The hepatic stellate cell has surprised and engaged physiologists, pathologists, and hepatologists for over 130 years, yet clear evidence of its role in hepatic injury and fibrosis only emerged following the refinement of methods for its isolation and characterization. The paradigm in liver injury of activation of quiescent vitamin A-rich stellate cells into proliferative, contractile, and fibrogenic myofibroblasts has launched an era of astonishing progress in understanding the mechanistic basis of hepatic fibrosis progression and regression. But this simple paradigm has now yielded to a remarkably broad appreciation of the cell's functions not only in liver injury, but also in hepatic development, regeneration, xenobiotic responses, intermediary metabolism, and immunoregulation. Among the most exciting prospects is that stellate cells are essential for hepatic progenitor cell amplification and differentiation. Equally intriguing is the remarkable plasticity of stellate cells, not only in their variable intermediate filament phenotype, but also in their functions. Stellate cells can be viewed as the nexus in a complex sinusoidal milieu that requires tightly regulated autocrine and paracrine cross-talk, rapid responses to evolving extracellular matrix content, and exquisite responsiveness to the metabolic needs imposed by liver growth and repair. Moreover, roles vital to systemic homeostasis include their storage and mobilization of retinoids, their emerging capacity for antigen presentation and induction of tolerance, as well as their emerging relationship to bone marrow-derived cells. As interest in this cell type intensifies, more surprises and mysteries are sure to unfold that will ultimately benefit our understanding of liver physiology and the diagnosis and treatment of liver disease.

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Hepatic Stellate Cells: Protean, Multifunctional, and Enigmatic Cells of the Liver

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes.

Osteogenic differentiation of purified, culture‐expanded human mesenchymal stem cells in vitro

Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the con- centration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immu- nologically tolerated "tissue" for wound hea ing and as a model for studying fibroblast function are discussed.

Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative

Mechanically challenged tissue-engineered organs, such as blood vessels, traditionally relied on synthetic or modified biological materials for structural support. In this report, we present a novel approach to tissue-engineered blood vessel (TEBV) production that is based exclusively on the use of cultured human cells, i.e., without any synthetic or exogenous biomaterials. Human vascular smooth muscle cells (SMC) cultured with ascorbic acid produced a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. After maturation, the tubular support was removed and endothelial cells were seeded in the lumen. This TEBV featured a well-defined, three-layered organization and numerous extracellular matrix proteins, including elastin. In this environment, SMC reexpressed desmin, a differentiation marker known to be lost under standard culture conditions. The endothelium expressed von Willebrand factor, incorporated acetylated LDL, produced PGI2, and strongly inhibited platelet adhesion in vitro. The complete vessel had a burst strength over 2000 mmHg. This is the first completely biological TEBV to display a burst strength comparable to that of human vessels. Short-term grafting experiment in a canine model demonstrated good handling and suturability characteristics. Taken together, these results suggest that this novel technique can produce completely biological vessels fulfilling the fundamental requirements for grafting: high burst strength, positive surgical handling, and a functional endothelium.

A completely biological tissue-engineered human blood vessel

Recently, increasingly more microsatellites, or simple sequence repeats (SSRs) have been found and characterized within protein-coding genes and their untranslated regions (UTRs). These data provide useful information to study possible SSR functions. Here, we review SSR distributions within expressed sequence tags (ESTs) and genes including protein-coding, 3'-UTRs and 5'-UTRs, and introns; and discuss the consequences of SSR repeat-number changes in those regions of both prokaryotes and eukaryotes. Strong evidence shows that SSRs are nonrandomly distributed across protein-coding regions, UTRs, and introns. Substantial data indicates that SSR expansions and/or contractions in protein-coding regions can lead to a gain or loss of gene function via frameshift mutation or expanded toxic mRNA. SSR variations in 5'-UTRs could regulate gene expression by affecting transcription and translation. The SSR expansions in the 3'-UTRs cause transcription slippage and produce expanded mRNA, which can be accumulated as nuclear foci, and which can disrupt splicing and, possibly, disrupt other cellular function. Intronic SSRs can affect gene transcription, mRNA splicing, or export to cytoplasm. Triplet SSRs located in the UTRs or intron can also induce heterochromatin-mediated-like gene silencing. All these effects caused by SSR expansions or contractions within genes can eventually lead to phenotypic changes. SSRs within genes evolve through mutational processes similar to those for SSRs located in other genomic regions including replication slippage, point mutation, and recombination. These mutational processes generate DNA changes that should be connected by DNA mismatch repair (MMR) system. Mutation that has escaped from the MMR system correction would become new alleles at the SSR loci, and then regulate and/or change gene products, and eventually lead to phenotype changes. Therefore, SSRs within genes should be subjected to stronger selective pressure than other genomic regions because of their functional importance. These SSRs may provide a molecular basis for fast adaptation to environmental changes in both prokaryotes and eukaryotes.

Microsatellites Within Genes: Structure, Function, and Evolution

The localization of acidic glycosaminoglycans in the subtractions of the skin ofbullfrog tadpole has been studied by colorimetric analysis combined with two dimen-sional electrophoresis, enzymedigestion and chemical treatment. Dermatan sulfatewasfound to be concentrated in the basal lamellar fraction and heparan sulfate,hepaiin and chondroitin sulfate were in the epithelial.It is well known that acidic glycosaminoglycans in mammalian skins are mainlycomposed of dennatan sulfate and hyaluronic acid. The presence of chondroitinsuifates, heparan sulfate1, and of heparin* in the skins is also reported. However, inthe case of anuran skins, Lipson and Silbert3-4 and Lipson et al.s reported that bull-frog (Rana catesbeiana) tadpole skins in early larval stages contain essentially onlyhyaluronic acid and that dennatan sulfate becomes a predominant glycosamino-glycan with a concomitant reduction in the amount of hyaluronic acid during meta-morphosis. Therefore, our interest was focused to elucidate acidic glycosaminoglycanconstituents in the basal lamella of anuran larval skin where collagen fibers weremostly localized.Recently wereported a micro method for the analysis of acidic glycosamino-glycans by two dimensional electrophoresis on cellulose acetate strips6-7. The use ofthis method provided us with a simple and rapid way to detect and analyze eachacidic glycosaminoglycan component in a sample to be examined.The present paper describes the local distribution of acidic glycosaminoglycansin anuran skins in early larval stages by using a colorimetric analysis combined withtwo dimensional electrophoresis on cellulose acetate strips, enzyme digestion andchemical treatment.Bullfrog (Rana catesbeiana} tadpoles in early metamorphic stages (hind limblength : tail length, less than O.I) were placed in ice water for about 1 h, then theback skins of the anesthetized tadpoles were excised with scissors. The back skins thusobtained were placed in0.5 M NaOHsolution at 5 °C, stirred for 16 h, and neutralizedwith 1 M HCI solution. After adjusting the pH to 7.8 by adding an equal volume of0.1 MTris-HCl buffer containing 5 mMCaCl2, the solution was boiled for 0.5 h and

Distribution of acidic glycosaminoglycansin tadpole backskin

A method for the separation of collagenchains has beendeveloped by employing two-dimensionalgel electrophoresiswhich consists of isoelectricfocusing in a 4°/o polyaery1amide gel-6 M iirea-2°/» Pharmalyte with a pH gradient ranging 7 to 10 in the first dimension and SDS-polyactylamideslab gel (5°/», plus 3 M urea) electrophoresisin the second dimension. The method gives reproducible results and can be employedfor the separation of \-chains of types I, II, III and V (AB) collagensas low as 10 :«gand for the estimation of their isoelectric points. Apparent isoelectric points of «1(I), u2, alJII), «1(III), aA and oB-chainsof bovine collagenswerefound to be 8.8,9.2, 8.7,9.1, 8.6 and 8.3 respectively.

Two-dimensional Electrophoresis of the "-Chainsof Collagens and its Applicationto the Determinationof their Isoelectric Points

The gene expression of human type 1 procollagen was investigated in cirrhotic human liver by using in situ hybridization with nonradioactive DNA probes. Using in situ hybridizaiton can provide direct evidence for the cell type capable for type I collagen synthesis in tissues. T-T dimerized DNA probes were used and DNAs hybridized in situ were detected immunohistochemically using specific antibodies against T-T dimer. The data demonstrated that type I collagen is synthesized in hepatocyles and stellate cells in pseudolobules and in fibroblasts in Glissons capsules in cirrhotic human livers. we indicated hepatocytes morphologically and functionally by using immunohistochemical localization of albumin, which ws used as a marker of hepatocyte, since albumin is synthesized exclusively by hepatocytes. © 1992 Wiley-Liss, Inc.

Simultaneous expression of type I procollagen mRNA and albumin in cirrhotic human liver.

A two-dimensionalmapping method developedfor collagen chains has been applied to the CNBr-pepridesof rat collagentypes i, II and III and bovine type V i'AB)collagen. The method consists of isoelectricfocusing in a 4°.'o polyacrylamide gcl-6M urea-2°'o Pharmalyte\virh a pH gradient ranging 5 to 10 in the first dimensionand SDS-polyacrylamide slab gel (15°'a") electrophoresisat pH 8.8 in the second dimension. Characteristic peptide maps were obtained for each type of (/-chain and can be used for identification of isolated collagen chains. Applicationof this method to an authentic mixture of collagentypes and a fraction Isolated from rat skin has indicated that the method is sensitive enough to resolvecollagentypeswith a sampleas low as 40 «g per a-chain.

Two-dimensional Electrophoresis Patternsof the CNBr Peptidesof CollagenTypesI, II, HIand V

The acidic glycosaminoglycans in urine, serum and myocardial tissue of aged patients with myocardial infarction have been analyzed by two-dimensional electrophoresis on cellulose acetate membranes in order to study the mechanism of repair of myocardium after infarction. Patients tested 4 years or more after infarction showed excretion of urinary glycosaminoglycans within the range normally encountered for the aged. However, in cases of acute myocardial infarction, the excretion of glycosaminoglycans was found to be increased 3 to 4 times in the early acute phase and then gradually to decrease to within the normal range during recovery. A significant increase in excretion of low sulfated chondroitin sulfate was observed in the acute phase. Low sulfated chondroitin sulfate was the major glycosaminoglycan in sera from the patients, regardless of stages. In normal myocardial tissue, hyaluronic acid was the major component and heparan sulfate, chondroitin sulfate and dermatan sulfate were the minor components. In myocardium from patients with acute infarction, a decrease in heparan sulfate was observed, while in that of old infarction, dermatan sulfate was the main component and in advanced aneurysm, increase in chondroitin sulfate was observed along with dermatan sulfate. These findings suggest that the repair process of infarcted myocardium is very similar to that occurring during healing of hepatic fibrosis and damaged arterial walls.

Ryu-Ichiro Hata

Papers

Distribution of acidic glycosaminoglycansin tadpole backskin

Two-dimensional Electrophoresis of the "-Chainsof Collagens and its Applicationto the Determinationof their Isoelectric Points

Simultaneous expression of type I procollagen mRNA and albumin in cirrhotic human liver.

Two-dimensional Electrophoresis Patternsof the CNBr Peptidesof CollagenTypesI, II, HIand V

Acidic glycosaminoglycans in urine, serum and myocardium of the aged patients with myocardial infarction