EP1341915A1

EP1341915A1 - Use of cells derived from embryonic stem cells for increasing transplantation tolerance and for repairing damaged tissue

Info

Publication number: EP1341915A1
Application number: EP01995556A
Authority: EP
Inventors: Michael Bader; Bert Binas; Giuxuan Chai; Fred FÄNDRICH; Detlev Ganten; Xiongbin Lin
Original assignee: Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Current assignee: Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Priority date: 2000-12-04
Filing date: 2001-12-04
Publication date: 2003-09-10
Also published as: WO2002046401A1; US20040208857A1; JP2004519437A; DE10061334A1

Abstract

The invention relates to the use of cells from cell lines, which are derived from early embryonic stages, for the donor-specific increase in transplantation tolerance and for repairing damaged tissue. Areas of application of the invention include the field of medicine and the pharmaceutical industry. The aim of the invention is to produce a donor-specific immunotolerance in order to prevent a rejection of the transplanted tissue due to an immune response and thus to be able to limit the administration of immunosuppressives. In order to produce a donor-specific immunotolerance, embryonic stem cell-like cell lines (ECL) are obtained from blastocysts and are transfected with genetic material of the donor, which codes for the MHC haplotypes. The cells produced in such a manner are administered to the recipient before the transplantation for producing an immunotolerance against the tissue to be transplanted or for regenerating already damaged tissue.

Description

Use of cells derived from embryonic stem cells to increase transplant tolerance and to restore destroyed tissue

Description:

The invention relates to the use of cells from cell lines, which are derived from early embryonic stages, for the donor-specific increase of the transplant tolerance and for the restoration of destroyed tissue. Areas of application of the invention are medicine and the pharmaceutical industry.

State of the art:

In transplant medicine, the development of increasingly powerful immunosuppressive drugs such as prednisone, cyclosporin, tacrolimus, mycophenolate mefetil and antilymphocyte antibodies has increased patient survival and the length of stay of the grafts by an average of one year. The routine use of these drugs has made clinical transplantation a standard treatment chosen for most non-malignant terminal diseases of the heart, kidneys and liver. However, an improvement in the early survival of the grafts was not achieved without significant infectious morbidity and non-immune side effects (Gaber et al., Transplantation 66: 29-37, 1998). In addition, a better early survival could not be translated into a better long-term survival of the graft, since chronic rejection continued to affect the grafts after the first year at a frequency that has not changed substantially in the past 20 years (Cecka and Terasaki, Clinical Transplants 1997, Los Angeles, UCLA Tissue Typing Laboratory, 1998). In addition, long-term monitoring of the clinical course of transplantations (Pirsch and Friedman, J. Gen. Intern. Med. 9: 29-37, 1994) increasingly showed late morbidity and mortality due to the further need for non-specific immunosuppression.

The term immune tolerance (hereinafter referred to as tolerance), which can generally be described as the absence of an immune response after administration or intake of a specific antigen (AG), has a central role in transplantation medicine. From a transplant immunologist's point of view, tolerance can be defined as persistent tissue persistence in the absence of a deleterious immune response that can be achieved without ongoing therapeutic intervention. In this In context, it is important to note that tolerance is not an innate characteristic of an individual, but is acquired (Owen, Science 102: 400-401, 1945; Billingham et al., Nature 172: 603-606, 1953). It is also known that the tolerant state that exists at birth changes constantly, especially when the body is confronted with new antigens in the course of life. For example, the immune system must be able to tolerate certain "foreign" antigens, such as physiological hormones released during puberty and pregnancy (Fowlkes and Ramsdell, Curr. Opin. Immunol. 5: 873-879, 1993). The fact that fetal life can develop and survive in a host that is not adapted to major histocompatibility complex (MHC) is another example of nature's ability to distinguish not only between foreign and non-foreign, but also between dangerous and harmless can (Vacchio and Jiang, Grit. Rev. Immunol. 19: 461-480, 1999).

In allogeneic hematopoietic stem cell transplantation (CD34 ⁺ ), successful transplantation in hosts not adapted to MHC is only achieved if the recipient is sublethally myeloablated or irradiated.

Disadvantages of previous organ or tissue transplants

In order to prevent the graft from being rejected by an immune reaction, strong immunosuppressive agents are currently being administered which, however, in turn cause an increased susceptibility to infections. For example, ubiquitous germs, which pose no danger to a normally functioning immune system, can cause serious illnesses in an immunosuppressive state. With a blood stem cell transplant (CD34 ⁺ ), for example, a successful transplant into a recipient not adapted to MHC is only achieved if the recipient's bone marrow has been removed beforehand or destroyed by X-ray radiation.

Task:

The invention is based on the object of generating a donor-specific immune tolerance in order to prevent rejection of the transplanted tissue by an immune reaction and thus to be able to restrict the administration of immunosuppressive agents.

Description of the invention:

The invention is implemented according to claim 1, the subclaims are preferred variants. To create a donor-specific immune tolerance, embryonic stem cell-like cell lines (ECL) are obtained from blastocysts and with the donor's genetic material that codes for the MHC haplotypes. transfected. The cells produced in this way are finally administered to the recipient prior to the transplantation in order to generate immune tolerance to the tissue to be transplanted or to renew already damaged tissue.

The use of the cells from the ECLs as "tolerance vectors" is opened by a lack of MHC antigen expression and the associated immunogenic inactivity of the ECL. Research has shown that cells from ECLs can be transplanted and survive in the long term, producing hematopoietic cells of different origins. In addition, these hematopoietic cells derived from ECL have generated a constant mixed chimerism (donor and recipient hematopoietic cells coexist in the same host) and thus create the basis for long-term allograft acceptance. As a result, they can either be used as an ideal means of creating tolerance, or alternatively they can be used in a situation in which the parenchymal injury of a particular organ has to be remedied.

The invention is explained in more detail below.

Three rat breeds were chosen for ECL isolation, Wistar Kyoto (WKY), Sprague Dawley (SD) and ACI.

Blastocysts obtained from these were planted on mitomycin-inactivated embryo fibroblasts from mice (MEF) or rats (REF) as providers. The MEF turned out to be better and both, MEF and REF, were clearly superior to gelatin. If inner cell mass (ICM) growths formed after the blastocysts had grown to the supply layer, they could usually be easily expanded in groups of ES-like cell clumps (primary clumps) for about 10 days (primary growth). After that, the cells largely differentiated into a mixture of different cell types and were soon overgrown by round, loosely attached, endoderm-like cells. Only a small part of the cells from the primary clump survived and was expanded to a cell line.

WKY blastocysts grew very rapidly, showed strong primary growth, and more than 10% of the embryos achieved permanent cell lines (Table 1). SD blastocysts grew with some delay, achieved a moderate number of primary clumps, and the effectiveness of cell line generation was poor. ACI blastocysts took the longest time to grow, produced a very small number of primary clumps, and no cell line could be generated by this breed. These results suggest that the rate at which blastocysts bind to the host cell layer and the number of primary clumps during the greatest primary growth are positively related to the effectiveness of ECL derivation (Table 1). It is interesting that the Hybrid blastocysts of the WKY genotype that ACI beats in producing the ECLs, but not the SD genotype (Table 1).

The use of the cells from the cell lines, which were generated from the embryonic stem cells, as "tolerance vectors" to bring about donor-specific immune tolerance also requires the expression of the donor-specific MHC antigens. This property is achieved according to the invention in that cells of the ECLs are transfected with the genes of the donor which code for the MHC antigens. This can be done by (i) fusing the ECL with a given somatic cell or cell line that contains the MHC genes of interest, (ii) transfecting the ECL with a given MHC-encoding plasmid, by (iii) Creation of transgenic rats with an MHC-encoding plasmid and the generation of ECL from this breed or by (iv) peptide loading the ECL with MHC allopeptides of class I, which are responsible for the highly polymorphic 0.1 helix of a specific MHC class I antigen encode. There are different variants for the administration of the transfected cells, e.g. via the portal vein, by intraperitoneal, subcutaneous or intravenous injection.

In clinical practice of organ transplantation, this creates the possibility of modifying the recipient's alloreaction by administering ECLs that express the donor-specific MHC antigens. An exact phenotyping and morphological characterization of the descendants derived from ECL makes it possible to search for similar cells with stem cell characteristics in the adult host. This enables a better understanding of the plasticity of the stem cells derived from an adult tissue, which, as an alternative to the ECL, share the ECL properties and can be used in a similar form.

EXAMPLES

1. Isolation and cultivation of the rat ECL

Mouse embryo fibroblasts (MEF) or rat embryo fibroblasts (REF) are prepared from 13-14 day pregnant animals that have been mitotically inactivated by 3-5 treatments with mitomycin C (10 μg / ml) for 2 or 1 hours, washed with phosphate buffered saline ( PBS) and planted in Nunc 4-wells. The blastocysts are flushed with PBS / 20% FCS (fetal calf serum) or a culture medium from the uterus of rats 4.5 days pregnant, planted on inactivated embryo fibroblasts and 3-4 days in DMEM / 15% FCS / 2,500 μ / ml LIF ( "Leukemia inhibiting factor", ESGRO, Life Technologies) with additives (Iannaccone et al, Dev. Biol. 1994; 163: 288-292) left untreated in a medium of 6% CO ₂ / air. During this time, the blastocysts develop and tie themselves to the supplier, and the ICM begins to grow, with the Efficiency depends on the genetic background. Outgrowths with an ES-cell-like appearance are picked up and broken up into several lumps by suction in drawn glass capillaries with a somewhat smaller diameter than the outgrowths and applied to fresh supply plates. Picking up and breaking up occurs either daily or every other day. Crumbled colonies are reset in rows until a small number of clean, steadily growing ES-like clumps are obtained. The clump population is then expanded to several dozen, stored in 35 mm dishes and slightly trypsinized into a mixture of individual cells and small aggregates. The rat ES cells produced were passaged every or every other day by trypsinization (0.05% trypsin, 0.02% EDTA-4Na, 1% chicken serum, in Ca / Mg-free PBS). The species identity of the resulting cell lines is checked by PCR using Renin gene primers (Brenin et al., Transplant. Proc. 1997; 29: 1761-1765) in order to rule out contamination by mouse ES cells.

2. Intraportal injection of WKY-derived ECL into allogeneic host rats

A first series of experiments examined the fate of a single intraportal injection of WKY-derived 1.0 x 10 ⁶ ECL into allogeneic DA (RTl. ^Avl ) host rats that had received no immunosuppressive or myeloablative treatment. The investigations show that these cells can survive long-term (> 150 days) in DA host rats. It was found that the ECL and its descendants can produce a state of constantly mixed chimerism (hematopoietic cells of the donor and the recipient coexist in the same host). It was also found that these cells differentiate into hematopoietic cells that express class II MHC antigens (Ox-3) and B cell lineage markers (Ox-45). The monoclonal antibody (mAb) Ox-3 is a specific antibody of a (WKY) MHC class II donor that binds to class II MHC epitopes that are expressed on WKY but not to DA MHC positive cells class II. Flow cytometric determination of double-stained leukocytes (WBC) showed that 3 to 5% of the WBC taken from DA rats (100 days after the ECL injection) expressed Ox-3 ⁺ cells, with 15 - 20% of the splenic lymphocyte population contained Ox-3 ⁺ . These results demonstrate the fact that ECL can produce hematopoietic cells. Accordingly, Ox-3 ⁺ cells were detected histomorphologically (10-15%) in the interstitial space of the recipient (DA) hearts, which were selectively destroyed 100 days after a single intraportal injection of 1.0 x 10 ⁶ ECL derived from WKY (see Fig.l). The stable chimeric state of these animals provides the basis for examining the fate of the WKY cardiac allograft transplanted in DA rats seven days after the intraportal ECL injection. Kaplan Meier diagrams show that pretreatment of DA rats with 1.0 x 10 ⁶ ECL intraportally and heart transplantation (HTx) seven days later led to long-term (> 150 days) non-rejection allograft acceptance, whereas untreated DA rats caused WKY Reject allografts acutely (see Fig. 2). At the same time, CAP rat heart transplants were rejected from DA rats injected with WKY-ECL within 12.4 ± 1.4 days, demonstrating the immune competence of these rats.

3. Co-cultivation of ECL with somatic cell lines

Primary in vitro studies have shown that the ECL cells described above assume differentiation into astrocytes or cardiomyocytes and hepatocytes by co-cultivation with somatic cells of neuronal or entodermal origin. The embryonic cell lines described are therefore also suitable for the treatment of organ-specific diseases of the central nervous system (e.g. as dopaminergic cells for the treatment of Parkinson's disease, as hepatocytes for the treatment of cirrhosis of the liver or as cardiomyocytes for the treatment of fresh heart attacks). The forthcoming isolation of the signal proteins necessary for these specific forms of differentiation is of great clinical importance, since it could enable problem-free programming of the ECLs for the desired cell population. The goal is therefore the exact sequencing of these functional proteins in order to enable their recombinant production. The large sequence homology between rat and human protein would also provide information on the analog production of human functional proteins. The associated therapeutic application possibilities include both the use of the ECL-derived somatic cell lines described above and the functional proteins derived therefrom for future clinical use on all indication levels of tissue engineering for organ replacement, for gene therapy and for the treatment of metabolic diseases in the area of the CNS, the liver and the heart.

Claims

claims

1. A method for generating a donor-specific immune tolerance to transplanted tissue, characterized in that the recipient is administered before the transplantation cells which are from early embryonic stages, e.g. Blastocysts have been derived ("embryonic stem cell like cell lines", ECL).

2. The method according to claim 1, characterized in that the administered cells have been transfected with MHC genes and / or reporter genes.

3. The method according to claim 2, characterized in that the cells have been transfected with the donor-specific MHC genes.

4. The method according to claim 3, characterized in that the transfection is carried out by a fusion of the ECL with a somatic cell or cell line which have the donor-specific MHC genes.

5. The method according to claim 3, characterized in that the transfection is carried out with a specific MHC-coding plasmid.

6. The method according to claim 3, characterized in that the transfection takes place by creating transgenic non-human mammals with an MHC-coding plasmid and the production of ECL from these transgenic animals.

7. The method according to claim 3, characterized in that the transfection by peptide loading of the ECL with MHC allopeptides of class I, which code for the highly polymorphic α-helix of a specific MHC antigen of class I, takes place.

8. The method according to claim 2, characterized in that the administered cells have been transfected with a LacZ plasmid.

9. The method according to any one of claims 1-8, characterized in that cells from human cell lines are used.

10. The method according to any one of claims 1-9, characterized in that the cells are fed 3-7 days before the transplant.

11. The method according to any one of claims 1-10, characterized in that the cells are supplied intravenously.

12. The method according to claim 11, characterized in that the cells are supplied intraportally.

13. The method according to any one of claims 1-10, characterized in that the cells are supplied subcutaneously.

14. The method according to any one of claims 1-10, characterized in that the cells are supplied intraperitoneally.

15. The method according to claim 1, characterized in that the ECL cell line is programmed as a starting cell for differentiation into neuronal cells with a specific transmitter function (e.g. dopamine).

16. The method according to claim 1, characterized in that the ECL cell line is programmed as a starting cell for differentiation in hepatocytes to support the liver-specific metabolisms.

17. The method according to claim 1, characterized in that the ECL cell line is programmed as a starting cell for differentiation in cardiomyocytes for regeneration of the heart muscle function.

18. The method according to claim 1, characterized in that the signal proteins found as a result of the co-cultivation with specific differentiation potential for neuronal cells (dopamine-producing cells), hepatocytes and cardiomyocytes are produced as recombinant proteins.