Three Special Issues, so far, have been dedicated to overall MSC prospective biology, from cell regulation to tissue regeneration [...]

Mesenchymal Stem Cells (MSC) represent a captivating field of research attempting to address the vast variety of disease burdens, which at present lack efficient treatment [...]

Calcium carbonate (CC)-hydroxyapatite (HAP) porous microparticles have gained a lot of popularity as a promising material for clinical applications. The objective of this study is to evaluate the effects of CC-HAP microparticles on osteoblast-like cells to be used as a bone-regeneration biomaterial. In this study, the different concentrations of conditioned media were used to compare the effects of released ions from CC-HAP microparticles. The material’s characteristics demonstrated that the immersion in cell culture medium did not change the crystal phases of CC-HAP. The decrease of calcium ions in cell culture medium is due to the dissolution-precipitation reactions on the material surfaces, which made more crystalline surfaces. The atomic absorption spectroscopy measurement demonstrated that the dissolution-precipitation reactions on the material surfaces in cell culture medium happened in 3 days and were stable between 3 to 5 days. The conditioned media immersed in cell culture medium for 4 days were used for further experiments. Cell evaluations demonstrated that excessive adding of CC -HAP could inhibit cell behaviors such as cell adhesion, proliferation, and differentiation. The cell adhesion indicated by the number of vinculin-positive focal adhesions per cell decreased with the increase of the CC-HAP concentrations. The cells cultured with CC-HAP proliferated at a lower rate than the control without CC-HAP. One of the reasons for the inhibition of cell proliferation was thought to be less formation of focal adhesions with higher concentrations of CC-HAP. The excessive adding of CC-HAP had an inhibitory effect on osteoblast differentiation. The results of this study revealed that the conditioned media prepared by immersion of CC-HAP porous microparticles in cell culture media had effects on the behaviors of osteoblast-like cells such as cell adhesion, proliferation, and differentiation.

Bone graft materials are widely used in orthopedic and maxillofacial surgeries. The controlled resorbability of the graft material is essential for bone regeneration. Hydroxyapatite and biphasic calcium phosphate bone grafts have poor resorption and limited bone conductive effects. Histology analyses of bone biopsy from SCPC grafted human extraction sockets showed complete bone regeneration and graft resorption in absence of osteoclasts and macrophages. The hypothesis of the present study is that bioactive SCPC inhibits osteoclast’s activity due to the presence of resorbable silica phase in the material. Our objective is to analyze the effect of SCPC dissolution products on the resorption activity of osteoclasts. The conditioned medium was prepared by immersion of SCPC resorbable bioactive SCPC porous granules (Shefabone, Inc, USA) in cell culture medium at various ratios at 37°C for 3 days. The concentration of Si ions released from the SCPC granules into cell culture medium was measured using ICP-OES. Osteoclast precursors derived from human bone marrow were seeded on bone slices and cultured in the conditioned medium containing 10% FBS and osteoclast induction factors. Osteoclast differentiation and resorption were evaluated by TRAP staining and measurement of the volume of resorption pits on the bone slices. Mature multinuclear giant TRAP-positive osteoclasts were observed on the bone substrates after 14 days incubation in control medium containing osteoclast induction factors. In conditioned medium, the number of multinuclear TRAP-positive cells was significantly decreased as the concentration of SCPC dissolved silica increased. The dissolution of silica from SCPC into the culture medium correlates well with down regulation of osteoclast differentiation and the rapid bone regeneration in human bone defects.

Mesenchymal stem cells (MSC) have piqued worldwide interest for their extensive potential to treat a large array of clinical indications, their unique and controversial immunogenic and immune modulatory properties allowing ample discussions and debates for their possible applications. Emerging data demonstrating that the interaction of biomaterials and physical cues with MSC can guide their differentiation into specific cell lineages also provide new interesting insights for further MSC manipulation in different clinical applications. Moreover, recent discoveries of some regulatory molecules and signaling pathways in MSC niche that may regulate cell fate to distinct lineage herald breakthroughs in regenerative medicine. Although the advancement and success in the MSC field had led to an enormous increase in the amount of ongoing clinical trials, we still lack defined clinical therapeutic protocols. This review will explore the exciting opportunities offered by human and animal MSC, describing relevant biological properties of these cells in the light of the novel emerging evidence mentioned above while addressing the limitations and challenges MSC are still facing.

Adeno-associated virus (AAV) vectors are one of the most frequently applied gene transfer systems in research and human clinical trials. Since AAV vectors do not possess an integrase activity, application is restricted to terminally differentiated tissues if transgene expression is required long term. To overcome this limitation and to generate AAV vectors that persist episomally in dividing cells, AAV vector genomes were equipped with a scaffold/matrix attachment region (S/MAR). After a mild antibiotic selection, cells transduced with AAV-S/MAR established colonies that maintained long-term transgene expression (>50 population doublings) from replicating AAV vector episomes in the absence of further selection. Unexpectedly, with a lesser but still significant efficiency, the control vector (AAV-ΔS/MAR), a standard single-stranded AAV vector, also established stable transgene-expressing colonies, most of which were maintained as replicating episomes rather than integrated vector genomes. Thus, based on the result in HeLa cells, it is concluded that AAV vector genomes per se possess the ability to establish episomal maintenance in proliferating cells, a feature that can be enhanced by incorporation of a foreign genomic element such as an S/MAR element.

Graft rejection and graft-versus-host disease are leading causes of transplant related mortality despite advancements in immunosuppressive therapy. Mesenchymal stem cells (MSCs) offer a promising addition to immunosuppressive drugs (ISD), while NK-cells are increasingly used as effector cells in graft-versus-leukemia. Combined therapy of ISD, NK-cells and/or MSCs is used in clinical practice. Here, we examined the effects of MSCs and selected ISD (tacrolimus, cyclosporin A, mycophenolic acid, dexamethasone) treatment on early NK-cell activation. We assessed STAT4 and STAT5 phosphorylation triggered by IL-12 and IL-2, respectively. Furthermore, we determined IFNγ, perforin production and the expression pattern of selected NK-cell receptors. Of all drugs tested, only dexamethasone inhibited NK-cell STAT4 and STAT5 phosphorylation. All ISD, with the exception of MPA, significantly inhibited IFNγ, and only dexamethasone inhibited upregulation of early activation markers CD69 and CD25 (IL-2 condition only). MSCs inhibited IL-2 induced NK cell STAT5 phosphorylation, IFNγ production and CD69 upregulation, and IL-12 induced IFNγ and perforin production. While MSCs mediated inhibition of CD69 expression was cell contact dependent, inhibition of IFNγ and perforin production, as well as STAT5 phosphorylation was cell-contact independent. Importantly, dexamethasone augmented MSCs mediated inhibition of both IL-12 and IL-2 induced CD69 expression and IFNγ production, as well as IL-2 induced STAT5 phosphorylation. Taken together, these novel insights may help the design of future NK-cell and MSCs based immunotherapy.

The use of fetal bovine serum (FBS) for the culture and expansion of mesenchymal stromal cells (MSCs) limits their possible clinical applications. Although some recent studies recommended substituting FBS with human platelet lysate (HPL) for the expansion of MSCs for clinical use, the functional capacity of the expanded cells has only been partially explored. 10% FBS and two other commercial FBS‐containing media (MesenCult and MesenPro) were compared with 10% HPL‐containing medium for their ability to support MSCs expansion and immunomodulation. We demonstrate that HPL sustained MSC proliferation and expansion in vitro. However, the cumulative cell numbers recovered were comparable with those obtained in MesenPro medium. Moreover, we show that HPL alters the expression of some relevant MSC surface molecules, namely the DNAM‐1 ligands PVR and Nectin‐2, the NKG2D ligand ULBP3, the adhesion molecules CD49d and αvβ3 and the fibroblast‐associated protein. In addition, MSCs cultured in HPL displayed impaired inhibitory capacity on T‐cell proliferation to alloantigen and NK‐cell proliferation and cytotoxicity. Finally, they showed decreased constitutive PGE2 production while IL‐6, IL‐8 and RANTES secretion were upregulated. These results imply some limitations in the use of HPL for the expansion of MSCs to be used as immunomodulators in clinical applications.

The umbilical cord blood derived endothelial progenitor cells (EPCs) contribute to vascular regeneration in experimental models of ischemia. However, their ability to participate in cardiovascular tissue restoration has not been elucidated yet. We employed a novel co-culture system to investigate whether human EPCs have the capacity to integrate into living and ischemic cardiac tissue, and participate to neovascularization. EPCs were cocultured with either living or ischemic murine embryonic ventricular slices, in the presenceor absence of a pro-angiogenic growth factor cocktail consisting of VEGF, IGF-1, EGF and bFGF. Tracking of EPCs within the co-cultures was performed by cell transfection with green fluorescent protein or by immunostaining performed with anti-human vWF, CD31, nuclei and mitochondria antibodies. EPCs generated vascular tube-like structures in direct contact with the living ventricular slices. Furthermore, the pro-angiogenic growth factor cocktail reduced significantly tubes formation. Co-culture of EPCs with the living ventricular slices in a transwell system did not lead to vascular tube-like structures formation, demonstrating that the direct contact is necessary and that the soluble factors secreted by the living slices were not sufficient for their induction. No vascular tubes were formed when EPCs were co-cultured with ischaemic ventricular slices, even in the presence of the pro-angiogenic cocktail. In conclusion, EPCs form vascular tube-like structures in contact with living cardiac tissue and the direct cell-to-cell interaction is a prerequisite for their induction. Understanding the cardiac niche and micro-environmental interactions that regulate EPCs integration and neovascularization is essential for applying these cells to cardiovascular regeneration.

After 7years of hard work in Europe, resulting in a PhD degree and several international awards, it was hard to go back home to achieve any professional success, immigration was my only option! However, one week after my PhD exams, the Egyptian revolution exploded. Although I was not in Egypt, yet they were the most stressful days of my life! Between January 25th and 28th (2011) my life changed. I booked one way ticket to join Egyptians on the “Friday of Rage”. It was a strange feeling, hard to believe and explain, I couldn’t think except to be back to struggle for my country with my family, either for freedom or to die. I was blocked in the airport, no internet and no cell phone, trying to follow the news on the TV screens, I saw my house set on fire. Finally released from the airport, surprising my parents with my arrival. Thank GOD they were fine! Egyptians continued the struggle against the Mubarak Regime for 2 more weeks, then “WE MADE IT”, Mubarak resigned. My perplexity was resolved; I am home to face a lifetime challenge of “reforms”. I started dreaming of educational and health reforms. I was able to win a European capacity building fund through which I travelled to Germany with two of my female students to train them and be back forming a research team. In the meantime, I applied for WIT mentorship scheme, I was accepted and then surprisingly selected to become a Mentor too! The breaking news followed, winning the national award as the best Egyptian young scientist. My dreams started to come true! I requested a visit to my Mentor, she kindly invited me to give a talk about my research work after which I was fascinated to find a marvelous, enthusiastic international group working in the same research line. Fruitful discussion was initiated and several points of collaboration were proposed. Through her support, we were able to write a joint proposal to be funded by the Netherlands and we applied for the call. Finally I was back Cairo again, university reforms started to take place after the revolution and we were able to change the regulations. For the first time in history, Cairo University (the oldest University in the Middle East) had undergone fair presidential elections. Through WIT and the mentor-ship scheme I am looking forwards to collaboration and staff exchange that will build and raise Cairo University to international standards. Our revolution for liberty and justice is still ongoing until Presidential electionsFigure: [Egyptian Revolution]

Background: Human platelet lysate (HPL) has gained increased interest as a substitute to fetal calf serum (FCS) for clinical grade expansion of mesenchymal stem cells (MSCs). MSCs are known to support hematopoiesis and have been frequently co-infused with hematopoietic stem cells (HSCs) to promote early engraftment after HSC transplantation. In this study we investigated the ability of HPL-expanded MSCs to support HSC in -vitro, as compared to FCS-based expansion media Methods: Growth, expansion potential and surface phenotype of MSCs cultured with 10% HPL, as a supplement, was compared to a standard culture condition containing 10% FCS, as well as to two other FCS-based commercially available media; MesenCult and MesenPro. Differently expanded MSCs were co-cultured with cord blood CD34selected hematopoietic stem cells. The latter cells were then evaluated for survival, proliferation, stemness and maturation phenotype while MSCs were evaluated for their secreted cytokine profile. Results: MSCs were efficiently generated from all culture conditions. While HPL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity, they failed to support CD34+ progenitors in culture; furthermore, the number of these progenitors declined markedly with time as compared to MSCs expanded in other conditions. Progenitors co-cultured with HPL-MSC had an earlier commitment rate tested by the co-expression of CD34, CD38, CD33 and CD14 markers. In addition, HPL supplemented media increased the production of IL-6, IL-8 and RANTES by MSCs which may play a role in their altered function Conclusion: Substituting FCS with HPL for MSCs expansion is accompanied by decreased proliferation and stemness of HSCs cocultured with the expanded cells. Therefore, MSCs expansion conditions clearly define functional capacity. More standardization of MSCs culture conditions is warranted before clinical application.

1Cairo/Cairo/EGYPT, 2Clinical And Experimental Immunology, Giannina Gaslini Institute, University of Genova, Genova/ITALY

The umbilical cord blood derived endothelial progenitor cells (EPCs) contribute to vascular regeneration in experimental models of ischaemia. However, their ability to participate in cardiovascular tissue restoration has not been elucidated yet. We employed a novel coculture system to investigate whether human EPCs have the capacity to integrate into living and ischaemic cardiac tissue, and participate to neovascularization. EPCs were cocultured with either living or ischaemic murine embryonic ventricular slices, in the presence or absence of a pro-angiogenic growth factor cocktail consisting of VEGF, IGF-1, EGF and bFGF. Tracking of EPCs within the cocultures was performed by cell transfection with green fluorescent protein or by immunostaining performed with anti-human vWF, CD31, nuclei and mitochondria antibodies. EPCs generated vascular tube-like structures in direct contact with the living ventricular slices. Furthermore, the pro-angiogenic growth factor cocktail reduced significantly tubes formation. Coculture of EPCs with the living ventricular slices in a transwell system did not lead to vascular tube-like structures formation, demonstrating that the direct contact is necessary and that the soluble factors secreted by the living slices were not sufficient for their induction. No vascular tubes were formed when EPCs were cocultured with ischaemic ventricular slices, even in the presence of the pro-angiogenic cocktail. In conclusion, EPCs form vascular tube-like structures in contact with living cardiac tissue and the direct cell-to-cell interaction is a prerequisite for their induction. Understanding the cardiac niche and micro-environmental interactions that regulate EPCs integration and neovascularization are essential for applying these cells to cardiovascular regeneration.