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AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for clinical indications. In this study, we focus on the less studied adeno-associated virus AAV3B and generate a collection of AAV plasmid substrates that assemble virion particles deficient specifically in VP1, VP2, or VP1 and 2 structural subunits. Using a collection of biological and structural assays, we observed that virions devoid of VP1, VP2, or VP1 and 2 efficiently assembled virion particles, indistinguishable by cryoelectron microscopy (cryo-EM) from that of wild type (WT), but unique in virion transduction (WTVP2VP1VP1 and 2 mutants). We also observed that the missing structural subunit was mostly compensated by additional VP3 protomers in the formed virion particle. Using cryo-EM analysis, virions fell into three classes, namely full, empty, and partially filled, based on comparison of density values within the capsid. Further, we characterize virions described as "broken" or "disassembled" particles, and provide structural information that supports the particle dissolution occurring through the two-fold symmetry sites. Finally, we highlight the unique value of employing cryo-EM as an essential tool for release criteria with respect to AAV manufacturing.

A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community.eTOCThe report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation. This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 to J.G.-C.; and RTICC: RD12/0036/0027 to J.G-C, RD12/0036/0020 to S.M.) and the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C) in Spain. A.A. was supported by Juan de la Cierva program of the Spanish Plan Nacional (MINECO) and Sara Borrell program of the ISCIII/FEDER. A.Al. was supported by the “Miguel Servet” program of the ISCIII/FEDER. We gratefully acknowledge support from Asociación Pablo Ugarte (CIF G86121019) and AFANION (CIF G02223733). The experiments were approved by the appropriate committees. Sí

Mesenchymal stem cells (MSCs), together with hematopoietic stem cells (HSCs), are the most frequently used cell type for cell-based therapeutics. As for other cell types intended for research and translational use, it is important to establish correctly typed cell lines from human tissue donations. Here, we describe methods for isolating, culturing, and identifying MSCs from various tissues obtained through human tissue donation. The methods have been used in the context of a biobank, prepared as standard operating procedures (SOPs), ensuring traceability and reproducibility of cell production.

The recognition of the therapeutic potential of Multipotent Mesenchymal Stromal Cells (MSCs) is one of the most exciting recent advances in cell therapy. In just ten years, since the description of the multilineage potential of MSCs by Pittenger et al in 1999 until now, MSCs are being used in more than 150 clinical trials as therapeutic agents. The potential of these cells for cell-based therapies relies on several key properties: (1) their capacity to differentiate into several cell lineages; (2) their lack of immunogenicity and their immunomodulatory properties; (3) their ex vivo expansion potential; (4) their ability to secrete soluble factors which regulate crucial biological functions such as proliferation and differentiation over a broad spectrum of target cells; and (5) their ability to home to damaged tissues and tumor sites. Based on these properties MSCs are being exploited worldwide for a wide range of potential clinical applications including cell replacement strategies, treatment of graft-versus-host disease, autoimmune diseases and rejection after solid organ transplantation as well as their use as vehicles to deliver anti-cancer therapies. Importantly, the low inherent immunogenicity of MSCs means that they could be used not only for autologous but also for allogeneic cell therapies. In addition, increasing evidence has revealed a complex relationship between MSCs and cancer. Thus, solid evidence has placed MSCs transformed with specific mutations as the most likely cell of origin for certain sarcomas, and MSCs have been reported to both, inhibit or promote tumor growth depending on yet undefined conditions. Here we will thoroughly discuss the different potential clinical applications of MSC as well as the role of MSCs on sarcomagenesis and the control of tumor growth.

The recognition of the therapeutic potential of Multipotent Mesenchymal Stromal Cells (MSCs) is one of the most exciting recent advances in cell therapy. In just ten years, since the description of the multilineage potential of MSCs by Pittenger et al in 1999 until now, MSCs are being used in more than 150 clinical trials as therapeutic agents. The potential of these cells for cell-based therapies relies on several key properties: (1) their capacity to differentiate into several cell lineages; (2) their lack of immunogenicity and their immunomodulatory properties; (3) their ex vivo expansion potential; (4) their ability to secrete soluble factors which regulate crucial biological functions such as proliferation and differentiation over a broad spectrum of target cells; and (5) their ability to home to damaged tissues and tumor sites. Based on these properties MSCs are being exploited worldwide for a wide range of potential clinical applications including cell replacement strategies, treatment of graft-versus-host disease, autoimmune diseases and rejection after solid organ transplantation as well as their use as vehicles to deliver anti-cancer therapies. Importantly, the low inherent immunogenicity of MSCs means that they could be used not only for autologous but also for allogeneic cell therapies. In addition, increasing evidence has revealed a complex relationship between MSCs and cancer. Thus, solid evidence has placed MSCs transformed with specific mutations as the most likely cell of origin for certain sarcomas, and MSCs have been reported to both, inhibit or promote tumor growth depending on yet undefined conditions. Here we will thoroughly discuss the different potential clinical applications of MSC as well as the role of MSCs on sarcomagenesis and the control of tumor growth.

Human adipose-derived mesenchymal stem cells (hASCs) are mesenchymal stem cells (MSCs) with reduced immunogenicity and capability to modulate immune responses. Whereas the immunosuppressive activity of bone marrow-MSCs has received considerable attention during the last few years, the specific mechanisms underlying hASC-mediated immunosuppression have been poorly studied. Recent studies comparing both cell types have reported differences at transcriptional and proteomic levels, suggesting that hASCs and bone marrow-MSCs, while having similarities, are quite different. This suggests that different mechanisms of immunosuppression may apply. Here, we report that hASCs inhibit peripheral blood mononuclear cells (PBMCs), and CD4(+) and CD8(+) T cell proliferation in both cell-cell contact and transwell conditions, which is accompanied by a reduction of proinflammatory cytokines. We demonstrate that hASCs do not constitutively express immunomodulatory factors. Conditioned supernatants from hASCs stimulated by IFN-gamma, PBMCs, or activated PBMCs highly inhibited PBMC proliferation, indicating that inhibitory factors are released upon hASC activation. Many factors have been involved in MSC-mediated immunosuppression, including IFN-gamma, IL-10, hepatocyte growth factor, prostaglandin E2, transforming growth factor-beta1, indoleamine 2,3-dioxygenase (IDO), nitric oxide, and IL-10. Using pharmacological inhibitors, neutralizing antibodies, and genetically modified hASCs that constitutively express or silence IDO enzyme, we demonstrate that, in the case of hASCs, the IFN-gamma/IDO axis is essential. Taken together, our data support the key role of IDO in the therapeutic use of hASC on immunomediated diseases.

Mesenchymal stem cells (MSCs) from adult somatic tissues may differentiate in vitro and in vivo into multiple mesodermal tissues including bone, cartilage, adipose tissue, tendon, ligament or even muscle. MSCs preferentially home to damaged tissues where they exert their therapeutic potential. A striking feature of the MSCs is their low inherent immunogenicity as they induce little, if any, proliferation of allogeneic lymphocytes and antigen-presenting cells. Instead, MSCs appear to be immunosuppressive in vitro. Their multi-lineage differentiation potential coupled to their immuno-privileged properties is being exploited worldwide for both autologous and allo-geneic cell replacement strategies. Here, we introduce the readers to the biology of MSCs and the mechanisms underlying immune tolerance. We then outline potential cell replacement strategies and clinical applications based on the MSCs immunological properties. Ongoing clinical trials for graft-versus-host-disease, haematopoietic recovery after co-transplantation of MSCs along with haematopoietic stem cells and tissue repair are discussed. Finally, we review the emerging area based on the use of MSCs as a target cell subset for either spontaneous or induced neoplastic transformation and, for modelling non-haematological mesenchymal cancers such as sarcomas.