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.

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.

Specific association of human telomerase activity with immortal cells and cancer.

Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.

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.

Shortened telomeres in the expanded CD28-CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis.

OBJECTIVE: To test the hypothesis that the expanded population of non-proliferative CD28-CD8+ T cells in HIV disease have shortened telomeres, thereby providing evidence that increased rounds of CD8+ cell division occur during HIV disease, possibly leading to replicative senescence and exhaustion of CD8+ T-cell responses. DESIGN: CD8+ cells play a central role in control of HIV infection. In late HIV disease, an expanded population of CD28-CD8+ cells with reduced proliferative potential has been documented. A similar population of CD28-CD8+ cells has been identified in ageing humans, where telomere length measurements have suggested that these cells have reached the irreversible state of replicative senescence. METHODS: CD8+ cells from HIV-infected and control subjects were sorted by flow cytometry into CD28+ and CD28- fractions. Telomere lengths were determined as mean terminal restriction fragment (TRF) lengths by Southern hybridization. RESULTS: The TRF lengths of sorted CD28-CD8+ cells in HIV-infected subjects ranged between 5 and 7 kilobases (kb) and were significantly shorter than TRF lengths of CD28-CD8+ cells in uninfected subjects (P = 0.003). The TRF length in CD28-CD8+ cells from HIV-infected subjects was the same as that observed for centenarian peripheral blood mononuclear cells and is compatible with a state of replicative senescence. CONCLUSIONS: The shortened telomeres in the CD28-CD8+ cells in HIV-infected subjects and the poor proliferative potential of these cells identifies CD8+ cell replicative senescence as a newly described feature of HIV disease. Our results provide a mechanism for the loss of CD8+ cell control of viral replication that accompanies advanced HIV disease. Replicative senescence may contribute to exhaustion of the T-cell response as a result of chronic HIV disease. Whether this phenomenon occurs in other chronic viral infections is unknown.

Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells.

PURPOSE: To investigate the relation of senescence-related beta-galactosidase activity and telomere shortening to replicative senescence in cultured human retinal pigment epithelial (RPE) cells. METHODS: A human RPE cell line was serially passaged until 80% of cells were nondividing in a 72-hour 5-bromo-2'-deoxyuridine (BrdU) labeling study. Early- and late-passage cells were double-stained for BrdU and senescence-related beta-galactosidase activity (pH 6). The average chromosomal telomere length at several population doublings was estimated by Southern blot analysis after double digestion of DNA with RsaI and HinfI and using a telomere-specific probe. RESULTS: BrdU-beta-galactosidase double-staining revealed an inverse correlation between the number of BrdU-labeled nuclei and beta-galactosidase-labeled cells as a function of population doubling level (PDL). At PDL 58, only 20% of all cells labeled for BrdU, whereas 57% stained for beta-galactosidase. The mean terminal restriction fragment length (TRF) was reduced from 10 kb in early (PDL 12) cultures to 4 kb in late (PDL 57) cultures. CONCLUSIONS: Senescence-related beta-galactosidase activity and mean TRF length may prove useful in studying the senescence of RPE cells in vitro. These techniques may be valuable in determining senescence of the retinal pigment epithelium in vivo, where senescent RPE cells could be involved in the development of age-related maculopathy and age-related macular degeneration.

Re-expression of senescent markers in deinduced reversibly immortalized cells.

We have developed a simian virus 40 (SV40) T-antigen immortalized human cell line, 1MR90-D305.2H4 (IDH4), in which the expression of T-antigen is controlled by the mouse mammary tumor virus (MMTV) promoter and thus regulated by steroids such as dexamethasone. Studies on the regulation of proliferation by T-antigen led to the formulation of a two-stage model for human cell immortalization, in which a mortality stage 1 mechanism (M1) was the target of T-antigen action, and an independent mortality stage 2 mechanism (M2) produced crisis and prevented T-antigen from directly immortalizing cells. Rarely, a cell expressing T-antigen escaped crisis (e.g., M2) and was capable of indefinite proliferation. This model predicted that the deinduction of T-antigen in IDH4 cells would lead to the reexpression of the M1 mechanism, and thus a reexpression of the senescent phenotype. Our study confirms the prediction that, in the absence of steroids, IDH4 cells express a variety of morphological and biochemical markers characteristic of normal senescent human fibroblasts.

Altered expression of plasminogen activator and plasminogen activator inhibitor during cellular senescence.

Fibroblast senescence is associated with a loss of proliferative potential and an alteration in extracellular gene expression. Because the expression of extracellular gene products are frequently growth state dependent, we undertook a comparative study of the regulation of the components of the plasminogen activation system in young and senescent cells under controlled conditions of growth. Young and senescent cells were compared in quiescent and activated growth conditions for the secretion of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2). Whereas young cells showed decreased levels of PAI-1 in the secreted and extracellular matrix pools upon serum deprivation, senescent cells showed a more constitutive pattern of gene expression, with no noticeable decrease of the levels in a low concentration of serum. RNA analysis revealed that senescent lung and skin cells, independent of the growth state, constitutively express levels of u-PA and PAI-1 comparable to the expression levels in young mitotically growing cells. These expression levels are down-regulated in quiescent young cells. In contrast, both t-PA and PAI-2 were markedly overexpressed in senescent skin lung cells under all growth conditions. Total plasminogen activator activity in conditioned medium was 50-fold higher in senescent-cell medium compared to young when cultured in 0.5% fetal calf serum (FCS) for five days, with the majority of the activity co-migrating on zymograms with u-PA. Increases in PAI-1 was also observed in senescent human umbilical vein endothelial cells. In summary, cells of various types display alterations in plasminogen activator activity during replicative senescence. The inappropriate over-expression of plasminogen activator activity in vivo may be expected to lead to a progressive disruption of extracellular matrix maintenance. Thus, our observations suggest that cellular replicative senescence is associated with an altered expression of several genes regulating tissue maintenance which, in turn, could lead to degenerative changes in tissue in age-related disease(s).

The cellular and molecular biology of skin aging.

BACKGROUND: The dramatic alterations in the appearance of the integument with increasing age are due in part to a progressive destruction of the delicate architecture of the connective tissue components of the dermis. Both collagenous and elastic components display a degeneration consistent with the overexpression of proteolytic activity. Recent advances in the field of molecular gerontology, using in vitro models of cellular aging, are yielding clues as to the fundamental causes of dermal aging. OBSERVATIONS: Dermal fibroblasts possess a finite replicative capacity of 50 to 100 doublings, then cease replicating in response to growth factors. Cells cultivated to the end of their replicative lifespan in vitro display alterations consistent with their playing a role in aging in vivo. In particular, senescent dermal fibroblasts overexpress metalloproteinase activities that may explain the age-related atrophy of extracellular matrix architecture. CONCLUSIONS: The recent discovery of a structural change in the telomeric region of the genome with cellular aging and new insights into DNA damage checkpoint mechanisms offer new opportunities to uncover both the molecular mechanisms regulating cellular aging and possibly to devise new strategies to manipulate these molecular events for therapeutic effect.

Carcinoma of the nasopharynx. A 25-year study.

Between 1958-1983, 79 patients with a diagnosis of epithelial tumor of the nasopharynx received definitive irradiation at Thomas Jefferson University Hospital. Seventy-two percent of the patients had a Stage IV lesion. The dose to the nasopharynx was over 6,000 cGy in all but four patients. The 5- and 10-year actuarial survivals were 33% and 19% respectively. The 5-year disease-free survival was 33%. Histology had no bearing on survival. Survival was influenced by the stage of primary tumor and nodes. Advanced nodal disease correlated with distant metastasis, being present in 13/15 cases with hematogenous spread.

The projected supply of registered nurses, 1990.

The number of employed registered nurses (RNs) in the United States stood at an all-time high of 1.3 million in 1980. This paper, using life table techniques, develops a population base of all living graduates from data on graduations from basic nursing education programs between 1928 and 1980, and estimates that there are some 1.9 million graduates now living. The number of graduates is projected to rise to some 2.4 million by the end of 1990, of whom 1.7 million will be active in the profession. Factors taken into account include recent increases in admissions, which rose from 79,000 in 1970 to 112,000 in 1980; the extent to which older women are entering the profession; the rapid growth of 2-year associate degree programs, which now account for half of all admissions; and increased labor force participation, with 68 percent of all living graduates in the labor force in 1980, compared to 60 percent in 1970. It is projected that the movement to advanced education among RNs will continue so that, by 1990, the proportion with baccalaureate or higher degrees will have risen from 29 to 36 percent of the employed RNs. By 1990 the largest number of active RNs will be in their 30s. Graduates with diplomas will have a median age of 45; those with associate degrees, 35; and those with baccalaureate or higher degrees, 32 years.

Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming.

AIM: To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. MATERIALS & METHODS: An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. RESULTS: We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09-21.1 kb) but markedly shorter TRF lengths (6.4-12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines. In order to verify these results in an isogenic background, we generated six iPS cell clones from the hES-derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells, had telomerase activity, expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells. Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However, the clone EH3, with relatively high levels of telomerase activity, progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. CONCLUSION: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.

Aspects of the workplace and return to work for persons with brain injury in supported employment.

This prospective study examined the effect of work environments on return to work for persons with brain injuries. Participants (n = 37) were individuals placed into supported employment by six placement agencies. All were assessed using the Vocational Integration Index (VII), an observational instrument for rating the opportunities for integration (Job Scale) and the extent to which an employee benefits from those opportunities (Consumer Scale). Individuals who retained their jobs for 6 months (n = 19) had been rated higher on all subscales and total scores for the VII, with seven of eight subscales statistically significant. Findings are discussed in regard to improving employment outcomes for persons with severe brain injuries.

An assessment of the potential use of anionic dextrans as a plasma substitute.

Several problems exist when dextrans are used as plasma substitutes. High molecular weight dextrans can cause red cell aggregation and increased blood viscosity. Low molecular weight dextrans, although shown to improve circulation and promote flow, are removed rather rapidly from the circulation due to high premeation rates across capillary walls. In the present study, a small anionic charge is introduced onto the dextran to make it electrostatically negative. Since capillary walls have been shown to retain negatively charged solutes in preference to neutral solutes, the anionic dextran should retain its effectiveness for longer periods of time compared to similar sized neutral dextran. Studies were done on eight unanaesthetized dogs to compare the relative disappearance rates of dextran and anionic dextran (carboxymethyl dextran) from the circulation. It was shown that anionic dextrans do remain in the circulation over a longer period of time compared to neutral dextrans.

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.

Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity.

The atrophy of extracellular matrix is a common event during the aging of connective tissues. In this study, we tested the hypothesis that the altered ability of senescent cells to be modulated by serum growth factors correlated with a loss of regulation of collagenase synthesis. We examined the levels of immunoreactive procollagenase and collagenase inhibitor (the tissue inhibitor of metalloproteinases, TIMP) associated with young and senescent fibroblasts cultured in vitro. Young fibroblasts cultured in low (0.5%) concentrations of fetal bovine serum respond to increased (10%) serum by increasing levels of procollagenase and TIMP beginning 4.0 h after serum stimulation. In contrast, senescent fibroblasts constitutively produce relatively high levels of procollagenase even when cultured in low levels of serum and do not respond to serum stimulation by increasing procollagenase synthesis. In addition, senescent fibroblasts constitutively express a relatively small amount of TIMP which is not induced upon serum stimulation. This altered expression of collagenase and TIMP appears unique to the senescent phenotype and not merely a result of growth inhibition, since young cells growth arrested by density-dependent growth inhibition displayed a temporal pattern of procollagenase and TIMP expression upon serum stimulation similar to that of subconfluent young cultures. An assay of net collagenase activity revealed a greater than 20-fold elevation of activity in trypsin-activated extracts from senescent versus young fibroblasts when cultured in a low concentration of fetal bovine serum. These results demonstrate for the first time a direct correlation between alterations in the molecular pathways regulating connective tissue homeostasis and those of replicative senescence. The increased collagenolytic activity of senescent compared to young fibroblasts cultured in the presence of a low serum concentration suggests that aging fibroblasts may become increasingly fibroclastic causing many of the age-associated alterations in dermal collagen observed during aging in vivo.

Characterization of the serological cross-reactivity between glycoproteins of the human immunodeficiency virus and equine infectious anaemia virus.

The reported serological relatedness between the major glycoproteins of human immunodeficiency virus (HIV gp120) and equine infectious anaemia virus (EIAV gp90) was examined using purified antigens in radioimmunoprecipitation (RIP), radioimmunoassay (RIA) and immunoblot assays with reference serum from acquired immunodeficiency syndrome (AIDS) patients, an anti-gp120 goat serum and EIAV-infected horse serum. To assess the contributions of glycoprotein oligosaccharide and peptide components to any observed reactivities, antigens treated with endoglycosidase F to remove carbohydrate were assayed in parallel with the intact glycoprotein. The results of the experiments indicated that the reactivity observed for each antigen was dependent on the immunoassay employed. The RIP and RIA analyses demonstrated that HIV gp120 is equally reactive with the AIDS patient serum, the goat anti-gp120 serum and the EIAV-infected horse serum, whereas the EIAV gp90 reacted only with the horse serum. In immunoblot assays, the HIV gp120 reacted with AIDS patient serum, but not with the EIAV-infected horse serum. Deglycosylation of the HIV gp120 evidently increased its reactivity with the AIDS patient serum, had no significant effect on its reactivity with the goat antiserum, and essentially abolished its reactivity with the EIAV reference serum. Thus, it appears that the serological cross-reactivity observed between HIV gp120 and sera from EIAV-infected horses can be attributed to the oligosaccharide rather than the peptide components of the viral glycoprotein. These studies also emphasize the necessity of employing several assay procedures in assessing lentivirus antigenicity.