Multicellular living organisms and unmodified parts thereof and – Nonhuman animal
Reexamination Certificate
1994-08-12
2002-09-24
Baker, Anne-Marie (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
C800S009000, C800S010000, C800S011000
Reexamination Certificate
active
06455756
ABSTRACT:
TECHNICAL FIELD
The field of this invention is immunocompromised mammals comprising xenogeneic tissues which are capable of long term reconstitution of myeloid and lymphoid cells.
BACKGROUND
Hematopoiesis is a continuous process of differentiation and amplification, replacing billions of mature lymphoid and myeloid cells in the normal human every day. This process depends on the continuous turnover of hematopoietic stem cells, which have the capacity for self-maintenance, extensive proliferation, and multipotentiality. These characteristics have been studied in great detail in the murine system, particularly through the uses of sequential bone marrow transplants and genetic marking using chromosomal rearrangements or retroviruses. Similar studies with human hematopoietic stem cells have lagged, primarily due to a lack of an equivalent model for long-term multipotential differentiation.
Scientists have recently succeeded in demonstrating human hematopoietic progenitor engraftment and differentiation in immunodeficient mice. Of particular interest has been the use of such mice for studying the tissue, its response to drugs and changes in the environment of the tissue. Various aspects of the human tissue may be studied in an environment simulating the natural environment using such chimeric animals.
Significant advances have been made in understanding the earliest events in human hematopoietic development by transplanting human cells or tissues into immunocompromised mice and observing human hematopoiesis for prolonged periods. However, each of the prior art systems is limited in its ability to study concomitant human mature T-cell, mature B-cell and myeloid cell production from a common stem cell pool. Implants of human fetal thymus and liver are limited in the extent to which myeloid and B-cells will develop. It is therefore of interest to develop a chimeric animal which is capable of long term reconstitution of myeloid, as well as both B- and T-lineages of the hematopoietic system.
Relevant Literature
A description of the SCID-hu mouse may be found in J. M. McCune et al. (1988)
Science
241:1632-1639; R. Namikawa et al. (1990)
J. Exp. Med.
172:1055-1063 and J. M. McCune et al. (1991)
Ann. Rev. Immunol.
9:395-429. Implantation of functional bone marrow is described in S. Kyoizumi et al. (1992)
Blood
79:1704. European patent application no. 469 632 discloses the use of immunocompromised mammals with a thy-liv implant.
Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice is described in T. Lapidot et al. (1992)
Science
255:1137; J. Nolta et al. (1994)
Blood
83:3041; and S. Kyoizumi et al (1993)
Blood
81:1479-1488. The proliferation and engraftment of immature cord blood progenitors in such mice is further discussed in J. Vormeer et al. (1994)
Blood
83:2489.
Immunocompromised mouse strains are described in S. Nonoyama et al. (1993)
J. Immunol
150:3817-3824; I. Gerling et al. (1994) Diabetes 43:433-440; Bosma, et al. (1983)
Nature
301:52; and P. Mombaerts et al. (1992)
Cell
68:869-877.
SUMMARY OF THE INVENTION
Immunocompromised hosts are provided, comprising xenogeneic functioning hematolymphoid tissue comprising bone marrown spleen and optionally, thymus tissue. The tissue grows to form a hybrid organ structure capable of producing B-lineage lymphocytes, T-lineage lymphocytes and myeloid cells.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for the production of human hematopoietic cells with a plurality of lineages in an immunocompromised heterologous mammalian host, particularly a mouse, for extended periods of time. The method comprises combining non-dispersed bone and spleen fragments in juxtaposition, optionally together with a thymus fragment, in an appropriate site in an immunocompromised host. The chimeric animal is useful for studying human hematopoiesis and pathogenesis in an experimental setting.
The co-implantation of human bone and spleen tissue is sufficient to support the growth of hematopoetic progenitors which, in the absence of a thymus implant, are able to mature into myeloid lineage cells as evidenced by expression of CD33, including granulocytes, as evidenced by the expression of CD14 and CD15; monocytes; and B lineage cells, as evidenced by the expression of CD19 and CD20. Myeloid cells may include neutrophils, monocytes and macrophages, eosinophils, basophils and mast cells, and progenitors thereof. Some T cell subsets are also present in the bone and spleen implant, as shown by the expression of CD4. However, maturation of T cell progenitors to provide all T cell subsets, particularly those that express CD8, requires the presence of a co-implantation of thymus, which provides stromal and epithelial cells necessary for differentiation. When thymus tissue is present, a subset of progenitor cells derived from the bone/spleen co-implant are able to differentiate into T cells, including CD4
+
CD8
+
, CD4
+
CD8
−
and CD4
−
CD8
+
subsets.
The host animal is engrafted with both spleen and bone, where the tissue implants are normally contiguous to provide a continuous source of hematopoietic progenitor cells. The spleen tissue appears to amplify to partially or wholly surround the growing human fetal bone and thymus to form a hybrid tissue, which provides a continuous source of myeloid cells, B-cells and other lymphoid progenitor cells. To provide for maturation of T cells, human thymus is also engrafted, in close proximity, usually in contact with the bone and spleen tissue. The thymus tissue may have a different HLA allotype from the spleen and bone. Differences in HLA have shown that mature T-cells are derived from stem cells present in the spleen/bone graft. Such animals are useful in determining the contribution that thymic stromal and epithelial elements, or hematopoietic progenitor cells make to T-cell maturation. The hybrid organ, containing bone, thymus and spleen (BTS), is vascularized, and able to survive in the host for long periods of time. The hybrid tissue may be used after at least about 3 weeks, more usually after at least about 6 weeks, and the hybrid tissue will remain functional for at least about 9 months, or more.
A suitable site for implantation must be able to accomodate the size of the implanted tissue and to keep the implanted tissues in close proximity. Of particular interest is subcutaneous implantation. The position of the subcutaneous implant on the body of the host is not critical, but the area of the mammary fat pads may conveniently be used. The tissue will be implanted, conveniently by incision of the host skin and placement with a trocar, etc.
The BTS tissue transplant may be only one of other tissues transplanted into the host. For example, in addition to the BTS implant, other hematopoietic components may be included, such as stem cells, lymph nodes, embryonic yolk sac, fetal liver, pancreatic tissue, appendix tissue, tonsil tissue and the like, which may serve in the development of a hematopoietic system in the immunocompromised host for a variety of purposes. Sites for introduction of additional tissue may include under the spleen capsule, abdominal wall muscle, under the renal capsule, in the eye, the peritoneum, the peritoneal lining, brain, subcutaneous, vascular system, spinal cord, membranous sacs or capsules of various tissue, the retroperitoneal space, reproductive organs, etc.
Introduction of the secondary tissue may be achieved by injection, implantation, or joining blood vessels (and other vessels if necessary) of the donor and host, using intravenous catheters, trocars, and/or surgical incision, or the like. The tissue or cells of interest will generally be normal, e.g. non-transformed and non-malignant tissue or cells. With various organs one may include native surrounding tissue with the organ tissue itself. The surrounding tissue may comprise connective tissue, or portions of blood and lymphatic vessels. In some cases, whole organ grafts may be transplanted by anastomosing donor and
Chen Benjamin P.
Fraser Christopher C.
Baker Anne-Marie
Golightly Douglas A.
Maigs J. Timothy
Novartis AG
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