Hyaluronan improves photoreceptor differentiation and maturation in human retinal organoids.

Human stem cell-derived organoids enable both disease modeling and serve as a source of cells for transplantation. Human retinal organoids are particularly important as a source of human photoreceptors; however, the long differentiation period required and lack of vascularization in the organoid often results in a necrotic core and death of inner retinal cells before photoreceptors are fully mature. Manipulating the in vitro environment of differentiating retinal organoids through the incorporation of extracellular matrix components could influence retinal development. We investigated the addition of hyaluronan (HA), a component of the interphotoreceptor matrix, as an additive to promote long-term organoid survival and enhance retinal maturation. HA treatment had a significant reduction in the proportion of proliferating (Ki67+) cells and increase in the proportion of photoreceptors (CRX+), suggesting that HA accelerated photoreceptor commitment in vitro. HA significantly upregulated genes specific to photoreceptor maturation and outer segment development. Interestingly, prolonged HA-treatment significantly decreased the length of the brush border layer compared to those in control retinal organoids, where the photoreceptor outer segments reside; however, HA-treated organoids also had more mature outer segments with organized discs structures, as revealed by transmission electron microscopy. The brush border layer length was inversely proportional to the molar mass and viscosity of the hyaluronan added. This is the first study to investigate the role of exogenous HA, viscosity, and polymer molar mass on photoreceptor maturation, emphasizing the importance of material properties on organoid culture. STATEMENT OF SIGNIFICANCE: Retinal organoids are a powerful tool to study retinal development in vitro, though like many other organoid systems, can be highly variable. In this work, Shoichet and colleagues investigated the use of hyaluronan (HA), a native component of the interphotoreceptor matrix, to improve photoreceptor maturation in developing human retinal organoids. HA promoted human photoreceptor differentiation leading to mature outer segments with disc formation and more uniform and healthy retinal organoids. These findings highlight the importance of adding components native to the developing retina to generate more physiologically relevant photoreceptors for cell therapy and in vitro models to drive drug discovery and uncover novel disease mechanisms.

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells.

As a metabolic organ, the liver plays a variety of roles, including detoxification. It has been difficult to obtain stable supplies of hepatocytes for transplantation and for accurate hepatotoxicity determination in drug discovery research. Human pluripotent stem cells, capable of unlimited self-renewal, may be a promising source of hepatocytes. In order to develop a stable supply of embryonic stem cell (ESC)-derived hepatocytes, we have purified human ESC-derived hepatic progenitor cells with exposure to cytocidal puromycin by using their ability to metabolize drugs. Hepatic progenitor cells stably proliferated at least 220-fold over 120 days, maintaining hepatic progenitor cell-like properties. High drug-metabolizing hepatic progenitor cells can be matured into liver cells by suppressing hepatic proliferative signals. The method we developed enables the isolation and proliferation of functional hepatic progenitors from human ESCs, thereby providing a stable supply of high-quality cell resources at high efficiency. Cells produced by this method may facilitate cell therapy for hepatic diseases and reliable drug discovery research.

Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture.

BACKGROUND: The liver plays an important role in various metabolic processes, including protein synthesis, lipid and drug metabolisms and detoxifications. Primary culture of hepatocytes is used for the understanding of liver physiology as well as for the drug development. Hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. Alternatively, systems using animal models and hepatocellular carcinoma cells have been established, but interspecies differences, variation between human cell sources and limited hepatic functions are among the challenges faced when using these models. Therefore, there is still a need for a highly stable method to purify human hepatocytes with functional sufficiency. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply. METHODS: We first established a growth culture system for hepatocytes derived from patients with drug-induced liver injury using fetal mouse fibroblasts and EMUKK-05 medium. We then evaluated the morphology, proliferative capacity, chromosome stability, gene and protein expression profiles, and drug metabolic capacity of hepatocytes in early, middle and late passages with and without puromycin. In addition, hepatic maturation in 3D culture was evaluated from morphological and functional aspects. RESULTS: In our culture system, the stable proliferation of human hepatocytes was achieved by co-culturing with mouse fetal fibroblasts, resulting in dedifferentiation into hepatic progenitor-like cells. We purified human hepatocytes by selection with cytocidal puromycin and cultured them for more than 60 population doublings over a span of more than 350 days. Hepatocytes with high expression of cytochrome P450 genes survived after exposure to cytocidal antibiotics because of enhanced drug-metabolizing activity. CONCLUSIONS: These results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.

Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI).

BACKGROUND: Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI). METHODS: To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations. RESULTS: The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen. CONCLUSIONS: iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.

Correction: Quantification of nematic cell polarity in three-dimensional tissues.

[This corrects the article DOI: 10.1371/journal.pcbi.1008412.].

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells.

Ammonia has a cytotoxic effect and can therefore be used as a selection agent for enrichment of zone I hepatocytes. However, it has not yet been determined whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed an ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human embryonic stem cells (ESCs) and from induced pluripotent stem cells (iPSCs). The resistance to cytotoxicity or cell death by ammonia is likely attributable to the metabolism of ammonia in the cells. In addition to ammonia metabolism-related genes, ammonia-selected hepatocytes showed increased expression of the cytochrome P450 genes. Additionally, the ammonia-selected cells achieved immortality or at least an equivalent life span to human pluripotent stem cells without affecting expression of the liver-associated genes. Ammonia treatment in combination with in vitro propagation is useful for obtaining large quantities of hepatocytes.

Quantification of nematic cell polarity in three-dimensional tissues.

How epithelial cells coordinate their polarity to form functional tissues is an open question in cell biology. Here, we characterize a unique type of polarity found in liver tissue, nematic cell polarity, which is different from vectorial cell polarity in simple, sheet-like epithelia. We propose a conceptual and algorithmic framework to characterize complex patterns of polarity proteins on the surface of a cell in terms of a multipole expansion. To rigorously quantify previously observed tissue-level patterns of nematic cell polarity (Morales-Navarrete et al., eLife 2019), we introduce the concept of co-orientational order parameters, which generalize the known biaxial order parameters of the theory of liquid crystals. Applying these concepts to three-dimensional reconstructions of single cells from high-resolution imaging data of mouse liver tissue, we show that the axes of nematic cell polarity of hepatocytes exhibit local coordination and are aligned with the biaxially anisotropic sinusoidal network for blood transport. Our study characterizes liver tissue as a biological example of a biaxial liquid crystal. The general methodology developed here could be applied to other tissues and in-vitro organoids.

Liquid-crystal organization of liver tissue.

Functional tissue architecture originates by self-assembly of distinct cell types, following tissue-specific rules of cell-cell interactions. In the liver, a structural model of the lobule was pioneered by Elias in 1949. This model, however, is in contrast with the apparent random 3D arrangement of hepatocytes. Since then, no significant progress has been made to derive the organizing principles of liver tissue. To solve this outstanding problem, we computationally reconstructed 3D tissue geometry from microscopy images of mouse liver tissue and analyzed it applying soft-condensed-matter-physics concepts. Surprisingly, analysis of the spatial organization of cell polarity revealed that hepatocytes are not randomly oriented but follow a long-range liquid-crystal order. This does not depend exclusively on hepatocytes receiving instructive signals by endothelial cells, since silencing Integrin-ß1 disrupted both liquid-crystal order and organization of the sinusoidal network. Our results suggest that bi-directional communication between hepatocytes and sinusoids underlies the self-organization of liver tissue.

A Predictive 3D Multi-Scale Model of Biliary Fluid Dynamics in the Liver Lobule.

Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury.

RNAi-nanoparticulate manipulation of gene expression as a new functional genomics tool in the liver.

BACKGROUND & AIMS: The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models. METHODS: Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2). RESULTS: The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1(-/-)Mst2(-/-) liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53(LSL/LSL) mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth. CONCLUSION: Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver.

A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture.

A prerequisite for the systems biology analysis of tissues is an accurate digital three-dimensional reconstruction of tissue structure based on images of markers covering multiple scales. Here, we designed a flexible pipeline for the multi-scale reconstruction and quantitative morphological analysis of tissue architecture from microscopy images. Our pipeline includes newly developed algorithms that address specific challenges of thick dense tissue reconstruction. Our implementation allows for a flexible workflow, scalable to high-throughput analysis and applicable to various mammalian tissues. We applied it to the analysis of liver tissue and extracted quantitative parameters of sinusoids, bile canaliculi and cell shapes, recognizing different liver cell types with high accuracy. Using our platform, we uncovered an unexpected zonation pattern of hepatocytes with different size, nuclei and DNA content, thus revealing new features of liver tissue organization. The pipeline also proved effective to analyse lung and kidney tissue, demonstrating its generality and robustness.

Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes.

An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

Nanoparticle-formulated siRNA targeting integrins inhibits hepatocellular carcinoma progression in mice.

Integrins play an important role during development, regulating cell differentiation, proliferation and survival. Here we show that knockdown of integrin subunits slows down the progression of hepatocellular carcinoma (HCC). Using nanoparticulate delivery of short interfering RNAs targeting ß1 and αv integrin subunits, we downregulate all integrin receptors in hepatocytes. Short-term integrin knockdown (2 weeks) does not cause apparent structural or functional perturbations of normal liver tissue. Alterations in liver morphology accumulate on sustained integrin downregulation (7 weeks). The integrin knockdown leads to significant retardation of HCC progression, reducing proliferation and increasing tumour cell death. This tumour retardation is accompanied by reduced activation of the MET oncogene as well as expression of its mature form on the cell surface. Our data suggest that transformed proliferating cells from HCC are more sensitive to knockdown of integrins than normal quiescent hepatocytes, highlighting the potential of small interfering RNA-mediated inhibition of integrins as an anti-cancer therapeutic approach.

Rab5 is necessary for the biogenesis of the endolysosomal system in vivo.

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Inhibition of Stabilin-2 elevates circulating hyaluronic acid levels and prevents tumor metastasis.

Hyaluronic acid (HA) has been implicated in the proliferation and metastasis of tumor cells. However, most previous studies were conducted on extracellular matrix or pericellular HA, and the role of circulating HA in vivo has not been studied. HA is rapidly cleared from the bloodstream. The scavenger receptor Stabilin-2 (Stab2) is considered a major clearance receptor for HA. Here we report a dramatic elevation in circulating HA levels in Stab2-deficient mice without any overt phenotype. Surprisingly, the metastasis of B16F10 melanoma cells to the lungs was markedly suppressed in the Stab2-deficient mice, whereas cell proliferation was not affected. Furthermore, administration of an anti-Stab2 antibody in Stab2(+) mice elevated serum HA levels and prevented the metastasis of melanoma to the lung, and also suppressed spontaneous metastasis of mammary tumor and human breast tumor cells inoculated in the mammary gland. Administration of the antibody or high-dose HA in mice blocked the lodging of melanoma cells to the lungs. Furthermore, HA at high concentrations inhibited the rolling/tethering of B16 cells to lung endothelial cells. These results suggest that blocking Stab2 function prevents tumor metastasis by elevating circulating HA levels. Stab2 may be a potential target in antitumor therapy.

New innovative instruments facilitate both direct-vision and endoscopic-assisted mini-mitral valve surgery.

OBJECTIVE: The efficacy of new, innovative, original instruments, including a left atrial retractor, silicon annuloplasty ring sizer, modified Cosgrove aortic clamp, and reusable clip for fixing knots of polytetrafluoroethylene (Gore-Tex; WL Gore & Associates, Inc, Flagstaff, Ariz) suture, to allow surgical exposure in an ideal operative setting of mini-mitral valve repair surgery was verified. METHODS: Since 1998, a great deal of innovation has contributed to establishing mitral valve repair via right minithoracotomy as a routine surgical approach for mitral valve insufficiency in 252 cases. During the last 2 years, a newly launched left atrial retractor system attachable to the minithoracotomy spreader has been used. An additional retractor for the posterior wall of the left atrium was attached to the minithoracotomy spreader. The retractor moves flexibly and can be fixed in any favorable position to realize optimal exposure of the mitral valve. A 5 blade size was available depending on the left atrial size and target legion. By using the smallest size, even papillary muscles were exposed easily and clearly. Furthermore, a flexible silicon ring sizer, which could easily pass thorough a narrow working port without tissue damage, was used for sizing the annuloplasty ring. For the surgical technique, multiple chordal reconstructions by the loop technique with polytetrafluoroethylene (Gore-Tex CV-5 sutures) were applied. A reusable clip for fixing knots made it easy to tie the Gore-Tex suture in the correct position without slipping. RESULTS: No operative mortality occurred. There were 2 conversions to sternotomy for correction of aortic dissection (1) and for coronary artery bypass grafting (1). There were 2 early reoperations for failure of mitral valve repair. The mean aortic crossclamp time was 163.5 ± 41.6 minutes. Annuloplasty with a ring or band was performed in all cases except one. The loop technique was used in 173 cases. Among them, a combination of the loop technique and resection and suture technique was used in 56 cases. CONCLUSIONS: Newly innovated mini-mitral valve surgical instruments and techniques facilitate both direct-vision and endoscopic-assisted approaches and accomplish a favorable surgical outcome even in the complex pathology of mitral valve insufficiency.

Development of stabilin2+ endothelial cells from mouse embryonic stem cells by inhibition of TGFbeta/activin signaling.

To understand the endothelial cell (EC) development, arterial, venous, and lymphatic EC (LEC) have been successfully induced from embryonic stem cells (ESC). However, tissue-specific EC, such as hepatic sinusoidal EC (HSEC), have never been generated from ESC. Based on the findings that TGFbeta/activin signaling negatively regulates differentiation of both LEC and HSEC, and that HSEC and LEC are distinguishable by the expression of marker genes, we assessed the role of TGFbeta/activin signaling in EC development from ESC. Here we show that the inhibition of TGFbeta/activin signaling by a TGFbeta receptor I (TGFbetaRI) kinase inhibitor increased the expression of Lyve1 and stabilin2 but not podoplanin in CD31+CD34+ EC derived from ESC. EC generated by the inhibition of TGFbetaRI signaling also exhibited stronger endocytic activity than control EC, indicating that their phenotype is similar to fetal HSEC. Our results reveal that TGFbeta/activin signaling negatively regulates the early events of HSEC differentiation.

p75 Neurotrophin receptor is a marker for precursors of stellate cells and portal fibroblasts in mouse fetal liver.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are 2 distinct mesenchymal cells in adult liver. HSCs in sinusoids accumulate lipids and express p75 neurotrophin receptor (p75NTR). HSCs and PFs play pivotal roles in liver regeneration and fibrosis. However, the roles of mesenchymal cells in fetal liver remain poorly understood. In this study, we aimed to characterize mesenchymal cells in mouse fetal liver. METHODS: We prepared an anti-p75NTR monoclonal antibody applicable for flow cytometry and immunohistochemistry. p75NTR(+) cells isolated from fetal liver by flow cytometry were characterized by reverse-transcription polymerase chain reaction, immunohistochemistry, and cell cultivation. Lipid-containing cells were visualized by Oil-red O staining. RESULTS: p75NTR(+) cells in fetal liver were clearly distinct from endothelial cells and showed characteristics of mesenchymal cells. At embryonic day (E) 10.5, p75NTR(+) cells were present at the periphery of the liver bud in close contact with endothelial cells, and spread over the liver at E11.5. With the formation of the liver architecture, they began to localize to 2 distinct areas, parenchymal and portal areas, and lipid-containing p75NTR(+) cells increased accordingly. p75NTR(+) cells around portal veins were adjacent to cholangiocytes and expressed Jagged1, a crucial factor for the commitment of hepatoblasts to cholangiocytes. By cultivation, p75NTR(+) cells showed features of adult HSCs with markedly increased expression of glial fibrillary acidic protein and alpha-smooth muscle actin. CONCLUSIONS: p75NTR(+) mesenchymal cells in fetal liver include progenitors for HSCs and PFs, and the anti-p75NTR monoclonal antibody is useful for their isolation.

Development of murine hepatic sinusoidal endothelial cells characterized by the expression of hyaluronan receptors.

Endothelial cells (ECs) display distinct structural and functional characteristics depending on the tissue and developmental stage; however, the development of tissue-specific ECs remains poorly understood. Here, we describe the development of hepatic sinusoids in mice based on the expression of hyaluronan receptors Stab2 and Lyve-1. Flk-1(+) cells in and around the liver bud begin to express Stab2 at embryonic day (E) 9.5, before the formation of vascular lumen. Hepatic sinusoidal endothelial cells (HSECs) begin to express Lyve-1 at E10.5, and both markers continue to be expressed in HSECs thereafter. Although HSECs and lymphatic ECs (LECs) are known to share functional and phenotypic characteristics, we clearly show that HSECs can be distinguished from LECs by the expression of molecular markers and higher endocytotic activity. Our results provide new insight into the development of tissue-specific ECs and phenotypic criteria to distinguish HSECs from other types of ECs, including LECs.

Serial analysis of gene expression in sinusoidal endothelial cells from normal and injured mouse liver.

Here we describe gene expression profiles of mouse liver sinusoidal endothelial cells (LSECs) revealed by serial analysis of gene expression (SAGE). We prepared SAGE libraries of LSECs from normal and injured liver by CCl(4) administration, and we obtained 32,867 tags from normal and 37,493 tags from injured liver, representing 6011 unique transcripts. CCl(4) administration upregulated several genes related to cell growth and differentiation (Cdkn1a, Irf1, Il4ra, etc.), whereas it downregulated genes related to cell growth or protein transport (Kdr, Igfbp4, Ap1b1, etc.). To identify genes preferentially expressed in LSEC, we compared our SAGE libraries with 77 publicly available libraries generated from various mouse tissues and cell lines. We identified 23 genes, including Stab2 and uncharacterized genes, as possible markers for LSEC, which will be useful to analyze the specific role for LSECs in normal as well as regenerating liver.