A study of liver regeneration using fetal rat liver tissue transplanted into the spleen

The liver morphology of fetal hepatic tissue transplanted into an ectopic location was investigated over one year period. Fetal liver fragments prepared from a maternal rat on the 18th or 19th day of pregnancy were injected into the splenic parenchyma of syngeneic rats using a 21 gauge needle. Histologically, the fetal liver did not essentially show any apparent lobular architecture or cord structure. The transplanted fetal hepatic tissues survived and formed hepatic cords in the spleen instead of undergoing degeneration and necrosis. Three characteristic features became complete during the 4 weeks following transplantation, namely; clumps of hepatocytes with obvious hepatic cords and sinusoids, markedly proliferating bile ducts and proliferating individual hepatocytes. Macroscopic nodules of the hepatocytes on the spleen were seen at about 6 months after transplantation. When the differentiation of the transplanted fetal hepatic tissue was compared with the development of a normal neonatal liver after birth, it was delayed by only about one week, while there was no proliferation of bile ducts in the normal neonatal liver. This experimental model provides a useful system for investigating liver regeneration and the mechanism of cell growth.     

A study of liver regeneration using fetal rat liver tissue transplanted into the spleen

The liver morphology of fetal hepatic tissue transplanted into an ectopic location was investigated over one year period. Fetal liver fragments prepared from a maternal rat on the 18th or 19th day of pregnancy were injected into the splenic parenchyma of syngeneic rats using a 21 gauge needle. Histologically, the fetal liver did not essentially show any apparent lobular architecture or cord structure. The transplanted fetal hepatic tissues survived and formed hepatic cords in the spleen instead of undergoing degeneration and necrosis. Three characteristic features became complete during the 4 weeks following transplantation, namely; clumps of hepatocytes with obvious hepatic cords and sinusoids, markedly proliferating bile ducts and proliferating individual hepatocytes. Macroscopic nodules of the hepatocytes on the spleen were seen at about 6 months after transplantation. When the differentiation of the transplanted fetal hepatic tissue was compared with the development of a normal neonatal liver after birth, it was delayed by only about one week, while there was no proliferation of bile ducts in the normal neonatal liver. This experimental model provides a useful system for investigating liver regeneration and the mechanism of cell growth.     

小鼠胎肝干细胞抗原阳性细胞移植后向神经组织细胞分化

目的了解小鼠胎肝干细胞是否具有向神经组织细胞分化的潜能。方法　采用免疫磁珠法分离雄性胎鼠肝脏的千细胞抗原1阳性(Sca-1 )细胞;将8-10周的C57BL/6J雌性小鼠用致死剂量(10.0Gy)放射线照射,然后尾静脉注射Sca-1 细胞2.0×103。Sca-1 细胞移植后2、4、6个月后,将受者脑组织作免疫组织化学和FISH双染色检测。结果移植后雌性受鼠脑组织内存在大量Y染色体阳性细胞;统计比较移植后2、4、6个月时,受者脑组织中Y染色体阳性细胞百分数无明显差别。免疫组织化学分析显示,这些Y染色体阳性细胞表达神经组织细胞特异标志,如神经元核特异蛋白(Neu N)、神经纤维细丝蛋白(NF-M)、微管蛋白Ⅲ(TuJ-1)或者胶质纤维酸性蛋白(GFAP) 等。Y染色体阳性细胞约占脑组织总细胞数的(4.5±0.5)%.其中Y染色体和Neu N均阳性的细胞为(1.2±0.3)%、Y染色体和GFAP均阳性的细胞为(1.0±0.2)%。结论经尾静脉移植小鼠胎肝Sca 1 细胞能迁移进入经致死剂量全身照射的雌性小鼠脑组织,并且能分化成神经组织细胞,且能长期存活(存活至少6个月以上)。这为神经组织细胞发育分化机制研究和神经系统疾病的治疗提供了理论依据和研究材料     

Morphology and function of isolated hepatocytes transplanted into rat spleen

Hepatocytes isolated by the collagenase digestive method were transplanted into the spleens of syngeneic rats. Morphology and function of the hepatocytes in the spleen were investigated for 12 to 17 months after transplantation. The transplanted hepatocytes proliferated and reconfigured in the spleen without direct perfusion of portal venous blood and with the presence of an intact host liver. Fourteen to 17 months after transplantation, the hepatocytes which had formed a demarcated nodule occupied approximately 40% of the area of the splenic parenchyma without undifferentiation on microscopic examination. However, the weight of the hepatized spleen did not increase beyond the weight of a normal spleen and the weight of the host liver that had normal morphology also did not differ from a normal liver. Light and electron microscopic studies demonstrated differentiated cord structure and normal architecture for each heptocyte. Furthermore, the hepatized spleen synthesized albumin and glycogen as demonstrated by immunofluorescence and histochemical studies. Ammonia tolerance and indocyanine green clearance tests revealed functioning hepatocytes in the spleen proper. These results indicate that our experimental model lends itself well to investigations in cell growth mechanism and that hepatocellular transplantation has potential clinical application to compensate for impaired hepatic function.     

Histologic differentiation of human fetal pancreatic explants transplanted into nude mice

The transplantation of human fetal pancreas has been suggested as a means of treatment of insulin-dependent diabetes in man. We have obtained human fetal pancreata during the second trimester of pregnancy and transplanted 1-mm3 explants subcutaneously (s.c.) into both diabetic and nondiabetic nude mice, some of the tissue being cultured in vitro before implantation. These implants coalesced and grew. They were removed at intervals up to 37 wk later and showed selective differentiation of endocrine tissue that normally occurs in the fetus and neonate, with formation of bipolar, mantle, and mature islets. There was growth of this endocrine tissue with significantly more islets than in the freshly stained fetal pancreas assuming an average dimension larger than 150 micron, which is the reported mean diameter of a neonatal islet. Duct and fibrous tissue remained viable, but there was no definitive acinar tissue seen. The pancreata uncultured before implantation reached a larger size than that attained by those implants cultured before being transplanted, the difference probably being the amount of ductular and mesenchymal tissue still present. Of those glands cultured before transplantation, the longer the period of culture, the smaller the size the implants reached. Culture beyond 3 wk in vitro made it difficult to macroscopically locate the implant. These data show that, in human fetal pancreas removed from its usual environment, both selective differentiation of the endocrine component and growth of the islets can occur.     

Evidence of metabolic activity of adult and fetal rat hepatocytes transplanted into solid supports.

This study was undertaken to assess the metabolic effect of fetal and adult hepatocyte transplantation in the Gunn rat, genetically incapable of bilirubin conjugation. A comparison was made between fetal and adult hepatocytes transplanted into the spleen, and those injected into polytetrafluoroethylene (PTFE) solid supports that had previously been implanted intraperitoneally. Between 4 and 12 weeks after intrasplenic transplantation of adult liver cells, serum bilirubin was significantly decreased when compared with control animals (39.6 +/- 5.6%; P less than 0.01 vs. controls). Intrasplenic transplantation of fetal hepatocytes resulted in a maximal decrease of 33.2 +/- 9.1% at 8 weeks postoperatively (P less than 0.02 vs. controls). Similar declines of serum bilirubin levels were found after transplantation of adult or fetal liver cells into the solid supports. At 12 weeks after transplantation, bilirubin conjugates were detectable in the bile of all animals that underwent intrasplenic hepatocyte transplantation and in 60% of those that underwent the solid support procedure, whereas none could be detected in control animals. Histological evidence of surviving cells was obtained in all but one animal at 12 weeks, and confirmed at 12 months postoperatively. It is concluded that the PTFE solid support technique offers an attractive alternative to the intrasplenic route, and that both fetal and adult hepatocytes, transplanted in either way still exert their conjugating activity after 12 weeks.     

The isolated perfused rat spleen. An original method for studying the function of hepatocytes transplanted into the spleen

The aim of this study was to assess directly the function of isolated hepatocytes 1 year after transplantation into the spleen, using an original model of isolated rat-spleen perfusion. Three specific liver functions, albumin synthesis, indocyanine-green clearance, and antipyrine oxidation, were studied. Five x 10(6) isolated hepatocytes were injected into the spleen of syngenic Wistar-Furth rats. One year later, splenectomy was performed, and the splenic pedicle was carefully isolated in order to allow a selective ex vivo perfusion for 3 hr. De novo albumin synthesis was studied by qualitatively using immunoelectrophoresis and autoradiography, and quantitatively using (35S)-methionine incorporation in albumin. De novo albumin synthesis was observed in spleens containing transplanted hepatocytes but not in controls (P less than 0.001); (35S)-methionine incorporation was significantly higher in spleens containing transplanted hepatocytes than in controls (132 +/- 67 cpm/spleen/hr vs. 14 +/- 6 cpm/spleen/hr, P less than 0.001). Antipyrine clearance was significantly higher in spleens with transplanted hepatocytes than in controls (67.4 +/- 4.9 microliters/min/g vs. 0.2 +/- 0.4 microliters/min/g, P less than 0.01). No statistically significant difference was observed with indocyanine-green clearance (4.2 +/- 6.0 microliters/min/g, vs. 5.2 +/- 5.1 microliters/min/g, P greater than 0.05); this was probably due to the absence of compartmentation between the sinusoid and biliary sectors in this model. In conclusion, using this original isolated rat-spleen perfusion model, it was directly observed that 1 year after transplantation, intrasplenic hepatocytes can perform two liver-specific functions, i.e., de novo albumin synthesis and antipyrine clearance.     

Anatomic and physiologic characteristics of transplanted fetal rat intestine.

Avascular segments of fetal rat intestine transplanted to the subcutaneous tissues of host syngeneic rats will become vascularized and grow. This study more fully characterizes this tissue, which we call "neogut," and compares it to normal rat small intestine. Anatomy was studied with light microscopy and scanning and transmission electron microscopy; transport and electrophysiologic parameters were measured in full-thickness pieces of tissue mounted in Ussing chambers; motility patterns, including slow wave and spike activity, were recorded. Subtle anatomic differences (shortened villi and microvilli) were noted in neogut compared to normal small bowel. Both neogut and normal rat ileum demonstrated net mucosal to serosal transport of d-glucose; the magnitudes of the electrophysiologic parameters (PD, Isc, and G) were less in neogut than in ileum. Slow-wave frequency of neogut was slightly less than native small bowel while spike activity was increased. These data show that neogut has structural and physiologic characteristics similar to normal rat small bowel and offers hope that this tissue may provide a nutritionally useful accessory gut for the patient with critical short-gut syndrome.     

Beta1 integrin as a xenoantigen in fetal porcine mesencephalic cells transplanted into the rat brain

Xenografts of fetal porcine mesencephalic cells implanted into the rat striatum are generally rejected within several weeks. The fetal donor mesencephalon predominantly consists of neurons, but also contains microglial and endothelial cells, which are more immunogenic. In the present work, we investigated the occurrence of donor endothelial cells in grafts of porcine mesencephalic cells implanted into the rat striatum. Pig endothelial cells were monitored by immunochemical methods, using a monoclonal antibody (mAb) that recognizes a peptidic epitope of the porcine beta1 integrin, and isolectin IB4, for the staining of the Galalpha1,3Gal epitope. The analysis also involved the detection of the pig hyaluronate receptor CD44, and the cell adhesion molecule CD31. The anti-beta1 integrin mAb revealed endothelial-like cells in grafts of porcine mesencephalic cells as soon as 1 week after implantation. A similar staining pattern was obtained with the IB4 lectin. Unlike aortic endothelial cells, these pig brain-derived endothelial-like cells were not recognized by the anti-CD44 antibody. They also failed to express the CD31 adhesion molecule, a fact which suggests that they remained poorly mature, even in grafts maintained during 45 days in immunosuppressed rats. Interestingly, a strong expression of beta1 integrin immunoreactivity was noticed in a large proportion (80%) of the cells freshly dissociated from the fetal pig mesencephalic tissue. The immunoreactivity decreased progressively after transplantation of the cells into the rat brain. This observation suggests that dissociated neuroblasts are capable of a temporary expression of beta1 integrin. This molecule is known to participate in the process of cell sorting and migration in the developing brain. Hence, its expression could be the hallmark of a rescue mechanism triggered by the disruption of the cell/matrix interactions during the dissociation of the fetal mesencephalon. This disruption might account for part of the dramatic cell death process that occurs during the manipulation of the donor tissue.     

Immunohistological observations of rat fetal pancreas allografts transplanted into unmodified and cyclosporine-treated recipients

Administration of CsA (15 mg/kg/day) prolonged the survival of DA (RT1a) rat fetal pancreas transplanted to the renal subcapular site of both PVG (RT1c) and Lewis (RT1(1] recipients. Sections of fetal pancreas examined 40 days after transplantation into allogeneic CsA-treated recipients showed growth and development of the fetal pancreas tissue, and the presence of numerous insulin-containing islets. CsA treatment prevented the induction of MHC antigen within allografts. Whereas at day 4, both rejecting and CsA treated grafts showed donor class I MHC expression on duct epithelium and islet cells, only rejecting grafts displayed class I MHC induction on acinar cells. Rejecting grafts showed strong induction of class II MHC antigen expression on duct epithelium from day 4 onward but this was completely prevented by CsA treatment. Islet cells in both rejecting and CsA treated allografts remained class II-negative throughout. CsA also resulted in a reduction in the day 6 cellular infiltrate of allografts (median area leukocyte infiltrate reduced from 43% to 10%) with a marked decrease in the number of MRC OX-8-positive cells. These results show a favorable effect of CsA on rat fetal pancreas allografts with a reduction in MHC antigen expression within the graft and prolonged survival of insulin-rich endocrine tissue.     

Transplantation of amniotic epithelial cells into fetal rat liver by in utero manipulation

It has been hoped that amniotic epithelial cells would be a gene carrier to neural and hepatic tissue, because of 1) the presence of neural and hepatic stem-like cells, 2) the ability to cryopreserve them, 3) long-term survival in the transplanted site, and 4) few ethical problems concerning procurement. But transplantation of a sufficient number of cells to adult tissue needs large-scale cell supply and may lead to vascular embolism. We attempted transplantation of amniotic epithelial cells into fetal liver, because 1) the fetal liver is at the proliferative stage, 2) the number of cells required is small, and 3) the fetal stage is advantageous for the induction of immunological tolerance. Amniotic epithelial cells from day 18.5-20.5 fetuses were transfected with adenoviral AdlacZ and harvested to inject into fetal rat liver of the syngeneic strain (day 18.5-20.5). The efficacy of cell transplantation into the liver increased in the order: intraplacental < intraumbilical vein < intrahepatic route. LacZ-transfected amniotic cells (1-8 x 10(5) cells), hepatocytes (5 x 10(5) cells), or AdlacZ vector solution (1.7 x 10(7) pfu) were injected through the uterine membrane into the liver. Transplanted cells formed a cellular mass and survived for up to 14 days after birth, whereas lacZ-transfected cells were rapidly decreased after the injection of AdlacZ vector or rat hepatocytes as a gene carrier so that the use of amniotic epithelial cells as a gene carrier will result in long-term expression of exogenous genes in the liver.     

Promoting effect of endogenous TSH on carcinogenesis of rat thyroid transplanted into the spleen

In an attempt to clarify a modifying effect of TSH on thyroid tumorigenesis, we carried out intrasplenic thyroid autotransplantation in rats treated with a thyroid carcinogen, diisopropanolnitrosamine (DIPN). Experiments comprised 3 groups of Wistar male rats: Group I (effective no., 15), total thyroidectomy plus hemithyroid transplantation 2 weeks after subcutaneous injection of DIPN at a dose of 2.4g/kg BW; group II (effective no., 22), DIPN alone; group III (effective no., 18), transplantation alone. The thyroid lesions of all rats at week 28 were examined by histopathology and enzyme histochemistry for gamma-glutamyl transpeptidase (GGT). Periodical radioimmunoassay for serum T3, T4 and TSH revealed low levels of T3 & T4 for at least 10 weeks and increased level of TSH until week 18 after the transplantation. Altered foci (AF) occurred in 36% and 6% of rats in group I and group II, respectively, whereas neoplastic nodules (NN) were found in 53% of rats only in group I. Both AF and NN displayed a positive reaction for GGT which was considered to be a useful marker for preneoplastic and neoplastic thyroid lesions. The present data are indicative that increased serum level of TSH for a significant period may exert a promoting effect on thyroid tumorigenesis.     

Blood flow to transplanted fetal rat intestine

Avascular fetal rat intestine becomes vascularized and grows and develops certain properties of normal small intestine when transplanted into the subcutaneous tissues of host syngeneic rats. We quantified the blood flow to this transplanted intestine (called "neogut") to clarify the role of angiogenesis in its growth and development. Additionally, we correlated blood flow and the growth of neogut into either patent tubular segments or large saccular segments with associated strictures. Blood flow was studied using 141Ce or 85Sr labeled microspheres. Blood flows are expressed as mean +/- SEM in (ml/min X g). Neogut blood flow (0.19 +/- 0.02) was greater than that of adjacent subcutaneous tissue (0.03 +/- 0.00, P less than 0.01) but less than that of native small bowel (0.85 +/- 0.07, P less than 0.01). The blood flow to the large saccular segments (0.10 +/- 0.01) was less than that to the tubular portions (0.27 +/- 0.02, P less than 0.01). The weight of neogut was comparable to that of native small bowel. These findings demonstrate that neogut implants stimulate angiogenesis to a degree which may be sufficient for it to absorb nutrients. Furthermore, ischemia is present in large saccular neogut segments with associated strictures. Additional investigation appears warranted to see if neogut can serve as a clinically useful small bowel substitute.     

Can hepatocytes proliferate when transplanted into the spleen? Demonstration by autohistoradiography in the rat

The aim of this work was to study hepatocyte multiplication after transplantation into the spleen, in order to apply this technique to the treatment of chronic liver disease. Hepatocytes isolated by an in situ collagenase perfusion technique in Wistar Furth rats were injected into the splenic parenchyma of three groups of syngeneic rats: controls with normal liver (group 1), 75% hepatectomies (group 2), and end-to-side portacaval shunts (group 3). The proliferation of transplanted hepatocytes was studied by autohistoradiography after the intraperitoneal administration of 0.6 microCi/g body weight of [3H]-thymidine, at 1, 3, 7 and 15 days after transplantation of hepatocytes. Significant incorporation of [3H]-thymidine by the transplanted hepatocytes during the study period was observed mostly in groups 2 and 3. The incorporation, although delayed was sustained and of greatest magnitude in the portacaval-shunted animals. The ability of transplanted hepatocytes to proliferate in the spleen, particularly after a portacaval shunt, indicates that this procedure may have therapeutic applications in the treatment of chronic liver disease.     

HLA-DR typing of fetal human spleen and liver lymphoblastoid cells transformed by Epstein-Barr virus

Because few HLA-DR-positive cells are present in the fetal spleen and liver, full HLA typing cannot be performed. However, B lymphocyte precursors can be transformed with Epstein-Barr virus to produce lymphoblastoid cells which express HLA-A, B, and DR antigens. Successful transformation was achieved, usually with spleen and liver, in nine fetuses aged from 15 to 18 weeks, mostly within 7 to 14 days of initiation of the cultures. Spleen-derived lymphoblasts were more suitable for typing because of their greater homogeneity and higher viability. Tissues from two 13-week fetuses from prostaglandin-induced abortions and from a spontaneously aborted 22-week fetus could not be transformed. This is probably attributable to prolonged ischemia before the tissues were obtained but, in the 13-week fetuses, absence of B lymphocyte precursors was not excluded. HLA-DR typing may be useful in obtaining well matched donor-recipient pairs in fetal pancreatic islet transplantation.