Galanin System in the Human Bile Duct and Perihilar Cholangiocarcinoma.

BACKGROUND: Perihilar cholangiocarcinoma (pCCA) is characterised by poor outcomes. Early diagnosis is essential for patient survival. The peptide galanin (GAL) and its receptors GAL1-3 are expressed in various tumours. Detailed characterisation of the GAL system in pCCA is lacking. Our study sought to characterise GAL and GAL1-3 receptor (GAL1-3-R) expression in the healthy human bile duct, in cholestasis and pCCA. METHODS: Immunohistochemical staining was performed in healthy controls (n = 5) and in the peritumoural tissues (with and without cholestasis) (n = 20) and tumour tissues of pCCA patients (n = 33) using validated antibodies. The score values of GAL and GAL1-3-R expression were calculated and statistically evaluated. RESULTS: GAL and GAL1-R were expressed in various bile duct cell types. GAL2-R was only slightly but still expressed in almost all the examined tissues, and GAL3-R specifically in cholangiocytes and capillaries. In a small pCCA patient cohort (n = 18), high GAL expression correlated with good survival, whereas high GAL3-R correlated with poor survival. CONCLUSIONS: Our in-depth characterisation of the GAL system in the healthy human biliary duct and pCCA in a small patient cohort revealed that GAL and GAL3-R expression in tumour cells of pCCA patients could potentially represent suitable biomarkers for survival.

The enhancer landscape predetermines the skeletal regeneration capacity of stromal cells.

Multipotent stromal cells are considered attractive sources for cell therapy and tissue engineering. Despite numerous experimental and clinical studies, broad application of stromal cell therapeutics is not yet emerging. A major challenge is the functional diversity of available cell sources. Here, we investigated the regenerative potential of clinically relevant human stromal cells from bone marrow (BMSCs), white adipose tissue, and umbilical cord compared with mature chondrocytes and skin fibroblasts in vitro and in vivo. Although all stromal cell types could express transcription factors related to endochondral ossification, only BMSCs formed cartilage discs in vitro that fully regenerated critical-size femoral defects after transplantation into mice. We identified cell type-specific epigenetic landscapes as the underlying molecular mechanism controlling transcriptional stromal differentiation networks. Binding sites of commonly expressed transcription factors in the enhancer and promoter regions of ossification-related genes, including Runt and bZIP families, were accessible only in BMSCs but not in extraskeletal stromal cells. This suggests an epigenetically predetermined differentiation potential depending on cell origin that allows common transcription factors to trigger distinct organ-specific transcriptional programs, facilitating forward selection of regeneration-competent cell sources. Last, we demonstrate that viable human BMSCs initiated defect healing through the secretion of osteopontin and contributed to transient mineralized bone hard callus formation after transplantation into immunodeficient mice, which was eventually replaced by murine recipient bone during final tissue remodeling.

Chondrocyte Isolation from Loose Bodies-An Option for Reducing Donor Site Morbidity for Autologous Chondrocyte Implantation.

Loose bodies (LBs) from patients with osteochondritis dissecans (OCD) are usually removed and discarded during surgical treatment of the defect. In this study, we address the question of whether these LBs contain sufficient viable and functional chondrocytes that could serve as a source for autologous chondrocyte implantation (ACI) and how the required prolonged in vitro expansion affects their phenotype. Chondrocytes were isolated from LBs of 18 patients and compared with control chondrocyte from non-weight-bearing joint regions (n = 7) and bone marrow mesenchymal stromal cells (BMSCs, n = 6) obtained during primary arthroplasty. No significant differences in the initial cell yield per isolation and the expression of the chondrocyte progenitor cell markers CD44 + /CD146+ were found between chondrocyte populations from LBs (LB-CH) and control patients (Ctrl-CH). During long-term expansion, LB-CH exhibited comparable viability and proliferation rates to control cells and no ultimate cell cycle arrest was observed within 12 passages respectively 15.3 ± 1.1 mean cumulative populations doublings (CPD). The chondrogenic differentiation potential was comparable between LB-CH and Ctrl-CH, but both groups showed a significantly higher ability to form a hyaline cartilage matrix in vitro than BMSC. Our data suggest that LBs are a promising cell source for obtaining qualitatively and quantitatively suitable chondrocytes for therapeutic applications, thereby circumventing donor site morbidity as a consequence of the biopsies required for the current ACI procedure.

Hydrogen Sulfide Metabolizing Enzymes in the Intestinal Mucosa in Pediatric and Adult Inflammatory Bowel Disease.

Hydrogen sulfide (H2S) is a toxic gas that has important regulatory functions. In the colon, H2S can be produced and detoxified endogenously. Both too little and too much H2S exposure are associated with inflammatory bowel disease (IBD), a chronic intestinal disease mainly classified as Crohn's disease (CD) and ulcerative colitis (UC). As the pathogenesis of IBD remains elusive, this study's aim was to investigate potential differences in the expression of H2S-metabolizing enzymes in normal aging and IBD. Intestinal mucosal biopsies of 25 adults and 22 children with IBD along with those of 26 healthy controls were stained immunohistochemically for cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST). Expression levels were calculated by multiplication of the staining intensity and percentage of positively stained cells. Healthy adults showed an overall trend towards lower expression of H2S-metabolizing enzymes than healthy children. Adults with IBD also tended to have lower expression compared to controls. A similar trend was seen in the enzyme expression of children with IBD compared to controls. These results indicate an age-related decrease in the expression of H2S-metabolizing enzymes and a dysfunctional H2S metabolism in IBD, which was less pronounced in children.

A functional corona around extracellular vesicles enhances angiogenesis, skin regeneration and immunomodulation.

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell-derived extracellular vesicles (EVs). Here we demonstrate that nano-sized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration (TFF) and subtractive tandem mass-tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein-based flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super-resolution microscopy, electron microscopy and zeta-potential shift, and served as a proof-of-concept. Understanding EV corona formation will improve rational EV-inspired nano-therapy design.

Batch Effects during Human Bone Marrow Stromal Cell Propagation Prevail Donor Variation and Culture Duration: Impact on Genotype, Phenotype and Function.

Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

Synergy of Human Platelet-Derived Extracellular Vesicles with Secretome Proteins Promotes Regenerative Functions.

Platelet-rich plasma is a promising regenerative therapeutic with controversial efficacy. We and others have previously demonstrated regenerative functions of human platelet lysate (HPL) as an alternative platelet-derived product. Here we separated extracellular vesicles (EVs) from soluble factors of HPL to understand the mode of action during skin-organoid formation and immune modulation as model systems for tissue regeneration. HPL-EVs were isolated by tangential-flow filtration (TFF) and further purified by size-exclusion chromatography (SEC) separating EVs from (lipo)protein-enriched soluble fractions. We characterized samples by tunable resistive pulse sensing, western blot, tandem mass-tag proteomics and super-resolution microscopy. We evaluated EV function during angiogenesis, wound healing, organoid formation and immune modulation. We characterized EV enrichment by TFF and SEC according to MISEV2018 guidelines. Proteomics showed three major clusters of protein composition separating TSEC-EVs from HPL clustering with TFF soluble fractions and TFF-EVs clustering with TSEC soluble fractions, respectively. HPL-derived TFF-EVs promoted skin-organoid formation and inhibited T-cell proliferation more efficiently than TSEC-EVs or TSEC-soluble fractions. Recombining TSEC-EVs with TSEC soluble fractions re-capitulated TFF-EV effects. Zeta potential and super-resolution imaging further evidenced protein corona formation on TFF-EVs. Corona depletion on SEC-EVs could be artificially reconstituted by TSEC late fraction add-back. In contrast to synthetic nanoparticles, which commonly experience reduced function after corona formation, the corona-bearing EVs displayed improved functionality. We conclude that permissive isolation technology, such as TFF, and better understanding of the mechanism of EV corona function are required to realize the complete potential of platelet-based regenerative therapies.

Self-assembly of differentiated progenitor cells facilitates spheroid human skin organoid formation and planar skin regeneration.

Self-assembly of solid organs from single cells would greatly expand applicability of regenerative medicine. Stem/progenitor cells can self-organize into micro-sized organ units, termed organoids, partially modelling tissue function and regeneration. Here we demonstrated 3D self-assembly of adult and induced pluripotent stem cell (iPSC)-derived fibroblasts, keratinocytes and endothelial progenitors into both, planar human skin in vivo and a novel type of spheroid-shaped skin organoids in vitro, under the aegis of human platelet lysate. Methods: Primary endothelial colony forming cells (ECFCs), skin fibroblasts (FBs) and keratinocytes (KCs) were isolated from human tissues and polyclonally propagated under 2D xeno-free conditions. Human tissue-derived iPSCs were differentiated into endothelial cells (hiPSC-ECs), fibroblasts (hiPSC-FBs) and keratinocytes (hiPSC-KCs) according to efficiency-optimized protocols. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicated their stem/progenitor potential. Triple cell type floating spheroids formation was promoted by human platelet-derived growth factors containing culture conditions, using nanoparticle cell labelling for monitoring the organization process. Planar human skin regeneration was assessed in full-thickness wounds of immune-deficient mice upon transplantation of hiPSC-derived single cell suspensions. Results: Organoids displayed a distinct architecture with surface-anchored keratinocytes surrounding a stromal core, and specific signaling patterns in response to inflammatory stimuli. FGF-7 mRNA transfection was required to accelerate keratinocyte long-term fitness. Stratified human skin also self-assembled within two weeks after either adult- or iPSC-derived skin cell-suspension liquid-transplantation, healing deep wounds of mice. Transplant vascularization significantly accelerated in the presence of co-transplanted endothelial progenitors. Mechanistically, extracellular vesicles mediated the multifactorial platelet-derived trophic effects. No tumorigenesis occurred upon xenografting. Conclusion: This illustrates the superordinate progenitor self-organization principle and permits novel rapid 3D skin-related pharmaceutical high-content testing opportunities with floating spheroid skin organoids. Multi-cell transplant self-organization facilitates development of iPSC-based organ regeneration strategies using cell suspension transplantation supported by human platelet factors.

Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production.

Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet's coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.

Heparin Differentially Impacts Gene Expression of Stromal Cells from Various Tissues.

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.

Evaluation of modified Interferon alpha mRNA constructs for the treatment of non-melanoma skin cancer.

Application of in vitro transcribed (IVT) messenger ribonucleic acid (mRNA) is an increasingly popular strategy to transiently produce proteins as therapeutics in a tissue or organ of choice. Here, we focused on the skin and aimed to test if whole human skin tissue explant technology can be used to evaluate the expression efficacy of different IVT Interferon alpha (IFN-α) mRNA constructs in situ, after biolistic delivery. Skin explants were viable and intact for at least five days based on histologic analysis and TUNEL staining. Using GFP reporter mRNA formulations, we found mostly epidermal expression after biolistic delivery. Two out of five sequence-optimized IFN-α mRNA variants resulted in significantly improved IFN-α protein expression in human skin compared to native IFN-α mRNA transfection. IFN-α secretion analysis of the surrounding culture media confirmed these results. We provide a proof-of-concept that IFN-α mRNA delivery into intact human full thickness skin explants can be utilized to test mRNA sequence modifications ex vivo. This approach could be used to develop novel mRNA-based treatments of common epidermal skin conditions including non-melanoma skin cancer, where IFN-α protein therapy has previously shown a strong therapeutic effect.

Selection of Tissue Factor-Deficient Cell Transplants as a Novel Strategy for Improving Hemocompatibility of Human Bone Marrow Stromal Cells.

Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation. Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC. Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells. Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.

Synergistic effects of dendritic cell targeting and laser-microporation on enhancing epicutaneous skin vaccination efficacy.

Due to its unique immunological properties, the skin is an attractive target tissue for allergen-specific immunotherapy. In our current work, we combined a dendritic cell targeting approach with epicutaneous immunization using an ablative fractional laser to generate defined micropores in the upper layers of the skin. By coupling the major birch pollen allergen Bet v 1 to mannan from S. cerevisiae via mild periodate oxidation we generated hypoallergenic Bet-mannan neoglycoconjugates, which efficiently targeted CD14+ dendritic cells and Langerhans cells in human skin explants. Mannan conjugation resulted in sustained release from the skin and retention in secondary lymphoid organs, whereas unconjugated antigen showed fast renal clearance. In a mouse model, Bet-mannan neoglycoconjugates applied via laser-microporated skin synergistically elicited potent humoral and cellular immune responses, superior to intradermal injection. The induced antibody responses displayed IgE-blocking capacity, highlighting the therapeutic potential of the approach. Moreover, application via micropores, but not by intradermal injection, resulted in a mixed TH1/TH17-biased immune response. Our data clearly show that applying mannan-neoglycoconjugates to an organ rich in dendritic cells using laser-microporation is superior to intradermal injection. Due to their low IgE binding capacity and biodegradability, mannan neoglycoconjugates therefore represent an attractive formulation for allergen-specific epicutaneous immunotherapy.

Characterization of light lesion paradigms and optical coherence tomography as tools to study adult retina regeneration in zebrafish.

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.

Fgf signaling is required for photoreceptor maintenance in the adult zebrafish retina.

Fibroblast growth factors (Fgf) are secreted signaling molecules that have mitogenic, patterning, neurotrophic and angiogenic properties. Their importance during embryonic development in patterning and morphogenesis of the vertebrate eye is well known, but less is known about the role of Fgfs in the adult vertebrate retina. To address Fgf function in adult retina, we determined the spatial distribution of components of the Fgf signaling pathway in the adult zebrafish retina. We detected differential expression of Fgf receptors, ligands and downstream Fgf targets within specific retinal layers. Furthermore, we blocked Fgf signaling in the retina, by expressing a dominant negative variant of Fgf receptor 1 conditionally in transgenic animals. After blocking Fgf signaling we observe a fast and progressive photoreceptor degeneration and disorganization of retinal tissue, coupled with cell death in the outer nuclear layer. Following the degeneration of photoreceptors, a profound regeneration response is triggered that starts with proliferation in the inner nuclear layer. Ultimately, rod and cone photoreceptors are regenerated completely. Our study reveals the requirement of Fgf signaling to maintain photoreceptors and for proliferation during regeneration in the adult zebrafish retina.

Heterogeneity and Fgf dependence of adult neural progenitors in the zebrafish telencephalon.

Adult telencephalic neurogenesis is a conserved trait of all vertebrates studied. It has been investigated in detail in rodents, but very little is known about the composition of neurogenic niches and the cellular nature of progenitors in nonmammalian vertebrates. To understand the components of the progenitor zones in the adult zebrafish telencephalon and the link between glial characteristics and progenitor state, we examined whether canonical glial markers are colocalized with proliferation markers. In the adult zebrafish telencephalon, we identify heterogeneous progenitors that reside in two distinct glial domains. We find that the glial composition of the progenitor zone is linked to its proliferative behavior. Analyzing both fast-cycling proliferating cells as well as slowly cycling progenitors, we find four distinct progenitor types characterized by differential expression of glial markers. Importantly, a significant proportion of progenitors do not display typical radial glia characteristics. By blocking or activating Fgf signaling by misexpression of a dominant negative Fgf-receptor 1 or Fgf8a, respectively, we find that ventral and dorsal progenitors in the telencephalon also differ in their requirement for Fgf signaling. Together with data on the expression of Fgf signaling components in the ventricular zone of the telencephalon, this suggests that Fgf signaling directly regulates proliferation of specific subsets of adult telencephalic progenitors in vivo. Taken together our results show that adult neural progenitor cells are heterogeneous with their respect to distribution into two distinct glial domains and their dependence upon Fgf signaling as a proliferative cue in the zebrafish telencephalon.

Expression of marker genes during early ear development in medaka.

Induction of the otic placode involves a number of regulatory interactions. Early studies revealed that the induction of this program is initiated by instructive signals from the mesendoderm as well as from the adjacent hindbrain. Further investigations on the molecular level identified in zebrafish Fgf3, Fgf8, Foxi1, Pax8, Dlx3b and Dlx4b genes as key players during the induction phase. Thereafter an increasing number of genes participates in the regulatory interactions finally resulting in a highly structured sensory organ. Based on data from zebrafish we selected medaka genes with presumptive functions during early ear development for an expression analysis. In addition we isolated Foxi1 and Dlx3b gene fragments from embryonic cDNA. Altogether we screened the spatio-temporal distribution of more than 20 representative marker genes for otic development in medaka embryos, with special emphasis on the early phases. Whereas the spatial distribution of these genes is largely conserved between medaka and zebrafish, our comparative analysis revealed several differences, in particular for the timing of expression.