Co-Transplantation of Marginal Mass Allogeneic Islets with 3D-Culture-Derived Adult Human Skin Cells Improves Glycemia in Diabetic Mice

Background. Pancreatic islets transplantation represents a promising therapeutic option for severe type 1 diabetes (T1D). Maintenance of long-term viability of transplanted islets still requires improvement. Stem cell use represents an option to repair and replace damaged islets or, alternatively, b cells in T1D. Mesenchymal stem cells (MSC) have been proposed as adjuvants for islet transplantation, facilitating grafting and improving their functionality. Aggregation of stem cells has gained interest in providing physiological interactions between cells and enhancing the in situ concentration of modulators of inflammation and immunity.Methods. We established a hanging-drop culture by the spontaneous aggregation of adult human skin fibroblast-like cells as spheroids. Adult skin spheroid-derived cells (SphCs) were characterized in vitro and in vivo. We assessed the potential benefit of SphCs as adjuvants to improve islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model. We characterized the secretome of SphCs by mass spectrometry-based proteomics.Results. SphCs were characterized as multipotent progenitors by their surface expression of markers analyzed by flow cytometry and multilineage germ differentiation capacity. Coculture of SphCs with anti-CD3-stimulated mouse splenocytes diminished the proliferation of T-CD4+ lymphocytes and biased splenocyte cytokine secretion through an increase in the Th2/Th1 ratio. SphCs conditioned media attenuated apoptosis of islets induced by inflammatory cytokine challenge in vitro. Administration (i.t.) of SphCs showed the absence of tumorigenicity in immune-deficient mice.SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in an experimental diabetic mouse model without pharmacological immunosuppression. SphCs’ protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influence islets such as cytoprotection, cellular stress, metabolism, and survival.Conclusions. Hanging-drop cell culture methodology might contribute to the development of an efficient way to improve transplantation outcome by reducing undesirable consequences of pharmacological immunosuppression as well as the number of allogeneic islets required to achieve normoglycemia in T1D transplanted patients. Further studies might determine whether the identified proteins sustain immunomodulation and/or cytoprotective effects in transplanted allogeneic islets.

PE38-based gene therapy of HER2-positive breast cancer stem cells via VHH-redirected polyamidoamine dendrimers

Breast cancer stem cells (BCSCs) resist conventional treatments and cause tumor recurrence. Almost 25% of breast cancers overexpress human epidermal growth factor receptor-2 (HER2). Here we developed a novel multi-targeted nanosystem to specifically eradicate HER2+ BCSCs. Plasmids containing CXCR1 promoter, PE38 toxin, and 5′UTR of the basic fibroblast growth factor-2 (bFGF 5'UTR) were constructed. Polyamidoamine (PAMAM) dendrimers functionalized with anti-HER2 VHHs were used for plasmid delivery. Stem cell proportion of MDA-MB-231, MDA-MB-231/HER2+ and MCF-10A were evaluated by mammosphere formation assay. Hanging drop technique was used to produce spheroids. The uptake, gene expression, and killing efficacy of the multi-targeted nanosystem were evaluated in both monolayer and spheroid culture. MDA-MB-231/HER2+ had higher ability to form mammosphere compared to MCF-10A. Our multi-targeted nanosystem efficiently inhibited the mammosphere formation of MDA-MB-231 and MDA-MB-231/HER2+ cells, while it was unable to prevent the mammosphere formation of MCF-10A. In the hanging drop culture, MDA-MB-231/HER+ generated compact well-rounded spheroids, while MCF-10A failed to form compact cellular masses. The multi-targeted nanosystem showed much better uptake, higher PE38 expression, and subsequent cell death in MDA-MB-231/HER2+ compared to MCF-10A. However, the efficacy of our targeted toxin gene therapy was lower in MDA-MB-231/HER2+ spheroids compared with that in the monolayer culture. the combination of the cell surface, transcriptional, and translational targeting increased the stringency of the treatment.

O-190 Comparison between effects of exposure to platinum-based chemotherapeutics (cisplatin and carboplatin) on Sertoli cell number and functions in immature human testicular tissues

Study question Does exposure to either cisplatin or carboplatin have a damaging effect on the Sertoli cell population in the immature human testicular tissues? Summary answer Exposure to cisplatin or carboplatin did not appear to have a major effect on Sertoli cell number or function in the immature human testicular tissues What is known already Long-term survival rates for children with cancer are more than 80%. However, childhood cancer treatment may result in subsequent infertility. Cisplatin is one of the most commonly used drugs for childhood cancers. Carboplatin, a second generation platinum drug, is administered at 10-times the dose of cisplatin and is believed to be less gonadotoxic. In our recent publication we have shown that exposure to both cisplatin and carboplatin acutely reduce the germ cell number in immature human testicular tissues. However, it is not known how cisplatin and carboplatin affect Sertoli cell number and function. Study design, size, duration In-vitro culture of human fetal and pre-pubertal testicular tissues was utilised. Tissue pieces were cultured for 1-3 days prior to exposure to clinically-relevant doses of chemotherapeutics or vehicle control for 24hrs in two sets of experiments: 1) 0.5 or 1 μg/ml cisplatin and culture ended at 24 and 96hrs post-exposure (fetal only); 2) 0.5 μg/ml cisplatin or 5 ­μg/ml carboplatin until 72 (both fetal and pre-pubertal) and 240hrs post-­exposure (fetal only). Participants/materials, setting, methods Testicular tissue fragments from second trimester human fetal (14-22 gestational weeks; n = 3-6) or pre-pubertal patients (1-8years old; n = 5) were cultured in a ‘hanging drop’ system.Quantification of Sertoli cell number (cells per cord/tubular area (mm2)) was performed on sections stained for expression of SOX9. Culture medium was collected to measure levels (ng/ml) of Anti-Mullerian hormone (AMH) and Inhibin B using ELISA. Statistical analysis was performed using two-way ANOVA to account for inter-individual variation between fetuses/patients. Main results and the role of chance Quantification of positively stained Sertoli cells showed that exposure to both doses of cisplatin had no effect on Sertoli cell number at 24 and 96hrs post-exposure. No changes in AMH and inhibin B levels were observed at these time-points. Comparison between cisplatin- or carboplatin-exposed human fetal testicular tissues showed no difference in Sertoli cell numbers at either 72hrs or 240hrs post-exposure. No difference in Sertoli cell number was observed in pre-pubertal testicular tissues exposed to either cisplatin or carboplatin at 72hrs post-exposure. Limitations, reasons for caution Human fetal and pre-pubertal testis tissue is of limited availability, thus, sample sizes used in this study were relatively low. ‘Hanging drop’ culture might not recapitulate all in-vivo aspects of immature testis microenvironment. Wider implications of the findings Exposure to cisplatin or carboplatin did not affect Sertoli cell number in the immature human testicular tissues. Taken together with our recent publication, this suggests that these two platinum-based chemotherapeutic agents cause direct damage to germ cells. Functionality of Sertoli cells in chemotherapy-exposed tissues need to be further investigated. Trial registration number not applicable

Tailored generation of insulin producing cells from canine mesenchymal stem cells derived from bone marrow and adipose tissue

The trend of regenerative therapy for diabetes in human and veterinary practices has conceptually been proven according to the Edmonton protocol and animal models. Establishing an alternative insulin-producing cell (IPC) resource for further clinical application is a challenging task. This study investigated IPC generation from two practical canine mesenchymal stem cells (cMSCs), canine bone marrow-derived MSCs (cBM-MSCs) and canine adipose-derived MSCs (cAD-MSCs). The results illustrated that cBM-MSCs and cAD-MSCs contain distinct pancreatic differentiation potential and require the tailor-made induction protocols. The effective generation of cBM-MSC-derived IPCs needs the integration of genetic and microenvironment manipulation using a hanging-drop culture of PDX1-transfected cBM-MSCs under a three-step pancreatic induction protocol. However, this protocol is resource- and time-consuming. Another study on cAD-MSC-derived IPC generation found that IPC colonies could be obtained by a low attachment culture under the three-step induction protocol. Further, Notch signaling inhibition during pancreatic endoderm/progenitor induction yielded IPC colonies through the trend of glucose-responsive C-peptide secretion. Thus, this study showed that IPCs could be obtained from cBM-MSCs and cAD-MSCs through different induction techniques. Also, further signaling manipulation studies should be conducted to maximize the protocol’s efficiency.

A Microfluidic Hanging-Drop-Based Islet Perifusion System for Studying Glucose-Stimulated Insulin Secretion From Multiple Individual Pancreatic Islets

Islet perifusion systems can be used to monitor the highly dynamic insulin release of pancreatic islets in glucose-stimulated insulin secretion (GSIS) assays. Here, we present a new generation of the microfluidic hanging-drop-based islet perifusion platform that was developed to study the alterations in insulin secretion dynamics from single pancreatic islet microtissues at high temporal resolution. The platform was completely redesigned to increase experimental throughput and to reduce operational complexity. The experimental throughput was increased fourfold by implementing a network of interconnected hanging drops, which allows for performing GSIS assays with four individual islet microtissues in parallel with a sampling interval of 30 s. We introduced a self-regulating drop-height mechanism that enables continuous flow and maintains a constant liquid volume in the chip, which enables simple and robust operation. Upon glucose stimulation, reproducible biphasic insulin release was simultaneously observed from all islets in the system. The measured insulin concentrations showed low sample-to-sample variation as a consequence of precise liquid handling with stable drop volumes, equal flow rates in the channels, and accurately controlled sampling volumes in all four drops. The presented device will be a valuable tool in islet and diabetes research for studying dynamic insulin secretion from individual pancreatic islets.

A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation

ABSTRACT Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from in vitro studies under static or flow conditions, in vivo observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant Staphylococcus aureus (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new in vitro system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection. IMPORTANCE The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant Staphylococcus aureus (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their in vivo location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting.