Generation of histocompatible tissues using nuclear transplantation

Abstract

Nuclear transplantation (therapeutic cloning) could theoretically provide a limitless source of cells for regenerative therapy. Although the cloned cells would carry the nuclear genome of the patient, the presence of mitochondria inherited from the recipient oocyte raises questions about the histocompatibility of the resulting cells. In this study, we created bioengineered tissues from cardiac, skeletal muscle, and renal cells cloned from adult bovine fibroblasts. Long-term viability was demonstrated after transplantation of the grafts into the nuclear donor animals. Reverse transcription-PCR (RT-PCR) and western blot analysis confirmed that the cloned tissues expressed tissue-specific mRNA and proteins while expressing a different mitochondrial DNA (mtDNA) haplotype. In addition to creating skeletal muscle and cardiac "patches", nuclear transplantation was used to generate functioning renal units that produced urinelike fluid and demonstrated unidirectional secretion and concentration of urea nitrogen and creatinine. Examination of the explanted renal devices revealed formation of organized glomeruli- and tubule-like structures. Delayed-type hypersensitivity (DTH) testing in vivo and Elispot analysis in vitro suggested that there was no rejection response to the cloned renal cells. The ability to generate histocompatible cells using cloning techniques addresses one of the major challenges in transplantation medicine.

Staged in vitro reconstitution and implantation of engineered rat kidney tissue

“…It has also been shown that adult ''progenitorlike'' cells from the injured mouse kidney can form tubular epithelial structures in vitro and migrate to multiple compartments of the developing kidney in organ culture (14). These types of cells, or possibly others that have recently been described (10,49,52), may be more suitable.…”

“…In vitro propagation of the branching ureteric bud or subdivided progenitor tissue has been proposed as a method for generating a number of kidneys from a single progenitor tissue (9). Cellular components and scaffolding have been combined to create cell-based filtering devices and nephron components (10,11). Additional cell-based strategies have included the use of adult renal stem-like cells (12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

Recent Applications of Polymeric Biomaterials and Stem Cells in Tissue Engineering and Regenerative Medicine

“…146 Bovine skin fibroblasts from adult Holstein steers were obtained by ear notch, and single donor cells were isolated and microinjected into the perivitelline space of donorenucleated oocytes (nuclear transfer). The resulting blastocysts were implanted into progestin-synchronized recipients to allow for further in vivo growth.…”

Somatic Cell Dedifferentiation/Reprogramming for Regenerative Medicine

“…Even though ES cells derived from SCNT have the nuclear genome of the donor cells, mitochondrial DNA inherited by the oocyte could lead to immunogenicity after transplantation. Lanza et al (44) evaluated the histocompatibilty of nuclear transfer-generated tissue; the nucleus of a bovine skin fibroblast was microinjected into an enucleated oocyte. Even if the blastocyst was implanted, the purpose was to produce cardiac, renal and skeletal muscle cells, which were then harvested, expanded in vitro and seeded onto biodegradable scaffolds.…”