Title Participants Abstract "Pituitary cell differentiation from stem cells and other cells: toward restorative therapy for hypopituitarism?" "Christophe Willems, Hugo Vankelecom" "The pituitary gland, key regulator of our endocrine system, produces multiple hormones that steer essential physiological processes. Hence, deficient pituitary function (hypopituitarism) leads to severe disorders. Hypopituitarism can be caused by defective embryonic development, or by damage through tumor growth/resection and traumatic brain injury. Lifelong hormone replacement is needed but associated with significant side effects. It would be more desirable to restore pituitary tissue and function. Recently, we showed that the adult (mouse) pituitary holds regenerative capacity in which local stem cells are involved. Repair of deficient pituitary may therefore be achieved by activating these resident stem cells. Alternatively, pituitary dysfunction may be mended by cell (replacement) therapy. The hormonal cells to be transplanted could be obtained by (trans-)differentiating various kinds of stem cells or other cells. Here, we summarize the studies on pituitary cell regeneration and on (trans-)differentiation toward hormonal cells, and speculate on restorative therapies for pituitary deficiency." "Mesenchymal stem cell secreted platelet derived growth factor exerts a pro-migratory effect on resident Cardiac Atrial appendage Stem Cells" "Severina WINDMOLDERS, Astrid De Boeck, Remco KONINCKX, Annick Daniëls, Olivier De Weyer, Marc Bracke, Marc HENDRIKX, Karen HENSEN, Jean-Luc RUMMENS" "Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors, but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stemcells (CSCs). This study aimed to test the pro-migratory effect ofMSC conditionedmedium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay, we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts, perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells, specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs), including a clonogenic potential of~21.5% and expression of pluripotency associated genes like Oct-4, Nanog, c-Myc and Klf-4. Similar to CASCs,migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and ImatinibMesylate, a PDGF receptor inhibitor, suggested a role for the PDGFAA/PDGF receptor α axis in enhancing the migration process of CASCs. In conclusion, our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies inwhichMSC secreted factors, like PDGF-AA, can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury." "Co-transplantation of mesenchymal stem cells improves spermatogonial stem cell transplantation efficiency in mice" "Prashant Kadam, Elissavet Ntemou, Yoni Baert, Sven Van Laere, Dorien Van Saen, Ellen Goossens" "Background: Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. Methods: A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n = 10), MSCs (MSCT, n = 10), a combination of SSCs and MSCs (MS-SSCT, n = 10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10). Results: The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs. Conclusion: Co-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven." "Can mesenchymal stem cells improve spermatogonial stem cell transplantation efficiency?" "Prashant Kadam, Dorien Van Saen, Ellen Goossens" "Improved treatments have led to an increased survival rate in cancer patients. However, in pre-pubertal boys, these gonadotoxic treatments can result in the depletion of the spermatogonial stem cell (SSC) pool causing lifelong infertility. SSC transplantation has been proposed as a promising technique to preserve the fertility of these patients. In mice, this technique has resulted in live-born offspring, but the efficiency of colonization remained low. This could be because of a deficient microenvironment, leading to apoptosis of the transplanted SSCs. Interestingly, mesenchymal stem cells (MSCs), being multipotent and easy to isolate and multiply in vitro, are nowadays successfully and widely used in regenerative medicine. Here, we shortly review the current understanding of MSC and SSC biology, and we hypothesize that a combined MSC-SSC transplantation might improve the efficiency of SSC colonization and differentiation as paracrine factors from MSCs may contribute to the SSC niche." "Complicity in stem cell research: the case of induced pluripotent stem cells" "Katrien Devolder" "Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells" "Petra HILKENS, Pascal GERVOIS, Yanick FANTON, J Vanormelingen, Wendy MARTENS, Tom STRUYS, Constantinus POLITIS, Ivo LAMBRICHTS, Annelies BRONCKAERS" "Dental pulp stem cells (DPSCs) are an attractive alternative mesenchymal stem cell (MSC) source because of their isolation simplicity compared with the more invasive methods associated with harvesting other MSC sources. However, the isolation method to be favored for obtaining DPSC cultures remains under discussion. This study compares the stem cell properties and multilineage differentiation potential of DPSCs obtained by the two most widely adapted isolation procedures. DPSCs were isolated either by enzymatic digestion of the pulp tissue (DPSC-EZ) or by the explant method (DPSC-OG), while keeping the culture media constant throughout all experiments and in both isolation methods. Assessment of the stem cell properties of DPSC-EZ and DPSC-OG showed no significant differences between the two groups with regard to proliferation rate and colony formation. Phenotype analysis indicated that DPSC-EZ and DPSC-OG were positive for CD29, CD44, CD90, CD105, CD117 and CD146 expression without any significant differences. The multilineage differentiation potential of both stem cell types was confirmed by using standard immuno(histo/cyto)chemical staining together with an in-depth ultrastructural analysis by means of transmission electron microscopy. Our results indicate that both DPSC-EZ and DPSC-OG could be successfully differentiated into adipogenic, chrondrogenic and osteogenic cell types, although the adipogenic differentiation of both stem cell populations was incomplete. The data suggest that both the enzymatic digestion and outgrowth method can be applied to obtain a suitable autologous DPSC resource for tissue replacement therapies of both bone and cartilage." "Tumour-promoting properties of mesenchymal stem cells in multiple myeloma: a potential risk for therapeutical use after haematopoietic stem cell transplantation in myeloma patients?" "Song Xu, Benjamin Van Camp" "Mesenchymal stem cells (MSCs) have recently been used in several pre-clinical and clinical studies to support hematopoiesis after hematopoietic stem cell transplantation (HSCT), to control graft versus host disease after allogeneic HSCT or for gene therapy in cancer. Although it is believed that MSCs have therapeutical potential for these particular applications,several studies have shown that MSCs might also play an active role in the pathogenesis and progression of tumors.Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells (PCs), which are predominantly localized in the bone marrow (BM). Mesenchymal stem cells (MSCs) give rise to most bone marrow stromal cells that interact with MM cells. However, the direct effect of MSCs on the growth control of MM cells has not been addressed. In the present study, we showed by in vitro migration assays that human MSCs are attracted by MM cells and that CCL25 is a major MM cell-produced chemoattractant stimulating chemotaxis of MSCs through CCR9. By coculture experiments we found that MSCs favor the proliferation of stroma-dependent MM cells through secretion of soluble factors and cell to cell contact.This growth promoting effect was also demonstrated by intrafemoral co-engraftment experiments in the in vivo mouse myeloma model 5T33MM. In addition we demonstrated that MSCs protect MM cells in vitro against spontaneous and Bortezomib-induced apoptosis. The tumor-promoting effects of MSCs correlates with their capacity to activate in MM cells AKT and ERK activities, accompanied with increased expresson of CyclinD2, CDK4 and Bcl-XL, and decreased cleaved caspase-3 and PARP expression. In turn, MM cells were found to upregulate interleukin-6 (IL-6), interleukin-10 (IL-10), insulin growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF) and dickkopf homolog 1 (DKK1) expression in MSCs. Finally, we demonstrated that intraveneous injection of in vitro expanded murine MSCs (mMSCs) in 5T33MM mice results in a signifi cantly shorter survival as compared to the control group, being injected with MM cells without mMSCs (p" "An mRNA mix redirects dendritic cells towards an antiviral program, inducing anticancer cytotoxic stem cell and central memory CD8+ T cells" "Wout de Mey, Hanne Locy, Kirsten De Ridder, Phaedra De Schrijver, Dorien Autaers, Asma Lakdimi, Arthur Esprit, Lorenzo Franceschini, Kris Thielemans, Magali Verdonck, Karine Breckpot" "Dendritic cell (DC)-maturation stimuli determine the potency of these antigen-presenting cells and, therefore, the quality of the T-cell response. Here we describe that the maturation of DCs via TriMix mRNA, encoding CD40 ligand, a constitutively active variant of toll-like receptor 4 and the co-stimulatory molecule CD70, enables an antibacterial transcriptional program. Besides, we further show that the DCs are redirected into an antiviral transcriptional program when CD70 mRNA in TriMix is replaced with mRNA encoding interferon-gamma and a decoy interleukin-10 receptor alpha, forming a four-component mixture referred to as TetraMix mRNA. The resulting TetraMixDCs show a high potential to induce tumor antigen-specific T cells within bulk CD8+ T cells. Tumor-specific antigens (TSAs) are emerging and attractive targets for cancer immunotherapy. As T-cell receptors recognizing TSAs are predominantly present on naive CD8+ T cells (TN), we further addressed the activation of tumor antigen-specific T cells when CD8+ TN cells are stimulated by TriMixDCs or TetraMixDCs. In both conditions, the stimulation resulted in a shift from CD8+ TN cells into tumor antigen-specific stem cell-like memory, effector memory and central memory T cells with cytotoxic capacity. These findings suggest that TetraMix mRNA, and the antiviral maturation program it induces in DCs, triggers an antitumor immune reaction in cancer patients." "Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers" "Paolo De Coppi" "BACKGROUND INFORMATION: In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. RESULTS: Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. CONCLUSIONS: This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential." "Spermatogonial stem cells. An update on spermatogonial stem cell banking and transplantation" "Ellen Goossens, Dorien Van Saen, Mieke Geens, Liang Ning, Herman Tournaye" "Like every other adult stem cell in the human body, spermatogonial stem cells (SSCs) have the capacity to either renew themselves or to start differentiation, i.e. spermatogenesis. Due to these properties, several options for preservation and re-establishment of the spermatogenic process exist. Prevention of sterility has become an important issue in reproductive medicine. For adult men, sterility after cancer treatment can be circumvented by banking sperm samples. For pre-pubertal patients, however, this is not an option, since spermatogenesis has not yet started. Currently, spermatogonial stem cell transplantation (SSCT) is considered the most promising tool for fertility restoration in pre-pubertal cancer patients. In these patients, testicular tissue could be removed and cryopreserved before starting any cancer treatment. When the boy is cured, SSCT can be applied for fertility restoration. However, before such an application can be applied, both the safety and the efficiency of the procedure have to be assured. In the mouse, we have shown that sperm obtained after SSCT were able to fertilize and produce offspring in-vivo and after assisted reproduction. However, it was also observed that fertilizing potential was lower with transplanted males compared to control mice. Moreover, the litter sizes were smaller and the fetal length and weight were significantly lower in the first-generation offspring from transplanted animals, whereas subsequent generations did not show those abnormalities. This observation may be suggestive for imprinting disorders. The efficiency of SSCT depends on the number of SSCs injected in the recipient's tubules. Since only the SSCs can relocate to the basement membrane and initiate colonization, enriching the proportion of SSCs may improve tranplantation efficiency. Although, SSCT could prove important for fertility preservation, this technique may not be without any risk. Testicular cell suspensions from cancer patients may be contaminated with cancerous cells. It is obvious that reintroduction of malignant cells into an otherwise cured patient must be omitted. Magnetic Activated Cell Sorting and Fluoresence Activated Cell Sorting are two strategies that can be used to decontaminate the cell suspension from malignant cells or to enrich the cell suspension for SSCs. Xenogeneic transplantation and xenografting are two other hypothetical methods to preserve fertility. Until now, human spermatogonial stem cells are reported only to survive in the murine testis and differentiation to spermatozoa has not yet been observed. Pre-pubertal murine tissue could be grafted successfully, with spermatogenesis observed in almost all the grafts, but adult murine and adult human grafts were lost because of sclerosis or atrophy. Although xenografting of human pre-pubertal tissue may be within reach, xenogeneic transplantation and xenografting should not be used in a clinical application because of the ethical and biological concerns inherent to these approaches. These techniques could however be useful to test the risk of malignant contamination of the testicular tissue. When SSCT becomes available for clinical use, efficient protocols for the cryopreservation of SSCs and testicular tissue will be of great benefit. By using a non-controlled freezing protocol, the survival rate of SSCs was higher compared with other testicular cells, which resulted in an enrichment of SSCs in the final suspension, but an important loss of functionality of spermatogonial stem cells was found after freezing and thawing. An alternative way to preserve SSCs is to freeze the whole testicular tissue instead of cell suspensions. The structure of the tissue can be well preserved and especially the spermatogonia survive. An alternative to cryopreservation could be in-vitro culture of SSCs. This approach may be applied to generate spermatozoa in-vitro from cultured spermatogonial stem cells, which, in turn, could be used for intracytoplasmic sperm injection. Recently, it was reported that mouse SSCs could be expanded in-vitro with maintenance of functionality. When this approach would be feasible with human SSCs, it may improve the efficiency of cryopreservation, either by increasing the number of SSCs before freezing or after thawing. Since mouse SSCs can be cultured over long time periods, long-term in-vitro culture may even become an alternative to cryostorage."