Hepatic differentiation of mouse embryonic stem cells in a bioreactor using polyurethane/spheroid culture.

BACKGROUND: We have previously developed a hybrid artificial liver (HAL) using polyurethane foam (PUF)/hepatocyte spheroid culture. The PUF-HAL has been successfully scaled up to a clinical level. However, one of the most difficult problems for clinical application of HALs is obtaining a cell source. We now focused our attention on embryonic stem (ES) cells as a potential source for HAL. In this study, we investigated the differentiation of mouse ES (mES) cells into functional hepatocytes in the PUF-HAL module. METHODS: The PUF-HAL module included a cylindrical PUF block having many capillaries for medium flow. mES cells were immobilized in the module. To induce hepatic differentiation, growth factors were added to the culture medium. We evaluated cell density, gene expression analysis, and liver-specific functions. RESULTS: mES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of PUF. mES cells proliferated by 20 days, achieving a high cell density (about 1 x 10(8) cells/cm3 PUF). Differentiating ES cells expressed endodermal-specific genes such as alpha-fetoprotein, albumin, and tryptophan 2, 3-deoxygenase. The activity of ammonia removal of mES cells per unit volume of the module was detectable by 15 days and increased with culture time. Maximal expression levels were comparable to those of primary (porcine and human) hepatocytes. SUMMARY: mES cells immobilized in the PUF module expressed liver-specific functions at high level, because of high cell density in culture and hepatic differentiation. These results indicated that PUF module-immobilized mES cells may be useful as a biocomponent of HALs.

Hepatic differentiation of embryonic stem cells in HF/organoid culture.

OBJECTIVE: The use of embryonic stem cells (ES cells) has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional mature cells from ES cells in large quantities. We applied HF/organoid culture, where cultured cells formed cylindrical multicellular aggregates (organoids) in the lumen of hollow fibers, to mouse and cynomolgus monkey ES cells for hepatic differentiation. MATERIALS AND METHODS: ES cells were injected into hollow fibers. The hollow fibers were centrifuged to induce organoid formation and cultured in medium including factors for hepatic differentiation. To determine the characteristics of cells in the bundle, we evaluated gene expression and liver-specific functions. RESULTS: ES cells immobilized inside hollow fibers proliferated and formed cylindrical organoids. In mouse ES cell cultures, the expression of mRNAs of hepatocyte-specific genes increased with culture time. Ammonia removal activity detected at 15 days increased with culture time. Albumin secretion activity detected at 12 days increased by 21 days. In cynomolgus monkey ES cell cultures, ES cells showed spontaneous ammonia removal functions. The maximum levels of these functions per unit volume of the hollow fibers were roughly comparable to those of primary hepatocyte-organoids. CONCLUSIONS: ES cells differentiated into hepatocyte-like cells using the organoid culture technique. The results indicated that the combination of ES cells and an organoid culture technique was useful to obtain mature hepatocytes.

Digoxin transport by renal proximal tubule cells is enhanced by adhesive synthetic RGD peptide.

INTRODUCTION: The dialyzer apparatus has been widely used as an artificial kidney in medical treatment. However, side effects such as amyloidosis have occurred during long-term treatment. Therefore, we focused on developing a hybrid artificial kidney with a filtration and reabsorption apparatus, but it was found that cells spread extensively and it is difficult to maintain a uniform monolayer with a regular cell shape on a collagen-coated substrate. The purpose of this study was to improve cell adhesion, uniform stable monolayer formation and active transport function by immobilization of arginine-glycine-aspartic acid (RGD) on the culture substratum. MATERIALS AND METHODS: Polycarbonate semipermeable membranes were coated with collagen, fibronectin, laminin and synthetic polypeptide, including RGD (Pronectin F). Cell adhesion and digoxin transport were estimated using a renal proximal tubule cell line that overexpressed the P-glycoprotein gene. RESULTS AND DISCUSSION: Under initial and confluent conditions, immobilized cell density in Pronectin F-coated wells was higher than that under other conditions. Transepithelial electrical resistance and digoxin transport activity on Pronectin F-coated membranes were the highest of all conditions. This might have been caused by uniform cell morphology and high cell density.

Differentiation effects by the combination of spheroid formation and sodium butyrate treatment in human hepatoblastoma cell line (Hep G2): a possible cell source for hybrid artificial liver.

The aim of this study was to investigate the feasibility of human hepatoblastoma cell line (Hep G2), which differentiates by spheroid formation, and treatment with sodium butyrate (SB) as a cell source for hybrid artificial liver (HAL). Hep G2 spontaneously formed spheroids in polyurethane foam (PUF) within 3 days of culture and restored weak ammonia removal activity. Treatment with SB, which is a histone deacetylase inhibitor, further increased the ammonia removal activity of Hep G2 spheroids in a concentration-dependent manner. The activation of ornithine transcarbamylase--a urea cycle enzyme--was significantly related to the upregulation of ammonia removal by spheroid formation, but scarcely contributed to the further upregulation following SB treatment. In contrast with ammonia removal, treatment with SB reduced the albumin secretion of Hep G2 spheroids in a concentration-dependent manner. In the PUF-HAL module in a circulation culture, the ammonia removal rate and albumin secretion rate (per unit volume of the module) of Hep G2 spheroids treated with 5 mM SB were almost the same as those of primary porcine hepatocyte spheroids. These results suggest that simultaneous use of spheroid formation and SB treatment in Hep G2 is beneficial in enhancing the functions of human hepatocytes with potential applications in regenerative medicine and drug screening.

Predominance of macrophage-colony formation in human cord blood.

To clarify understanding of the characteristics of granulopoiesis during early infancy, we investigated the colony-forming unit in culture (CFUC), in cord blood (CB), in peripheral blood (PB), and in bone marrow (BM) of infants. The macrophage-colonies (M-colonies) were always predominant in the study of colony formation by CB and PB cells from infants, regardless of the source of the CSF used. This result contradicts previous reports which concluded that the colonies were mainly granulocytic type. In the colony formation by BM cells from one-month-old infants, the ratio of M-colonies was significantly higher than that of adult BM cells. The CSF produced by CB and PB mononuclear cells (MNC) from infants stimulated neutrophil-colony formation in the adult BM cells.

The effect of hydrocortisone on human granulopoiesis in vitro with cytochemical analysis of colonies.

In order to clarify the regulation of granulopoiesis by hydrocortisone in humans, we investigated the effect of glucocorticoids on the formation of granulocyte and/ or macrophage colonies. By means of the dual esterase staining techniques applied to whole mount preparations of agar culture dishes, we examined the granulocyte-macrophage colony type. It was revealed that hydrocortisone stimulated the formation of neutrophil-containing colonies and inhibited macrophage colony formation. There was a significant increase in neutrophil colonies when the cells were preincubated with hydrocortisone for only 24 h. Delayed addition of hydrocortisone to the cultures was less effective in increasing the proportion of neutrophil colonies than addition at the beginning of culture. Moreover, addition of hydrocortisone to T-lymphocyte- and phagocyte-depleted bone marrow cells also increased the number of neutrophil colonies and decreased macrophage colonies in the presence of CSF. These results suggest that hydrocortisone may affect granulocyte-macrophage precursors (CFUGM) in an early period of their differentiation.

Characterization of hemopoietic precursor cells in juvenile-type chronic myelocytic leukemia.

In order to study the pathogenesis of juvenile-type chronic myelocytic leukemia (CML), we examined the colony-forming capacity and colony composition in the bone marrow (BM) and peripheral blood (PB) of three children with juvenile-type CML. Large numbers of granulocytes and macrophage colonies were formed by BM and PB cells. Whole agar culture staining revealed that especially macrophage colonies increased in comparison with normal controls. After removal of carbonyl iron-laden cells with a magnet or deprivation of cells adherent to glass from BM cells, the number of macrophage colonies markedly reduced in comparison with the number of colonies formed by untreated BM cells, suggesting that some of the macrophage colony-forming cells (M-CFC) may have phagocytic and/or adherent activity. Radiation sensitivity and thymidine suicide rate of these M-CFC were not different from those of granulocyte colony-forming cells (G-CFC). The predominance of M-CFC in juvenile-type CML may be one of the reflections of fetal-type myelopoiesis since M-CFC are predominant in cord blood and PB in the neonatal period. Moreover, considerable numbers of erythroid-colony-forming units (CFU-E) were present in PB of all patients. It may be concluded that juvenile-type CML is a panmyelopathy with the predominance of M-CFC.

Synthesis and transport characterization of alginate/aminopropyl-silicate/alginate microcapsule: application to bioartificial pancreas.

To develop a novel type of immunoisolation membrane for a microcapsule-shaped bioartificial pancreas, we attempted to use a sol-gel synthesized silicate. An aminopropyl-silicate membrane derived from 3-aminopropyltrimethoxysilane and tetramethoxysilane was formed on Ca-alginate gel beads via electrostatic interaction. The positively charged amino groups remaining on the surface of the resultant gel beads were neutralized by immersion in an aqueous Na-alginate solution. From measurements of the partition coefficients and effective diffusivities of different substances to the gel beads, it was found that the aminopropyl-silicate membrane prepared under optimized composition of silicon alkoxide precursors successfully rejected gamma-globulin, giving good permeability to substances having a low molecular weight. Islets could be encapsulated within the newly developed microcapsules while retaining their ability to secrete insulin.

Newly developed aminopropyl-silicate immunoisolation membrane for a microcapsule-shaped bioartificial pancreas.

An aminopropyl-silicate membrane, synthesized from tetramethoxysilane (TMOS) and 3-aminopropyltrimethoxysilane (APTrMOS) by the sol-gel method, was formed onto Ca-alginate gel beads via electrostatic interactions. The permeability of the membrane could be controlled easily by changing the molar ratio of both the precursors ([APTrMOS]/[TMOS]). The aminopropyl-silicate membrane prepared at a molar ratio of 2.40 rejected gamma-globulin and BSA successfully, whereas it permeated ovalbumin. This result indicates that the molecular weight cutoff point of this newly developed aminopropyl-silicate membrane is approximately 60 kDa.

Evaluating the performance of a hybrid artificial liver support system with a recoverable hepatic failure rat model.

To evaluate the performance of an artificial liver, we created a recoverable hepatic failure rat model. This involves a 30-60 minute warm ischemia, via clamping, of one-third of the liver with a partial (two-thirds) hepatectomy. Variations on this method provide for the possibility of several modes of hepatic failure. Survival time of the rats was prolonged (35%) by applying our hybrid artificial liver. However, the extracorporeal circulation is a considerable burden to the rat. Therefore, we need to apply the hybrid artificial liver intermittently and repeatedly.

Efficacy of a polyurethane foam/spheroid artificial liver by using human hepatoblastoma cell line (Hep G2).

We invesigated the availability of human hepatoblastoma cell line (Hep G2), compared with human primary hepatocytes (HH) and porcine primary hepatocytes (PH), as a cell source for the hybrid artificial liver support system (HALSS) by using polyurethane foam (PUF). All three kinds of hepatocytes spontaneously formed spherical multicellular aggregates (spheroids) of 100-200 microm diameter in the pores of PUF within 3 days of culture. In a PUF stationary culture, Hep G2 spheroids recovered the ammonia removal activity that was lost in monolayer culture, although the removal for each unit cell number was about one tenth that of HH spheroids and about one eighth of PH spheroids. The synthesis activities of albumin and fibrinogen of each unit cell number of Hep G2 were also upregulated by PUF spheroid culture, and were about twice as high as in monolayer culture. The albumin secretion activity of Hep G2 spheroids was almost the same as that of PH spheroids. HH scarcely secreted these proteins in this experiment, probably because they were cultured in a serum-free medium. In the PUF module in a circulation culture, HH had high ammonia removal and low synthesis activities similar to stationary culture. Hep G2 proliferated to a high cell density, such as about 4.8 x 10(7) cells/cm3-module at 10 days of culture. Although Hep G2 spheroids had low ammonia removal activity in each cell, the removal rate in the PUF module was almost the same as for PH at 7 days of culture because of the high cell density culture by cell proliferation. The albumin secretion rate by Hep G2 in the PUF module also increased with cell proliferation and was about 10 times higher than the initial for the rate for PH at 7 days of culture. These results suggest that Hep G2 is a potential cell source PUF-HALSS.

Polyurethane foam/spheroid culture system using human hepatoblastoma cell line (Hep G2) as a possible new hybrid artificial liver.

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we used human hepatocytes with replication capacity (fetal hepatocytes, Hep G2, and Huh 7) in a polyurethane foam (PUF)/spheroid culture system in vitro, and analyzed liver functions such as ammonia removal and albumin synthesis capacity; results were compared to those of porcine hepatocytes. Human fetal hepatocytes, Hep G2, and Huh 7 formed spheroids spontaneously within 24 h in a PUF/spheroid culture system; ammonia removal activity (micromol/10(6) nuclei/h) was upregulated, as was albumin synthesis activity (microg/10(6) nuclei/day). In particular, Hep G2 spheroids demonstrated high ammonia removal and albumin synthesis activities: 85% of the ammonia removal activity and 171.7% of the albumin synthesis activity of porcine hepatocytes in the monolayer culture. These results indicate the possibility of the development of a multicapillary PUF (MC-PUF) packed-bed culture system of hepatocyte spheroids as a HALSS using Hep G2.

Ultrasonic aspiration in coronary artery surgery.

With the aid of an ultrasonically powered aspiration device, the coronary artery to undergo distal anastomosis during aortocoronary bypass grafting can be exposed in the beating heart prior to institution of cardiopulmonary bypass and systemic administration of heparin. The thick adipose tissue and bridging muscles of the superficial layer are removed with almost no bleeding, leaving the vascular elements intact. The optimal site for anastomosis of the coronary artery is readily exposed and confirmed, and aortic cross-clamp time is minimized.

Stereoselective synthesis of a blood group A type glycopeptide present in human blood mucin.

N,N-Dimethyl-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1-->3)-O- [(alpha-L-fucopyranosyl)-(1-->2)]-O-(beta-D-galactopyranosyl)-(1-->3)-O- (2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1-->3)-L-serine, a core I glycotetraosyl peptide structure and a predominant substructure in complex glycan-glycoproteins present in human blood group A ovarian mucin, was synthesized for the first time. The title compound was synthetically accomplished via the following key manoeuvres: regio- and stereo-controlled construction of the alpha-GalNAc-(1-->3)-Gal synthon, stereoselective glycosylation generating a alpha-GalN3-(1-->3)-Ser glycopeptide synthon and alpha-selective fucosylation towards an acceptor which was derived from glycosylation of the latter two synthons. An alternative route to that of the latter, to synthesize a fully protected equivalent of the title compound, involving the coupling of a alpha-GalNAc-(1-->3)-beta-Gal-(1-->3)-GalN3X synthon to an aglycon serine derivative, is described herein.

Evaluation of left ventricular posterior wall movement after open mitral commissurotomy by echocardiogram: with reference to the effect of papilloplasty.

Left ventricular posterior wall movement in 20 patients with mitral stenosis (MS) was measured using M-mode echocardiogram in order to evaluate the improvement of myocardial function after open mitral commissurotomy (OMC) and compared between the cases with (10 patients) and without (10 patients) papilloplasty. The maximum left ventricular end-diastolic posterior wall velocity (LVPEVdmax) was increased from 71 +/- 12 to 90 +/- 16 mm/s in OMC patients (p less than 0.01) and from 59 +/- 19 to 101 +/- 28 mm/s in OMC + P patients (p less than 0.001). The maximum left ventricular systolic posterior wall velocity (LVPWVsmax) showed an increase from 51 +/- 9 to 62 +/- 10 mm/s in OMC patients (p less than 0.02) and from 48 +/- 10 to 69 +/- 8 mm/s in the OMC + P group (p less than 0.001). The mean LVPWVs increased from 35 +/- 8 to 48 +/- 8 mm/s in the OMC + P group (p less than 0.01). These parameters correlate fairly well with stroke volume index (SVI), ejection fraction (EF), and fractional shortening (FS) derived from internal LV dimensions. Thus, the posterior wall movement may prove to be useful as an index for evaluating the improvement of LV function after OMC.

Aneurysm of patent ductus arteriosus in an adult case: findings of cardiac catheterization, angiography, and pathology.

A 42-year-old male was admitted to our hospital for evaluation of a left precordial continuous murmur. Results of catheterization revealed a step-up of oxygen content in the pulmonary arteries and the calculated left-to-right shunt flow was 40% of the pulmonary arterial flow. Furthermore, aortography unexpectedly revealed an aneurysm of the patent ductus arteriosus. During surgery, the aneurysm was discovered to arise from the frontal wall of the ductus arteriosus, and histological observation showed focal necrosis and mucoid degeneration of the media of the aneurysmal wall in contrast to a thickened intimal fibroelastosis of the adjacent ductal wall. This is presumed to be the first adult case of patent ductal aneurysm ever reported in which antemortem diagnosis and surgical treatment were successfully conducted. Our case may suggest that the fragility of the ductal wall following the structural change in an incomplete closing process is considered as a potential pathogenesis of ductal aneurysm formation.

Temporary arteriovenous fistula for venous reconstruction using synthetic graft: a clinical and experimental investigation.

This report discusses venous reconstructive surgery combined with a temporary distal arteriovenous fistula creation for ilio-femoral vein obstruction. One patient who had long standing, high degree edema of the left lower extremity due to postphlebitic syndrome and four patients who had acute occlusion of the iliac vein were treated by means of extra-anatomical femoro-femoral vein bypass grafting combined with temporary distal femoro-saphenous arteriovenous (a-v) fistula creation. An expanded polytetrafluoroethylene (EPTFE) graft was selected for this procedure instead of a cross-over saphenous vein. The surgically created a-v fistula was effective in keeping the implanted grafts patent for a long time in three out of the five patients. In animal experiments, the bilateral iliac veins of mongrel dogs were replaced with several kinds of synthetic graft. Femoro-femoral a-v fistulae were made only on the left side. In 26 animals, 84.6% of the synthetic grafts on the a-v fistula side were patent, whereas only 11.5% of the grafts on the control side were patent. This investigation clearly demonstrates that the increased blood flow velocity through the created distal a-v fistula preserved the patency of synthetic vein grafts.

Left ventricular hypertrophy due to aortic bypass grafting with a long prosthesis.

Progressive left ventricular hypertrophy was observed following operations in which long bypasses were inserted from the ascending aorta to the abdominal aorta for aneurysm exclusion in cases of thoracic aortic aneurysm. This effect was not observed after operations in which there was grafting with a short prosthesis for abdominal aortic aneurysm, wrapping and trans-section of the dissecing aneurysm. Clinical and experimental studies of the hemodynamic aspects suggest that the left ventricular hypertrophy was caused by an increase in systolic blood pressure at the aortic root due to the low distensibility of the long graft, a decrease in the Windkessel effect by exclusion of the proximal aorta and energy loss by turbulence formation of the proximal anastomotic area. Therefore, the distensibility or compliance at the long prosthesis seems to be very important in cases with long grafts of the aorta.

Modulation of immunologic reactions between cultured porcine hepatocytes and human sera.

In the clinical application of a hybrid artificial liver system using porcine hepatocytes, some immunologic reactions occur between human serum and porcine hepatocytes. In this study, we investigated the immunologic mechanisms of the cytotoxic reactions, and we tried to inactivate the human serum cytotoxicity by heating the serum or the addition of nafamostat mesilate (NM). Immunologic reaction between human serum and porcine hepatocytes by evaluating the immunochemical response against human IgM, IgG, and C3 was investigated. The immunochemical analysis of inactivation by heated human serum (56 degrees C, 30 min) and adding NM were performed. The evaluation of serum cytotoxicity was as follows: when porcine hepatocytes were cultured with heating the human serum or the addition of NM, the survival ratio was observed. Immunochemical reactions against human C3 were all positive, but positive reaction against human IgM occurred in only one case (5%); those against human IgG were all negative. Both heating the serum and adding NM inhibited the immunochemical reaction of human C3. The inhibition of human C3 with NM was dependent on that concentration. Both heating of the serum and adding NM to the medium decreased damage of porcine hepatocytes. An immunologic reaction between human serum and porcine hepatocytes in a porcine bioartificial liver clearly occurred, and this reaction was controlled by heating the serum and adding NM. We believe that NM is useful in the clinical application of our hybrid artificial liver system.

Development of co-culture system of hepatocytes with bone marrow cells for expression and maintenance of hepatic functions.

In this study, a co-culture system of hepatocytes and bone marrow cells (BMCs) was developed and characterized for the expression and maintenance of ammonia metabolism and albumin secretion activities. A culture medium supplemented with epidermal growth factor, insulin, L-proline, hydrocortisone and 20% (v/v) heat-inactivated fetal bovine serum was developed. In addition to adhesive bone marrow cells, the co-existence of non-adhesive bone marrow cells was effective in expressing liver-specific functions for at least 3 weeks. On the other hand, experiments with Transwell in which cultured cells were separated by a semi-permeable membrane, suggested that soluble factors secreted by BMCs are the key components in the functional enhancement of cells. Furthermore, direct contact between hepatocytes and BMCs enhanced the formation of spheroids and the expression of liver specific functions. These results indicate that this co-culture system is promising in, for example, bioartificial liver, regenerative medicine, and liver function simulator applications.