Extension of cell life-span and telomere length in animals cloned from senescent somatic cells.

The potential of cloning depends in part on whether the procedure can reverse cellular aging and restore somatic cells to a phenotypically youthful state. Here, we report the birth of six healthy cloned calves derived from populations of senescent donor somatic cells. Nuclear transfer extended the replicative life-span of senescent cells (zero to four population doublings remaining) to greater than 90 population doublings. Early population doubling level complementary DNA-1 (EPC-1, an age-dependent gene) expression in cells from the cloned animals was 3.5- to 5-fold higher than that in cells from age-matched (5 to 10 months old) controls. Southern blot and flow cytometric analyses indicated that the telomeres were also extended beyond those of newborn (<2 weeks old) and age-matched control animals. The ability to regenerate animals and cells may have important implications for medicine and the study of mammalian aging.

Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer.

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.

Prospects for the use of nuclear transfer in human transplantation.

The successful application of nuclear transfer techniques to a range of mammalian species has brought the possibility of human therapeutic cloning significantly closer. The objective of therapeutic cloning is to produce pluripotent stem cells that carry the nuclear genome of the patient and then induce them to differentiate into replacement cells, such as cardiomyocytes to replace damaged heart tissue or insulin-producing beta cells for patients with diabetes. Although cloning would eliminate the critical problem of immune incompatibility, there is also the task of reconstituting the cells into more complex tissues and organs in vitro. In the review, we discuss recent progress that has been made in this field as well as the inherent dangers and scientific challenges that remain before these techniques can be used to harness genetically matched cells and tissues for human transplantation.

Human therapeutic cloning.

Somatic cell nuclear 'reprogramming' in livestock species is now routine in many laboratories. Here, Robert Lanza, Jose Cibelli and Michael West discuss how these techniques may soon be used to clone genetically matched cells and tissues for transplantation into patients suffering from a wide range of disorders that result from tissue loss or dysfunction.

Xenotransplantation of cells using biodegradable microcapsules.

BACKGROUND: The use of immunoisolation to protect transplanted cells from the immune system of the host has broad application to the treatment of major diseases such as diabetes and a wide range of other disorders resulting from functional defects of native cell systems. In most cases, limitations in functional cell longevity will necessitate periodic replenishment of the cells. We describe a hydrogel-based microcapsule that breaks down at a rate that can be adjusted to correspond to the functional longevity of the encapsulated cells. These injectable capsules can be engineered to degrade over several weeks to months for short-term drug delivery, or to remain intact and immunoprotective for more extended periods. When the supply of cells needs to be replenished, no surgery will be required to localize and remove the old capsules. METHODS: Porcine and bovine islets were immobilized in "composite" microcapsules fabricated from alginate and low-relative molecular mass (Mr) poly (L-lysine[PLL]) (Mr exclusion <120 Kd) and implanted into the peritoneum of normal and streptozotocin-induced diabetic rats. In addition to demonstrating long-term islet viability and function, a series of in vitro studies were carried out to determine the permeability and biodegradability of the microcapsules used in the present system. RESULTS: Xenogeneic islets implanted in nonimmunosuppressed rats remained in excellent condition indefinitely (>40 weeks)(viability was comparable to that of preimplant control specimens). In contrast, no islets survived in uncoated alginate spheres after 2 weeks postimplantation. By changing the concentration of the alginate, it was possible to vary the rate of capsule breakdown in rats from mechanically unstable (outer matrix <0.5-0.75% alginate) to stable for >1 year (> or =1.5% alginate). In addition to in vivo breakdown studies, the biodegradability of the capsular components was verified in vitro using a mixture of tritosomes (enzymes isolated from animal cells). CONCLUSIONS: We have designed a microcapsule system with controllable biodegradability which allows breakdown and absorption of implants when the cells die or become functionally inactive. These results may have application to other alginate-PLL encapsulation systems. The ability to cross species lines using these biodegradable microcapsules has the potential to expand dramatically the number of patients and the scope of diseases that can be successfully treated with cellular therapy.

Transplantation of islets using microencapsulation: studies in diabetic rodents and dogs.

Studies involving the transplantation of human islets in Type I diabetics have been of significant value both in documenting the potential importance of islet transplantation as a therapeutic modality, and in defining some of the problems which must be overcome before this approach can be used in large numbers of patients. The currently limited supply of adult human pancreatic glands, and the fact that chronic immunosuppression is required to successfully transplant islets into patients, indicate that techniques must be further developed and refined for allo- and xenografting of isolated islets from human and animal sources to diabetic patients. An increasing body of evidence using microencapsulation techniques strongly suggests that this will be achieved during the next few years. Data from our laboratory in rodents and dogs indicate that these systems can function for extended periods of time. In one study, insulin independence was achieved in spontaneously diabetic dogs by islet microencapsulation inside uncoated alginate gel spheres (Mr exclusion >600 kD). No synthetic materials or membrane coatings were employed in this study. Spheres containing canine islets were implanted into the peritoneum of 4 diabetic dogs. The animals received low-dose CsA (levels below readable limits by HPLC at 3 weeks). Implantation of these spheres completely supplanted exogenous insulin therapy in the dogs for 60 to >175 days. Blood glucose concentration averaged 122+/-4 mg/dl for these animals during the first 2 months. The glycosylated hemoglobin (HbAIC) levels during this period dropped from 6.7+/-0.5% to 4.2+/-0.2% (P<0.001). IVGTT K-values at 1 and 2 months postimplantation were 1.6+/-0.1 (P<0.002) and 1.9+/-0.1 (P<0.001), respectively compared with 0.71+/-0.3 before implantation. In a second group of studies, bovine islets were immobilized inside a new type of selectively permeable "microreactor" (Mr exclusion <150 kD) and implanted into the peritoneum of 33 STZ-induced diabetic rats without any immunosuppression. Diabetes was promptly reversed, and normoglycemia maintained for periods of several weeks to months. Immunohistochemical staining of microreactors recovered from these animals revealed well-granulated beta-cells consistent with functionally active insulin synthesis and secretion. To test further the secretory function of the islets, some of the explanted microreactors were incubated in media containing either basal or stimulatory concentrations of glucose. The islets responded with an approximately 3- to 5-fold average increase above basal insulin secretion. These results are encouraging, and may have important implications in assessing the potential role of these microencapsulation systems as therapy for human insulin-dependent diabetes.

Xenotransplantation of cells and tissues: application to a range of diseases, from diabetes to Alzheimer's.

The ability to cross species lines will dramatically expand the number of patients and the scope of human diseases that can be treated successfully with transplantation. In addition to whole organs, the transplantation of cells and tissues with specific differentiated functions represents an important conceptual and medical advance. In the USA alone, over 15 million patients suffer from diabetes, over 7 million patients suffer from neurodegenerative diseases, and millions more suffer from liver failure, AIDS, hemophilia and other disorders caused by tissue loss or dysfunction. Clinical trials using animal cells to treat many of these diseases are already under way, and it seems likely that this list will continue to grow as researchers identify new bioactive molecules and expand their understanding of the role different cells play in the human disease process.

Immunoisolation: at a turning point.

The principle of immunoisolation is to separate transplanted cells from the hostile immunological environment of the host by a selectively permeable membrane. Low-molecular-weight substances such as nutrients, electrolytes, oxygen and biotherapeutic agents are exchanged across the membrane, while immunocytes, antibodies and other transplant-rejection effector mechanisms are excluded. Here, Robert Lanza and William Chick review these systems.

Transplantation of pancreatic islets.

The currently limited supply of human pancreatic glands, and the fact that multiple glands may be required to isolate sufficient numbers of islets to treat a single patient, indicate that techniques must be further developed and refined for xenografting of isolated islets from animal sources to diabetic patients. An increasing body of evidence using immunoisolation techniques strongly suggests that this will be achieved during the next few years. Several different types of systems employing selectively permeable membranes and matrix supports for cells have been successfully tested in animals, including devices anastomosed to the vascular system as arteriovenous (AV) shunts, tubular membrane chambers, and spherical micro- and macrocapsules. Results in diabetic animals indicate that these systems can function for periods of several months to > year without the use of any immunosuppression. Our data suggest that this approach has the potential not only to allow the transplantation of islets across wide species barriers, but that it can be achieved using injectable microreactors fabricated from biodegradable polymers. The use of these various immunoisolation systems to transplant islets and other cells and tissues offers the opportunity to revolutionize current therapy for many human disease.

Encapsulated cell technology.

The potential therapeutic applications of encapsulated cells are enormous. In the US alone, it has been estimated that nearly half-a-trillion dollars are spent each year to care for patients who suffer tissue loss or dysfunction. Over 6 million patients suffer from neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease, over 14 million patients suffer from diabetes, and millions more from liver failure, hemophilia, and other diseases caused by the loss of specific vital cellular functions. It appears likely that by the end of the decade clinical trials of encapsulated cells to treat many of these diseases will become a reality. The Food and Drug Administration has already authorized studies to evaluate the safety and biological activity of several types of systems. A number of issues will have to be addressed, including the sourcing of raw materials, the design and building of manufacturing facilities, the scale-up and optimization process, storage and distribution of the product, and quality control.