Immortalized human stromal cell lines

Details Immortalized human stromal cell lines Immortalized human stromal cell lines

2000-12-21

2003-04-01

Chan, Christina (Department: 1644)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S366000, C435S377000, C435S404000, C435S405000

Reexamination Certificate

active

06541249

ABSTRACT:

BACKGROUND OF THE INVENTION
Hematopoietic cells are believed to originate in the bone marrow from primitive, undifferentiated precursors called totipotent stem cells. Totipotent stem cells give rise to more mature precursor cells called pluripotent stem cells. Pluripotent stem cells are able to self-renew and, thus, give rise to identical types of pluripotent stem cells. Further, pluripotent cells differentiate into two major stem cell lineages; myeloid stem cells or lymphoid stem cells. Once stem cells are committed to a given lineage they can only give rise to cells of that same lineage (these cells are called committed or dedicated “progenitors”). Of the two main lineages, myeloid cells may mature into red blood cells, granulocytes, monocytes, or megakaryocytes whereas lymphoid cells may mature into either B lymphocytes (B-cells) or T lymphocytes (T-cells).
The bone marrow microenvironment provides signals which regulate early stage stem cell maturation. These signals induce the progenitor cells to divide and differentiate. Thus, defining the functional components of the bone marrow microenvironment is a prerequisite to understanding how the proliferation and differentiation of progenitor cells is coordinately regulated. The cellular complexity of the marrow microenvironment has been demonstrated both in situ and in vitro by a variety of histochemical techniques (Lichtman, Exp. Hematol. 9:391 (1981), and Allen et al., Exp. Hematol. 12: 517 (1984)). The marrow microenvironment is comprised of both hematopoietic and stromal or mesenchymal derived cells. The stromal cells include endothelial cells that form the sinuses and adventitial reticular cells that have characteristics consistent with adipocytes, fibroblasts, and muscle cells (Charbord et al., Blood 66: 1138 (1985), and Charbord et al., Exp. Hematol. 18: 276 (1990)). Numerous advances in recent years have provided considerable information on the ontogeny and development of hematopoietic cells; however, ontogeny of the stromal components and their precise role in controlling hematopoiesis has proven elusive (Ogawa, Blood 81: 2844 (1993); Muller-Sieburg et al., Critical Rev. Immunol. 13: 115 (1993); and Dorshkind, Ann. Rev. Immunol. 8:111 (1990)).
Mesenchymal or “stromal” cells constitute a critical part of the bone marrow microenvironment. Stromal cells act (at least in part) by secreting soluble growth factors and cytokines that stimulate stem cell proliferation and differentiation. Some cytokines known to affect proliferation, differentiation, and function of hematopoietic cells include: bFGF (Huang et al., Nature, 360, 745 (1992)); FLT3L (Hannum et al., Nature, 368, 643 (1994); Lyman, Int. J. Hematol., 62, 63 (1995)); G-CSF (Ikebuchi et al., PNAS USA, 85, 3445 (1988)); GM-CSF (Brandt et al., J. Clin. Invest., 86, 932 (1990)); IL-1alpha (March, et al., Nature, 315, 641 (1985); Clemens, et al., Lymphokines and Interferons, A Practical Approach, 1, 272 (1987); IL-1beta (March, et al., Nature, 315, 641 (1985); Auron, et al., Proc. Natl. Acad. Sci. USA, 81, 7907 (1984); Clemens, et al., Lymphokines and Interferons, A Practical Approach, 1, 272 (1987); IL-1 and IL-6 (Brandt et al., J. Clin. Invest., 82, 1017 (1988); IL-3 (Leary et. al., Blood, 71, 1759(1988)); IL-7 (Goodwin, et al., Proc. Natl. Acad. Sci. USA, 86, 302 (1989); Yokota, et al., Proc. Natl. Acad. Sci. USA, 83, 5894 (1986)). IL-8 (Matsushima, et al., J. Exp. Med., 167, 1883 (1988); Schroeder, et al., J. Immunol., 139, 3474 (1987). IL-11 (Paul et al., PNAS USA, 87, 7512 (1990); Tsuji et al., PNAS USA, 87, 7512 (1990); LIF (Fletcher et. al., Blood, 76, 1098 (1990)); MCP-1 (Yoshimura, et al., FEBS Lett., 244, 487 (1989); Matsushima, et al., J. Exp. Med., 169, 1485 (1989)); M-CSF (Wong, et al., Science, 235, 1504 (1987); Halenbeck, et al., Biotechnology, 7, 710 (1989)); MIP-1alpha (Wolpe, et al., FASEB J., 3, 2565 (1989); Graham, et al., 344, 442 (1990)); SCF (Brandt et al., Blood, 79, 634 (1992); Zsebo et al., Cell, 63, 195 (1990)); TGF-betal (Tsang, et al., Cytokine, 7, 389 (1995)); TNF-alpha (Clemens, et al., Nature, 312, 724 (1984); Matthews, et al., Lymphokines and Interferons, A Practical Approach, 1, 221 (1987)); Tpo (Foster, et al., Proc. Natl. Acad. Sci. USA, 91, 13023 (1994); Avanzi, et al., Br. J. Haematol., 69, 359 (1988); Lok, et al., Stem Cells, 12, 586 (1994)).
Following initial evolution in the bone marrow, hematopoietic cells move to the peripheral circulation and into a variety organs (such as the thymus, liver, and spleen) where they further mature to terminally differentiated immune cells.
In contrast to the in vivo situation, long-term propagation of proliferating and/or differentiating hematopoietic stem cells in vitro is problematic. Due to the heterogeneous nature of the bone marrow microenvironment, it is extremely difficult to distinguish the subset of factors necessary and sufficient to recapitulate in vivo bone marrow conditions.
Long term cultures of marrow cells in vitro can approximate some conditions of the in vivo marrow microenvironment and have been informative with respect to the identification of growth factors, adhesion proteins and extracellular matrix proteins that mediate the interaction between the hematopoietic cells and the stromal elements (Muller-Sieburg et al., supra; Dorshkind, supra; Liesveld et al., Exp. Hematol. 9: 391 (1981); Kittler et al., Blood 79: 3168 (1992); Eaves et al., Blood 78: 110 (1991); Clark et al., Bailliere's Clin. Haematol. 5: 619 (1992); and Heinrich et al., Blood 82: 771 (1993)). One improvement to this system incorporated the use of stromal precursors, positive for the STRO-1 antigen, to initiate long term cultures (LTC); STRO-1 positive stromal precursors are devoid of myeloid components and less heterogeneous than primary cultures, but are still capable of supporting hematopoiesis (Simmons and Torok-Storb, Blood 78: 55-62 (1991)). However, both the STRO-1 initiated cultures and the primary LTC are too complex to delineate contributions from individual cell types. Moreover, primary cultures can be highly variable and change with time, further complicating the identification of stromal cells that have a role in controlling hematopoiesis.
Immortalized stromal cell lines have been used to circumvent some of these problems (Zipori et al., J. Cell Physiol. 118: 143 (1984); Zipori et al., J. Cell Physiol. 122: 81 (1985); Song et al., Exp. Hematol. 12:523 (1984)). In contrast to mouse cell lines, however, human cell lines undergo senescence unless first immortalized by transformation (for example, by introducing a retrovirus; see Lanotte et al., J. Cell Sci. 50: 281 (1981)). The first few human bone marrow stromal cell lines available were established using the SV40 virus large T antigen (Harigaya et al., Proc. Natl. Acad. Sci. USA 82: 3477 (1985); Tsai et al., J. Cell Physiol. 127: 137 (1986); Novotny et al., Exp. Hematol. 18: 775 (1990); Slack et al., Blood 75: 2319 (1990); Singer et al., Blood 70: 464 (1987); Cicutinni et al., Blood 80: 102 (1992); and Thalmeir et al., Blood 83: 1799 (1994)).
The ability to culture hematopoietic cells and their precursors (derived from the bone marrow, peripheral blood, or umbilical cord blood of a patient or donor) offers the potential to vastly improve immunosuppressive and immunodestructive protocols currently used in treating cancer and other life-threatening immune disorders. Ex vivo cultured hematopoietic cells, for example, may be used to reconstitute a patient's blood-clotting and infection-fighting functions following such therapies. Additionally, the ability to expand a desired population of hematopoietic cells in vitro would alleviate dependence on bone marrow aspiration and multiple aphereses as the only means of obtaining sufficient cell numbers for transplants.
Early work in the field of hematopoietic stem cell culture centered around the culture of murine bone marrow aspirates in agar gel or liquid medium. Unfractionated bone marrow (including stem cells, progenitor cells, more differentiated hematopoietic cells, and stromal elements) was

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