutritive medium for maintaining hematopoietic progenitor cells ex vivo, of a combination of thrombopoietin (TPO), stem cell factor (SCE), and flt3 ligand (Flt-3L; i.e., the ligand of the flt3 gene product) was useful for expanding primitive (i.e., relatively non-differentiated) human hematopoietic progenitor cells in vitro, and that those cells were capable of engraftment in SCID-hu mice (Luens et al., 1998, Blood 91:1206-1215). Nonetheless, a significant need remains for other methods for expanding multi-potential hematopoietic progenitor cells in a manner that preserves a population of the cells at an early stage of their differentiation/development.
The present invention provides compositions, kits, and methods which satisfy one or more of the needs described above.
BRIEF SUMMARY OF THE INVENTION
The invention includes a method of maintaining mammalian (e.g., human) hematopoietic progenitor cells in vitro. This method comprises maintaining the cells in vitro in a nutritive medium (e.g., for minutes, hours, or 3, 6, 9, 13, or more days) comprising murine prolactin-like protein E (mPLP-E) or murine prolactin-like protein F (mPIP-F; collectively mPLP-E/F). The mPLP-E/F can, for example, have the amino acid sequence SEQ ID NO: 2, and can be glycosylated at one or more of residues 6, 28, 80, 119, 127, and 234 of SEQ ID NO: 2. The nutritive medium can further comprise a cytokine selected from the group consisting of thrombopoietin, interleukin-3, interleukin-6, interleukin-11, leukemia inhibitory factor, kit ligand, stem cell factor, and flt3 ligand. The cells can, for example, be cells obtained from bone marrow, fetal cells, or cells obtained from cord blood. Use of this method can result in expansion of the cells. This method can also be used to induce proliferation, differentiation, or both, of mammalian hematopoietic progenitor cells in vitro.
The invention also includes a method of inhibiting differentiation of mammalian hematopoietic progenitor cells. This method comprises contacting the cells (e.g., in vitro) with an antibody substance which specifically binds with mPLP-E/F. In an alternative embodiment, the cells are contacted with an antibody substance which specifically binds with a cell surface receptor of the cells and which inhibits binding of mPLP-E/F with the receptor.
The invention further includes a method of inducing proliferation of mammalian hematopoietic progenitor cells. This method comprises contacting the cells with an antibody substance which inhibits binding of one of mPLP-E and mPLP-F with a receptor on the cells without significantly inhibiting an activity normally associated with binding of mPLP-E or mPLP-F with the receptor.
In another aspect, the invention includes a method of assessing whether a test compound is useful for modulating either of proliferation and differentiation of mammalian hematopoietic progenitor cells. This method comprises maintaining the cells in vitro in a nutritive medium comprising mPLP-E (or mPLP-F) and in the presence and absence of the test compound, whereby a difference between
proliferation or differentiation of the cells in the presence of the test compound and
proliferation or differentiation of the cells in the absence of the test compound is an indication that the test compound is useful for modulating proliferation or differentiation of mammalian hematopoietic progenitor cells.
REFERENCES:
Lefebvre et al., A Novel Murine Pregnancy Specific Hormone Acts as a Multilineage Survival Factor for Human Bone Marrow Progenitors. (Nov. 15, 1999) Blood vol. 19 No. 10 Suppl. 1 part 1 191a, abstract #837.*
Lin et al. Three Members of the Mouse Prolactin/Growth Hormone Family are Homologous to Proteins Expressed in the Rat (1997) Endochrinology, vol. 138, No. 12, pp. 5541-5549.*
Lin et al. A Novel Megakaryocyte Differentiation Factor from Mouse Placenta (1999) Trends in Cardiovasc. Med. vol. 9, No. 6, pp. 167-171.*
Lin et al., Induc
Cohen Isaac
Lefebvre Phil
Lin Jiandie
Linzer Daniel
Guttman Harry J
Morgan, Lewis & Bockius, L.L.P.
The Board of Trustees of Northwestern University
Weber Jon P.
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