Hepatic differentiation of amniotic epithelial cells.

UNLABELLED: Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Human amniotic epithelial cells (hAECs) from term placenta express surface markers and gene characteristics of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e., liver). Thus, hAECs could provide a source of stem cell-derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the livers of severe combined immunodeficient (SCID)/beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a mixture of growth factors, cytokines, and hormones are required for differentiation of hAECs into hepatocyte-like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone, and 17α-hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of retrorsine (RS)-treated SCID/beige mice, naïve hAECs differentiated into hepatocyte-like cells that expressed mature liver genes such as cytochromes, plasma proteins, transporters, and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. CONCLUSION: Amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes both in vitro and in vivo, thus representing a useful and noncontroversial source of cells for transplantation.

The growth pattern of transplanted normal and nodular hepatocytes.

Overt neoplasia is often the end result of a long biological process beginning with the appearance of focal lesions of altered tissue morphology. While the putative clonal nature of focal lesions has often been emphasized, increasing attention is being devoted to the possible role of an altered growth pattern in the evolution of carcinogenesis. Here we compare the growth patterns of normal and nodular hepatocytes in a transplantation system that allows their selective clonal proliferation in vivo. Rats were pre-treated with retrorsine, which blocks the growth of resident hepatocytes, and were then transplanted with hepatocytes isolated from either normal liver or hepatocyte nodules. Both cell types were able to proliferate extensively in the recipient liver, as expected. However, their growth pattern was remarkably different. Clusters of normal hepatocytes integrated in the host liver, displaying a normal histology; however, transplanted nodular hepatocytes formed new hepatocyte nodules, with altered morphology and sharp demarcation from surrounding host liver. Both the expression and distribution of proteins involved in cell polarity, cell communication, and cell adhesion, including connexin 32, E-cadherin, and matrix metalloproteinase-2, were altered in clusters of nodular hepatocytes. Furthermore, we were able to show that down-regulation of connexin 32 and E-cadherin in nodular hepatocyte clusters was independent of growth rate. These results support the concept that a dominant pathway towards neoplastic disease in several organs involves defect(s) in tissue pattern formation.

The microenvironments of multistage carcinogenesis.

Overt neoplasia is often the result of a chronic disease process encompassing an extended segment of the lifespan of any species. A common pathway in the natural history of the disease is the appearance of focal proliferative lesions that are known to act as precursors for cancer development. It is becoming increasingly apparent that the emergence of such lesions is not a cell-autonomous phenomenon, but is heavily dependent on microenvironmental cues derived from the surrounding tissue. Specific alterations in the tissue microenvironment that can foster the selective growth of focal lesions are discussed herein. Furthermore, we argue that a fundamental property of focal lesions as it relates to their precancerous nature lies in their altered growth pattern as compared to the tissue where they reside. The resulting altered tissue architecture translates into the emergence of a unique tumor microenvironment inside these lesions, associated with altered blood vessels and/or blood supply which in turn can trigger biochemical and metabolic changes fueling tumor progression. A deeper understanding of the role(s) of tissue and tumor microenvironments in the pathogenesis of cancer is essential to design more effective strategies for the management of this disease.

Repopulation by endogenous hepatocytes does not reconstitute liver mass in rats treated with retrorsine.

The retrorsine (RS)-based model for massive liver repopulation was laid on the hypothesis that transplanted cells can proliferate in the recipient liver if the growth capacity of endogenous hepatocytes is persistently impaired. In order to directly test this hypothesis, we examined the long-term response to 2/3 partial hepatectomy (PH) in rats pretreated with RS, according to the protocol for liver repopulation. Rats were given RS or saline and 4 weeks later they underwent PH; they were killed up to 16 weeks thereafter. Liver weights, liver DNA, and protein content were significantly lower in the RS group throughout the experimental time considered (e.g., at 16 weeks post-PH relative liver weight was 1.99 +/- 0.30% in RS group vs. 3.06 +/- 0.5% in controls). Regenerative nodules were present in RS-treated livers; they occupied about 3% of the liver at 2 weeks post-PH and this value increased to nearly 50% at 8 weeks and to > 95% at 16 weeks. In conclusion, RS-treated rat liver is unable to recover its original mass for several months following PH, despite the development of regenerative nodules. This long-lasting effect is likely to contribute to the growth of transplanted hepatocytes, leading to massive liver repopulation.

Aging is associated with increased clonogenic potential in rat liver in vivo.

Cancer increases with age and often arises from the selective clonal growth of altered cells. Thus, any environment favoring clonal growth per se poses a higher risk for cancer development. Using a genetically tagged animal model, we investigated whether aging is associated with increased clonogenic potential. Groups of 4-, 12-, 18-, and 24-month-old Fischer 344 rats were infused (via the portal vein) with 2x10(6) hepatocytes isolated from a normal syngenic 2-month-old donor. Animals deficient in dipeptidyl-peptidase type IV (DPP-IV-) enzyme were used as recipients, allowing for the histochemical detection of injected DPP-IV+ cells. Groups of animals were sacrificed at various times thereafter. No growth of DPP-IV+ transplanted hepatocytes was present after either 2 or 6 months in the liver of rats transplanted at young age, as expected. In striking contrast, significant expansion of donor-derived cells was seen in animals transplanted at the age of 18 months: clusters comprising 7-10 DPP-IV+ hepatocytes/cross-section were present after 2 months and were markedly enlarged after 6 months (mean of 88+/-35 cells/cluster/cross-section). These results indicate that the microenvironment of the aged liver supports the clonal expansion of transplanted normal hepatocytes. Such clonogenic environments can foster the selective growth of pre-existing altered cells, thereby increasing the overall risk for cancer development associated with aging.

Liver repopulation by transplanted hepatocytes and risk of hepatocellular carcinoma.

BACKGROUND: Transplantation of isolated hepatocytes in rats treated with retrorsine (RS) results in massive repopulation of the host liver. In this study, the long-term fate of hepatocytes transplanted into RS-treated recipients was followed for up to two years. METHODS: Dipeptidyl-peptidase type IV-deficient (DPPIV) Fischer 344 rats were given two injections of RS (30 mg/kg), followed by transplantation of 2 million hepatocytes, isolated from a syngenic, DPPIV donor. RESULTS: Extensive (91+/-7%) liver replacement by transplanted hepatocytes was observed in animals sacrificed 18 months posttransplantation. Similar levels of repopulation persisted at two years (87+/-5%). No evidence of preneoplastic and/or neoplastic evolution of the transplanted cell population was present in the RS-treated and repopulated livers at any time point considered. Furthermore, serum parameters related to hepatocyte function and integrity were in the normal range. In control groups given cell transplantation in the absence of prior treatment with RS, only small clusters of donor-derived, DPPIV hepatocytes were discerned. CONCLUSIONS: These results indicate that liver repopulation in this model is largely stable, persisting for up to two years and allowing for a normal liver function. In addition, no increased risk of neoplastic transformation appears to be associated with the process of liver repopulation for as long as over two thirds of the life span of the recipient animal.

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.

Genetic isolates in Corsica (France): linkage disequilibrium extension analysis on the Xq13 region.

Genetic isolates with a history of a small founder population, long-lasting isolation and population bottlenecks represent exceptional resources in the identification of genes involved in the pathogenesis of multifactorial diseases. In these populations, the disease allele reveals linkage disequilibrium (LD) with markers over significant genetic intervals, therefore facilitating disease locus identification. This study has been designed to examine the background LD extension in some subpopulations of Corsica. Our interest in the island of Corsica is due to its geographical and genetic proximity to the other Mediterranean island of Sardinia. Sardinian isolates in which the extension of the background LD is particularly high have been recently identified and are now the object of studies aimed at the mapping of genes involved in complex diseases. Recent evidence has highlighted that the genetic proximity between the populations of Corsica and Sardinia is particularly true for the internal conservative populations. Given these considerations, Sardinia and Corsica may represent a unique system to carry out parallel association studies whose results could be validated by comparison. In the present study, we have analyzed the LD extension on the Xq13 genomic region in three subpopulations of Corsica: Corte, Niolo and Bozio, all located in the mountainous north-center of the island. Our results show a strong degree of LD over long distance for the population of Bozio and to a less extent for the population of Niolo. Their LD extent is comparable to or higher than that reported for other isolates.

Normal hepatocyte transplantation delays the emergence of chemically induced preneoplastic nodules in rat liver.

Cancer often arises in a background of chronic tissue damage. It is also increasingly appreciated that such an injured tissue microenvironment might foster the selective emergence of altered cells, leading to neoplasia. Accordingly, reversal of chronic tissue damage could represent a potential strategy to counteract neoplastic disease. In these studies, we aim to investigate whether transplantation of normal cells in the context of an injured, neoplastic-prone microenvironment might impact on the evolution of the carcinogenic process. A rat model of chemically induced hepatocarcinogenesis was used. Animals were given a single dose of diethylnitrosamine (DENA), followed by two injections of retrorsine (RS), a pyrrolizidine alkaloid that imposes a persistent block on hepatocyte cell cycle. At the end of this protocol, rats were either given no further treatment or injected, via the portal circulation, with 4 million normal hepatocytes isolated from a syngenic donor. After 3 months, rats given DENA+RS alone displayed numerous discrete nodular lesions (up to 30 per liver), ranging 1 to 3 mm in size. On the other hand, in animals receiving DENA+RS and transplantation, donor hepatocytes were able to repopulate over 50% of the host liver, as expected. Most importantly, both the number and the size of hepatocyte nodules were greatly reduced in these animals (percent nodular area was 1.8 ± 0.3, down from a control value of 8.5 ± 2.8). The above data indicate that strategies aimed at reestablishing a normal tissue microenvironment might be relevant to the management of neoplastic disease.

Cancer as a disease of tissue pattern formation.

The diagnosis of neoplastic disease still lays its foundations on the detection of altered tissue morphology. Most importantly, cancer begins, at least in many cases as a disease with altered tissue pattern formation. It is therefore rather surprising that the issue regarding the possible mechanistic role of such property in the pathogenesis of cancer has received relatively little attention so far. To be more specific, we need to ask the following question: is altered tissue pattern formation a mere bystander, with its pervasive presence along the entire carcinogenic sequence, or does it play a role in fuelling this process? Pathways related to morphogenesis and to the establishment of cell polarity will be considered for their possible mechanistic involvement in early phases of neoplastic disease. Evidences and hypotheses relating altered tissue pattern formation to the emergence of the tumor microenvironment and to neoplastic progression will be discussed.

Liver repopulation and carcinogenesis: two sides of the same coin?

Liver repopulation by transplanted normal hepatocytes has been described in a number of experimental settings. Extensive repopulation can also occur from the selective proliferation of endogenous normal hepatocytes, both in experimental animals and in the human liver. This review highlights the intriguing association between clinical and experimental conditions related to liver repopulation and an increased risk for development of hepatocellular carcinoma. It is suggested that any microenvironment that is able to sustain the clonal growth of normal transplanted (or endogenous) hepatocytes is also geared to select for the emergence of rare resistant cells with an altered phenotype. Whereas the first pathway leads to liver repopulation with normal histology, the latter results in the growth of focal proliferative lesions and carries an increased risk of neoplastic disease. The implications of this association are discussed, both in terms of pathogenetic significance and possible therapeutic exploitation.

IRAK-M is involved in the pathogenesis of early-onset persistent asthma.

Asthma is a multifactorial disease influenced by genetic and environmental factors. In the past decade, several loci and >100 genes have been found to be associated with the disease in at least one population. Among these loci, region 12q13-24 has been implicated in asthma etiology in multiple populations, suggesting that it harbors one or more asthma susceptibility genes. We performed linkage and association analyses by transmission/disequilibrium test and case-control analysis in the candidate region 12q13-24, using the Sardinian founder population, in which limited heterogeneity of pathogenetic alleles for monogenic and complex disorders as well as of environmental conditions should facilitate the study of multifactorial traits. We analyzed our cohort, using a cutoff age of 13 years at asthma onset, and detected significant linkage to a portion of 12q13-24. We identified IRAK-M as the gene contributing to the linkage and showed that it is associated with early-onset persistent asthma. We defined protective and predisposing SNP haplotypes and replicated associations in an outbred Italian population. Sequence analysis in patients found mutations, including inactivating lesions, in the IRAK-M coding region. Immunohistochemistry of lung biopsies showed that IRAK-M is highly expressed in epithelial cells. We report that IRAK-M is involved in the pathogenesis of early-onset persistent asthma. IRAK-M, a negative regulator of the Toll-like receptor/IL-1R pathways, is a master regulator of NF- kappa B and inflammation. Our data suggest a mechanistic link between hyperactivation of the innate immune system and chronic airway inflammation and indicate IRAK-M as a potential target for therapeutic intervention against asthma.

Cyclin D1 is up-regulated in hepatocytes in vivo following cell-cycle block induced by retrorsine.

BACKGROUND/AIMS: We reported massive liver repopulation by transplanted hepatocytes in rats given retrorsine (RS), a pyrrolizidine alkaloid which blocks proliferation of resident cells. In these studies, molecular alterations induced by RS on hepatocyte cell cycle were investigated. METHODS: Animals were treated according to the protocol for liver repopulation, i.e. two injections of RS (30 mg/kg) followed by two-thirds partial hepatectomy (PH) and were sacrificed at various time points thereafter. Livers were analyzed for the expression of cell cycle-related genes. RESULTS: Prior to PH, increased cyclin D1 mRNA and protein levels were found in livers of RS-treated rats. Expression of PCNA was also increased; however, DNA synthesis was not significantly changed. Other cyclins, including cyclin B and cyclin E, were not induced. Cyclin D1 expression increased in controls post-PH and then declined by 48 h, as expected. By contrast, no such modulation of cyclin D1 levels was seen in RS group receiving PH and expression remained high at 48 h, without mitotic division. CONCLUSIONS: Exposure to RS is able to block cell cycle progression after cyclin D1 and PCNA induction, but prior to S phase. Such persistent block outside the resting phase may contribute to the selective replacement of resident cells during liver repopulation.