Scott F. Deamond

Longitudinal study of in vivo wound repair and in vitro cellular senescence of dermal fibroblasts

A longitudinal study was performed to confirm the inverse relationship between in vivo age and in vitro proliferative capacity previously observed in a cross-sectional study, and to investigate the relationship between the growth of dermal fibroblasts in vitro and a physiological function (i.e., wound repair) that is known to decline with age in vivo. Fibroblast cultures were generated from skin punch biopsies from 12 male hamsters beginning at 1 month of age and at 6-months interval thereafter until the natural death of the animal. All cultures from all individuals exhibited finite proliferative capacity, and an inverse relationship was observed between donor age and maximum in vitro proliferative capacity. In addition, a direct correlation between the in vitro proliferative capacity of the dermal fibroblasts in vitro and the repair efficiency of the biopsy site was observed. However, these changes in the in vitro proliferative capacity and in vivo wound repair efficiency were not progressive beyond 12-18 months of age and were not indicative at any age of an individuals ultimate lifespan. This study provides evidence that in vitro proliferative capacity of dermal fibroblasts and in vivo wound repair may be comparable phenomena that share a common mechanism. However, the nonprogressive nature and the lack of correlation between these phenomena and the individuals ultimate lifespan indicate that their use as biological markers of aging is limited to animals younger than the mean lifespan of the species.

In vitro senescence of Syrian hamster mesenchymal cells of fetal to aged adult origin. Inverse relationship between in vivo donor age and in vitro proliferative capacity

Normal diploid Syrian hamster dermal mesenchymal cell strains, regardless of the age of the tissue of origin, exhibit in vitro cellular senescence. The frequency of spontaneous escape from senescence and conversion to a permanent cell line is less than 5% among replicate flasks. The overall pattern of senescence of cells of fetal, neonatal, young adult (6 months) and aged adult (24 months) origin is similar in terms of the morphological changes and proliferative changes indicated by the reduction of saturation density, cloning efficiency and [3H]thymidine labeling index and by the increase in population doubling time and cell volume. However, the average maximum cumulative population doubling level is characteristic for each cell type: 13-day gestation fetal cells, 28.6; neonatal cells, 18.7; young adult cells, 13.8; aged adult cells, 11.1. Thus, the in vitro proliferative capacity of Syrian hamster mesenchymal cells is inversely related to the in vivo age of the donor.

Intrabody tissue-specific delivery of antisense conjugates in animals: ligand-linker-antisense oligomer conjugates.

Publisher Summary The selective inhibition of gene expression through specific oligonucleotide binding to key mRNA target sequences is the goal of antisense strategies. However, from the beginning, antisense strategies have faced several obstacles, such as in vivo stability of the oligonucleotide, cellular uptake, efficiency of hybridization of the antisense agent to the mRNA target, selectivity of oligonucleotide binding, and inhibition of gene expression. Of these factors, the low and nonselective cellular uptake of antisense oligonucleotides has hindered its therapeutic usefulness most severely. A versatile method to specifically and efficiently deliver an antisense oligonucleotide to the intracellular medium of a particular cell within a single organ would have a pronounced effect on the realization of effective antisense therapies. This chapter describes an approach for the ligand-directed delivery of antisense oligonucleotides. Paramount to this approach is the design and synthesis of a molecular scaffold symbolized as “A-L-P” with “A” representing a unique ligand, specific for a receptor on the surface of the target cell. The “P” represents the “payload” portion, typically an oligonucleotide, which is linked uniquely to the ligand through the linker (the “L” portion). The methods describe A-L-P neoglycoconjugate formation, the cellular uptake of these ligand-linker-oligonucleotide conjugates via receptor-mediated endocytosis, their in vivo biodistribution, and their effectiveness toward the inhibition of expression of an integrated hepatitis B virus (HBV).

Age-related differences in promoter-induced extension of in vitro proliferative life span of Syrian hamster fibroblasts

The proliferative capacity of Syrian hamster dermal fibroblasts has been previously shown to be inversely related to the age of the donor (Mech. Ageing and Dev., 34 (1986) 151). The present study demonstrates an inverse correlation between in vivo age and the in vitro morphological and proliferative response of Syrian hamster dermal fibroblasts to the tumor promoter phorbol-12,13-didecanoate. Treatment of fetal fibroblasts with promoter increased the proliferative life span of the cultures by approximately 2-fold, but did not increase the frequency of conversion to established cell lines. Neonatal and young adult fibroblasts exhibited intermediate responses to promoter treatment, showing 54.9% and 33.1% extension, respectively. In contrast, promoter treatment had no significant effect on aged adult fibroblasts. Maximal extension required continual treatment beginning in primary culture or at passage 1. Promoter-induced extension of proliferative life span appears to be mediated through the prolonged maintenance of small, highly proliferative cells that are present in primary cultures of these cells.

NMR relaxation study of water protons in Syrian hamster fetal cells at 300 MHz

TheT1 andT2 relaxation times of water protons in two cell types in culture derived from Syrian hamster fetuses (normal primary or secondary fetal cells vs BP6T tumor cells derived from the normal cells transformed by carcinogens) were measured at 7.05 Tesla magnetic field (proton frequency =300 MHz). TheT1/T2 ratios and the correlation time, τc, calculated from theT1/T2 ratio of cellular water protons, are significantly different in these two fibroblastic cell types of the same biological origin and with similar morphologies and growth rates in culture.

Longevity and age-related pathology of LVG outbred golden syrian hamsters (Mesocricetus auratus)

A colony of male Lakeview Golden (LVG) Syrian hamsters has been maintained for the last nine years as a source of various tissues for cellular aging studies. Observations on this colony also yielded data on survival time and physical and pathological manifestations of aging in this strain. Based on 150 spontaneous deaths, the median life span was found to be 19.5 months. The maximum life span was 36 months and the minimum 6 months. A cross-sectional pathological survey of sacrificed and spontaneously dying members of the population revealed a low rate of neoplasia and a variety of degenerative lesions that increased with age. These observations of a varied pathology and a low frequency of neoplasia provide justification for the continued development of the male LVG Syrian hamster as an animal model system for use in studies on the mechanism of both in vivo and in vitro aging.

High-frequency 1H NMR studies of the effects of growth factors and phorbol esters on normal Syrian hamster diploid fibroblast cells.

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T1), and spin-spin (T2) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T1 values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T2 values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T1 and T2 were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T2 values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted in smaller T2 values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T2 values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.

Studies of the T1 and T2 of intracellular water as a function of frequency in normal and transformed fetal cells

Frequency-dependent values of the spin-lattice relaxation time (T1) and the spin-spin relaxation time (T2) have been obtained for intracellular water in normal and transformed Syrian hamster fetal fibroblasts. Values of T1 and T2 were obtained for normal and transformed cells at 24.3 (0.57 T), 100 (2.4 T), 300 (7.0 T), and 400 MHz (9.4 T). At each frequency, values of T1 were the same for both normal and transformed cells, whereas values of T2 were lower for one passage of transformed cells. As expected, T1 increased with frequency. However, T2 decreased with frequency for both normal and transformed cells. The frequency dependence of T2, was similar for all cells; thus, the ability of T2 to make a distinction between normal and transformed cells did not change with field.