Metformin improves healthspan and lifespan in mice.

Metformin is a drug commonly prescribed to treat patients with type 2 diabetes. Here we show that long-term treatment with metformin (0.1% w/w in diet) starting at middle age extends healthspan and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with metformin mimics some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced low-density lipoprotein and cholesterol levels without a decrease in caloric intake. At a molecular level, metformin increases AMP-activated protein kinase activity and increases antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation. Our results indicate that these actions may contribute to the beneficial effects of metformin on healthspan and lifespan. These findings are in agreement with current epidemiological data and raise the possibility of metformin-based interventions to promote healthy aging.

Age-related retention of fiber cell nuclei and nuclear fragments in the lens cortices of multiple species.

PURPOSE: To determine the differences between species in the retention of lens fiber cell nuclei and nuclear fragments in the aging lens cortex and the relationship of nuclear retention to lens opacity. For this purpose old human, monkey, dog, and rat lenses were compared to those of three strains of mouse. We also investigated possible mechanisms leading to nuclear retention. METHODS: Fixed specimens of the species referred to above were obtained from immediate on site sacrifice of mice and rats, or from recently fixed lenses of other species, dogs, monkeys, and humans, obtained from collaborators. The retention of undegraded nuclei and nuclear fragments was graded 1-4 from histologic observation. All species lenses were examined microscopically in fixed sections stained with hematoxylin and eosin (H&E) or 4',6-diamidino-2-phenylindole (DAPI). Slit lamp observations were made only on the mice and rats before sacrifice and lens fixation. Values of 0 to 4 (clear lens to cataract) were given to degree of opacity. MRNA content in young versus old C57BL/6 mouse lenses was determined by quantitative PCR (qPCR) for DNase II-like acid DNase (DLAD) and other proteins. DLAD protein was determined by immunofluorescence of fixed eye sections. RESULTS: In old C57BL/6 and DBA mice and, to a lesser degree, in old CBA mice and old Brown Norway (BN) rats lenses were seen to contain a greatly expanded pool of unresolved whole nuclei or fragments of nuclei in differentiating lens fiber cells. This generally correlated with increased slit lamp opacities in these mice. Most old dog lenses also had an increase in retained cortical nuclei, as did a few old humans. However, a second rat strain, BNF1, in which opacity was quite high had no increase in retained nuclei with age nor did any of the old monkeys, indicating that retained nuclei could not be a cause of opacity in these animals. The nuclei and nuclear fragments were located at all levels in the outer cortex extending inward from the lens equator and were observable by the DAPI. These nuclei and nuclear fragments were seen from 12 months onward in all C57BL/6 and DBA/2 mice and to a lesser degree in the CBA, increasing in number and in space occupancy with increasing age. Preliminary results suggest that retention of nuclei in the C57BL/6 mouse is correlated with an age-related loss of DLAD from old lenses. CONCLUSIONS: A very marked apparently light refractive condition caused by retained cortical nuclei and nuclear fragments is present in the lens cortices, increasing with age in the three strains of mice examined and in one of two strains of rats (BN). This condition was also seen in some old dogs and a few old humans. It may be caused by an age-related loss of DLAD, which is essential for nuclear DNA degradation in the lens. However, this condition does not develop in old BNF1 rats, or old monkeys and is only seen sporadically in humans. Thus, it can not be a universal cause for age related lens opacity or cataract presence, although it develops concurrently with opacity in mice. This phenomenon should be considered when using the old mouse as a model for human age-related cataract.

Age-related cataract in dogs: a biomarker for life span and its relation to body size.

Clinical data from 72 dog breeds of varying size and life expectancy were grouped according to breed body mass and tested for prevalence at ages 4 to 5, ages 7 to 10, and lifetime incidence of non-hereditary, age-related cataract (ARC). The incidence of ARC was found to be directly related to the relative life expectancies in the breed groups: The smallest dog breeds had a lower ARC prevalence between ages 4 and 5 than mid-size breeds and these, in turn, a lower prevalence than the giant breeds. A similar sequence was evident for ages 7 to 10 and for overall lifetime incidence of ARC. These differences became more significant when comparing small and giant breeds only. We could also confirm the inverse relationship between body size and life expectancy in these same sets of dog breeds. Our results show that body size, life expectancy, and ARC incidence are interrelated in dogs. Given that ARC has been shown to be at least partially caused by oxidative damage to lens epithelial cells and the internal lens, we suggest that it can be considered not only as a general biomarker for life expectancy in the canine and possibly other species, but also for the systemic damages produced by reactive oxygen species. This suggests new approaches to examine the gene expression pathways affecting the above-noted linkages.

X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts.

PURPOSE: To compare age-related cataractous (ARC) changes in unirradiated mice lenses to those induced by head-only X-irradiation of 3 month-old mice. METHODS: lens epithelial cells (LECs) as well as partially degraded cortical DNA were visualized in fixed sections using 4',6-diamidino-2-phenylindole (DAPI) staining, and in fresh lenses using the vital stain Hoechst 33342. reactive oxygen species (ROS) activity was also visualized directly in fresh lenses using the vital dye Dihydrorhodamine (DHR). In fixed lenses an antibody specific for 8-OH Guanosine (8-OH-G) lesions was used to visualize DNA oxidative adducts from ROS damage. Alpha smooth muscle actin was visualized using specific antibodies to determine if myofibroblasts were present. Fluorescence was quantified using Laser Scanning Confocal Microscopy (LSCM). The degree of lens opacity and cataract formation was determined by slit lamp, or from digitalized images of light reflections taken with a low magnification light microscope. RESULTS: Using DNA- and ROS-specific vital fluorescent dyes, and laser scanning confocal microscopy we have previously described 4 changes in the aging rodent lenses: 1) a significantly decreased density of surface LECs in lenses from old compared to younger mice and rats; 2) a very large increase in retained cortical nuclei and DNA fragments in the secondary lens fibers of old rodent lenses; 3) increased cortical ROS in old rodent lenses; 4) increased cataract concomitantly with the cortical DNA and ROS increases. In the current study we report that these same 4 changes also occur in an accelerated fashion in mice given head-only X-irradiation at 3 months of age. In addition to vital staining of fresh lenses, we also examined sections from fixed eyes stained with DAPI or hematoxylin and eosin (H&E) and found the same loss of surface LECs and accumulation of undigested nuclei and debris in secondary lens fibers occur with age or following X-irradiation. In addition sections from fixed-eyes were examined for ROS damage to DNA with antibodies specific for 8-OH-G lesions. The frequency of 8-OH-G lesions increased dramatically in lenses from old unirradiated mice over 24 months of age, and similarly in X-irradiated lenses by 9-11 months post irradiation. The accumulation of cortical nuclei was not the result of conversion or invasion by myofibroblasts as tested by antibodies to a marker for such cells, alpha smooth muscle actin. CONCLUSIONS: X-irradiation damage induces a large decrease in surface LECs over a period of 3-11 months post X-irradiation of young mice. These changes are similar in extent to those seen in 24-29 months-old control mouse lenses with age-related cataracts. In 24+ month-old unirradiated mice the secondary lens fibers are not able to degrade nuclei or nuclear DNA efficiently and accumulate large numbers of cortical nuclei and nuclear fragments as well as ROS and 8-OHG lesions. X-irradiated lenses develop the same abnormalities in a more accelerated fashion. The extensive loss of LECS and accumulation of undegraded nuclei, ROS, and ROS damage may play a causal role in cataract generation in both unirradiated old mice and in previously irradiated young adult mice.

Disruption of protein kinase A in mice enhances healthy aging.

Mutations that cause a reduction in protein kinase A (PKA) activity have been shown to extend lifespan in yeast. Loss of function of mammalian RIIbeta, a regulatory subunit of PKA expressed in brain and adipose tissue, results in mice that are lean and insulin sensitive. It was therefore hypothesized that RIIB null (RIIbeta(-/-)) mice would express anti-aging phenotypes. We conducted lifespan studies using 40 mutant and 40 wild type (WT) littermates of equal gender numbers and found that both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the 80% lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). There was no difference in either median or 80% lifespan in female genotypes. WT mice of both genders became increasingly obese with age, while mutant mice maintained their lean phenotype into old age. Adiposity was found to correlate with lifespan for males only. 50% of male mice between 30 and 35 g, corresponding to about 5% body fat, for either genotype lived over 1000 days. No male mouse outside of this weight range achieved this lifespan. During their last month of life, WT mice began losing weight (a total of 8% and 15% of body weight was lost for males and females, respectively), but RIIbeta(-/-) male mice maintained their lean body mass to end of life. This attenuation of decline was not seen in female mutant mice. Old male mutant mice were insulin sensitive throughout their life. Both genders showed modestly lower blood glucose levels in old mutants compared to WT. Male mutants were also resistant to age-induced fatty liver. Pathological assessment of tissues from end of life male mutant mice showed a decrease in tumor incidence, decreased severity of renal lesions, and a trend towards a decrease in age-related cardiac pathology. These findings help establish the highly conserved nature of PKA and suggest that disruption of PKA affects physiological mechanisms known to be associated with healthy aging.

Resveratrol delays age-related deterioration and mimics transcriptional aspects of dietary restriction without extending life span.

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.

Radiation cataracts: mechanisms involved in their long delayed occurrence but then rapid progression.

PURPOSE: This study was directed to assess the DNA damage and DNA repair response to X-ray inflicted lens oxidative damage and to investigate the subsequent changes in lens epithelial cell (LEC) behavior in vivo that led to long delayed but then rapidly developing cataracts. METHODS: Two-month-old C57Bl/6 female mice received 11 Grays (Gy) of soft x-irradiation to the head only. The animals' eyes were examined for cataract status in 30 day intervals by slit lamp over an 11 month period post-irradiation. LEC migration, DNA fragment, free DNA retention, and reactive oxygen species (ROS) presence were established in the living lenses with fluorescent dyes using laser scanning confocal microscopy (LSCM). The extent and removal of initial LEC DNA damage were determined by comet assay. Immunohistochemistry was used to determine the presence of oxidized DNA and the response of a DNA repair protein in the lenses. RESULTS: This treatment resulted in advanced cortical cataracts that developed 5-11 months post-irradiation but then appeared suddenly within a 30 day period. The initially incurred DNA strand breaks were repaired within 30 min, but DNA damage remained as shown 72 h post-irradiation by the presence of the DNA adduct, 8-hydroxyguanosine (8-OHG), and a DNA repair protein, XRCC1. This was followed months later by abnormal behavior by LEC descendant cells with abnormal differentiation and migration patterns as seen with LSCM and fluorescent dyes. CONCLUSIONS: The sudden development of cortical cataracts several months post-irradiation coupled with the above findings suggests an accumulation of damaged descendants from the initially x-irradiated LECs. As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O(2) with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins. The events seen in this study indicate the retention and transmission of progenitor cell DNA damage in descendant LEC. The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.

Reduction in glutathione peroxidase 4 increases life span through increased sensitivity to apoptosis.

Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme that plays an important role in detoxification of oxidative damage to membrane lipids. Because oxidative stress is proposed to play a causal role in aging, we compared the life spans of Gpx4 heterozygous knockout mice (Gpx4(+/-) mice) and wild-type mice (WT mice). To our surprise, the median life span of Gpx4(+/-) mice (1029 days) was significantly longer than that of WT mice (963 days) even though the expression of Gpx4 was reduced approximately 50% in all tissues of Gpx4(+/-) mice. Pathological analysis revealed that Gpx4(+/-) mice showed a delayed occurrence of fatal tumor lymphoma and a reduced severity of glomerulonephritis. Compared to WT mice, Gpx4(+/-) mice showed significantly increased sensitivity to oxidative stress-induced apoptosis. Our data indicate that lifelong reduction in Gpx4 increased life span and reduced/retarded age-related pathology most likely through alterations in sensitivity of tissues to apoptosis.

Reduced age-related cataracts among elderly persons who reach age 90 with preserved cognition: a biomarker of successful aging?

Tissue damage due to oxidative stress has been implicated in aging, memory loss, and cataract formation. We hypothesized that persons who achieved exceptional longevity with preserved cognition (successful aging [SAG]) would exhibit a lower rate of age-related cataract (ARC) than the general population. The age-specific rates of ARC for a group of 100 (50 male, 50 female) elderly persons who reached at least age 90 years with preserved cognition were compared to the corresponding rates of ARC reported in five population-based studies. The principal finding of this report was that the SAG group manifested a significant reduction in the age-specific rate and lifetime cumulative incidence of ARC compared to the general population. Steroid use, alcohol consumption, gout, and skin lesions resulting from excessive sun exposure emerged as risk factors. Our findings suggest that the progressive development of lens opacities may be reflective of degenerative events occurring more generally throughout the body.

Cellular debris and ROS in age-related cortical cataract are caused by inappropriate involution of the surface epithelial cells into the lens cortex.

PURPOSE: To quantify changes in the lens epithelial cells and underlying lens cortex responsible for age-related cortical cataract (ARCC) in the rat. METHODS: Freshly isolated lenses were stained vitally for DNA with Hoechst 33342. Reactive oxygen species (ROS) and mitochondria were visualized and quantified by dihydrorhodamine 123 (DHR). The fluorescence was quantified using Laser Scanning Confocal Microscopy (LSCM) of vitally stained lenses. Cortical DNA was verified as such by DNAse I digestion. Cataract reflections were determined from digitalized images of light reflections taken with a low magnification light microscope, or with the LSCM. RESULTS: The anterior surface epithelia of old rat lenses were full of gaps and ragged in appearance with a decrease of over 50% in lens epithelial cell (LEC) density. The surface LECs were frequently seen to have involuted into the cortex at inappropriate sites, forming deposits full of DNA, nuclear and mitochondrial debris, and abundant ROS. These involutions frequently originated near open gaps in the surface epithelia, where they appear to have detached from the capsular membrane. Cortical cataracts in the rat lenses were seen to co-localize with these LEC involutions, as had been seen previously in mice with ARCC. CONCLUSIONS: ARCC in rats co-localized with inappropriate accumulations of nuclei, mitochondria, DNA, and expression of ROS in debris filled foci. These were the result of both involution of surface LECs into areas of cortical ARCC, and by an extension of the normal bow region deep into the anterior and posterior of cataractous lenses. These results were in complete agreement with our previous studies on ARCC in mice.

Accumulation of DNA, nuclear and mitochondrial debris, and ROS at sites of age-related cortical cataract in mice.

PURPOSE: Lenses from young and old mice were analyzed by laser scanning confocal microscopy (LSCM) with vital dyes, to determine whether age-related subcapsular and cortical cataracts were linked to the failure of lens fiber cells to degrade nuclei, DNA, and mitochondria properly and whether they result in the overproduction of reactive oxygen species (ROS) at the same sites. RESULTS: As opposed to the clear DNA-free subcapsular and cortical areas of young adult mouse lenses, these areas in cataractous old mouse lenses were found to contain accumulations of nuclei, nuclear fragments, aggregated mitochondria, and amorphous DNA as cortical inclusions (P < 0.001 between young and old lenses). These inclusions correlated spatially with age-related cataracts and with the presence of ROS. The source of such undegraded material was a large expansion of transition nuclei in the bow region and also direct involution of surface lens epithelial cells (LECs) into the underlying cortex, frequently leaving bare patches devoid of nuclei on the surface of the anterior epithelium. METHODS: Live lenses were stained vitally for DNA with Hoechst 33342. ROS and mitochondria were stained and quantified with dihydrorhodamine 123 (DHR). In fixed lenses, DNA was stained with propidium iodide (PI) or 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI). The intensity and position of each probe's fluorescence was determined by LSCM. Cataract localization was ascertained by digitalized microscopy of reflected light. CONCLUSIONS: In aged mice, most subcapsular and cortical cataracts colocalize with accumulations of nuclei, mitochondria, and DNA, These effects are accompanied at the same sites by the production of ROS. The condition is due to the failure of lens fiber cells in the bow region to differentiate properly into the clear fiber state and to the improper involution of cells from the anterior epithelium directly into the underlying cortex, resulting in cataractous opacities.

Age-related cataract progression in five mouse models for anti-oxidant protection or hormonal influence.

Five mouse models with known alterations of resistance to oxidative damage were compared by slit lamp examination for the presence and degree of advancement of age-related cataract in young adult and old animals along with wild type controls. A group of young and old normal C57BL/6Jax mice were examined first to constitute a standard, and they were found to exhibit age-related cataract development. Following this, four models on the C57BL/6 background with imposed genetic alterations affecting anti-oxidant enzyme presence or activity, and one outbred model in which a deletion blocked the growth hormone/IGF-1 axis, were similarly examined. There was no evidence of foetal or juvenile cataract development in any of these models, and an age-related severity for lens opacities was shown between young adult and old mice in all groups. Model 1, mice null for the anti-oxidant gene glutathione peroxidase-1 (GPX1) had significantly advanced cataracts in older mice vs. same age controls. In mouse model 2 hemizygous knockout of SOD2 (MnSOD) did not affect age-related cataract development. In model 3 combining the GPX1 and SOD2 deficiencies in the same animal did not advance cataract development beyond that of the GPX1 null alone. In model 4 the addition of anti-oxidant protection in the lens by transfection of human catalase targeted only to the mitochondria resulted in a significant delay in cataract development. The 5th model, growth hormone receptor knockout (GHR-/-) mice, also demonstrated a significant reduction in age-related cataract development, as well as dwarfism. These findings, in general, support the oxidative theory of age-related cataract development. The exception, the partial deletion of SOD2 in the hemizygous KO model, probably did not represent a sufficiently severe deprivation of anti-oxidant protection to produce pathologic changes in the lens.

Extension of murine life span by overexpression of catalase targeted to mitochondria.

To determine the role of reactive oxygen species in mammalian longevity, we generated transgenic mice that overexpress human catalase localized to the peroxisome, the nucleus, or mitochondria (MCAT). Median and maximum life spans were maximally increased (averages of 5 months and 5.5 months, respectively) in MCAT animals. Cardiac pathology and cataract development were delayed, oxidative damage was reduced, H2O2 production and H2O2-induced aconitase inactivation were attenuated, and the development of mitochondrial deletions was reduced. These results support the free radical theory of aging and reinforce the importance of mitochondria as a source of these radicals.

Mice transgenic for Alzheimer disease beta-amyloid develop lens cataracts that are rescued by antioxidant treatment.

Alzheimer disease is characterized by cerebral Abeta deposition, which we have recently discovered occurs also in the lens as cataracts in Alzheimer disease patients. Here we report the presence of significantly increased cataracts in the lenses of an Abeta-transgenic mouse model for Alzheimer disease and their amelioration upon treatment with EUK-189, a synthetic SOD/catalase mimetic. These data support an oxidative etiology for AD-associated lens cataracts and their potential to be treated preventatively with antioxidants.

Biomarkers of aging: from primitive organisms to humans.

Leading biologists and clinicians interested in aging convened to discuss biomarkers of aging. The goals were to come to a consensus, construct an agenda for future research, and make appropriate recommendations to policy makers and the public-at-large. While there was not total agreement on all issues, they addressed a number of questions, among them whether biomarkers can be identified and used to measure the physiological age of any individual within a population, given emerging information about aging and new technological advances. The hurdles to establishing informative biomarkers include the biological variation between individuals that makes generalizations difficult; the overlapping of aging and disease processes; uncertainty regarding benign versus pathogenic age-related changes; the point at which a process begins to do damage to the organism, and, if so, when does it occur; and when to distinguish critical damage from noncritical damage. Finally, and significantly, it is difficult to obtain funding for this research.

Quantitative trait locus mapping for age-related cataract severity and synechia prevalence using four-way cross mice.

PURPOSE: The goal of this study was to map mouse quantitative trait loci (QTL) that influence the development of murine age-related cataract and synechia, by using a genetically heterogeneous mouse population bred by a four-way cross. METHODS: The test population consisted initially of 510 mice bred as the progeny of (BALB/cJ x C57BL/6J)F1 females and (C3H/HeJ x DBA/2J)F1 males. Each mouse was examined by slit lamp at 18 and 24 months of age and scored for degree of lens opacity on a 0 to 4+ scale, and the presence or absence of additional anterior chamber disease was noted. The presence of synechia was confirmed by histology. Each mouse was genotyped at 96 maternal and 92 paternal loci, and the significance of association between genotype and eye lesions was tested by permutation analysis. RESULTS: Significant QTL with effects on lens opacity at 24 months were detected on mouse chromosomes 4, 11, and 12. The effects were additive, and severe cataracts were seen in 80% of the mice with all three high-risk alleles, but in only 28% of the mice with all three low-risk alleles. The risk of synechia was associated with paternal chromosome 1 and on both the maternal and paternally inherited chromosome 4. Mice with all three high-risk alleles had a 68% risk of synechia, compared with a 0% incidence in mice with all three counteralleles. CONCLUSIONS: A four-way cross population of mice can be used to map polymorphic loci that influence cataract severity and synechia prevalence in late life. The results provide a first step toward identification of the individual genes involved and may help to guide the search for homologous human genes.

Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging.

Mice heterozygous for the Sod2 gene (Sod2+/- mice) have been used to study the phenotype of life-long reduced Mn-superoxide dismutase (MnSOD) activity. The Sod2+/- mice have reduced MnSOD activity (50%) in all tissues throughout life. The Sod2+/- mice have increased oxidative damage as demonstrated by significantly elevated levels of 8-oxo-2-deoxyguanosine (8oxodG) in nuclear DNA in all tissues of Sod2+/- mice studied. The levels of 8oxodG in nuclear DNA increased with age in all tissues of Sod2+/- and wild-type (WT) mice, and at 26 mo of age, the levels of 8oxodG in nuclear DNA were significantly higher (from 15% in heart to over 60% in liver) in the Sod2+/- mice compared with WT mice. The level of 8oxodG was also higher in mitochondrial DNA isolated from liver and brain in Sod2+/- mice compared with WT mice. The increased oxidative damage to DNA in the Sod2+/- mice is associated with a 100% increase in tumor incidence (the number of mice with tumors) in old Sod2+/- mice compared with the old WT mice. However, the life spans (mean and maximum survival) of the Sod2+/- and WT mice were identical. In addition, biomarkers of aging, such as cataract formation, immune response, and formation of glycoxidation products carboxymethyl lysine and pentosidine in skin collagen changed with age to the same extent in both WT and Sod2+/- mice. Thus life-long reduction of MnSOD activity leads to increased levels of oxidative damage to DNA and increased cancer incidence but does not appear to affect aging.

Intraclonal plasticity for bone, smooth muscle, and adipocyte lineages in bone marrow stroma fibroblastoid cells.

Bone marrow stroma fibroblastoid cells (BMSFC) develop from a single clone of cells within each of the in vitro fibroblastoid colonies (CFU-F) derived from either murine or human bone marrow. All of the clones represented by these colonies displayed antigenic and product markers for osteoblast, smooth muscle, and adipocyte lineages when tested separately for each marker. Separate sets of fibroblastoid colonies derived from the same individual donor's culture tested positive with antibodies specific for smooth muscle-specific heavy chain myosin (SMMHC), smooth muscle alpha actin-1, bone sialoprotein, osteocalcin, or alkaline phosphatase, and developed von Kossa-positive deposits shown by X-ray microanalysis and electron diffraction to be hydroxyapatite. Individual cells were positive for both SMMHC and osteocalcin. All cells in the multiple clones tested were capable of metabolizing a fatty acid to form intracellular lipid droplets. PCR transcripts obtained from the human cell cultures that provided these BMSFC clones were consistent with the immunocytochemical findings. Transcripts for PPAR (gamma)-2 and Cbfa-1 were dependent upon the culture medium content, suggesting an osteoblast/adipocyte differentiation switch point. Cell lineage specificity for markers and RNA transcripts was determined by comparison to skin fibroblast controls. These findings demonstrate a high degree of interlineage plasticity in vitro for BMSFC.