Kidney Therapeutic Cloning and Stem Cells

Approximately 70,000 people in the United States are on a transplant wait list due to renal disease. The two currently available options for such patients are dialysis and renal allotransplantation. Both options are life-sustaining, but both are associated with significant morbidity and mortality. Dialysis is often poorly tolerated, and transplantation is burdened with severe donor shortages as well as the complications that accompany immuno-suppression. This has motivated researchers to develop alternative solutions for end-stage renal disease. Previous methods of tissue engineering of renal tissue involved extracorpo-real systems comprising biologic and synthetic components. Somatic cell nuclear transfer theoretically can reduce or eliminate the immune response of allogenic grafts.

Genetically identical renal tissue has been produced applying the principles of tissue engineering and therapeutic cloning in a large animal model, the cow (Bos Taurus) (14).

Stem cells were created by isolating and microinjecting single bovine skin fibro-blast donor cells into the perivitelline space of donor enucleated oocytes (nuclear transfer). Renal cells were isolated from a cloned metanephros and expanded until the desired number of cells was obtained. The renal cells were then seeded onto scaffolds consisting of three collagen-coated cylindrical polycarbonate membranes. Renal devices were constructed by connecting catheters to the ends of three membranes. The catheters functioned as a collecting system that drained into a reservoir, thereby creating a renal neo-organ with a mechanism for collecting excreted fluid. The devices were then subcutaneously implanted back into the same steer from which the genetic material was derived. The renal devices were explanted after 12 weeks and studied.

On gross inspection, the renal units appeared intact, and yellow, urine-like fluid was seen in the reservoirs. Chemical analysis of this fluid suggested unidirectional secretion of urea nitrogen and creatinine, as well as filtration and reabsorption of glucose and other electrolytes (Fig. 3). Histological examination of the retrieved implants demonstrated extensive vascularization of the renal units, and self-assembly into glomeruli and tube-like structures. A continuum between the glomeruli, tubules, and polycarbonate membrane was observed that allowed passage of urine into the collecting system reservoir. Renal specific proteins were detected in the renal units with immunohistochemical analysis and Western blot analysis. Reverse transcription-polymerase chain reaction analysis confirmed the transcription of renal-specific ribose nucleic acid in the cloned specimens. The cloned renal cells also produced both erythropoietin and 1, 25-dihydroxyvitamin D3, important endocrine metabolites produced by a normal kidney.

FIGURE3 ■ (A) Illustration of renal unit and units retrieved three months after implantation. (B) Unseeded control. (C) Seeded with allogeneic control cells. (D) Seeded with cloned cells, showing the accumulation of urine-like fluid.

The goal of therapeutic cloning, as described above, is to produce tissues that are genetically identical to those of the donor. Other researchers have demonstrated that animals produced by somatic cell nuclear transfer inherit their mitochondria entirely or in part from the recipient oocyte and not from the donor cell (14). Theoretically, these foreign mitochondrial proteins derived from the oocyte could produce an immune response from the donor after transplantation. We investigated the possibility of an immune response to the cloned renal units described above with delayed-type hypersensitivity testing in vivo and Elispot analysis of interferon-gamma-secreting T-cells in vitro. Neither test demonstrated an immune response to the cloned renal tissue, suggesting that rejection will not necessarily occur in the presence of oocyte-derived mitochondrial deoxyribonucleic acid. This finding offers the possibility that renal tissue can be derived via nuclear transfer and that these tissue may be implanted in a host without the histocompatibility issues that plague other forms of allotransplantation.

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