Optimization of the technique to isolate fetal hepatocytes, and assessment of their functionality by transplantation

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative less immunogenic and more resistant to both cryopreservation and ischemic injury. In the present study, we describe the method for isolation of FH and the relationship between the transplantability of FH into the spleen of analbuminemic rats and expression of albumin mRNA. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Nagase analbuminemic rats (NAR), a strain which bears a mutation that determines the impossibility of the normal splicing of the albumin mRNA were used as recipients. The transplanted FH immediately migrated to the liver via portal vein, and anchored there. To assess the functional state of the transplanted cells, one month after transplantation, the expression of the albumin gene was studied in the liver of the recipients.     

Hepatic proliferation in Gunn rats transplanted with hepatocytes: effect of retrorsine and tri-iodothyronine

Hepatocyte transplantation would offer an attractive alternative to liver transplantation in the treatment of inborn errors of liver metabolism. However, a major problem in most transplantation studies to date has been the limited growth of transplanted cells in the recipient organ. We performed a strategy for selective proliferation of transplanted cells by interfering with the proliferative capacity of resident hepatocytes, using the pyrrolizidine alkaloid retrorsine and then transplanting liver cells in conjunction with repeated administration of triiodothyronine, an inducer of hepatocyte proliferation in rats. In the present study, foetal and adult syngeneic hepatocyte transplantation into spleen was performed in retrorsine-treated hyperbilirubinemic Gunn rats. In parallel, repeated injections of triiodothyronine were given to recipients. Rats were sacrificed at 1, 7, 30 and 90 days after transplantation and blood and bile samples were taken to assess the functionality of transplanted cells. The proliferative activity of transplanted hepatocytes was evaluated using proliferating cell nuclear antigen labelling index. In summary, both adult and foetal hepatocyte transplantation were effective in correcting a metabolic abnormality in Gunn rats for as long as 3 months. The RS/T3 model, as a measure to increase graft function, could represent an important advance to future clinical application of hepatocyte transplantation.     

Disappearance of GFP-Positive Hepatocytes Transplanted into the Liver of Syngeneic Wild-Type Rats Pretreated with Retrorsine

Background and Aim

Green fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models. The differing results from various previously reported studies using GFP could be attributed to the immunogenicity of GFP.

Methods

Hepatocytes were obtained from GFP-expressing transgenic (Tg) Lewis rats and were transplanted into the livers of wild-type Lewis rats after they had undergone a partial hepatectomy. The proliferation of endogenous hepatocytes in recipient rats was inhibited by pretreatment with retrorsine to enhance the proliferation of the transplanted hepatocytes. Transplantation of wild-type hepatocytes into GFP-Tg rat liver was also performed for comparison.

Results

All biopsy specimens taken seven days after transplantation showed engraftment of transplanted hepatocytes, with the numbers of transplanted hepatocytes increasing until day 14. GFP-positive hepatocytes in wild-type rat livers were decreased by day 28 and could not be detected on day 42, whereas the number of wild-type hepatocytes steadily increased in GFP-Tg rat liver. Histological examination showed degenerative change of GFP-positive hepatocytes and the accumulation of infiltrating cells on day 28. PCR analysis for the GFP transgene suggested that transplanted hepatocytes were eliminated rather than being retained along with the loss of GFP expression. Both modification of the immunological response using tacrolimus and bone marrow transplantation prolonged the survival of GFP-positive hepatocytes. In contrast, host immunization with GFP-positive hepatocytes led to complete loss of GFP-positive hepatocytes by day 14.

Conclusion

GFP-positive hepatocytes isolated from GFP-Tg Lewis rats did not survive long term in the livers of retrorsine-pretreated wild-type Lewis rats. The mechanism underlying this phenomenon most likely involves an immunological reaction against GFP. The influence of GFP immunogenicity on cell transplantation models should be considered in planning in vivo experiments using GFP and in interpreting their results.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

An in vivo model for monitoring trans-differentiation of bone marrow cells into functional hepatocytes

The plasticity of bone marrow cells (BMCs) remains controversial. The present study found that persistent injury induces efficient trans-differentiation of BMCs into functional hepatocytes. Mice with liver cirrhosis induced by carbon tetrachloride were injected with 1 x 10(5) non-treated green fluorescent protein (GFP)-positive BMCs via the tail vein. In these mice, transplanted GFP-positive BMCs efficiently migrated into the peri-portal area of liver lobules after one day, repopulating 25% of the recipient liver by 4 weeks. In contrast, no GFP-positive BMCs were detected following transplantation into control mice with undamaged livers. BMCs trans-differentiated into functional mature hepatocytes via immature hepatoblasts. Serum albumin levels were significantly elevated to compensate for chronic liver failure in BMC transplantation. These results reveal that recipient conditions and microenvironments represent key factors for successful cell therapy using BMCs.     

Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation

Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)^–/– mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.     

Cell transplantation causes loss of gap junctions and activates GGT expression permanently in host liver

Cell transplantation into hepatic sinusoids, which is necessary for liver repopulation, could cause hepatic ischemia. To examine the effects of cell transplantation on host hepatocytes, we transplanted Fisher 344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. Within 24 h of cell transplantation, areas of ischemic necrosis, along with transient disruption of gap junctions, appeared in the liver. Moreover, host hepatocytes expressed gamma-glutamyl transpeptidase (GGT) extensively, which was observed even 2 years after cell transplantation. GGT expression was not associated with alpha-fetoprotein activation, which is present in progenitor cells. Increased GGT expression was apparent after transplantation of nonparenchymal cells and latex beads but not after injection of saline, fragmented hepatocytes, hepatocyte growth factor, or turpentine. Some host hepatocytes exhibited apoptosis, as well as DNA synthesis, between 24 and 48 h after cell transplantation. Changes in gap junctions, GGT expression, DNA synthesis, and apoptosis after cell transplantation were prevented by vasodilators. The findings indicated the onset of ischemic liver injury after cell transplantation. These hepatic perturbations must be considered when transplanted cells are utilized as reporters for biological studies.     

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or α-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.     

In vivo distribution and gene expression of genetically modified hepatocytes after intrasplenic transplantation

To investigate the feasibility and efficacy of liver gene therapy mediated by intrasplenic transplanta-tion of genetically modified hepatocytes, the normal mouse liver cell line BNL CL. 2 cells were introduced with Neo-re-sistant (NeoR) gene or interleukin-2 (IL-2) gene in vitro, and transplanted intrasplenically into normal syngeneic mice (2 × 106 cell/mouse); subsequently, the expressions of the introduced genes in vivo were detected. The RT-PCR results showed that NeoR mRNA expressions were detectable in livers 24 h after transplantation and lasted over 11 weeks. Moreover, The NeoR mRNA was detected to be expressed temporarily in spleens (24 h- 1 week) and lungs (24-96 h) after transplantation. After intrasplenic transplantation of IL-2 gene-modified BNL CL.2 cells, the stable expressions of IL-2 mRNA in the livers of transplanted mice were detectable by RT-PCR (24 h-11 weeks), and certain levels of IL-2 (5-40 pg/mL) remained in the peripheral blood. When IL-2 gene-modified BNL CL. 2 cells were tran     

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future.     

Transplanted reporter cells help in defining onset of hepatocyte proliferation during the life of F344 rats

Transplanted hepatocytes integrate in the liver parenchyma and exhibit gene expression patterns that are similar to adjacent host hepatocytes. To determine the fate of genetically marked hepatocytes in the context of hepatocellular proliferation throughout the rodent life span, we transplanted Fischer 344 (F344) rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. The proliferative activity in transplanted hepatocytes was studied in animals ranging in age from a few days to 2 yr. Transplanted hepatocytes proliferated during liver development between 1 and 6 wk of age, each dividing an estimated two to five times. DNA synthesis in occasional cells was demonstrated by localizing bromodeoxyuridine incorporation. There was no evidence for transplanted cell proliferation between 6 wk and 1 yr of age. Subsequently, transplanted cells proliferated again, with increased sizes of transplanted cell clusters at 18 and 24 mo of age. The proliferative activity of transplanted cells was greater in rats entering senescence compared with during postnatal liver development. In old rats, some liver lobules were composed entirely of transplanted cells. We conclude that hepatocyte proliferation in the livers of very young and old F344 rats is regulated in a temporally determined, biphasic manner. The findings will be relevant to mechanisms concerning liver development, senescence, and oncogenesis, as well as to cell and gene therapy.     

利用人脐血单个核细胞重建急性肝损伤小鼠肝组织的实验研究

利用人脐血单个核细胞重建急性肝损伤小鼠肝组织,探索建立人-小鼠嵌合肝模型方法。15只SCID小鼠,以四氯化碳(CCL4)制备急性肝损伤模型,24h后行2/3肝切除,然后分为三个实验组细胞移植组(7只)、阴性对照组(3只)及空白对照组(5只);将人脐血单个核细胞悬液注入细胞移植组小鼠脾脏内,阴性对照组小鼠脾脏内注入等量磷酸盐缓冲液(PBS),空白对照组不注射细胞悬液和PBS。术后7d、14d及21d取小鼠肝组织观察病理变化、检测人白蛋白(ALB)及细胞角蛋白19(CK19),同时检测小鼠血清及肝组织匀浆中人ALB含量。全部小鼠表现出急性肝损伤组织学特征;细胞移植组小鼠术后7d、14d、21d肝组织内均见大量人ALB及CK19阳性表达细胞,血清及肝组织匀浆可检测出人ALB;阴性对照组小鼠肝组织未见人ALB及CK19阳性表达,血清及肝组织匀浆中未检测出人ALB。人脐血单个核细胞在部分肝切除的急性肝损伤小鼠肝组织内可大量分化为人肝细胞及胆管细胞,在建立模型方面已取得关键突破。     

Establishment of Alb-DsRed2 transgenic rat for liver regeneration research

Because serum albumin is specifically produced by mature hepatocytes, detection system of albumin producing cells could be a valuable tool to visualize liver regeneration or development. We have developed here an albumin enhancer/promoter-driven Alb-DsRed2 Tg rat that expresses DsRed2, having liver-specific reporter gene expression of red fluorescent protein. To study the transdifferentiation of bone marrow cells (BMCs) into albumin producing cells, BMCs from the Alb-DsRed2 Tg rat were injected into rats having acute liver damage caused by 2-acetylaminofluorene plus carbon tetrachloride and chronic liver damage by repeated administration of CCl(4). DsRed2-positive cells were generated in the recipient liver after BMC injection. The number of transdifferentiated DsRed2-positive cells in chronic liver injury model was increased comparing with that in acute injury model. We propose that the Alb-DsRed2 Tg rat is well suited to studying in vivo liver regeneration.     

Proliferation, differentiation and morphogenesis of fetal rat glandular stomach transplanted under the kidney capsule of syngeneic hosts

Undifferentiated glandular stomach tissue fragments from 16.5-day fetal rats were transplanted under the kidney capsule of syngeneic adult rats, and the proliferation, differentiation and morphogenesis of the transplanted tissues were investigated. Gastric epithelial cells began to invaginate 3–4 days after the transplantation and immature glands were formed after 1 week. During the period, there was a gradual increase in the expression of pepsinogen and cathepsin E, markers of cytodifferentiation of the stomach epithelia, both at protein and mRNA levels. Cathepsin E was weakly expressed in undifferentiated gastric epithelial cells at 16.5 days of gestation, and a higher level of the expression was observed in differentiated epithelia of the transplants. In contrast, the pepsinogen-producing cells first appeared around days 3–4 after transplantation and gradually increased in number to about 30% of the epithelial cells and became localized at the bottom of the gland. During the period of the experiment up to 1 month, the pepsinogen-producing cells were all positive for class III mucin and cathepsin E, indicating the immature character of these cells. In addition, no parietal cells were observed. When the tissue fragments were transplanted into adrenalectomized animals, the epithelial differentiation and morphogenesis was suppressed, but its proliferation was enhanced. The observed changes were reversed by hydrocortisone replacement. These results suggest that the development of the 16.5-day fetal stomach is regulated intrinsically to a certain extent by the genetic program of the cells involved and various gastric functions develop in the absence of luminal stimulation, stage-specific systemic hormonal change, neuronal regulation or other systemic influences, and that glucocorticoids modulate the developmental program of the fetal stomach tissues.     

Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: role of epidermal growth factor and hormones.

Sustained production of plasma proteins, notably albumin, is a reliable indicator of the differentiated state of hepatocytes. In this work, we have developed a fetal hepatocyte culture system where studying the regulation of albumin expression in proliferating liver cells. Our results show that under proliferative conditions (i.e., in the presence of EGF) fetal hepatocytes maintain albumin production above control quiescent non-treated cells. Glucagon and noradrenaline have no effect on the proliferation induced by EGF in cultured fetal hepatocytes; however, they act synergistically with the growth factor, increasing intracellular albumin levels. The maximum response is obtained by treatment of cells with EGF and noradrenaline. The stimulatory noradrenergic effect is mimicked by agents that increase cyclic AMP levels (forskolin plus IBMX). However, vasopressin or phorbol esters have no effect on albumin production, neither alone nor in combination with EGF. Dexamethasone, which does not alter the proliferative induction of EGF, increases albumin content. This effect is independent of the proliferative status of the cells and is not enhanced by glucagon, noradrenaline, or cyclic AMP increasing agents. The hormonal changes observed in albumin production partially correlate with changes in mRNA levels. This is the first time that cyclic AMP increasing agents are shown to act synergistically with EGF, increasing the expression of this liver specific gene.     

Zonal expression of hepatocytic marker enzymes during liver repopulation

Hepatocytes are metabolically specialised cells displaying distinctive gene expression patterns within the liver lobule. Here, we investigate whether pre-cultured adult rat hepatocytes adopt periportal and pericentral enzyme expression following their transplantation into the regenerating rat liver. Isolated primary rat hepatocytes, representing a mixture of both periportal and pericentral origin, lost expression of carbamoyl phosphate synthetase I (CPS I) and cytochrome P450 subtype 2B1 (CYP2B1) in culture as shown by immunofluorescence and Western blot analysis. Accordingly, urea synthesis and CYP2B1 enzyme activity decreased. Hepatocytes from DPPIV (CD26) wild type rats were cultured for 4 and 7 days, and then transplanted into the livers of CD26 deficient rats following prior treatment with retrorsine and partial hepatectomy to drive selective donor cell proliferation. CD26 positive donor cells engrafted in the periportal regions and grew in clusters expanding into the parenchyma as time proceeded. Ten weeks after transplantation, cells derived from donors surrounding the portal veins expressed CPS I, but not CYP2B1. The reverse was true for CD26 positive cells in close proximity to the central veins displaying immunoreactivity to CYP2B1, but no longer to CPS I. Hepatocytes lose their specific marker enzyme expression in culture. After transplantation, donor hepatocytes proliferate in the host parenchyma whilst acquiring the position-specific enzyme expression of the surrounding periportal and pericentral host hepatocytes. These results indicate the high degree of plasticity of gene expression in hepatocytes subjected to a change in microenvironment.     

Cholangiocyte marker-positive and -negative fetal liver cells differ significantly in their ability to regenerate the livers of adult rats exposed to retrorsine

We have used monoclonal antibodies against cell-surface developmental epitopes in combination with micromagnetic beads to isolate phenotypically defined subpopulations of cholangiocyte marker-positive fetal liver epithelial cells (CMP-FLEC). Differentiation potential was evaluated by injecting cell isolates from dipeptidyl peptidase IV (DPPIV) positive (DPPIV+) Fischer donor rats into the spleen of partially hepatectomized, DPPIV negative (DPPIV-) Fischer host rats exposed to retrorsine. At various time points, liver tissue was harvested and cells in DPPIV+ colonies were phenotyped by immunofluorescence and histochemical protocols. Functional differentiation and liver replacement were determined by comparing donor and host hepatocyte protein expression patterns and DPPIV enzyme activity in extracts from livers of host rats receiving CMP-FLEC. Our results showed that bipotentiality was retained during differentiation and maturation of CMP-FLEC, indicating that the acquisition of ductal morphology and phenotype were not indicative of lineage commitment. CMP-FLEC transplanted into the adult rat liver lost ductal and gained hepatocyte markers, and acquired protein expression patterns in 2D gels with a close similarity (>75% spot match) to host hepatocytes but differing significantly from the transplanted CMP-FLEC cell isolate (<25% spot match). The average size of donor hepatocyte colonies increased with time so that by 1 year, up to 70% of the host rat liver was replaced by CMP-FLEC derived DPPIV+ hepatocytes. Depletion of CMP-FLEC from fetal liver isolates resulted in a marked decrease in adult liver colonization, suggesting that a high percentage of the hepatocyte colonies in animals receiving total fetal liver isolates are derived from CMP-FLEC.     

Differentiation of a hepatic phenotype after heterotropic transplantation of heart, kidney, brain, and skin tissues into liver in F344 rats

While organ-specific stem cells with roles in tissue injury repair have been documented, their pathogenic significance in diseases and the factors potentially responsible for their activation remain largely unclear. In the present study, heart, kidney, brain, and skin samples from F344 transgenic rats carrying the GFP gene were transplanted into normal F344 rat liver one day after an intraperitoneal injection (i.p.) of carbon tetrachloride (CCl(4)) to test their differentiation capacity. The transplantation was carried out by female donors to male recipients, and vice versa. One week after transplantation, GFP antigen-positive cells with phenotypic characteristics of hepatocytes were noted. After two weeks, their extent increased, and at 4 weeks, large areas of strongly GFP-stained cells developed. All recipient livers had GFP antigen-positive hepatocyte cells. PCR analysis coupled with laser capture micro-dissection (LCM) revealed those cells to contain GFP DNA. Thus, our results indicate that tissue stem cells have multipotential ability, differentiating into hepatocytes when transplanted into an injured liver.