Syngeneic implantation of mouse hepatic progenitor cell-derived three-dimensional liver tissue with dense collagen fibrils.

BACKGROUND: Liver transplantation is a therapy for irreversible liver failure; however, at present, donor organs are in short supply. Cell transplantation therapy for liver failure is still at the developmental stage and is critically limited by a shortage of human primary hepatocytes. AIM: To investigate the possibility that hepatic progenitor cells (HPCs) prepared from the portal branch-ligated hepatic lobe may be used in regenerative medicine, we attempted to enable the implantation of extracellular matrices containing organoids consisting of HPC-derived hepatocytes and non-parenchymal cells. METHODS: In vitro liver organoid tissue has been generated by accumulating collagen fibrils, fibroblasts, and HPCs on a mesh of polylactic acid fabric using a bioreactor; this was subsequently implanted into syngeneic wild-type mice. RESULTS: The in vitro liver organoid tissues generated transplantable tissues in the condensed collagen fibril matrix and were obtained from the mouse through partial hepatectomy. CONCLUSION: Liver organoid tissue was produced from expanded HPCs using an originally designed bioreactor system. This tissue was comparable to liver lobules, and with fibroblasts embedded in the network collagen fibrils of this artificial tissue, it is useful for reconstructing the hepatic interstitial structure.

Tau pathology in aged cynomolgus monkeys is progressive supranuclear palsy/corticobasal degeneration- but not Alzheimer disease-like -Ultrastructural mapping of tau by EDX.

Concomitant deposition of amyloid -beta protein (Aß) and neuronal tau as neurofibrillary tangles in the human brain is a hallmark of Alzheimer disease (AD). Because these deposits increase during normal aging, it has been proposed that aging brains may also undergo AD-like changes. To investigate the neuropathological changes that occur in the aging primate brain, we examined 21 brains of cynomolgus monkeys (7-36 years old) for Aß- and tau-positive lesions. We found, 1) extensive deposition of Aß in brains of cynomolgus monkeys over 25 years of age, 2) selective deposition of 4-repeat tau as pretangles in neurons, and as coiled body-like structures in oligodendroglia-like cells and astrocytes, 3) preferential distribution of tau in the basal ganglia and neocortex rather than the hippocampus, and 4) age-associated increases in 30-34 kDa AT8- and RD4-positive tau fragments in sarkosyl-insoluble fractions. We further labeled tau-positive structures using diaminobezidine enhanced with nickel, and visualized nickel-labeled structures by energy-dispersive X-ray (EDX) analysis of ultrathin sections. This allowed us to distinguish between nickel-labeled tau and background electron-dense structures, and we found that tau localized to 20-25 nm straight filaments in oligodendroglia-like cells and neurons. Our results indicate that the cytopathology and distribution of tau deposits in aged cynomolgus brains resemble those of progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) rather than AD. Thus, even in the presence of Aß, age-associated deposition of tau in non-human primates likely does not occur through AD-associated mechanisms.

Repeated folding stress-induced morphological changes in the dermal equivalent.

BACKGROUND/PURPOSE: Repeated mechanical stresses applied to the same region of the skin are thought to induce morphological changes known as wrinkle. However, the underlying mechanisms are not fully understood. To study the mechanisms, we examined effects of repeated mechanical stress on the dermal equivalent. METHODS: We developed a novel device to apply repeated folding stress to the dermal equivalent. After applying the mechanical stress, morphological changes of the dermal equivalent and expression of several genes related to extracellular matrix turn over and cell contraction were examined. RESULTS: The repeated folding stress induced a noticeable decrease in the width of the dermal equivalent. The mechanical stress altered orientations of collagen fibrils. Hydroxyproline contents, dry weights and cell viability of the dermal equivalents were not affected by the mechanical stress. On the other hand, Rho-associated coiled-coil-containing kinase (ROCK) specific inhibitor Y27632 completely suppressed the decrease in the width of the dermal equivalent. CONCLUSION: The present results revealed that either degradation of collagen or changes in the number of cells were not responsible for the decrease in the width of the dermal equivalent and indicate that the repeated mechanical stress induces unidirectional contraction in the dermal equivalent through the RhoA-ROCK signaling pathway.

Characterization of a liver organoid tissue composed of hepatocytes and fibroblasts in dense collagen fibrils.

The adult liver is wrapped in a connective tissue sheet called the liver capsule, which consists of collagen fibrils and fibroblasts. In this study, we set out to construct a liver organoid tissue that would be comparable to the endogenous liver, using a bioreactor. In vitro liver organoid tissue was generated by combining collagen fibrils, fibroblasts, and primary murine hepatocytes or Hep G2 on a mesh of poly-lactic acid fabric using a bioreactor. Then, the suitability of this liver organoid tissue for transplantation was tested by implanting the constructs into partially hepatectomized BALB/cA-nu/nu mice. As determined by using scanning and transmission electron microscopes, the liver organoid tissues were composed of densely packed collagen fibrils with fibroblasts and aggregates of oval or spherical hepatocytes. Angiogenesis was induced after the transplantation, and blood vessels connected the liver organoid tissue with the surrounding tissue. Thus, a novel approach was applied to generate transplantable liver organoid tissue within a condensed collagen fibril matrix. These results suggested that a dense collagen network populated with fibroblasts can hold a layer of concentrated hepatocytes, providing a three-dimensional microenvrionment suitable for the reestablishment of cell-cell and cell-extracellular matrix (ECM) interactions, and resulting in the maintenance of their liver-specific functions. This liver organoid tissue may be useful for the study of intrahepatic functions of various cells, cytokines, and ECMs, and may fulfill the fundamental requirements of a donor tissue.

Fragility of reconstituted type V collagen fibrils with the chain composition of α1(V)α2(V)α3(V) respective of the D-periodic banding pattern.

The triple-helical domains of two subtypes of type V collagen were prepared from human placenta, one with the chain composition of [α1(V)](2)α2(V) (Vp112) and the other with the chain composition of α1(V)α2(V)α3(V) (Vp123) with limited pepsin treatment. In order to characterize the triple-helical domain of the type Vp123 collagen molecule, the reconstituted aggregate structure formed from the pepsin-treated collagen was compared by using transmission electron microscopy. The diameter of the fibrils reconstituted from types pepsin-treated type Vp123 collagen and type Vp112 collagen was highly uniform and less than the D-periodicity at all the temperatures examined, suggesting that the major triple-helical domain of both subtypes has a potency to limit their lateral growth. Both fibrils were approximately 45 nm in width and showed the D-periodic banding pattern along their axes at 34°C. In contrast to type Vp112, the reconstituted type Vp123 fibrils showed no banding pattern along their axes when they were reconstituted at 37°C. The banded fibrils once reconstituted from type Vp123 at 34°C tend to lose their characteristic pattern within 60 min when they were incubated at 37°C. One explanation is that a slightly higher content of hydrophobic residues of type Vp123 collagen than those of type V112p collagen augmented the intermolecular interaction that disturbs the D-periodicity governed essentially by electrostatic interactions. Taken together with recent data in Col5a3 gene-targeted mice, the results suggest that type V123 collagen exists not only as a periodic banded fibril but also as nonfibrillar meshwork structures.

The transformation suppressor gene Reck is required for postaxial patterning in mouse forelimbs.

The membrane-anchored metalloproteinase-regulator RECK has been characterized as a tumor suppressor. Here we report that mice with reduced Reck-expression show limb abnormalities including right-dominant, forelimb-specific defects in postaxial skeletal elements. The forelimb buds of low-Reck mutants have an altered dorsal ectoderm with reduced Wnt7a and Igf2 expression, and hypotrophy in two signaling centers (i.e., ZPA and AER) that are essential for limb outgrowth and patterning. Reck is abundantly expressed in the anterior mesenchyme in normal limb buds; mesenchyme-specific Reck inactivation recapitulates the low-Reck phenotype; and some teratogens downregulate Reck in mesenchymal cells. Our findings illustrate a role for Reck in the mesenchymal-epithelial interactions essential for mammalian development.

Versican/PG-M is essential for ventricular septal formation subsequent to cardiac atrioventricular cushion development.

Versican (Vcan)/proteoglycan (PG)-M is a large chondroitin sulfate proteoglycan which forms a proteoglycan/hyaluronan (HA) aggregate in the extracellular matrix (ECM). We tried to generate the Vcan knockout mice by a conventional method, which resulted in mutant mice Vcan(Δ3/Δ3) whose Vcan lacks the A subdomain of the G1 domain. The Vcan knockout embryos died during the early development stage due to heart defects, but some Vcan(Δ3/Δ3) embryos survived through to the neonatal period. The hearts in Vcan(Δ3/Δ3) newborn mice showed normal cardiac looping, but had ventricular septal defects. Their atrioventricular canal (AVC) cushion was much smaller than those of wild-type (WT) embryos, and the extracellular space for cardiac jelly was narrow. The Vcan deposition in the Vcan(Δ3/Δ3) AVC cushion had decreased, whereas the HA deposition was maintained and condensed. In the tip of ventricular septa, both Vcan and HA had decreased. The cell proliferation based on the number of Ki67-positive cells had remarkably increased in both the AVC cushion and ventricular septa, compared with that of WT embryos. Vcan(Δ3/Δ3) seemed to have endocardial and mesenchymal mixed characteristics. When the ex vivo explant culture of these regions was performed on the collagen gel, hardly any migration to make sufficient space for the ECM construction was apparent. Our results suggest that the proteoglycan aggregates are necessary in both the AVC cushion and ventricular septa to fuse interventricular septa, and the Vcan A subdomain plays an essential role for the interventricular septal formation by constituting the proteoglycan aggregates.

Atomic identification of fluorescent Q-dots on tau-positive fibrils in 3D-reconstructed pick bodies.

Pick body disease, characterized by the presence of Pick bodies, is distinguished from neurofibrillary tangles in Alzheimer disease on the basis of their smooth, spherical shape. Quantum dots (QDs) are nanometer-scale, water-soluble fluorophores that are detectable both as a fluorescent signal by light microscopy and as electron-dense particles under electron microscopy. In this study, tau-positive Pick bodies were immunofluorescently labeled with QD nanocrystals composed of cadmium selenide for three-dimensional (3D) reconstruction and subsequently subjected to electron microscopic observation to identify QD immunolabeling on the same Pick body for comparison in detail. The identity of the QD nanocrystals, which label the tau-positive fibrils, was confirmed by the presence of both cadmium and selenium on these nanocrystals, demonstrated as parallel peaks corresponding to these atoms on energy-dispersive X-ray spot analysis under super-resolution scanning transmission electron microscopy. This confirmation of the specificity of the QD labeling through both its fluorescence and energy-dispersive X-ray spectra reinforces the reliability of the labeling. In addition, this exact comparison of the same structure by electron microscopy and 3D light microscopy demonstrates how its ultrastructural details are related to its surrounding structures on a 3D basis, providing further insights into how molecules woven into specific pathological ultrastructures are at work in situ.

Morphological and histological evaluations of 3D-layered blood vessel constructs prepared by hierarchical cell manipulation.

Three-dimensional (3D)-layered blood vessel constructs consisting of human umbilical artery smooth muscle cells (SMCs) and human umbilical vascular endothelial cells (ECs) were fabricated by hierarchical cell manipulation, and their basic morphology, histology and blood compatibility were evaluated in relation to the EC layers. For the hierarchical cell manipulation, fibronectin-gelatin (FN-G) nanofilms were prepared on the surface of SMC layers to provide a cell adhesive nano-scaffold for the second layer of cells. The layer number of blood vessel constructs was easily controllable from 2 to 7 layers, and the histological evaluation, scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated a hierarchical blood vessel analogous morphology. The immunefluorescence staining revealed homogeneous and dense tight-junction of the uppermost EC layer. Furthermore, the nano-meshwork morphology of the FN-G films like a native extracellular matrix was observed inside the blood vessel constructs by SEM. Moreover, a close association between actin microfilaments and the nano-meshworks was observed on the SMC surface by TEM. The blood compatibility of the blood vessel constructs, 4-layered SMC/1-layered EC (4L-SMC/1L-EC), was clearly confirmed by inhibition of platelet adhesion, whereas the blood vessel constructs without EC layers (4L-SMC) showed high adhesion and activation of the platelet. The 3D-blood vessel constructs prepared by hierarchical cell manipulation technique will be valuable as a blood vessel model in the tissue engineering or pharmaceutical fields.

Pale neurites, premature α-synuclein aggregates with centripetal extension from axon collaterals.

Progressive aggregation of α-synuclein (αS) from pale bodies (PBs) and extension from Lewy neurites (LNs) are candidate mechanisms for Lewy body (LB) formation. To identify how aggregation of αS is related to its extension along neurites, 60-µm-thick brainstem sections of Parkinson disease (PD) patients were prepared for three-dimensional (3D) reconstruction of αS-positive neurites with neurofilament (NF) and thiazin red (TR), a fluorochrome with an affinity to solid aggregates. This demonstrated 3D layering of αS surrounded by NF with the aggregates probed by TR in the center, corresponding to the eosinophilic core of mature LBs. This eosinophilic/TR-positive profile, characteristically absent in PBs, premature counterpart of LBs, was similarly absent in some LNs. We would like to refer these premature LNs as "pale neurites" (PNs). Their premature nature was evidenced by 3D fluoroprofiling with quantum dots (QDs) and subsequent electron microscopic identification (3D-oriented immunoelectron microscopy) as loosely packed αS (QDs)-positive filaments. Quantification of LNs, frequently extended around branching axons, demonstrated that LNs are initiated at axon collaterals to extend centripetally into proximal segments. This branching-oriented extension of αS is related to its selective predisposition to systems with highly divergent axons, preferentially affected in PD, which may explain barely somatotopic manifestations of PD.

Key role of heparan sulfate chains in assembly of anchoring complex at the dermal-epidermal junction.

Epidermal basement membrane forms anchoring complex composed of hemidesmosomes, anchoring filaments, lamina densa and anchoring fibrils to link epidermis to dermis. However, the anchoring complex is rarely formed in skin equivalent models, probably because of degradation of extracellular matrix (ECM) proteins and heparan sulfate chains by matrix metalloproteinases (MMPs) and heparanase, respectively. To explore the roles of ECM proteins and heparan sulfate in anchoring complex assembly, we used specific inhibitors of MMPs and heparanase, and the formation of anchoring complex was analysed in terms of polarized deposition of collagen VII, BP180 and ß4 integrin at the dermal-epidermal junction (DEJ) by means of immunohistochemistry and transmission electron microscopy (TEM). The deposition of collagen VII was polarized to the basal side by the addition of MMP inhibitor, and the staining intensity was increased by combined treatment with MMP inhibitor and heparanase inhibitor, which enhanced anchoring fibril formation as observed by TEM. BP180 was polarized to the basal side by heparanase inhibitor, which protects HS chains, but not by MMP inhibitor. MMP inhibitor improved the polarization of ß4 integrin. Hemidesmosomes were formed in the presence of each inhibitor, as observed by TEM, and formation was greatly enhanced by the combined treatment. These findings suggest that heparan sulfate chains, in addition to ECM proteins at the DEJ, play an important role in the assembly of anchoring complex, especially hemidesmosomes and anchoring fibrils.

Characterization of dense artificial connective tissues generated in a newly designed bioreactor.

Dense connective tissues were generated simultaneously by accumulating collagen fibrils and fibroblasts on stainless steel mesh using a bioreactor system that we designed. The advantage of our system is that the artificial connective tissues can be generated within 24 hr in the absence of inhibitors against matrix metalloproteinases. The fibroblasts were suspended in 200 mL of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.5 mg/mL type I collagen. The mixed solution was circulated in two types of bioreactors with cylindrical or vertical configurations to generate luminal or parenchymal tissues, respectively. The gelatin zymography showed that MMPs were first detected in the media after 8 hr from the start of circulation and reached the highest levels on day 3. Glossy white aggregates, 1-3 mm in thickness, depending on the circulation period, accumulated on mesh grids. Fibroblasts were embedded in the network of collagen fibrils and possessed oval nuclei with or without prominent cell processes to form a bipolar shape. We could not observe distended cisternae of the endoplasmic reticula, the Golgi apparatus, or exploded mitochondria, showing hypoxic degenerative alterations of fibroblasts in dense connective tissues. The artificial tissues generated by our system will be useful for biological studies and transplantation therapy.

Delay of cell cycle progression and induction death of cancer cells on type I collagen fibrils [corrected].

Here, we report the behavior of three kinds of human cancer cell lines (Caco-2, MCF-7, HT-1080) on type I collagen substrates, which are in two-dimensional coated collagen or three-dimensional fibrils form. All tested cells on coated collagen adhered and proliferated. However, in the case of collagen fibrils, the proliferation of cancer cells was suppressed. Furthermore, Akt activation, which is known as a cell-survival signal, was inhibited in cells on collagen fibrils. But the activation of ERK1/2 was not completely inhibited. In Caco-2 cells, delay of cell cycle progression and cell death occurred at the same time. Thus, cell division and cell death occurred at equivalent rates on the collagen fibrils, and cell growth seemed to be stopped. These results imply that the fibril form of collagen plays a potential role in inhibiting the growth of cancer cells.

Mucosal remodeling in long-standing ulcerative colitis with colorectal neoplasia: significant alterations of NCAM+ or alpha-SMA+ subepithelial myofibroblasts and interstitial cells.

Evidence has been provided in ulcerative colitis (UC) that early genomic instability of both epithelial and stromal cells is important for colorectal tumorigenesis, as well as remodeling and morphological alterations of mucosal crypts. To clarify roles of stromal cells in tumor development in UC, the present study focused on heterogeneous phenotypes of subepithelial myofibroblasts and interstitial cells, in association with mucosal remodeling. To clarify the relationship of alterations to tumorigenesis, mucosa of resected rectae from patients with UC (n= 49) and sporadic cancer (n= 10) were analyzed on immunohistochemistry and also on immunoelectron microscopy. Heterogeneous phenotypes of neural cell adhesion molecule (NCAM)+ and/or alpha-smooth muscle actin (alpha-SMA)+ subepithelial myofibroblasts and interstitial cells were demonstrated, corresponding to colonic stellate cells. Decrease of NCAM+ subepithelial myofibroblasts and interstitial cells, and increase of alpha-SMA+ interstitial cells were significant in UC with neoplasia as compared to without neoplasia. alpha-SMA+ muscularis mucosae was significantly more thickened in tumor cases. Deposits of Masson's trichrome+ and type III and I collagen in the muscularis mucosae and lamina propria appeared to increase in relation to the numbers of alpha-SMA+ interstitial cells. Mucosal remodeling with alterations of NCAM+ or alpha-SMA+ subepithelial and interstitial cells may play a critical role in UC-associated tumorigenesis.

Negligible contribution of bone marrow-derived cells to collagen production during hepatic fibrogenesis in mice.

BACKGROUND & AIMS: Recent studies have reported that bone marrow (BM)-derived cells migrating into fibrotic liver tissue exhibit a myofibroblast-like phenotype and may participate in the progression of liver fibrosis. However, their contribution to collagen production has not been fully verified yet. We revisited this issue by using 2 mechanistically distinct liver fibrosis models introduced into transgenic collagen reporter mice and their BM recipients. METHODS: BM of wild-type mice was replaced by cells obtained from transgenic animals harboring tissue-specific enhancer/promoter sequences of alpha2(I) collagen gene (COL1A2) linked to enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC) gene. Liver fibrosis was introduced into those mice by repeated carbon tetrachloride injections or ligation of the common bile duct. Activation of COL1A2 promoter was assessed by confocal microscopic examination detecting EGFP signals and luciferase assays of liver homogenates. RESULTS: The tissue-specific COL1A2 enhancer/promoter was activated in hepatic stellate cells following a single carbon tetrachloride injection or during primary culture on plastic. A large number of EGFP-positive collagen-expressing cells were observed in liver tissue of transgenic COL1A2/EGFP mice in both liver fibrosis models. In contrast, there were few EGFP-positive BM-derived collagen-producing cells detected in fibrotic liver tissue of COL1A2/EGFP recipients. Luciferase assays of liver tissues from COL1A2/LUC-recipient mice further indicated that BM-derived cells produced little collagen in response to fibrogenic stimuli. CONCLUSIONS: By using a specific and sensitive experimental system, which detects exclusively BM-derived collagen-producing cells, we conclude an unexpectedly limited role of BM-derived cells in collagen production during hepatic fibrogenesis.

Keratinocyte differentiation and proliferation are regulated by adhesion to the three-dimensional meshwork structure of type IV collagen.

We examined the behavior of human foreskin keratinocytes (HFKs) on reconstituted type IV collagen gel. HFKs survived for several days and the upper layer cells expressed a differentiation marker, involucrin. Apoptosis was induced after involucrin expression while cell proliferation was suppressed. On molecular type IV collagen, integrins shifted from alpha 2 beta 1 to alpha 3 beta 1 during HFK culture. On type IV collagen gel, HFKs initially expressed integrin alpha 2 beta 1, and later expressed integrin alpha 3 beta 1 in the presence of alpha 2 beta 1 did not disappear. Using synthetic peptides, we examined integrin alpha2-mediated adhesion to type IV collagen gel. Addition of synthetic peptide dose-dependently inhibited cell adhesion both on type IV collagen gel and on molecular type IV collagen. On type IV collagen gel, weaker phosphorylation of focal adhesion kinase, paxillin, and Akt was observed compared with the molecular forms. Based on these observations, we think type IV collagen gel is a novel culture substrate that mimics the physiological environment for HFKs.

Intercellular accumulation of type V collagen fibrils in accordance with cell aggregation.

We reported previously that human fibroblasts form clumps when cultured on a dish coated with reconstituted type V collagen fibrils. Essentially all the type V collagen fibrils, initially coated on the dish, were recovered in the cell clumps that had eventually formed during the culture. We interpreted that type V collagen fibrils adhere to cells more strongly than to the dish and are detached by cell movements. In this study, type V collagen was suspended with fibroblasts to examine the fate of the type V collagen fibrils and to determine whether the fibrils affect the behaviour of the cells directly adherent to the dish. The added type V collagen accumulated in the intercellular space concomitantly with the local aggregation of fibroblasts. scanning electron microscope examination indicated that type V collagen fibrils were found in the vicinity of cells in cultures without ascorbic acid where essentially no collagen secretion takes place. These results indicate that type V collagen forms fibrils and the fibrils are accumulated in the intercellular spaces. The accumulated type V collagen fibrils work as a cementing material for cell clump formation. This phenomenon is discussed in relation to the possible involvement of type V collagen fibrils in tissue organization.

Collagen-type specificity of glycoprotein VI as a determinant of platelet adhesion.

Of the two physiologically important platelet collagen receptors, glycoprotein (GP) VI is the receptor responsible for platelet activation. However, its reactivities towards different types of vascular collagen have not been directly and quantitatively analysed with collagen preparations of defined composition, although the other major platelet collagen receptor integrin alpha(2)beta(1) was shown to react with collagen types I-VI and VIII under either static or flow conditions. We analysed the collagen type specificity of GPVI binding to identify the physiological contribution of the various vascular collagens and how platelet reactivity towards the various collagens may be affected by fibril size. We used two methods to analyse the binding of recombinant GPVI (GPVI-Fc(2)) to different types of bovine collagen: binding to collagen microparticles in suspension and binding to immobilized collagen. GPVI-Fc(2) bound to type I-III collagens that can form large fibrils, but not to type V that only forms small fibrils. The apparent GPVI binding to types IV and V could be ascribed to type I collagen that was a contaminant in each of these preparations. Kinetic analyses of the binding data showed that type III collagen fibrils have both a higher Kd and Bmax than types I and II. Flow adhesion studies demonstrated that type III collagen supports the formation of larger platelet aggregates than type I. Our present results suggest that the physiological importance of type III collagen is to induce thrombus formation. Furthermore, these studies indicate that GPVI mainly binds to collagen types that can form large collagen fibrils.