The potential of embryonal day (ED) 14 fetal liver epithelial progenitor (FLEP) cells from Fischer (F)344 rats to repopulate the normal and retrorsine-treated liver was studied throughout a 6-month period in syngeneic dipeptidyl peptidase IV (DPPIV-) mutant F344 rats. In normal liver, FLEP cells formed: 1) hepatocytic clusters ranging in size up to approximately 800 to 1000 cells; 2) bile duct structures connected to pre-existing host bile ducts; and 3) mixed clusters containing both hepatocytes and bile duct epithelial cells. Liver repopulation after 6 months was moderate (5 to 10%). In retrorsine-treated liver, transplanted cells formed large multilobular structures containing both parenchymal and bile duct cells and liver repopulation was extensive (60 to 80%). When the repopulating capacity of ED 14 FLEP cells transplanted into normal liver was compared to adult hepatocytes, three important differences were noted: 1) FLEP cells continued to proliferate at 6 months after transplantation, whereas adult hepatocytes ceased proliferation within the first month; 2) both the number and size of clusters derived from FLEP cells gradually increased throughout time but decreased throughout time with transplanted mature hepatocytes; and 3) FLEP cells differentiated into hepatocytes when engrafted into the liver parenchyma and into bile epithelial cells when engrafted in the vicinity of the host bile ducts, whereas adult hepatocytes did not form bile duct structures. Finally, after transplantation of ED 14 FLEP cells, new clusters of DPPIV+ cells appeared after 4 to 6 months, suggesting reseeding of the liver by transplanted cells. This study represents the first report with an isolated fetal liver epithelial cell fraction in which the cells exhibit properties of tissue-determined stem cells after their transplantation into normal adult liver; namely, bipotency and continued proliferation long after their transplantation.
BackgroundAtrial fibrillation is associated with higher mortality. Identification of causes of death and contemporary risk factors for all‐cause mortality may guide interventions.Methods and ResultsIn the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF) study, patients with nonvalvular atrial fibrillation were randomized to rivaroxaban or dose‐adjusted warfarin. Cox proportional hazards regression with backward elimination identified factors at randomization that were independently associated with all‐cause mortality in the 14 171 participants in the intention‐to‐treat population. The median age was 73 years, and the mean CHADS 2 score was 3.5. Over 1.9 years of median follow‐up, 1214 (8.6%) patients died. Kaplan–Meier mortality rates were 4.2% at 1 year and 8.9% at 2 years. The majority of classified deaths (1081) were cardiovascular (72%), whereas only 6% were nonhemorrhagic stroke or systemic embolism. No significant difference in all‐cause mortality was observed between the rivaroxaban and warfarin arms (P=0.15). Heart failure (hazard ratio 1.51, 95% CI 1.33–1.70, P<0.0001) and age ≥75 years (hazard ratio 1.69, 95% CI 1.51–1.90, P<0.0001) were associated with higher all‐cause mortality. Multiple additional characteristics were independently associated with higher mortality, with decreasing creatinine clearance, chronic obstructive pulmonary disease, male sex, peripheral vascular disease, and diabetes being among the most strongly associated (model C‐index 0.677).ConclusionsIn a large population of patients anticoagulated for nonvalvular atrial fibrillation, ≈7 in 10 deaths were cardiovascular, whereas <1 in 10 deaths were caused by nonhemorrhagic stroke or systemic embolism. Optimal prevention and treatment of heart failure, renal impairment, chronic obstructive pulmonary disease, and diabetes may improve survival.Clinical Trial Registration URL: https://www.clinicaltrials.gov/. Unique identifier: NCT00403767.
Recent studies have shown that nondividing primary cells, such as hepatocytes, can be efficiently transduced in vitro by human immunodeficiency virus-based lentivirus vectors. Other studies have reported that, under certain conditions, the liver can be repopulated with transplanted hepatocytes. In the present study, we combined these procedures to develop a model system for ex vivo gene therapy by repopulating rat livers with hepatocytes and hepatoblasts transduced with a lentivirus vector expressing a reporter gene, green fluorescent protein (GFP). Long-term GFP expression in vivo (up to 4 months) was achieved when the transgene was driven by the liver-specific albumin enhancer/promoter but was silenced when the cytomegalovirus (CMV) enhancer/promoter was used. Transplanted cells were massively amplified (ϳ10 cell doublings) under the influence of retrorsine/partial hepatectomy, and both repopulation and continued transgene expression in individual cells were documented by dual expression of a cell transplantation marker, dipeptidyl peptidase IV (DPPIV), and GFP. In this system, maintenance or expansion of the transplanted cells did not depend on expression of the transgene, establishing that positive selection is not required to maintain transgene expression following multiple divisions of transplanted, lentivirus-transduced hepatic cells. In conclusion, fetal hepatoblasts (liver stem/progenitor cells) can serve as efficient vehicles for ex vivo gene therapy and suggest that liver-based genetic disorders that do not shorten hepatocyte longevity or cause liver damage, such as phenylketonuria, hyperbilirubinemias, familial hypercholesterolemia, primary oxalosis, and factor IX deficiency, among others, might be amenable to treatment by this approach. (HEPATOLOGY 2003;37:994-1005.)
Background & Aims Highly proliferative fetal liver stem/progenitor cells (FLSPC) repopulate livers of normal recipients by cell competition. We investigated the mechanisms by which FLSPC repopulate livers of older, compared with younger rats. Methods Fetal liver cells were transplanted from DPPIV+ F344 rats into DPPIV− rats of different ages (2, 6, 14, or 18 months); liver tissues were analyzed 6 months later. Cultured cells and liver tissues were analyzed by reverse transcription PCR, immunoblot, histochemistry, laser-capture microscopy, and TUNEL analyses. Results We observed 4–5-fold increases in liver repopulation when FLSPC were transplanted into older, compared with younger, rats. mRNA levels of cyclin-dependent kinase inhibitors increased progressively in livers of older rats; hepatocytes from 20-month old rats had 6.1-fold higher expression of p15INK4b and were less proliferative, in vitro, than hepatocytes from 2-month old rats. Expression of p15INK4b in cultured hepatocytes was upregulated by activin A, which increased in liver during aging. Activin A inhibited proliferation of adult hepatocytes, whereas FLSPC were unresponsive because they had reduced expression of activin receptors (e.g. ALK-4). In vivo, expanding cell clusters derived from transplanted FLSPC had lower levels of ALK-4 and p15INK4b and increased levels of Ki-67, compared with the host parenchema. Liver tissue of older rats had 3-fold more apoptotic cells than of younger rats. Conclusions FLSPC, resistant to activin A signaling, repopulate livers of older rats; hepatocytes in older rats have less proliferation, because of increased activin A and p15INK4b levels, and increased apoptosis than of younger rats. These factors and cell types might be manipulated to improve liver cell transplantation strategies in patients with liver diseases in which activin A levels are increased.
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