Adult Human Tissues Research Articles

Neonatal Nav1.5 protein expression in normal adult human tissues and breast cancer

Expression of the neonatal splice variant of the voltage-gated sodium channel α-subunit (VGSC) subtype Nav1.5 (nNav1.5), encoded by the gene SCN5A, was shown earlier to be upregulated in human breast cancer (BCa), both in vitro and in vivo. Channel activity promoted BCa invasion of Matrigel®in vitro and metastasis in vivo. Consequently, expression of nNav1.5 has been proposed as a functional biomarker of BCa cells with metastatic potential. Here, we have determined immunohistochemically both nNav1.5 and total VGSC (tVGSC) protein expression in a range of adult human tissues. Some VGSC protein was expressed in normal colon, small intestine, stomach, prostate, bladder and breast. As expected, high levels of VGSC protein were expressed in brain, skeletal muscle and cardiac muscle. On the other hand, nNav1.5 protein was not expressed in any of the normal tissues tested except breast where a low-level of protein was present. In comparison to normal breast, nNav1.5 protein expression in BCa was consistently widespread and occurred at a significantly higher level. We also questioned whether there was any relationship between the nNav1.5 protein expression and the estrogen receptor (ERα) status of BCa and obtained the following results. First, all cases lacking nNav1.5 were positive for ERα. Second, in all ERα-negative tissues, nNav1.5 protein was expressed in plasma membrane. Third, however, in ERα-positive cases, nNav1.5 protein expression was observed in both plasma membrane and cytoplasm. In conclusion, nNav1.5 protein has a restricted expression pattern among human tissues. High level expression occurs in BCa and associates with ERα status. These results further support the proposition that nNav1.5 is a novel biomarker of metastatic BCa.

Short-term storage of human testicular tissue: effect of storage temperature and tissue size

During short-term storage, before cryopreservation, testicular tissue quality can be affected by storage medium, duration, temperature and tissue size. We previously established the best storage medium and time for short-term maintenance of tissue. In this study, three different storage temperatures (4°C, room temperature, 37°C) and four tissue sizes (~6 mm3; ~ 15 mm3; ~ 50 mm3 or ~80 mm3) were assessed over the course of a fixed period of 3 days. Storing human testicular tissue at 37°C caused a significant increase in the number of apoptotic cells per tubule (P = 0.002), compared with fresh control, but this was not the case at 4°C or room temperature. Temperature did not affect viability, tissue morphology or number of spermatogonia in samples. The morphology of the testicular tissue was optimally preserved when stored as large fragments (~50 mm3: P = 0.018; ~ 80 mm3: P = 0.018). Tissue size did not significantly affect viability, number of spermatogonia or apoptotic cells. Adult human testicular tissue can be preserved at 4°C or room temperature without altering tissue morphology, Sertoli cell morphology, number of spermatogonia or number of apoptotic cells. The tissue does not need to be extensively dissected as tissue morphology is better maintained in larger fragments.

The HIV-1 viral synapse signals human foreskin keratinocytes to secrete thymic stromal lymphopoietin facilitating HIV-1 foreskin entry.

The complexity of signal transduction resulting from the contact of human immunodeficiency virus type 1 (HIV-1)-infected cells and mucosal cells has hampered our comprehension of HIV-1 mucosal entry. Such process is driven efficiently only by viral synapse contacts, whereas cell-free HIV-1 remains poorly infectious. Using CD4+ T-cells expressing only HIV-1 envelope inoculated on human adult foreskin tissues, we designed methodologies to identify the signals transduced in foreskin keratinocytes following HIV-1-envelope-dependent viral synapse formation. We find that the viral synapse activates the MyD88-independent TLR-4-nuclear factor (NfκB) signaling pathway in keratinocytes and the subsequent secretion of cytokines including thymic stromal lymphopoietin (TSLP), a cytokine linking innate and T-helper type 2-adaptive immune responses. Moreover, the viral synapse upregulates the non-coding microRNA miR-375, known to control TSLP, and transfection of keratinocytes with anti-miR-375 blocks significantly TSLP secretion. Thus, the secretion of TSLP by keratinocytes is induced by the viral synapse in a miR-375 controlled manner. At the tissue level, these signals translate into the epidermal redistribution of Langerhans cells and formation of conjugates with T-cells, recapitulating the initial events observed in human foreskin infection by HIV-1. These results open new possibilities for designing strategies to block mucosal HIV-1 transmission, the major pathway by which HIV-1 spreads worldwide.

Prospective Isolation and Comparison of Human Germinal Matrix and Glioblastoma EGFR + Populations with Stem Cell Properties

SummaryCharacterization of non-neoplastic and malignant human stem cell populations in their native state can provide new insights into gliomagenesis. Here we developed a purification strategy to directly isolate EGFR+/− populations from human germinal matrix (GM) and adult subventricular zone autopsy tissues, and from de novo glioblastoma (GBM) resections, enriching for cells capable of binding EGF ligand (LBEGFR+), and uniquely compared their functional and molecular properties. LBEGFR+ populations in both GM and GBM encompassed all sphere-forming cells and displayed proliferative stem cell properties in vitro. In xenografts, LBEGFR+ GBM cells showed robust tumor initiation and progression to high-grade, infiltrative gliomas. Whole-transcriptome sequencing analysis confirmed enrichment of proliferative pathways in both developing and neoplastic freshly isolated EGFR+ populations, and identified both unique and shared sets of genes. The ability to prospectively isolate stem cell populations using native ligand-binding capacity opens new doors onto understanding both normal human development and tumor cell biology.

Comparison of global gene expression profiles of microdissected human foetal Leydig cells with their normal and hyperplastic adult equivalents

Do human adult Leydig cells (ALCs) within hyperplastic micronodules display characteristics of foetal LCs (FLCs)? The gene expression profiles of FLCs and all ALC subgroups were clearly different, but there were no significant differences in expressed genes between the normally clustered and hyperplastic ALCs. LCs are the primary androgen producing cells in males throughout development and appear in chronologically distinct populations; FLCs, neonatal LCs and ALCs. ALCs are responsible for progression through puberty and for maintenance of reproductive functions in adulthood. In patients with reproductive problems, such as infertility or testicular cancer, and especially in men with high gonadotrophin levels, LC function is often impaired, and LCs may cluster abnormally into hyperplastic micronodules (defined as clusters of >15 LCs in a cross-section). A genome-wide microarray study of LCs microdissected from human foetal and adult tissue samples (n=12). Additional tissue specimens (n=15) were used for validation of the mRNA expression data at the protein level. Frozen human tissue samples were used for the microarray study, including morphologically normal foetal (gestational week 10-11) testis samples, and adult testis specimens with normal LC distribution, LC micronodules or LC micronodules adjacent to hCG-producing testicular germ cell tumours. Transcriptome profiling was performed on Agilent whole human genome microarray 4×44 K chips. Microarray data pre-processing and statistical analysis were performed using the limma R/Bioconductor package in the R software, and differentially expressed genes were further analysed for gene set enrichment using the DAVID Bioinformatics software. Selected genes were studied at the protein level by immunohistochemistry. The transcriptomes of FLCs and ALCs differed significantly from each other, whereas the profiles of the normally clustered and hyperplastic ALCs were similar despite morphological heterogeneity. The study revealed several genes not known previously to be expressed in LCs during early development, including sulfotransferase family 2A member 1 (SULT2A1), WNT1-inducible signalling pathway protein 2 (WISP2), hydroxyprostaglandin dehydrogenase (HPGD) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), whose expression changes were validated at the protein level. The transcriptomic data are deposited in ArrayExpress (accession code E-MTAB-5453). The small number of biological replicates and the necessity of RNA amplification due to the scarcity of human tissues, especially foetal specimens, are the main limitations of the study. Heterogeneous subpopulations of LCs within micronodules were not discriminated during microdissection and might have affected the expression profiling. The study was constrained by the lack of availability of truly normal controls. Testis samples used as 'controls' displayed complete spermatogenesis and were from patients with germ cell neoplasia but with undetectable hCG and normal hormone levels. The changes in LC morphology and function observed in patients with reproductive disorders possibly reflect subtle changes in the expression of many genes rather than regulatory changes of single genes or pathways. The study provides new insights into the development and maturation of human LCs by the identification of a number of potential functional markers for FLC and ALC. The study was supported by research grants from the Danish Cancer Society, the Capital Region's Research Fund for Health Research, Rigshospitalet's research funds, the Villum Kann Rasmussen Foundation, the Danish Innovation Fund, ReproUnion, Kirsten and Freddy Johansen's foundation and the Novo Nordisk Foundation. None of the funding agencies had any influence on the study. The authors declare no conflicts of interest.

Influence of the Injury-to-Surgery Interval on the Healing Potential of Human Anterior Cruciate Ligament–Derived Cells

Background: Vascular CD34+ cells in anterior cruciate ligament (ACL) tissue have the potential for high proliferation and multilineage differentiation that can accelerate tendon-bone healing. While patient characteristics, such as age, can affect tendon-bone healing, the influence of elapsed time after injury on the healing process is unclear. Hypothesis: Cells obtained during the early phase after injury will exhibit a greater tendon-bone healing potential compared with chronic phase counterparts when applied to an immunodeficient rat model of ACL reconstruction. Study Design: Controlled laboratory study. Methods: Adult human ACL-ruptured tissue was harvested from patients undergoing arthroscopic primary ACL reconstruction and classified into 2 groups based on the time elapsed between injury and surgery: (1) early group (≤3 months from injury) and (2) chronic group (>3 months from injury). In addition, 76 ten-week-old female immunodeficient rats underwent ACL reconstruction, followed by intracapsular administration of one of the following: (1) ACL-derived cells from the early group (n = 5), (2) ACL-derived cells from the chronic group (n = 5), or (3) phosphate-buffered saline (PBS) only (n = 5). During the 8 weeks after surgery, histological (weeks 2, 4, 8), immunohistochemical (week 2), radiographic (weeks 0, 2, 4, 8), and biomechanical (week 8) analyses were performed to evaluate tendon-bone healing. Results: In the early group, the histological evaluation showed early healing, induction of endochondral ossification–like integration, and mature bone ingrowth. Micro–computed tomography showed that the tibial bone tunnels at week 4 and week 8 were significantly reduced in the early group compared with those in the chronic group and PBS group (P < .05). Moreover, biomechanical tensile strength was significantly greater in the early group than in the other groups (P < .05). An accelerated healing potential in the early group was further demonstrated by the enhancement of intrinsic angiogenesis/osteogenesis and human-derived vasculogenesis/osteogenesis. Conclusion: Compared with human ACL-derived cells obtained during the chronic phase, cells obtained during the early phase after injury have a greater tendon-bone healing potential when used in an immunodeficient rat model of ACL reconstruction. Clinical Relevance: During ACL reconstruction surgery, transplanting ACL remnant tissue in the early phase after injury could accelerate and enhance tendon-bone healing.

Engineering epithelial-stromal interactions in vitro for toxicology assessment.

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues.

P2.01-076 Drebrin: A New Targetable Molecular Marker of Lung Adenocarcinoma

Embryonic antigens, such as carcinoembryonic antigen (CEA) and alfa-fetoprotein (AFP), are routinely employed as serum and immunohistochemical tumor markers in clinical medicine. Since they are not expressed in adult human tissues, it is reasonable to conclude that embryonic antigens are extremely specific tumor markers. However, no systematic studies have yet identified clinically useful embryonic antigens for tumor diagnosis. In the present study, we developed a strategy for systematic identification of lung adenocarcinoma markers using monoclonal antibodies generated from embryonic swine tissue. Swine mRNA shows more than 80% homology with human mRNA, and embryonic swine tissue is thought to be a useful material for detection of human embryonic markers. In order to produce mouse monoclonal antibodies, we immunized BALB/c mice by injection of fetal swine lung nuclear fraction into the hock, and used a human lung adenocarcinoma nuclear fraction for the second immunization. We recovered lymph nodes from the mice, and fused mouse B lymphocytes with the murine myeloma cell line SP2/0 using polyethylene glycol. The resulting hybridomas were then selectively cultured. For selection of interesting hybridoma clones, we performed immunohistochemical staining using the supernatant from each one, with tissue microarray loading swine fetal and adult lung, human lung cancer and normal lung tissue. Immunohistochemical screening of 284-clones showed that the antibodies derived from four of them were strongly reactive with the cytoplasm and cytomembrane of fetal swine lung and human lung cancer. We then focused on one clone (B246) whose antibody reacted most clearly with human lung adenocarcinoma cells. Protein microarray analysis confirmed that B246 reacted specifically with “drebrin”, one of the actin binding proteins, originally identified in neuronal cells. There are two drebrin isoforms in human tissue: drebrin E (embryonic) is abundant in the developing neurons, and drebrin A (adult) is expressed in adult brain. We then examined the association of the drebrin expression pattern with the pathological features and prognosis of lung adenocarcinoma using 200 selected cases for which formalin-fixed and paraffin-embedded samples were available. Drebrin immunohistochemistry delineated the samples into those with strong (n=85) and weak (n=115) drebrin expression. Kaplan-Meier analysis demonstrated a significant difference in disease-free survival (DFS) between the groups with strong and weak drebrin expression (p=0.033). Drebrin is expressed specifically in lung adenocarcinoma and is associated with outcome. The present findings indicate that drebrin is a new marker of lung adenocarcinoma and indicative of prognosis.

Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems.

Isolation and Primary Culture of Adult Human Adipose-derived Stromal/Stem Cells.

Adipose-derived stromal/stem cells (ASCs) are multipotent cells that can be isolated from adipose tissue. Studies have shown that cells have the capacity to self-renew and differentiate into adipocyte, chondrocyte, myocyte, and osteoblast lineages. Thus, significant interest regarding their use for regenerative purposes to restore aging or damaged tissue has grown in recent decades. These cells have also been shown to immunomodulate the microenvironment and secrete abundant growth factors, which minimize inflammation and aid repair and regeneration. ASCs can be readily isolated from the stromal vascular fraction (SVF) of lipoaspirates. Given their ease of accessibility, bountiful source, and potential application in regenerative medicine and tissue engineering, there is growing interest in the characterization and utilization of ASCs. This protocol describes the isolation of ASCs from adult human adipose tissue as well as methods for culture maintenance including expansion and cryopreservation.

Phosphorylated mTOR Expression Profiles in Human Normal and Carcinoma Tissues.

Mammalian target of rapamycin (mTOR) is a key controller of cell growth and proliferation in normal tissues and solid tumors. In the present study, an immunohistochemical analysis of the expression pattern of phosphorylated mTOR (p-mTOR) was performed in human normal fetal and adult tissues and various carcinoma tissues. p-mTOR expression showed tissue and cell type specificity in normal and cancer tissues. In normal fetal and adult tissues, p-mTOR staining was observed in the intestinal crypt, intrahepatic bile ductule, pancreatic duct, distal nephron of the kidney, umbrella cell of urothelium, mesothelial cell, and choroid plexus. In cancer tissues, p-mTOR expression was higher in adenocarcinoma than in other types of cancers, in metastatic cancer than in primary cancer, and in the forefront of the infiltrating cancer cells. These results suggest that p-mTOR is implicated not only in cell proliferation but also in tubular morphogenesis in normal and cancer tissues. In addition, mTOR activation appears to be associated with cancer cell invasion and migration in solid tumors.

Efficient Induction of Functional Ameloblasts from Human Keratinocyte Stem Cells

Background: Although adult human tissue derived epidermal stem cells are capable of differentiating into enamelsecreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. In addition, whether the regenerated enamel does retain running pattern of prism identical to and acquires mechanical property comparable with human ones indeed warrant further study. Methods: Cultured hKSCs (human keratinocyte stem cells) were treated with FGF8 and SHH for 18hr or 36hr prior to being recombined with E13.5 mouse dental mesenchyme with implantation of FGF8 and SHH soaked agarose beads into reconstruct chimeric tooth germs. Recombinant tooth germs were subjected to kidney capsule culture in nude mice. Harvested samples at various time points were processed for histological, immunohistochemical, TUNEL, and Western blot analysis. Scanning electronic microscopy and Nanoindentation test were further employed to analyze prism running pattern and mechanical property of the regenerated enamel. Results: Treatment of hKSCs with both FGF8 and SHH prior to tissue recombination greatly enhanced the rate of tooth-like structure formation to about 70%. FGF8 and SHH dramatically enhanced stemness of cultured hKSCs. Scanning electron microscopic analysis revealed the running pattern of intact prisms of regenerated enamel is similar to that of human one. Nanoindentation test indicated that, although much softer than human child and adult mouse one, mechanical property of the regenerated enamel is improved as culture time extended. Conclusion: Application of FGF8 and SHH proteins in cultured hKSCs improves stemness but does not facilitate odontogenic fate of hKSCs, resulting in an enhanced efficiency of ameloblastic differentiation of hKSCs and tooth formation in human-mouse chimeric tooth germs.

Comparing Circadian Dynamics in Primary Derived Stem Cells from Different Sources of Human Adult Tissue.

Optimising cell/tissue constructs so that they can be successfully accepted and integrated within a host body is essential in modern tissue engineering. To do this, adult stem cells are frequently utilised, but there are many aspects of their environment in vivo that are not completely understood. There is evidence to suggest that circadian rhythms and daily circadian temporal cues have substantial effects on stem cell activation, cell cycle, and differentiation. It was hypothesised that the circadian rhythm in human adult stem cells differs depending on the source of tissue and that different entraining signals exert differential effects depending on the anatomical source. Dexamethasone and rhythmic mechanical stretch were used to synchronise stem cells derived from the bone marrow, tooth dental pulp, and abdominal subcutaneous adipose tissue, and it was experimentally evidenced that these different stem cells differed in their circadian clock properties in response to different synchronisation mechanisms. The more primitive dental pulp-derived stem cells did not respond as well to the chemical synchronisation but showed temporal clock gene oscillations following rhythmic mechanical stretch, suggesting that incorporating temporal circadian information of different human adult stem cells will have profound implications in optimising tissue engineering approaches and stem cell therapies.

Adult human pancreas-derived cells expressing stage-specific embryonic antigen 4 differentiate into Sox9-expressing and Ngn3-expressing pancreatic ducts in vivo.

BackgroundTissue-specific stem/progenitor cells are found in various adult tissues and may have the capacity for lineage-specific differentiation, facilitating applications in autologous transplantation. Stage-specific embryonic antigen 4 (SSEA-4), an early embryonic glycolipid antigen, is expressed in cells derived from adult human pancreas exocrine tissue. Here, we examined the characteristics and lineage-specific differentiation capacity of SSEA-4+ cells.MethodsHuman adult partial pancreas tissues were obtained from different donors and cultured in vitro. SSEA-4+ and CA19-9+ cells were isolated from adult human pancreas exocrine cells using magnetic-activated cell sorting, and gene expression was validated by quantitative polymerase chain reaction. To confirm in-vivo differentiation, SSEA-4+ and CA19-9+ cells were transplanted into the dorsal subcutaneous region of mice. Finally, morphological features of differentiated areas were confirmed by immunostaining and morphometric analysis.ResultsSSEA-4-expressing cells were detected in isolated pancreas exocrine cells from adult humans. These SSEA-4+ cells exhibited coexpression of CA19-9, a marker of pancreatic duct cells, but not amylase expression, as shown by immunostaining and flow cytometry. SSEA-4+ cells exhibited higher relative expression of Oct4, Nanog, Klf4, Sox2, and c-Myc mRNAs than CA19-9+ cells. Pancreatic intralobular ducts (PIDs) were generated from SSEA-4+ or CA19-9+ cells in vivo at 5 weeks after transplantation. However, newly formed PIDs from CA19-9+ cells were less abundant and showed an incomplete PID morphology. In contrast, newly formed PIDs from SSEA-4+ cells were abundant in the transplanted area and showed a crowded morphology, typical of PIDs. Sox9 and Ngn3, key transcription factors associated with pancreatic development and regeneration, were expressed in PIDs from SSEA-4+ cells.ConclusionsSSEA-4-expressing cells in the adult human pancreas may have the potential for regeneration of the pancreas and may be used as a source of stem/progenitor cells for pancreatic cell lineage-specific differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0422-0) contains supplementary material, which is available to authorized users.

Diversity of human and mouse homeobox gene expression in development and adult tissues.

BackgroundHomeobox genes encode a diverse set of transcription factors implicated in a vast range of biological processes including, but not limited to, embryonic cell fate specification and patterning. Although numerous studies report expression of particular sets of homeobox genes, a systematic analysis of the tissue specificity of homeobox genes is lacking.ResultsHere we analyse publicly-available transcriptome data from human and mouse developmental stages, and adult human tissues, to identify groups of homeobox genes with similar expression patterns. We calculate expression profiles for 242 human and 278 mouse homeobox loci across a combination of 59 human and 12 mouse adult tissues, early and late developmental stages. This revealed 20 human homeobox genes with widespread expression, primarily from the TALE, CERS and ZF classes. Most homeobox genes, however, have greater tissue-specificity, allowing us to compile homeobox gene expression lists for neural tissues, immune tissues, reproductive and developmental samples, and for numerous organ systems. In mouse development, we propose four distinct phases of homeobox gene expression: oocyte to zygote; 2-cell; 4-cell to blastocyst; early to mid post-implantation. The final phase change is marked by expression of ANTP class genes. We also use these data to compare expression specificity between evolutionarily-based gene classes, revealing that ANTP, PRD, LIM and POU homeobox gene classes have highest tissue specificity while HNF, TALE, CUT and CERS are most widely expressed.ConclusionsThe homeobox genes comprise a large superclass and their expression patterns are correspondingly diverse, although in a broad sense related to an evolutionarily-based classification. The ubiquitous expression of some genes suggests roles in general cellular processes; in contrast, most human homeobox genes have greater tissue specificity and we compile useful homeobox datasets for particular tissues, organs and developmental stages. The identification of a set of eutherian-specific homeobox genes peaking from human 8-cell to morula stages suggests co-option of new genes to new developmental roles in evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0140-y) contains supplementary material, which is available to authorized users.

Drebrin: A new oncofetal biomarker associated with prognosis of lung adenocarcinoma

ObjectivesWith the aim of searching for novel oncofetal tumor biomarkers of lung adenocarcinoma other than carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), we developed a strategy involving monoclonal antibodies generated from embryonic tissue of miniature swine. Materials and methodsUsing immunohistochemistry, we selected suitable hybridoma clones that were reactive against swine fetal lung but not adult lung using tissue microarray loading of human normal lung, lung cancer, and fetal and adult swine tissues. ResultsThe selected clones included several that were uniquely reactive against both swine fetal lung and human lung adenocarcinoma, and protein microarray revealed that the antigen they recognized was “drebrin” (DBN1). We then examined the association between the pattern of drebrin expression and the clinicopathological characteristics of lung adenocarcinoma using surgically resected samples of human lung adenocarcinoma. Two hundred formalin-fixed and paraffin-embedded tumor samples were immunostained for drebrin using clone B246, one of the clones that were reactive against drebrin. The cases were divided into those with strong (n=85) and weak (n=115) drebrin expression. In terms of disease-free survival, cases showing strong drebrin expression had a significantly poorer prognosis than those with weak drebrin expression (p=0.033). ConclusionThe present findings indicate that “drebrin” is a unique oncofetal protein that can be applied as a new biomarker of lung adenocarcinoma.

Brown Adipose Tissue- What is Known and What to Be Known?

1. Abstract Adult human brown adipose tissue has been known for a long time as a vestigial organ with limited or no function that found with opulencein newborn and infants, helping them controlling their body thermogenesis without shivering, followed by its gradual disappearance with age (disparate form animals like rodents which keep BAT in adult life). Recently BAT existence, distribution and activity has been unraveled accidentally. There by researches on BAT demonstrated undeniable links to human metabolism and body composition. The fixed facts regarding body metabolism and energy homeostasis that have been granted for centuries are about to exhibit different perspectives. BAT discovery ignited a new line of research focusing on modulating energy expenditure and hence controlling many metabolic phenomena. Here we offer a brief review of what have been reported regarding BAT and it activity with pointing to novel challenges that need to be unveiled.

SOX2, OCT3/4 and NANOG expression and cellular plasticity in rare human somatic cells requires CD73

Endogenous Plastic Somatic (ePS) cells isolated from adult human tissues exhibit extensive lineage plasticity in vitro and in vivo. Here we visualize these rare ePS cells in a latent state, i.e. lacking SOX2, OCT3/4 and NANOG (SON) expression, in non-diseased breast specimens through immunohistochemical analysis of previously identified ePS-specific biomarkers (CD73+, EpCAM+ and CD90−). We also report a novel mechanism by which these latent ePS cells acquire SON expression and plasticity in vitro. Four extracellular factors are necessary for the acquisition of SON expression and lineage plasticity in ePS cells: adenosine (which is produced by the 5′ ecto-nucleotidase CD73 and activates in turn the PKA-dependent IL6/STAT3 pathway through the adenosine receptor ADORA2b), IL6, FGF2 and ACTIVIN A. Blocking any pathway component renders ePS cells incapable of SON expression and lineage plasticity. Notably, hESCs do not use adenosine or IL6 nor they express CD73 or ADORA2b and inhibition of adenosine signaling does not ablate their plasticity. Therefore, the data presented here delineate novel circuitry and physiological signals for accessing SON expression in rare, undifferentiated human cells.

A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids.

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro, and become more adult-like in their structure, cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al., 2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment, long-term survival, and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue, we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue, resulting in airway-like structures that were remarkably similar to the native adult human lung.

Glypican-3 Targeting Immunotoxins for the Treatment of Liver Cancer.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, yet no effective therapeutics exist. This review provides an overview of the recent development of recombinant immunotoxins for the treatment of glypican-3 (GPC3) expressing HCC. GPC3 is a cell surface heparan sulfate proteoglycan that is overexpressed in HCC, but is absent from normal adult human tissues. Treatment of HCC with anti-GPC3 immunotoxins represents a new therapeutic option. Using phage display and hybridoma technologies, three high affinity antibodies (HN3, HS20 and YP7) have been generated against GPC3. Two of these antibodies (HN3 and HS20) have demonstrated the ability to inhibit Wnt/Yap signaling, leading to a reduction in liver cancer cell proliferation. By combining the HN3 antibody capable of inhibiting Wnt/Yap signaling with the protein synthesis inhibitory domain of the Pseudomonas exotoxin, a recombinant immunotoxin that exhibits a dual inhibitory mechanism was generated. This immunotoxin was found to be highly effective in the treatment of human HCCs in mouse xenograft models. Engineering of the toxin fragment to reduce the level of immunogenicity is currently being explored. The development of immunotoxins provides opportunities for novel liver cancer therapies.