An important step towards a prevascularized islet microencapsulation device: in vivo prevascularization by combination of mesenchymal stem cells on micropatterned membranes.

Extrahepatic transplantation of islets of Langerhans could aid in better survival of islets after transplantation. When islets are transfused into the liver 60-70% of them are lost immediately after transplantation. An important factor for a successful extrahepatic transplantation is a well-vascularized tissue surrounding the implant. There are many strategies known for enhancing vessel formation such as adding cells with endothelial potential, the combination with angiogenic factors and / or applying surface topography at the exposed surface of the device. Previously we developed porous, micropatterned membranes which can be applied as a lid for an islet encapsulation device and we showed that the surface topography induces human umbilical vein endothelial cell (HUVEC) alignment and interconnection. This was achieved without the addition of hydrogels, often used in angiogenesis assays. In this work, we went one step further towards clinical implementation of the device by combining this micropatterned lid with Mesenchymal Stem Cells (MSCs) to facilitate prevascularization in vivo. As for HUVECs, the micropatterned membranes induced MSC alignment and organization in vitro, an important contributor to vessel formation, whereas in vivo (subcutaneous rat model) they contributed to improved implant prevascularization. In fact, the combination of MSCs seeded on the micropatterned membrane induced the highest vessel formation score in 80% of the sections.

Injectable Cell-Laden Polysaccharide Hydrogels: In Vivo Evaluation of Cartilage Regeneration.

Previously, 5% w/v hyaluronic acid-tyramine (HA-TA) and dextran-tyramine (Dex-TA) enzymatically cross-linked hybrid hydrogels were demonstrated to provide a mechanically stable environment, maintain cell viability, and promote cartilaginous-specific matrix deposition in vitro. In this study, 5% w/v hybrid hydrogels were combined with human mesenchymal stem cells (hMSCs), bovine chondrocytes (bCHs), or a combination of both in a 4:1 ratio and subcutaneously implanted in the backs of male and female nude rats to assess the performance of cell-laden hydrogels in tissue formation. Subcutaneous implantation of these biomaterials showed signs of integration of the gels within the host tissue. Histological analysis showed residual fibrotic capsules four weeks after implantation. However, enhanced tissue invasion and some giant cell infiltration were observed in the HA-TA/Dex-TA hydrogels laden with either hMSCs or bCHs but not with the co-culture. Moreover, hMSC-bCH co-cultures showed beneficial interaction with the hydrogels, for instance, in enhanced cell proliferation and matrix deposition. In addition, we provide evidence that host gender has an impact on the performance of bCHs encapsulated in HA-TA/Dex-TA hydrogels. This study revealed that hydrogels laden with different types of cells result in distinct host responses. It can be concluded that 5% w/v hydrogels with a higher concentration of Dex-TA (≥50%) laden with bCH-hMSC co-cultures are adequate for injectable applications and in situ cell delivery in cartilage regeneration approaches.

An ECHO of Cartilage: In Silico Prediction of Combinatorial Treatments to Switch Between Transient and Permanent Cartilage Phenotypes With Ex Vivo Validation.

A fundamental question in cartilage biology is: what determines the switch between permanent cartilage found in the articular joints and transient hypertrophic cartilage that functions as a template for bone? This switch is observed both in a subset of OA patients that develop osteophytes, as well as in cell-based tissue engineering strategies for joint repair. A thorough understanding of the mechanisms regulating cell fate provides opportunities for treatment of cartilage disease and tissue engineering strategies. The objective of this study was to understand the mechanisms that regulate the switch between permanent and transient cartilage using a computational model of chondrocytes, ECHO. To investigate large signaling networks that regulate cell fate decisions, we developed the software tool ANIMO, Analysis of Networks with interactive Modeling. In ANIMO, we generated an activity network integrating 7 signal transduction pathways resulting in a network containing over 50 proteins with 200 interactions. We called this model ECHO, for executable chondrocyte. Previously, we showed that ECHO could be used to characterize mechanisms of cell fate decisions. ECHO was first developed based on a Boolean model of growth plate. Here, we show how the growth plate Boolean model was translated to ANIMO and how we adapted the topology and parameters to generate an articular cartilage model. In ANIMO, many combinations of overactivation/knockout were tested that result in a switch between permanent cartilage (SOX9+) and transient, hypertrophic cartilage (RUNX2+). We used model checking to prioritize combination treatments for wet-lab validation. Three combinatorial treatments were chosen and tested on metatarsals from 1-day old rat pups that were treated for 6 days. We found that a combination of IGF1 with inhibition of ERK1/2 had a positive effect on cartilage formation and growth, whereas activation of DLX5 combined with inhibition of PKA had a negative effect on cartilage formation and growth and resulted in increased cartilage hypertrophy. We show that our model describes cartilage formation, and that model checking can aid in choosing and prioritizing combinatorial treatments that interfere with normal cartilage development. Here we show that combinatorial treatments induce changes in the zonal distribution of cartilage, indication possible switches in cell fate. This indicates that simulations in ECHO aid in describing pathologies in which switches between cell fates are observed, such as OA.

Low retinol levels differentially modulate bile salt-induced expression of human and mouse hepatic bile salt transporters.

UNLABELLED: The farnesoid X receptor/retinoid X receptor-alpha (FXR/RXRalpha) complex regulates bile salt homeostasis, in part by modulating transcription of the bile salt export pump (BSEP/ABCB11) and small heterodimer partner (SHP/NR0B2). FXR is activated by bile salts, RXRalpha by the vitamin A derivative 9-cis retinoic acid (9cRA). Cholestasis is associated with vitamin A malabsorption. Therefore, we evaluated the role of vitamin A/9cRA in the expression of human and mouse bile salt export pump (hBSEP/mBsep), small heterodimer partner (hSHP/mShp), and mouse sodium-dependent taurocholate co-transporting polypeptide (mNtcp). HBSEP and hSHP transcription were analyzed in FXR/RXRalpha-transfected HepG2 cells exposed to chenodeoxycholic acid (CDCA) and/or 9cRA. BSEP promoter activity was determined by luciferase reporter assays, DNA-binding of FXR and RXRalpha by pull-down assays. Serum bile salt levels and hepatic expression of Bsep, Shp, and Ntcp were determined in vitamin A-deficient (VAD)/cholic acid (CA)-fed C57BL/6J mice. Results indicated that 9cRA strongly repressed the CDCA-induced BSEP transcription in HepG2 cells, whereas it super-induced SHP transcription; 9cRA reduced DNA-binding of FXR and RXRalpha. The 9cRA repressed the CDCA-induced BSEP promoter activity irrespective of the exact sequence of the FXR-binding site. In vivo, highest Bsep messenger RNA (mRNA), and protein expression was observed in CA-fed VAD mice. Shp transcription was highest in CA-fed vitamin A-sufficient mice. Ntcp protein expression was strongly reduced in CA-fed VAD mice, whereas mRNA levels were normal. CA-fed control and VAD mice had similarly increased serum bile salt levels. CONCLUSION: We showed that 9cRA has opposite effects on bile salt-activated transcription of FXR/RXRalpha target genes. Vitamin A deficiency in CA-fed mice leads to high BSEP expression. Clearance of serum bile salts may, however, be limited because of post-transcriptional reduction of Ntcp. The molecular effects of vitamin A supplementation during cholestasis need further analysis to predict a therapeutic effect.

Coculturing Human Islets with Proangiogenic Support Cells to Improve Islet Revascularization at the Subcutaneous Transplantation Site.

While subcutaneous tissue has been proposed as a clinically relevant site for pancreatic islet transplantation, a major issue of concern remains, which is its poor vascular state. In an effort to overcome this limitation, we present an efficient and reproducible method to form human composite islets (CIs) with proangiogenic cell types in a controlled manner using nonadherent agarose microwell templates. In this study, we assessed the three-dimensional structure, function, and angiogenic potential of human CIs with human mesenchymal stromal cells (hMSCs), with or without human umbilical vein endothelial cells (HUVECs), and preconditioned hMSCs (PC-hMSCs) in EGM-2 under shear stress. Distinct cellular rearrangements could be observed in CIs, but islet functionality was maintained. In vitro angiogenesis assays found significantly enhanced sprout formation in case of CIs. In particular, the number of sprouts emanating from CIs with PC-hMSCs was significantly increased compared to other conditions. Subsequent in vivo assessment confirmed the proangiogenic potential of CIs. However, in contrast to our in vitro angiogenesis assays, CIs with hMSCs and HUVECs exhibited a higher in vivo angiogenic potential compared to control islets or islets combined with hMSCs or PC-hMSCs. These findings highlight the importance and necessity of verifying in vitro studies with in vivo models to reliably predict, in this case, revascularization outcomes. Regardless, we demonstrate here the therapeutic potential of CIs with proangiogenic support cells to enhance islet revascularization at a clinically relevant, although poorly vascularized, transplantation site.

An orthotopic mouse model for chondrosarcoma of bone provides an in vivo tool for drug testing.

Chondrosarcoma is a malignant cartilaginous tumor of the bone. Recently, mutations in isocitrate dehydrogenase-1 (IDH1) and isocitrate dehydrogenase-2 (IDH2) were identified in central chondrosarcomas. As chondrosarcomas are notoriously resistant to conventional treatment modalities, the need for model systems to screen new treatment options is high. We used two chondrosarcoma cell lines (CH2879 and SW1353) to generate a bioluminescent orthotopic chondrosarcoma mouse model. Cell lines were stably transduced with a lentiviral luciferase expression vector, and after clonal selection, luciferase-expressing clones were subcutaneously and orthotopically implanted in nude mice. Mice injected with CH2879 cells were treated with doxorubicin over a period of 6 weeks. Both cell lines resulted in tumor growth. CH2879 tumors were consistently larger than SW1353 tumors. No difference in size could be observed between subcutaneous and orthotopic tumors. Tumor growth could be monitored over time through assessment of luciferase activity, without harming the mice. Using this model, we show that doxorubicin does not have a significant effect on in vivo tumor growth. We describe an orthotopic chondrosarcoma mouse model that can be used to test new treatment strategies evolving from in vitro research.

A progressive familial intrahepatic cholestasis type 2 mutation causes an unstable, temperature-sensitive bile salt export pump.

BACKGROUND/AIMS: Progressive familial intrahepatic cholestasis type 2 (PFIC-2) patients have a defect in the hepatocanalicular bile salt secretion. The disease is caused by mutations in the bile salt export pump (BSEP). Ten different missense mutations have been described. In this study, we analysed the effect of the D482G PFIC-2 mutation on BSEP function. METHODS: Adenosine triphosphatase (ATPase) and taurocholate transport assays were performed with full-length mouse Bsep (mBsep) with and without the D482G mutation. The effect on expression and subcellular sorting was studied in HepG2 cells, stably expressing enhanced green fluorescent protein (EGFP)-tagged mBsep proteins. RESULTS: The D482G mutation did not significantly affect the taurocholate transport activity of mBsep, even though the bile salt-inducible ATPase activity of the mutant protein was slightly reduced. Protein expression and canalicular sorting were strongly affected by the D482G mutation. Mutant EGFP-mBsep protein was only partly glycosylated and detected in both the canalicular membrane and the cytoplasm. At 30 degrees C, the mutant mRNA and protein levels were strongly increased, and the protein was predominantly glycosylated and efficiently targeted to the canalicular membrane. CONCLUSIONS: These data suggest that PFIC-2 patients with the D482G mutation express a functional, but highly unstable, temperature-sensitive bile salt export pump.

Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump.

The bile salt export pump (BSEP or ABCB11) mediates the adenosine triphosphate-dependent transport of bile salts across the canalicular membrane of the hepatocyte. Mutations in the corresponding ABCB11 gene cause progressive familial intrahepatic cholestasis type 2. The aim of this study was to investigate the regulation of human ABCB11 gene transcription by bile salts. First, a 1.7-kilobase human ABCB11 promoter region was cloned. Sequence analysis for possible regulatory elements showed a farnesoid X receptor responsive element (FXRE) at position minus sign180. The farnesoid X receptor (FXR) functions as a heterodimer with the retinoid X receptor alpha (RXRalpha) and can be activated by the bile salt chenodeoxycholic acid (CDCA). Luciferase reporter gene assays showed that the ABCB11 promoter is positively controlled by FXR, RXRalpha, and bile salts in a concentration-dependent manner. Mutation of the FXRE strongly represses the FXR-dependent induction. Second, endogenous ABCB11 transcription regulation was studied in HepG2 cells, stably expressing the rat sodium-dependent taurocholate transporter (rNtcp) cells. ABCB11 expression was induced by adding bile salts to the culture medium, and this effect was maximized by combining it with cotransfection of rFxr and hRXRalpha. Reducing endogenous FXR levels using RNA interference fully repressed the bile salt-induced ABCB11 expression. In conclusion, these results show that FXR is required for the bile salt-dependent transcriptional control of the human ABCB11 gene and that the cellular amount of FXR is critical for the level of activation of ABCB11 transcription.