Clones of aging: When better fitness can be dangerous.

The biological and clinical significance of aberrant clonal expansions in aged tissues is being intensely discussed. Evidence is accruing that these clones often result from the normal dynamics of cell turnover in our tissues. The aged tissue microenvironment is prone to favour the emergence of specific clones with higher fitness partly because of an overall decline in cell intrinsic regenerative potential of surrounding counterparts. Thus, expanding clones in aged tissues need not to be mechanistically associated with the development of cancer, albeit this is a possibility. We suggest that growth pattern is a critical phenotypic attribute that impacts on the fate of such clonal proliferations. The acquisition of a better proliferative fitness, coupled with a defect in tissue pattern formation, could represent a dangerous mix setting the stage for their evolution towards neoplasia.

A comparative analysis on serious adverse events reported for COVID-19 vaccines in adolescents and young adults.

This study aims to assess the safety profile of COVID-19 vaccines (mRNA and viral vector vaccines) in teenagers and young adults, as compared to Influenza and HPV vaccines, and to early data from Monkeypox vaccination in United States. Methods: We downloaded data from the Vaccine Adverse Event Reporting System (VAERS) and collected the following Serious Adverse Events (SAEs) reported for COVID-19, Influenza, HPV and Monkeypox vaccines: deaths, life-threatening illnesses, disabilities, hospitalizations. We restricted our analysis to the age groups 12-17 and 18-49, and to the periods December 2020 to July 2022 for COVID-19 vaccines, 2010-2019 for Influenza vaccines, 2006-2019 for HPV vaccines, June 1, 2022 to November 15, 2022 for Monkeypox vaccine. Rates were calculated in each age and sex group, based on an estimation of the number of administered doses. Results: Among adolescents the total number of reported SAEs per million doses for, respectively, COVID-19, Influenza and HPV vaccines were 60.73, 2.96, 14.62. Among young adults the reported SAEs rates for, respectively, COVID-19, Influenza, Monkeypox vaccines were 101.91, 5.35, 11.14. Overall, the rates of reported SAEs were significantly higher for COVID-19, resulting in a rate 19.60-fold higher than Influenza vaccines (95% C.I. 18.80-20.44), 4.15-fold higher than HPV vaccines (95% C.I. 3.91-4.41) and 7.89-fold higher than Monkeypox vaccine (95% C.I. 3.95-15.78). Similar trends were observed in teenagers and young adults with higher Relative Risks for male adolescents. Conclusion: The study identified a risk of SAEs following COVID-19 vaccination which was markedly higher compared to Influenza vaccination and substantially higher compared to HPV vaccination, both for teenagers and young adults, with an increased risk for the male adolescents group. Initial, early data for Monkeypox vaccination point to significantly lower rates of reported SAEs compared to those for COVID-19 vaccines. In conclusion these results stress the need of further studies to explore the bases for the above differences and the importance of accurate harm-benefit analyses, especially for adolescent males, to inform the COVID-19 vaccination campaign.

Reuniting philosophy and science to advance cancer research.

Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer.

Cell competition, cooperation, and cancer.

Complex multicellular organisms require quantitative and qualitative assessments on each of their constitutive cell types to ensure coordinated and cooperative behavior towards overall functional proficiency. Cell competition represents one of the operating arms of such quality control mechanisms and relies on fitness comparison among individual cells. However, what is exactly included in the fitness equation for each cell type is still uncertain. Evidence will be discussed to suggest that the ability of the cell to integrate and collaborate within the organismal community represents an integral part of the best fitness phenotype. Thus, under normal conditions, cell competition will select against the emergence of altered cells with disruptive behavior towards tissue integrity and/or tissue pattern formation. On the other hand, the winner phenotype prevailing as a result of cell competition does not entail, by itself, any degree of growth autonomy. While cell competition per se should not be considered as a biological driving force towards the emergence of the neoplastic phenotype, it is possible that the molecular machinery involved in the winner/loser interaction could be hijacked by evolving cancer cell populations.

Aging and Cancer: The Waning of Community Bonds.

Cancer often arises in the context of an altered tissue landscape. We argue that a major contribution of aging towards increasing the risk of neoplastic disease is conveyed through effects on the microenvironment. It is now firmly established that aged tissues are prone to develop clones of altered cells, most of which are compatible with a normal histological appearance. Such increased clonogenic potential results in part from a generalized decrease in proliferative fitness, favoring the emergence of more competitive variant clones. However, specific cellular genotypes can emerge with reduced cooperative and integrative capacity, leading to disruption of tissue architecture and paving the way towards progression to overt neoplastic phenotypes.

Cell competition in liver carcinogenesis.

Cell competition is now a well-established quality control strategy to optimize cell and tissue fitness in multicellular organisms. While pursuing this goal, it is also effective in selecting against altered/defective cells with putative (pre)-neoplastic potential, thereby edging the risk of cancer development. The flip side of the coin is that the molecular machinery driving cell competition can also be co-opted by neoplastic cell populations to expand unchecked, outside the boundaries of tissue homeostatic control. This review will focus on information that begins to emerge regarding the role of cell competition in liver physiology and pathology. Liver repopulation by normal transplanted hepatocytes is an interesting field of investigation in this regard. The biological coordinates of this process share many features suggesting that cell competition is a driving force for the clearance of endogenous damaged hepatocytes by normal donor-derived cells, as previously proposed. Intriguing analogies between liver repopulation and carcinogenesis will be briefly discussed and the potential dual role of cell competition, as a barrier or a spur to neoplastic development, will be considered. Cell competition is in essence a cooperative strategy organized at tissue level. One facet of such cooperative attitude is expressed in the elimination of altered cells which may represent a threat to the organismal community. On the other hand, the society of cells can be disrupted by the emergence of selfish clones, exploiting the molecular bar codes of cell competition, thereby paving their way to uncontrolled growth.

Cancer as a disease of old age: changing mutational and microenvironmental landscapes.

Why do we get cancer mostly when we are old? According to current paradigms, the answer is simple: mutations accumulate in our tissues throughout life, and some of these mutations contribute to cancers. Although mutations are necessary for cancer development, a number of studies shed light on roles for ageing and exposure-dependent changes in tissue landscapes that determine the impact of oncogenic mutations on cellular fitness, placing carcinogenesis into an evolutionary framework. Natural selection has invested in somatic maintenance to maximise reproductive success. Tissue maintenance not only ensures functional robustness but also prevents the occurrence of cancer through periods of likely reproduction by limiting selection for oncogenic events in our cells. Indeed, studies in organisms ranging from flies to humans are revealing conserved mechanisms to eliminate damaged or oncogenically initiated cells from tissues. Reports of the existence of striking numbers of oncogenically initiated clones in normal tissues and of how this clonal architecture changes with age or external exposure to noxious substances provide critical insight into the early stages of cancer development. A major challenge for cancer biology will be the integration of these studies with epidemiology data into an evolutionary theory of carcinogenesis, which could have a large impact on addressing cancer risk and treatment.

Fecal Metaproteomic Analysis Reveals Unique Changes of the Gut Microbiome Functions After Consumption of Sourdough Carasau Bread.

Sourdough-leavened bread (SB) is acknowledged for its great variety of valuable effects on consumer's metabolism and health, including a low glycemic index and a reduced content of the possible carcinogen acrylamide. Here, we aimed to investigate how these effects influence the gut microbiota composition and functions. Therefore, we subjected rats to a diet supplemented with SB, baker's yeast leavened bread (BB), or unsupplemented diet (chow), and, after 4 weeks of treatment, their gut microbiota was analyzed using a metaproteogenomic approach. As a result, diet supplementation with SB led to a reduction of specific members of the intestinal microbiota previously associated to low protein diets, namely Alistipes and Mucispirillum, or known as intestinal pathobionts, i.e., Mycoplasma. Concerning functions, asparaginases expressed by Bacteroides were observed as more abundant in SB-fed rats, leading to hypothesize that in their colonic microbiota the enzyme substrate, asparagine, was available in higher amounts than in BB- and chow-fed rats. Another group of protein families, expressed by Clostridium, was detected as more abundant in animal fed SB-supplemented diet. Of these, manganese catalase, small acid-soluble proteins (SASP), Ser/Thr kinase PrkA, and V-ATPase proteolipid subunit have been all reported to take part in Clostridium sporulation, strongly suggesting that the diet supplementation with SB might promote environmental conditions inducing metabolic dormancy of Clostridium spp. within the gut microbiota. In conclusion, our data describe the effects of SB consumption on the intestinal microbiota taxonomy and functions in rats. Moreover, our results suggest that a metaproteogenomic approach can provide evidence of the interplay between metabolites deriving from bread digestion and microbial metabolism.

Time-restricted feeding delays the emergence of the age-associated, neoplastic-prone tissue landscape.

Aging increases the risk of cancer partly through alterations in the tissue microenvironment. Time-restricted feeding (TRF) is being proposed as an effective strategy to delay biological aging. In the present studies, we assessed the effect of long-term exposure to TRF on the emergence of the age-associated, neoplastic-prone tissue landscape. Animals were exposed to either ad libitum feeding (ALF) or TRF for 18 months and then transplanted with hepatocytes isolated from pre-neoplastic nodules. Both groups were continued ALF and the growth of transplanted cells was evaluated 3 months later. A significant decrease in frequency of larger size clusters of pre-neoplastic hepatocytes was seen in TRF-exposed group compared to controls. Furthermore, TRF modified several parameters related to both liver and systemic aging towards the persistence of a younger phenotype, including a decrease in liver cell senescence, diminished fat accumulation and up-regulation of SIRT1 in the liver, down-regulation of plasma IGF-1, decreased levels of plasma lipoproteins and up-regulation of hippocampal brain-derived growth factor (BDNF).These results indicate that TRF was able to delay the onset of the neoplastic-prone tissue landscape typical of aging. To our knowledge, this is the first investigation to describe a direct beneficial effect of TRF on early phases of carcinogenesis.

Development versus Evolution in Cancer Biology.

The terms 'development' and 'evolution' are both used to describe the unfolding of the carcinogenic process. However, there is increasing awareness of an essential difference in the meanings of these two terms with reference to cancer. We discuss evidence suggesting that the concepts of development and evolution are both pertinent to the description of carcinogenesis; however, they appropriately apply to distinct phases of a multistep process. Such a distinction bears important implications for the study and management of cancer.

Evidence of Amniotic Epithelial Cell Differentiation toward Hepatic Sinusoidal Endothelial Cells.

Amniotic epithelial cells (AECs) represent a useful and noncontroversial source for liver-based regenerative medicine, as they can differentiate into hepatocytes upon transplantation into the liver. However, the possibility that AECs can differentiate into other liver cell types, such as hepatic sinusoidal endothelial cells (HSECs), has never been assessed. In order to test this hypothesis, rat- and human-derived AECs (rAECs and hAECs, respectively) were subjected to endothelial cell tube formation assay in vitro. Moreover, to evaluate differentiation in vivo, the retrorsine (RS) model of liver repopulation was used. Pyrrolizidine alkaloids (including RS) are known to target both hepatocytes and endothelial cells, inducing cell enlargement and inhibition of cell cycle progression. rAECs and hAECs were able to form capillary-like structures when cultured under proangiogenic conditions. For in vivo experiments, rAECs were obtained from dipeptidyl peptidase type IV (DPP-IV, CD26) donors and were transplanted into the liver of recipient CD26 negative animals pretreated with RS. rAEC-derived cells were engrafted in between hepatocytes and resembled HSECs as assessed by morphological analysis and the pattern of expression of CD26. Donor-derived CD26+ cells coexpressed HSEC markers RECA-1 and SE-1, while they lacked expression of typical hepatocyte markers (i.e., cytochrome P450, hepatocyte nuclear factor 4α). As such, these results provide the first evidence that AECs can respond to proangiogenic signals in vitro and differentiate into HSECs in vivo. Furthermore, they support the conclusion that AECs possesses great plasticity and represents a promising tool in the field of regenerative medicine both in the liver and in other organs.

Regenerative Medicine: Shedding Light on the Link between Aging and Cancer.

The evidence linking aging and cancer is overwhelming. Findings emerging from the field of regenerative medicine reinforce the notion that aging and cancer are profoundly interrelated in their pathogenetic pathways. We discuss evidence to indicate that age-associated alterations in the tissue microenvironment contribute to the emergence of a neoplastic-prone tissue landscape, which is able to support the selective growth of preneoplastic cell populations. Interestingly, tissue contexts that are able to select for the growth of preneoplastic cells, including the aged liver microenvironment, are also supportive for the clonal expansion of normal, homotypic, transplanted cells. This suggests that the growth of normal and preneoplastic cells is possibly driven by similar mechanisms, implying that strategies based on principles of regenerative medicine might be applicable to modulate neoplastic disease.

A GC-MS untargeted metabolomics analysis in the plasma and liver of rats lacking dipeptidyl-peptidase type IV enzyme activity.

This study was achieved with the aim to find metabolic changes between Fischer rats with different dipeptidyl peptidase-type 4 (DPPIV) expression. The DPPIV is an enzyme expressed in several tissues and is critically involved in the regulation of meal-related insulin secretion in healthy individuals. The metabolic consequences of chronic DPPIV inhibition were analyzed in a surrogate animal model of genetic enzyme deficiency. Hyphenated gas chromatography-mass spectrometry (GC-MS) and multivariate data analysis techniques were used to study the metabolic aqueous fraction profile of 18 plasma and liver samples in two syngeneic rat strains differing in DPPIV activity (DPPIV+ vs. DPPIV-). The hyperglycemic response following oral glucose administration was attenuated in DPPIV- rats, as expected. Statistical significant differences between the two strains were observed among the low molecular weight polar metabolites analyzed from plasma and liver.These included a decrease in malic acid and glutamine and an increase in pyroglutamic acid, serine, and alanine in plasma of DPPIV- rats. In addition, palmitic acid, L-proline, and ribitol were decreased in the liver of DPPIV- strain. Such alterations were compatible with a normal phenotype. These results suggest that long-term exposure to DPPIV inhibitors looks compatible with an overall balanced metabolism.

Caloric restriction delays early phases of carcinogenesis via effects on the tissue microenvironment.

Caloric restriction (CR) is an effective and consistent means to delay aging and the incidence of chronic diseases related to old age, including cancer. However, the precise mechanisms responsible for the beneficial effect of CR on carcinogenic process are yet to be identified.In the present studies the hypothesis was tested that the CR might delay carcinogenesis via modulatory effects exerted on the age-associated, neoplastic-prone tissue microenvironment. Using a well characterized, orthotopic cell transplantation (Tx) system in the rat, preneoplastic hepatocytes isolated from liver nodules were injected into either old syngeneic rats fed ad libitum (AL) or animals of the same age given a CR diet (70% of AL feeding). Analysis of donor-derived cell clusters performed at 10 weeks post-Tx revealed a significant shift towards smaller class sizes in the group receiving CR diet. Clusters comprising more than 50 cells, including large hepatic nodules, were thrice more frequent in AL vs. CR animals. Incidence of spontaneous endogenous nodules was also decreased by CR. Markers of cell senescence were equally expressed in the liver of AL and CR groups. However, higher levels of SIRT1 and FOXO1 proteins were detected in CR-exposed livers, while expression of HDAC1 and C/EBPß were decreased. These results are interpreted to indicate that CR delays the emergence of age-associated neoplastic disease through effects exerted, at least in part, on the tissue microenvironment. Nutrient-sensing pathways might mediate such modulatory effect.

Aging promotes neoplastic disease through effects on the tissue microenvironment.

A better understanding of the complex relationship between aging and cancer will provide important tools for the prevention and treatment of neoplasia. In these studies, the hypothesis was tested that aging may fuel carcinogenesis via alterations imposed in the tissue microenvironment. Preneoplastic hepatocytes isolated from liver nodules were orthotopically injected into either young or old syngeneic rats and their fate was followed over time using the dipeptidyl-peptidase type IV (DPPIV) system to track donor-derived-cells. At 3 months post-Tx, the mean size of donor-derived clusters was 11±3 cells in young vs. 42±8 in old recipients. At 8 months post-Tx, no visible lesion were detected in any of 21 young recipients, while 17/18 animals transplanted at old age displayed hepatic nodules, including 7 large tumors. All tumors expressed the DPPIV marker enzyme, indicating that they originated from transplanted cells. Expression of senescence-associated ß-galactosidase was common in liver of 18-month old animals, while it was a rare finding in young controls. Finally, both mRNA and IL6 protein were found to be increased in the liver of aged rats compared to young controls. These results are interpreted to indicate that the microenvironment of the aged liver promotes the growth of pre-neoplastic hepatocytes.

Gas-phase fragmentation of the N-oxide and N-hydroxylated derivatives of retrorsine using liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

RATIONALE: We report the electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) analysis of a pyrrolizidine alkaloid extract containing both retrorsine [C18H25NO6] and its N-oxide [C18H25NO7] and N-hydroxyl [C18H26NO7] derivatives measured with a QqTOFMS hybrid instrument. METHODS: A solution of the pyrrolizidine alkaloid extract containing retrorsine and its N-oxide and N-hydroxyl derivatives was directly infused into an electrospray ionization-quadrupole-time-of-flight (ESI-QTOF) mass spectrometer and product ion scans of the protonated molecules of each species were acquired. Labile protons of each compound were deuterated and computational energy calculations of the proposed structures of the product ions were used to determine the fragmentation pathways of retrorsine and its N-oxide and N-hydroxyl derivatives. RESULTS: ESI-MS of the pyrrolizidine alkaloid extract containing retrorsine and its N-oxide and N-hydroxyl derivatives afforded the protonated retrorsine [M1 + H](+) at m/z 352.1760 and the protonated retrorsine N-oxide [M2 + H](+) at m/z 368.1631 in addition to the formation of the unexpected protonated N-hydroxyl radical [M3 + H](+•) at m/z 369.1686. CID-MS/MS of this series of protonated molecules allowed the evaluation of their gas-phase fragmentations and the establishment of their fragmentation pathways. It was also found that several product ions could be assigned to different structures. Deuterium exchange and computational energy calculations allowed us to determine the most probable structures for the characterized product ions. CONCLUSIONS: To our knowledge, the identification of the protonated retrorsine N-hydroxyl radical [M3 + H](+•) is reported for the first time. In addition, the MS/MS results can be used for the identification of retrorsine and its N-oxide and N-hydroxyl derivatives in different complex biological matrices.

The effect of environmental chemicals on the tumor microenvironment.

Potentially carcinogenic compounds may cause cancer through direct DNA damage or through indirect cellular or physiological effects. To study possible carcinogens, the fields of endocrinology, genetics, epigenetics, medicine, environmental health, toxicology, pharmacology and oncology must be considered. Disruptive chemicals may also contribute to multiple stages of tumor development through effects on the tumor microenvironment. In turn, the tumor microenvironment consists of a complex interaction among blood vessels that feed the tumor, the extracellular matrix that provides structural and biochemical support, signaling molecules that send messages and soluble factors such as cytokines. The tumor microenvironment also consists of many host cellular effectors including multipotent stromal cells/mesenchymal stem cells, fibroblasts, endothelial cell precursors, antigen-presenting cells, lymphocytes and innate immune cells. Carcinogens can influence the tumor microenvironment through effects on epithelial cells, the most common origin of cancer, as well as on stromal cells, extracellular matrix components and immune cells. Here, we review how environmental exposures can perturb the tumor microenvironment. We suggest a role for disrupting chemicals such as nickel chloride, Bisphenol A, butyltins, methylmercury and paraquat as well as more traditional carcinogens, such as radiation, and pharmaceuticals, such as diabetes medications, in the disruption of the tumor microenvironment. Further studies interrogating the role of chemicals and their mixtures in dose-dependent effects on the tumor microenvironment could have important general mechanistic implications for the etiology and prevention of tumorigenesis.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

Rat-derived amniotic epithelial cells differentiate into mature hepatocytes in vivo with no evidence of cell fusion.

Amniotic epithelial cells (AEC) derived from human placenta represent a useful and noncontroversial source for liver-based regenerative medicine. Previous studies suggested that human- and rat-derived AEC differentiate into hepatocyte-like cells upon transplantation. In the retrorsine (RS) model of liver repopulation, clusters of donor-derived cells engrafted in the recipient liver and, importantly, showed characteristics of mature hepatocytes. The aim of the current study was to investigate the possible involvement of cell fusion in the emergence of hepatocyte clusters displaying a donor-specific phenotype. To this end, 4-week-old GFP(+)/DPP-IV(-) rats were treated with RS and then transplanted with undifferentiated AEC isolated from the placenta of DPP-IV(+) pregnant rats at 16-19 days of gestational age. Results indicated that clusters of donor-derived cells were dipeptidyl peptidase type IV (DPP-IV) positive, but did not express the green fluorescent protein (GFP), suggesting that rat amniotic epithelial cells (rAEC) did not fuse within the host parenchyma, as no colocalization of the two tags was observed. Moreover, rAEC-derived clusters expressed markers of mature hepatocytes (eg, albumin, cytochrome P450), but were negative for the expression of biliary/progenitor markers (eg, epithelial cell adhesion molecule [EpCAM]) and did not express the marker of preneoplastic hepatic nodules glutathione S-transferase P (GST-P). These results extend our previous findings on the potential of AEC to differentiate into mature hepatocytes and suggest that this process can occur in the absence of cell fusion with host-derived cells. These studies support the hypothesis that amnion-derived epithelial cells can be an effective cell source for the correction of liver disease.

Cell-autonomous decrease in proliferative competitiveness of the aged hepatocyte.

BACKGROUND & AIMS: The regenerative potential of the liver declines with age, this might be dependent on a decrease in the intensity of the stimulus and/or an increased refractoriness of the target. In the present study, we compared the in vivo growth capacity of young and old hepatocytes transplanted into the same host. METHODS: We utilized the retrorsine (RS)-based model for liver repopulation, which provides a specific and effective stimulus for transplanted hepatocytes. Rats of the dipeptidyl-peptidase type IV (DPP-IV)-deficient strain were given RS and were injected with a mix of hepatocytes isolated from either a 2-month old or an 18-month old donor. To follow the fate of transplanted cells, they were each identified through a specific tag: young hepatocytes expressed the green fluorescent protein (GFP(+)), while those from old donors were DPP-IV-positive. RESULTS: At 1 month post-transplantation, DPP-IV-positive clusters (derived from old donor) were consistently smaller than those GFP(+) (young donor); the cross sectional area of clusters was decreased by 50%, while the mean volume was reduced to 1/3. Furthermore, when 2/3 partial hepatectomy (PH) was performed, the S-phase response of old hepatocyte-derived clusters was only 30-40% compared to that observed in cluster originating from young hepatocytes. No markers of cell senescence were expressed in clusters of transplanted hepatocytes. CONCLUSIONS: This is the first direct evidence in vivo that hepatocytes in the aged liver express a cell-autonomous decline in their replicative capacity and in their regenerative response to PH compared to those from a young animal.