Zonal Expression Research Articles

VSPACE: exploring virtual spatial representation of articular chondrocytes at the single-cell level.

vSPACE is a web-based application presenting a spatial representation of scRNAseq data obtained from human articular cartilage by emulating the concept of spatial transcriptomics technology, but virtually. This virtual 2D plot presentation of human articular cartage cells generates several zonal distribution patterns, for one or multiple genes at a time, revealing patterns that scientists can appreciate as imputed spatial distribution patterns along the zonal axis. vSPACE is implemented in Python Dash as a web-based toolbox designed for data visualization of zonal gene expression patterns in articular cartilage chondrocytes. This tool is freely accessible at: https://vspace.cse.uconn.edu/The source code and extra materials for this service can be downloaded from: https://github.com/zhacheny/vSPACE.

Cross-species variability in lobular geometry and cytochrome P450 hepatic zonation: insights into CYP1A2, CYP2D6, CYP2E1 and CYP3A4.

There is a lack of systematic research exploring cross-species variation in liver lobular geometry and zonation patterns of critical drug-metabolizing enzymes, a knowledge gap essential for translational studies. This study investigated the critical interplay between lobular geometry and key cytochrome P450 (CYP) zonation in four species: mouse, rat, pig, and human. We developed an automated pipeline based on whole slide images (WSI) of hematoxylin-eosin-stained liver sections and immunohistochemistry. This pipeline allows accurate quantification of both lobular geometry and zonation patterns of essential CYP proteins. Our analysis of CYP zonal expression shows that all CYP enzymes (besides CYP2D6 with panlobular expression) were observed in the pericentral region in all species, but with distinct differences. Comparison of normalized gradient intensity shows a high similarity between mice and humans, followed by rats. Specifically, CYP1A2 was expressed throughout the pericentral region in mice and humans, whereas it was restricted to a narrow pericentral rim in rats and showed a panlobular pattern in pigs. Similarly, CYP3A4 is present in the pericentral region, but its extent varies considerably in rats and appears panlobular in pigs. CYP2D6 zonal expression consistently shows a panlobular pattern in all species, although the intensity varies. CYP2E1 zonal expression covered the entire pericentral region with extension into the midzone in all four species, suggesting its potential for further cross-species analysis. Analysis of lobular geometry revealed an increase in lobular size with increasing species size, whereas lobular compactness was similar. Based on our results, zonated CYP expression in mice is most similar to humans. Therefore, mice appear to be the most appropriate species for drug metabolism studies unless larger species are required for other purposes, e.g., surgical reasons. CYP selection should be based on species, with CYP2E1 and CYP2D6 being the most preferable to compare four species. CYP1A2 could be considered as an additional CYP for rodent versus human comparisons, and CYP3A4 for mouse/human comparisons. In conclusion, our image analysis pipeline together with suggestions for species and CYP selection can serve to improve future cross-species and translational drug metabolism studies.

Generation and characterization of mature hepatocyte organoids for liver metabolic studies.

Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.

Passaged Articular Chondrocytes From the Superficial Zone and Deep Zone Can Regain Zone-Specific Properties After Redifferentiation

Background: Bioengineered cartilage is a developing therapeutic to repair cartilage defects. The matrix must be rich in collagen type II and aggrecan and mechanically competent, withstanding compressive and shearing loads. Biomechanical properties in native articular cartilage depend on the zonal architecture consisting of 3 zones: superficial, middle, and deep. The superficial zone chondrocytes produce lubricating proteoglycan-4, whereas the deep zone chondrocytes produce collagen type X, which allows for integration into the subchondral bone. Zonal and chondrogenic expression is lost after cell number expansion. Current cell-based therapies have limited capacity to regenerate the zonal structure of native cartilage. Hypothesis: Both passaged superficial and deep zone chondrocytes at high density can form bioengineered cartilage that is rich in collagen type II and aggrecan; however, only passaged superficial zone–derived chondrocytes will express superficial zone–specific proteoglycan-4, and only passaged deep zone–derived chondrocytes will express deep zone–specific collagen type X. Study Design: Controlled laboratory study. Methods: Superficial and deep zone chondrocytes were isolated from bovine joints, and zonal subpopulations were separately expanded in 2-dimensional culture. At passage 2, superficial and deep zone chondrocytes were seeded, separately, in scaffold-free 3-dimensional culture within agarose wells and cultured in redifferentiation media. Results: Monolayer expansion resulted in loss of expression for proteoglycan-4 and collagen type X in passaged superficial and deep zone chondrocytes, respectively. By passage 2, superficial and deep zone chondrocytes had similar expression for dedifferentiated molecules collagen type I and tenascin C. Redifferentiation of both superficial and deep zone chondrocytes led to the expression of collagen type II and aggrecan in both passaged chondrocyte populations. However, only redifferentiated deep zone chondrocytes expressed collagen type X, and only redifferentiated superficial zone chondrocytes expressed and secreted proteoglycan-4. Additionally, redifferentiated deep zone chondrocytes produced a thicker and more robust tissue compared with superficial zone chondrocytes. Conclusion: The recapitulation of the primary phenotype from passaged zonal chondrocytes introduces a novel method of functional bioengineering of cartilage that resembles the zone-specific biological properties of native cartilage. Clinical Relevance: The recapitulation of the primary phenotype in zonal chondrocytes could be a possible method to tailor bioengineered cartilage to have zone-specific expression.

Repetitive and zonal expression profiles of absorption-related genes in the gastrointestinal tract of ascidian Ciona intestinalis type A.

Intestinal absorption is essential for heterotrophic bilaterians with a tubular gut. Although the fundamental features of the digestive system were shared among chordates with evolution, the gut morphologies of vertebrates diverged and adapted to different food habitats. The ascidian Ciona intestinalis type A, a genome-wide research model of basal chordates, is used to examine the functional morphology of the intestines because of its transparent juvenile body. In the present study, the characteristic gene expression patterns (GEP) of Ciona absorptive proteins, e.g., brush border membrane enzymes for terminal digestion (lactase, maltase, APA, and APN) and transporters (SGLT1, GLUT5, PEPT1, and B0AT1), were investigated in juveniles and young adults, with a special reference to the absorption of other nutrients by pinocytosis- and phagocytosis-related proteins (megalin, cubilin, amnionless, Dab2, Rab7, LAMP, cathepsins, and MRC1). Whole-mount in situ hybridization revealed that these GEP showed multi-regional and repetitive features along the Ciona gastrointestinal tract, mainly in the stomach and several regions of the intestines. In young adults, many absorption-related genes, including pinocytosis-/phagocytosis-related genes, were also expressed between the stomach and mid-intestine. In the gastrointestinal epithelium, absorption-related genes showed zonal GEP along the epithelial structure. Comparisons of GEP, including other intestinal functions, such as nutrient digestion and intestinal protection, indicated the repetitive assignment of a well-coordinated set of intestinal GEP in the Ciona gastrointestinal tract.

Zonal expression of StARD1 and oxidative stress in alcoholic-related liver disease

Alcoholic-related liver disease (ALD) is one of the leading causes of chronic liver disease and morbidity. Unfortunately, the pathogenesis of ALD is still incompletely understood. StARD1 has emerged as a key player in other etiologies of chronic liver disease, and alcohol-induced liver injury exhibits zonal distribution. Here, we report that StARD1 is predominantly expressed in perivenous (PV) zone of liver sections from mice-fed chronic and acute-on-chronic ALD models compared to periportal (PP) area and is observed as early as 10 days of alcohol feeding. Ethanol and chemical hypoxia induced the expression of StARD1 in isolated primary mouse hepatocytes. The zonal-dependent expression of StARD1 resulted in the accumulation of cholesterol in mitochondria and increased lipid peroxidation in PV hepatocytes compared to PP hepatocytes, effects that were abrogated in PV hepatocytes upon hepatocyte-specific Stard1 KO mice. Transmission electron microscopy indicated differential glycogen and lipid droplets content between PP and PV areas, and alcohol feeding decreased glycogen content in both areas while increased lipid droplets content preferentially in PV zone. Moreover, transmission electron microscopy revealed that mitochondria from PV zone exhibited reduced length with respect to PP area, and alcohol feeding increased mitochondrial number, particularly, in PV zone. Extracellular flux analysis indicated lower maximal respiration and spared respiratory capacity in control PV hepatocytes that were reversed upon alcohol feeding. These findings reveal a differential morphology and functional activity of mitochondria between PP and PV hepatocytes following alcohol feeding and that StARD1 may play a key role in the zonal-dependent liver injury characteristic of ALD.

Peroxisome proliferator-activated receptor α agonist induces mouse hepatomegaly through the spatial hepatocyte enlargement and proliferation.

Peroxisome proliferator-activated receptor alpha (PPARα) activation-induced hepatomegaly is accompanied by hepatocyte hypertrophy around the central vein (CV) area and hepatocyte proliferation around the portal vein (PV) area. However, the molecular mechanisms underlying this spatial change of hepatocytes remains unclear. In this study, we examined the characteristics and possible reasons for the zonation distinction of hypertrophy and proliferation during PPARα activation-induced mouse liver enlargement. Mice were injected with corn oil or a typical mouse PPARα agonist WY-14643 (100 mg·kg-1·d-1, i.p.) for 1, 2, 3, 5 or 10 days. At each time point, the mice were sacrificed after the final dose, and liver tissues and serum were harvested for analysis. We showed that PPARα activation induced zonal changes in hepatocyte hypertrophy and proliferation in the mice. In order to determine the zonal expression of proteins related to hepatocyte hypertrophy and proliferation in PPARα-induced liver enlargement, we performed digitonin liver perfusion to separately destroy the hepatocytes around the CV or PV areas, and found that PPARα activation-induced increase magnitude of its downstream targets such as cytochrome P450 (CYP) 4 A and acyl-coenzyme A oxidase 1 (ACOX1) levels around the CV area were higher compared with those around the PV area. Upregulation of proliferation-related proteins such as cell nuclear antigen (PCNA) and cyclin A1 (CCNA1) after WY-14643-induced PPARα activation mainly occurred around the PV area. This study reveals that the zonal expression of PPARα targets and proliferation-related proteins is responsible for the spatial change of hepatocyte hypertrophy and proliferation after PPARα activation. These findings provide a new insight into the understanding of PPARα activation-induced liver enlargement and regeneration.

Atlas of the anatomical localization of atypical chemokine receptors in healthy mice.

Atypical chemokine receptors (ACKRs) scavenge chemokines and can contribute to gradient formation by binding, internalizing, and delivering chemokines for lysosomal degradation. ACKRs do not couple to G-proteins and fail to induce typical signaling induced by chemokine receptors. ACKR3, which binds and scavenges CXCL12 and CXCL11, is known to be expressed in vascular endothelium, where it has immediate access to circulating chemokines. ACKR4, which binds and scavenges CCL19, CCL20, CCL21, CCL22, and CCL25, has also been detected in lymphatic and blood vessels of secondary lymphoid organs, where it clears chemokines to facilitate cell migration. Recently, GPR182, a novel ACKR-like scavenger receptor, has been identified and partially deorphanized. Multiple studies point towards the potential coexpression of these 3 ACKRs, which all interact with homeostatic chemokines, in defined cellular microenvironments of several organs. However, an extensive map of ACKR3, ACKR4, and GPR182 expression in mice has been missing. In order to reliably detect ACKR expression and coexpression, in the absence of specific anti-ACKR antibodies, we generated fluorescent reporter mice, ACKR3GFP/+, ACKR4GFP/+, GPR182mCherry/+, and engineered fluorescently labeled ACKR-selective chimeric chemokines for in vivo uptake. Our study on young healthy mice revealed unique and common expression patterns of ACKRs in primary and secondary lymphoid organs, small intestine, colon, liver, and kidney. Furthermore, using chimeric chemokines, we were able to detect distinct zonal expression and activity of ACKR4 and GPR182 in the liver, which suggests their cooperative relationship. This study provides a broad comparative view and a solid stepping stone for future functional explorations of ACKRs based on the microanatomical localization and distinct and cooperative roles of these powerful chemokine scavengers.

The precipitation distribution set by eddy fluxes: the case of boreal winter

The latitudinal precipitation distribution shows a secondary peak in midlatitudes and a minimum in the subtropics. This minimum is widely attributed to the descending branch of the Eulerian Hadley cell. This study however shows that the precipitation distribution aligns more closely with the transformed Eulerian mean (TEM) vertical motion. In Northern Hemisphere winter, maximum TEM descent (ascent) and precipitation minimum (maximum) are collocated at ~20°N (~40°N). The subtropical descent is mostly driven by the meridional flux of zonal momentum by large-scale eddies, while the midlatitude ascent is driven by the meridional flux of heat by the eddies. When the poleward eddy momentum flux is sufficiently strong, however, the secondary precipitation peak shifts to 60°N corresponding to the location of the TEM ascent driven by the eddy momentum flux. Moisture supply for the precipitation is aided by evaporation which is enhanced where the TEM descending branch brings down dry air from the upper troposphere/lower stratosphere. This picture is reminiscent of dry air intrusions in synoptic meteorology, suggesting that the descending branch may embody a zonal mean expression of dry air intrusions. Moist air rises following the TEM ascending branch, suggesting that the ascending branch may be interpreted as a zonal mean expression of warm conveyor belts. This study thus offers a large-scale dynamics perspective of the synoptic description of precipitation systems. The findings here also suggest that future changes in the eddy momentum flux, which is poorly understood, could play a pivotal role in determining the future precipitation distribution.

Liver Acinus Dynamic Chip for Assessment of Drug-Induced Zonal Hepatotoxicity.

Zonation along the liver acinus is considered a key feature of liver physiology. Here, we developed a liver acinus dynamic (LADY) chip that recapitulates a key functional structure of the liver acinus and hepatic zonation. Corresponding to the blood flow from portal triads to the central vein in vivo, gradual flow of oxygen and glucose–carrying culture medium into the HepG2 cell chamber of the LADY chip generated zonal protein expression patterns in periportal (PP) zone 1 and perivenous (PV) zone 3. Higher levels of albumin secretion and urea production were obtained in a HepG2/HUVECs co-culture LADY chip than in HepG2 mono-culture one. Zonal expression of PEPCK as a PP marker and CYP2E1 as a PV marker was successfully generated. Cell death rate of the PV cells was higher than that of the PP cells since zonal factors responsible for metabolic activation of acetaminophen (APAP) were highly expressed in the PV region. We also found the co-culture enhanced metabolic capacity to process APAP, thus improving resistance to APAP toxicity, in comparison with HepG2 mono-culture. These results indicate that our LADY chip successfully represents liver zonation and could be useful in drug development studies as a drug-induced zonal hepatotoxicity testing platform.

Spatial transcriptomics reveals differences among genetic models of disruption in the Wnt‐beta‐catenin signaling in hepatocytes

Wnt‐beta‐catenin signaling plays key roles in liver biology. One such role is in regulating the process of metabolic zonation within a liver lobule where Wnt‐beta‐catenin axis controls the expression of many genes expressed in zone‐3 hepatocytes. The cell‐molecule circuitry of the Wnt‐beta‐catenin signaling in this process has been identified and consists of basal expression of Wnt2 and Wnt9b in endothelial cells lining the central vein, which then are secreted by the Wntless (Wls) protein to act in a paracrine manner on Frizzled‐LRP5‐6 co‐receptors on hepatocytes in zone‐3 to activate beta‐catenin. This axis has been revealed through the generation of key genetic knockout (KO) mice including endothelial cell specific Wnt2‐Wnt9b double KO (EC‐Wnt2/9b‐KO), endothelial cell specific Wls KO (prevents secretion of all Wnts from endothelial cells lining central vein) (EC‐Wls‐KO), hepatocyte‐specific‐LRP5‐6 DKO (HC‐LRP5‐6‐DKO) and hepatocyte‐specific‐beta‐catenin KO (HC‐beta‐Cat‐KO). In all 4 genetic mouse models, we have identified notable decrease to complete loss of many zone‐3 genes. However, it remains unclear if all the 4 KO mice are exactly identical in the loss of all well recognized zone‐3 hepatocyte specific targets or are there specific differences since disruption of the pathway occurs at different levels. Likewise, since zonal expression of genes is thought to be dynamic, it is of high interest to determine if loss of zone‐3 target gene expression perturbs the expression of zone‐1 and zone‐2 genes and if that occurs uniformly and similarly in all 4 genetic KO models. Using single cell spatial transcriptomics with Molecular CartographyTM, we are querying the expression of one hundred zonated genes across the hepatic lobule involved in various functions. The current study will allow us to for the first time identify any differences in target genes of Wnt‐beta‐catenin, Wnt‐independent‐beta‐catenin‐dependent, and Wnt‐dependent‐beta‐catenin‐independent target genes. At the same time, we will be able to address if loss of zone‐3 genes in each of the 4 KO models also indirectly perturbs the expression of genes normally expressed in zone‐1 and zone‐2 of the liver. Such novel information will be critical to both liver biologists and pathologists who study alterations in metabolic zonation in many liver pathologies including fatty liver disease and cholestatic liver injury.

Comparative study on a novel lobule structure of the zebrafish liver and that of the mammalian liver.

The mammalian liver has a lobule structure with a portal triad consisting of the portal vein, hepatic artery, and bile duct, which exhibits zonal gene expression, whereas those of teleosts do not have a portal triad. It remains to be demonstrated what kind of the unit structures they have, including their gene expression patterns. The aims of the present study were to demonstrate the unit structure of the teleost liver and discuss it in terms of evolution and adaptation in vertebrates and the use of teleosts as an alternative model for human disease. The zebrafish liver was examined as a representative of teleosts with respect to its morphological architecture and gene expression. A novel, polygonal lobule structure was detected in the zebrafish liver. In it, portal veins and central veins were distributed at the periphery and center, respectively. Sinusoids connected both veins. Anxa4-positive preductules were incorporated into the tubular lumen of two rows of hepatocytes in sections. Intrahepatic bile ducts resided randomly in the liver lobule. Zebrafish livers did not have zonal gene expression for metabolic pathways examined. The lobules of the zebrafish liver with preductules located in the tubular lumina of hepatocytes may resemble the oval cell reaction of injured livers of mammals and might convey bile to the intestine more safely than mammalian livers. The gene expression pattern in liver lobules and our liver lobule model of the zebrafish may be important to discuss data obtained in experiments using this animal as an alternative model for human disease.

Heterogeneous phenotypes of Pten-null hepatocellular carcinoma in hepatitis B virus transgenic mice parallels liver lobule zonal gene expression patterns

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The phenotypes of HCC are diverse, in part, due to mutations in distinct oncogenes and/or tumor suppressor genes. These genetic drivers of HCC development have generally been considered as major mediators of tumor heterogeneity. Using the liver-specific Pten-null HBV transgenic mouse model of chronic viral infection, a critical role for liver lobule zone-specific gene expression patterns in determining HCC phenotype and β-catenin-dependent HBV biosynthesis is demonstrated. These observations suggest that the position of the hepatocyte within the liver lobule, and hence its intrinsic gene expression pattern at the time of cellular transformation, make critical contributions to the properties of the resulting liver tumor. These results may explain why therapies targeting pathways modulated by specific identified tumor driver genes display variable treatment efficacy.

Subchondral Bone Microarchitectural and Mineral Properties and Expression of Key Degradative Proteinases by Chondrocytes in Human Hip Osteoarthritis.

Background: The purpose of this study was to investigate the relationship between the expression of key degradative enzymes by chondrocytes and the microarchitectural and mineral properties of subchondral bone across different stages of cartilage degradation in human hip osteoarthritis (OA). Methods: Osteochondral samples at different stages of cartilage degradation were collected from 16 femoral heads with OA. Osteochondral samples with normal cartilage were collected from seven femoral heads with osteoporosis. Microcomputed tomography was used for the investigation of subchondral bone microarchitecture and mineral densities. Immunohistochemistry was used to study the expression and distribution of MMP13 and ADAMTS4 in cartilage. Results: The microarchitecture and mineral properties of the subchondral plate and trabecular bone in OA varied with the severity of the degradation of the overlying cartilage. Chondrocytes expressing MMP13 and ADAMTS4 are mainly located in the upper zone(s) of cartilage regardless of the histopathological grades. The zonal expression of these enzymes in OA (i.e., the percentage of positive cells in the superficial, middle, and deep zones), rather than their overall expression (the percentage of positive cells in the full thickness of the cartilage), exhibited significant variation in relation to the severity of cartilage degradation. The associations between the subchondral bone properties and zonal and overall expression of these enzymes in the cartilage were generally weak or nonsignificant. Conclusions: Phenotypic changes in chondrocytes and remodelling of subchondral bone proceed at different rates throughout the process of cartilage degradation. Biological influences are more important for cartilage degradation at early stages, while biomechanical damage to the compromised tissue may outrun the phenotypic change of chondrocytes and is critical in the advanced stages.

Hepatocyte size fractionation allows dissection of human liver zonation.

Human hepatocytes show marked differences in cell size, gene expression, and function throughout the liver lobules, an arrangement termed liver zonation. However, it is not clear if these zonal size differences, and the associated phenotypic differences, are retained in isolated human hepatocytes, the "gold standard" for in vitro studies of human liver function. Here, we therefore explored size differences among isolated human hepatocytes and investigated whether separation by size can be used to study liver zonation in vitro. We used counterflow centrifugal elutriation to separate cells into different size fractions and analyzed them with label-free quantitative proteomics, which revealed an enrichment of 151 and 758 proteins (out of 5163) in small and large hepatocytes, respectively. Further analysis showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of previously described zonal markers and biological processes. We also found that the expression of zone-specific cytochrome P450 enzymes correlated with their metabolic activity in the different fractions. In summary, our results show that differences in hepatocyte size matches zonal expression patterns, and that our size fractionation approach can be used to study zone-specific liver functions in vitro.

OR19-01 IHC-Guided Capture of Adrenocortical Zones from Archival Formalin-Fixed, Paraffin-Embedded Adrenal Tissue Allows the Study Of RNA Expression Profiles

Introduction: Characterization of human adrenal RNA has relied on fresh tissue, or laser capture from OCT-embedded frozen tissue. These approaches have allowed identification of differences between zones of the adrenal cortex, but the need for fresh or frozen material has limited access for most researchers and has limited the number of samples that can be studied. In order to expand tissue availability and researcher access, we examined the use of archival formalin-fixed, paraffin-embedded (FFPE) adrenals for the capture and analysis of RNA from each adrenal zone.Methods: 13 normal adrenals were obtained from deceased renal donors. Following FFPE, tissue serial sections were examined for zonal histology. Immunohistochemistry (IHC) was used to guide the capture of specific adrenal zones from FFPE tissue. CYP11B2, CYP17A1, and CYB5A IHC was used to visualize the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR) respectively. Serial unstained sections were then dissected under a dissection microscope based on IHC-guided visualization of the ZG, ZF, and ZR. Following capture of zones and isolation of RNA, quantitative PCR (qPCR) was used to examine zonal expression of transcripts for the IHC markers. Using housekeeping genes for normalization, specific zonal markers identified successfully isolated zones.Results: Adrenals with clear zonal expression of CYP11B2 (ZG), HSD3B2 (ZG/ZF), and CYB5A (ZR) were included for study. Based on marker inclusion criteria, 3 ZG, 3 ZF, 3 ZR, and 3 medulla samples were studied. Using these samples, confirmatory markers of ZG (VSNL1), ZF (CYP17A1), ZR (SLC27A2), and medulla (TH) were analyzed by qPCR. Levels of these transcripts segregated as expected in the adrenal zones.Discussion: The use of archival normal and pathologic adrenal tissue for RNA analysis would increase the number of samples available to many research groups. Our data demonstrates the ability to capture and use FFPE tissue for the capture and analysis of RNA. Future comparison of normal adrenal zones to adrenal tumor phenotypes will improve our understanding of tumor biology and provide potential genetic markers for targeted therapeutics.

Directed differentiation of BMSCs on structural/compositional gradient nanofibrous scaffolds for ligament-bone osteointegration.

Nanofibrous scaffolds with structural and compositional gradients exhibit great potential to modulate zonal differentiation of stem cells for the regeneration of soft-to-hard tissue interface. Here, the response of bone marrow stem cells (BMSCs) to electrospun gradient nanofibrous scaffolds was investigated to demonstrate their potential capabilities for interfacial tissue regeneration. The electrospun scaffolds showed gradient distribution of BMP-2/nanoHA contents and the fiber orientations gradually changed from random to align. Biomimetic mineralization demonstrated that calcium and phosphorus elements can deposit onto the surface of the nanofibers in a gradient manner similar to nanoHA content. BMSCs cultured on the gradient nanofibrous scaffolds exhibited high cell viability and cell morphology gradually changed from disorder to highly align similar to the underlying fiber orientation. BMP-2/nanoHA content gradients in the nanofibrous scaffolds were found to effectively promote the zonal expression of bone-specific genes like osteocalcin (OCN), Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP). Immunofluorescent staining of osteopontin (OPN) and OCN further confirmed osteoblastic phenotypic maturation on the regions of the scaffolds with a higher level of nanoHA and BMP-2 contents after cultured 28days. These results indicated that the gradient nanofibrous scaffolds enable to guide zonal differentiation of BMSCs in vitro, which might be useful to realize multitissue regeneration in one construct for the regeneration of soft-to-hard tissue interface.

Localized Induction of Gene Expression in Embryonic Stem Cell Aggregates Using Holographic Optical Tweezers to Create Biochemical Gradients

Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses.Graphical abstract

Hyper-ammonia stress causes induction of inducible nitric oxide synthase gene and more production of nitric oxide in air-breathing magur catfish, Clarias magur (Hamilton).

Nitric oxide (NO) is an important signalling molecule that plays diverse physiological functions in several vertebrates including that of adaptation to various stressful stimuli. The air-breathing magur catfish (Clarias magur) is known to tolerate a very high external ammonia (HEA) stress in its natural habitats. We report here the possible induction of inducible nitric oxide (inos) gene and more generation of NO in magur catfish exposed to HEA. Exposure to HEA (25mM NH4Cl) for 14days led to the higher accumulation of NO in different tissues of magur catfish and also more efflux of NO from the perfused liver of NH4Cl-treated fish as a consequence of high build of toxic ammonia in body tissues. More synthesis and accumulation of NO in body tissues was associated with the induction of iNOS activity, which otherwise was not detectable in control fish. The stimulation of iNOS activity in HEA exposed fish was mainly due to induction of inos gene as evidenced by more expression of inos mRNA and also more abundance of iNOS protein in different tissues of magur catfish. Immunocytochemical analysis indicated the zonal specific expression of iNOS protein in different tissues of magur catfish. The augmentation of iNOS in the fish under HEA could be an adaptive strategy of the fish to defend against the ammonia stress through the generation of NO. Therefore, the present finding identifies the potential role of iNOS to enhance the adaptive capacity and survivability of catfish under various adverse environmental and pathological conditions that it faces in its natural habitats.

Three dimensional extrusion printing induces polymer molecule alignment and cell organization within engineered cartilage.

Proper cell-material interactions are critical to remain cell function and thus successful tissue regeneration. Many fabrication processes have been developed to create microenvironments to control cell attachment and organization on a three-dimensional (3D) scaffold. However, these approaches often involve heavy engineering and only the surface layer can be patterned. We found that 3D extrusion based printing at high temperature and pressure will result an aligned effect on the polymer molecules, and this molecular arrangement will further induce the cell alignment and different differentiation capacities. In particular, articular cartilage tissue is known to have zonal collagen fiber and cell orientation to support different functions, where collagen fibers and chondrocytes align parallel, randomly, and perpendicular, respectively, to the surface of the joint. Therefore, cell alignment was evaluated in a cartilage model in this study. We used small angle X-ray scattering analysis to substantiate the polymer molecule alignment phenomenon. The cellular response was evaluated both in vitro and in vivo. Seeded mesenchymal stem cells (MSCs) showed different morphology and orientation on scaffolds, as a combined result of polymer molecule alignment and printed scaffold patterns. Gene expression results showed improved superficial zonal chondrogenic marker expression in parallel-aligned group. The cell alignment was successfully maintained in the animal model after 7 days with distinct MSC morphology between the casted and parallel printed scaffolds. This 3D printing induced polymer and cell alignment will have a significant impact on developing scaffold with controlled cell-material interactions for complex tissue engineering while avoiding complicated surface treatment, and therefore provides new concept for effective tissue repairing in future clinical applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2190-2199, 2018.