Sessões PlenáriasAs sessões plenárias do Congresso da ISCT são as mais concorridas do evento por não ter coincidência com outra atividade do evento.
Abaixo serão colocadas todas que dispõem de texto realizado pelo próprio autor e disponibilizadas no site já citado anteriormente. www.celltherapy2011.org
Plenary Session 1: Presidential Plenary on Mesenchymal Stem Cells
Time: 9:00am – 10:30am
Chair: Edwin Horwitz
MSCs: Current Clinical Applications and Future ChallengesArmand Keating (Canadá)
In this talk the author discuss actual results and the future of cell therapy pointing the difference of replacement therapy and treatment with cell therapy. It was present many results in those fields that he considered very promising
Text by Mlton Artur Ruiz.
MSC from genetic stability to controls of safetyLuc Sensebé ( França)
In recent years, relevant data have indicated that mesenchymal stromal/stem cells (MSC) can be used as reparative/regenerative cells to treat a range of clinical conditions including immunological disorders as well as degenerative situations. A major safety concern is the genomic stability of mesenchymal stromal cells particularly related to the risk of cell transformation. As during ageing, cell expansion might be associated with replicative senescence, replicative stress, mutations, chromosomal abnormalities and other stochastic cell defects that could progressively alter cells and have to be investigated. Replicative senescence arises through different well known mechanisms such as telomere shortening, activation of pRB pathway through INK4a/ARF locus encoding p16ink4a and p19ink4a, and activation of p53 pathway (Krishnamurthy 2004, Campisi & d’Adda di Fagagna 2007). As previously demonstrated MSCs transformation is a rare, long, multistep process (Serakinci et al., 2004, Prockop et al, 2010). Recently, we showed that clinical-grade–cultured human MSCs, regardless of the presence of aneuploidy, reached senescence and never transformed (Tarte et al., 2010). However, the main control still used for the release of GMP clinical grade MSC remains karyotype that appears neither sufficient nor sensitive enough. To understand the changes and risks induced by culture process, it is mandatory to more deeply analyze genetic perturbations, not only the main molecules of replicative senescence checkpoint (p53, p21, p16, p19 and pRB) but also at a more subtle level defective DNA replication program, which has been shown as anticipating most gross cancer-associated genetic changes. Molecular targets involved in genetic stability of MSC should be defined to develop reliable quality controls for production of safe MSC according to GMP.
• Campisi J & d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. 2007 ; 8 :729-740
• Krishnamurthy J, Torrice C, Ramsey M.R,Kovalev G.I, Al-Regaiey K, Su L, Sharpless N.E. Ink4a/Arf expression is a biomarker of aging. J. Clin.Invest. 2004, 114:1299-1307
• Prockop DJ, Brenner M, Fibbe WE, Horwitz E, Le Blanc K, Phinney DG, Simmons PJ, Sensebe L, Keating A (2010). Defining the risks of mesenchymal stromal cell therapy. Cytotherapy 12:576-578
• Serakinci N, Guldberg P, Burns JS, Abdallah B, Schrodder H, Jensen T et al (2004). Adult human mesenchymal stem cell as a target for neoplastic transformation. Oncogene 23: 5095-8.
• Tarte K, Gaillard J, Lataillade JJ, Fouillard L, Becker M, Mossafa H et al (2010). Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation. Blood 115: 1549-53.
MISOT – Mesenchymal Stem Cells in Solid Organ Transplantation
Solid organ transplantation provides the definitive treatment for many end-stage diseases. However, life-long immunosuppression needed to prevent graft rejection causes clinically significant side effects. In fact, the overall success of solid organ transplantation as a curative therapy often depends on the occurrence and management of drug side effects. Cellular immunomodulatory therapies with MSC may therefore be suitable to reduce the dose of immunosuppressive drugs.
The MISOT study group has brought European investigators together and a variety of protocols to complement immunosuppressive pharmacotherapy with MSC have been suggested. To decide if patients undergoing organ transplantation can be safely treated with MSC and if these therapies yield a benefit for patient and graft survival, careful consideration of all available preclinical and clinical data has to be carried out.
Plenary Session 2 – Regenerative Medicine and Tissue Engineering
Time: 1:45pm – 3:15pm
Cartilage tissue engineering: are we ready to translate into the clinic?
Tissue engineered cartilage may be used to treat degenerative joint diseases such as osteoarthritis as well as diseases in other cartilaginous structures such as the airway. Knowing when we are ready to take our science out of the laboratory and into the clinic is a key problem for the tissue engineer. Articular cartilage repair using chondrocytes has been in widespread use for 20 years but using stem cells to create engineered cartilage that can be implanted into the joint as a mature tissue has not yet been considered ready for translation. Development of a more robust and predictable outcome of cartilage tissue engineering may help to provide the confidence need to use such implants therapeutically. Meniscal cartilage repair has not received as much research attention as articular cartilage and yet it is a significant medical problem with no therapy available other than removal of the torn tissue, resulting in a high risk of subsequent osteoarthritis. Stem cells may provide a good opportunity for a new approach to this old problem. Airway stenosis does not affect large numbers of patients but it can be life-threatening. Replacement of a severely damaged bronchus with tissue engineered trachea in one patient has shown the potential for whole organ replacement in the future. Each of these examples will be considered as an illustration of the complexity of knowing when we are ready to turn science into medicine.
Challenges in 3D tissue graft manufacturing
Despite the compelling clinical need to regenerate damaged tissues/organs, impressive advances in the field of tissue engineering have yet to result in viable engineered tissue products with widespread therapeutic adoption. The main challenges to be overcome have been identified in the yet not convincing benefit of the proposed therapies, combined with their high costs. Following the exemplifying paradigm of bone and cartilage regeneration, the lecture will highlight the bottlenecks of typical manufacturing strategies and will propose alternative bioreactor-based approaches for the manufacturing of 3D cellular grafts. The perspective will address issues related to quality standardization, process control and regulatory compliance in manufacturing cell-based products and highlight the need not only to automate, but also to streamline and simplify typical production processes. Examples will be given on the attractive paradigm to expand mesenchymal stem/progenitor cells from adult individuals directly in a “3D niche” environment, thereby maintaining a larger post-expansion differentiation capacity and bypassing the complex and costly serial cell passaging in monolayers. Finally, as a next generation paradigm, the lecture will propose and exemplify the concept of engineering regenerative strategies following principles of developmental biology, using the own body as the in vivo bioreactor.
Smart materials for programming stem cell fate in situ
A complex mixture of extracellular cues delivered by support cells is critical for adult stem cell maintenance and the regulation of self-renewal in their microenvironment, termed niche. Despite recent progress in the identification of relevant niche proteins and signaling pathways in mice, to date, many adult stem cell populations cannot be efficiently cultured in vitro without rapidly differentiating.
In this talk I will highlight recent efforts in my lab to develop and apply novel in vitro culture paradigms that allow fate decisions of hundreds of individual adult stem cells to be monitored under well-controlled conditions and in real time. For example, we have engineered microarrayed ‘artificial niches’ for hematopoietic stem cells (HSC) based on a combination of biomolecular hydrogel and microfabrication technologies that allow key biochemical characteristics of niches to be mimicked and the physiological complexity deconstructed into a smaller, experimentally amenable number of distinct signaling interactions. We have also built and applied microfluidic chips to sequentially capture single HSC after multiple divisions to assess their fate, and in particular the symmetry of division, by multigene single cell qRT-PCR.
The systematic deconstruction of a stem cell niche may serve as a broadly applicable paradigm for defining and reconstructing artificial niches to accelerate the transition of stem cell biology to the clinic.
Plenary Session 3 – Cardiovascular Cell Therapy
Time: 8:45am – 10:15am
Repair by bone marrow-derived cells: experimental and clinical insights
Synopsis not available.
Human iPSC models of cardiac disease
Much of what is known about the molecular pathways that lead to human cardiovascular disorders has come from studying animal models, particularly genetically modified mice. In some cases it is possible to translate genetic discoveries from humans to mice (e.g. non-sense mutations), but in most circumstances there are no direct correlates for human genetic variants such as single nucleotide polymorphisms (SNPs) or copy number variants. Therefore, it is imperative to replicate relevant features of human cardiovascular physiology in the context of the human genome. Recent advances in stem cell biology now raise the possibility of generating human models of cardiovascular physiology and disease.
Generating patient-specific cells and tissues has recently emerged with the demonstration that exogenous expression of four proteins in human skin fibroblasts (e.g. c-MYC, KLF4, OCT4, and SOX2) is sufficient to induce pluripotency in the cells. Although so-called induced pluripotent stem cells (iPSCs) are not perfectly equivalent to human ES cells, they retain important properties of ES cells such as the capacity for long-term propagation and the ability to differentiate into all human somatic cell types. Factor-based reprogramming enables us of the long-standing ambitions of stem cell biology: the ability to generate pluripotent cells from specific patients and figuratively, move a patient`s disease into the Petri dish. This will be discussed for human monogenetic cardiovascular diseases, e.g. LQT syndromes, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVC).
Recent advances describing the derivation of human iPSCs from peripheral, frozen blood brings the stem cell field an important step closer to bio-banked blood samples and eventual clinical use.
Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flügel L, Dorn T, Gödel A, Höhnke C, Hofmann F, Seyfarth M, Sinnecker D, Schömig A & Laugwitz K-L (2010). Patient-specific induced pluripotent stem cell models for long-QT syndrome. N Engl J Med., Epub Jul 21.
microRNAs in cardiovascular repair
MicroRNAs (miRs) are small non-coding RNAs, which control gene expression by either inducing mRNA degradation or by blocking translation, and play a crucial role in tissue homeostasis. In the cardiovascular system, miRs were shown to control cardiac hypertrophy, fibrosis and apoptosis, angiogenesis and vessel remodeling. In addition, miRs regulate stem cell maintenance and some miRs induced cell fate decisions. The presentation will provide an overview of involvement of miRs in cardiovascular lineage commitment and cell function. Particularly, the regulation and function of miRs in bone marrow derived cells that are used for cell therapy of cardiovascular diseases will be discussed.
Plenary Session 4 – T Cell ImmunotherapyFriday 5/20/2011
Time: 1:45pm – 3:15pm
Alloreactive T cells for the treatment of leukemia
Allogeneic hematopoietic stem cell transplantation allows the exploration of cellular immunotherapy strategies. Following engraftment of donor hematopoiesis in the patient, donor derived alloreactive T cells are capable of eliciting both graft versus host disease (GVHD) and graft versus leukemia (GVL) reactivity. Separation of immune responses resulting in GVHD from those involved in GVL reactivity is essential for improving the outcome of stem cell transplantation for hematological disorders.
T cell responses directed against polymorphic antigens expressed on normal non-hematopoietic tissues of the recipient are likely to be responsible for severe GVHD. In contrast, alloreactive donor derived T cell directed against antigens preferentially expressed on cells of hematopoietic origin may lead to profound reactivity against normal and malignant hematopoietic cells of recipient origin while preserving hematopoiesis of donor origin and sparing of patient derived non-hematopoietic tissues thereby limiting GVHD. Donor derived CD8 T cell responses directed against antigens recognized in the context of HLA class-I molecules will lead to strong cytotoxicity against target tissues. Since HLA class-I molecules are broadly expressed on most tissues, only CD8 T cells recognizing HLA class-I bound peptides derived from hematopoiesis specific proteins have been considered appropriate candidates for specific anti tumor reactivity after transplantation. However, not all T cell reactivity against antigens expressed on non hematopoietic tissues may lead to severe GVHD. The cellular activation state influenced by inflammatory circumstances in the non hematopoietic tissues is an essential factor in the ability of T cells to damage these tissues.
Since HLA class-II molecules show a more restricted tissue distribution, T cell responses against antigens presented in HLA class-II may also lead to relatively specific GVL responses. T cell responses recognizing antigens in the context of HLA-DQ and HLA-DP have been found to be associated with anti tumor effects with limited GVHD. However, inflammatory circumstances in the recipient may lead to upregulation of HLA class-II molecules on non-hematopoietic tissues resulting in GVHD. These results illustrate that both the specificity of the T cell responses, and the inflammatory environment will determine the balance between GVHD and GVL reactivity after allogeneic hematopoietic stem cell transplantation.
Dissection of cell therapy-induced cytotoxic T cell responses in melanoma
There is strong evidence that melanoma-reactive T cell responses induced by immunotherapeutic interventions such as anti-CTLA4 treatment or T cell therapy with tumor-infiltrating lymphocytes (TIL) can exert clinically meaningful effects. However, at present we do not know which cytotoxic T cell reactivities mediate cancer regression. Furthermore, as the number of potential melanoma-associated antigens to which these responses can be directed is very high, classical strategies to map cytotoxic T cell reactivity do not suffice. Knowledge of such reactivities would be useful to design more targeted strategies that selectively aim to induce immune reactivity against these antigens.
In the past years we have aimed to address this issue by designing MHC class I molecules occupied with UV-sensitive ‘conditional’ peptide ligands, thereby allowing the production of very large collections of pMHC complexes for T cell detection. Secondly, we have developed a ‘combinatorial coding’ strategy that allows the parallel detection of dozens of different T cell populations within a single sample. The combined use of MHC ligand exchange and combinatorial coding allows the high-throughput dissection of disease- and therapy-induced CTL immunity. We have now used this platform to monitor immune reactivity against a panel of over 200 melanoma-associated epitopes. Data on the composition of TIL products used for adoptive cell therapy and on the effect of TIL therapy on the tumor-reactive T cell repertoire in melanoma patients will be presented.
Translation of immunotherapy using WT1 and CMV specific TCR gene transfer into the clinic
Adoptive transfer of antigen-specific T cells is an effective form of immunotherapy for persistent virus infections and cancer. Major limitations of adoptive therapy are the inability to isolate antigen-specific T cells reproducibly and expand them to sufficient numbers ex vivo, whilst maintaining optimal function and specificity.
We and others have developed gene therapy approaches to overcome the problems related to poor tumour immunogenicity and lack of specificity of allogeneic T cell therapy. Retroviral gene transfer can generate large numbers of high avidity antigen-specific T cells. Retroviral transfer of cloned T cell receptor (TCR) genes reliably re-directs the antigen specificity of T cells. We have exploited the allo-reactive T cell repertoire to isolate high avidity T cells specific for the tumour-associated antigen WT1, which is highly expressed in MDS, AML, CML and ALL, together with a number of solid tumours. The genes encoding the T cell receptor of the WT1-specific, allo-restricted T cells were isolated and inserted into retroviral vectors for gene transfer into primary human T cells. In vitro, the gene modified T cells can kill primary human leukaemia cells and also autologous leukaemia cells expressing WT1 endogenously. Following adoptive transfer they can protect against the growth of autologous primary leukaemia cells in the xenogeneic NOD/SCID model. We can now manufacture GMP grade WT1 TCR-transduced T cells and aim to recruit patients into a Phase I study this year.
Reactivation of the latent human herpes virus, Cytomegalovirus (CMV) post allogeneic haematopoietic stem cell transplantation (Allo-HSCT) can result in significant morbidity and mortality unless treated promptly. Anti-viral therapy is usually effective, but has serious side effects, such as myelosuppression (Ganciclovir) or nephrotoxicity (Foscarnet). Cellular immunotherapy for CMV has been tested in Phase I/II trials in the UK and Europe. In these trials CMV-specific T cells were isolated from the peripheral blood of CMV seropositive donors and re-infused into recipients following CMV reactivation resulting in sustained anti-viral responses. It is clear that post-transplant recovery of CD8+ CMV-specific cytotoxic T-cells (CTL) abrogates the development of CMV-related disease. An advantage of cellular therapy for CMV reactivation is the transfer of immunological memory, which can reduce the number of subsequent reactivations. We have used TCR gene transfer to generate CMV pp65-specific T cells from donors who are CMV seronegative (therefore lack CMV-specific T cells), where the transplant recipient is CMV seropositive and at risk of CMV reactivation post transplant. Both CD8+ and CD4+ T cells expressing the MHC class I-restricted TCR display CMV-specific effector function in vitro and in vivo.
Plenary Session 5 – Cancer Stem CellsSaturday 5/21/2011
Time: 8:45am – 10:15am
Cancer stem cells from colorectal cancer cell linesSir Walter Bodmer
Colorectal cancer is one of the best defined cancers from a genetic point of view, both at the germ line and somatic levels. We work with a panel of more than 100 colorectal cancer derived cell lines that are extensively characterised with respect to their genetic/epigenetic make up and for whole genome mRNA expression. The lines provide invaluable models for studying the biology and somatic genetics of colorectal cancers, and enable in vitro investigation of the properties of colorectal cancer stem cells. We now can characterise the driving cancer stem cells in these lines and begin to identify the factors that control their differentiation, in particular, through having shown that hypoxia(1% oxygen) inhibits cancer stem cell differentiation in vitro.
Lgr5 Intestinal Stem Cells in self-renewal and cancerMarc van de Wetering
The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. Current models state that 4-6 crypt stem cells reside at the +4 position immediately above the Paneth cells in the small intestine; colon stem cells remain undefined. Lgr5/Gpr49 was selected from a panel of intestinal Wnt target genes for its restricted crypt expression. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several other tissues. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5+ve crypt base columnar cell (CBC) generated all epithelial lineages over a 14 month period, implying that it represents the stem cell of the small intestine and colon. The expression pattern of Lgr5 suggests that it marks stem cells in multiple adult tissues and cancers.
We have now established long-term culture conditions under which single crypts undergo multiple crypt fission events, whilst simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ve stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.
Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in in Lgr5+ve stem cells leads to their transformation within days. Transformed stem cells remain located at crypt bottoms, while fueling a growing microadenoma. These microadenomas display unimpeded growth and develop into macroscopic adenomas within 4-6 weeks. When APC is deleted in short-lived Transit Amplifying (TA) cells using a different Cre mouse, the growth of the induced microadenomas rapidly stalls. Even after 30 weeks, large adenomas are very rare in these mice. We conclude that stem cell-specific loss of APC results in progressively growing neoplasia. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the “cancer stem cell”-concept.
Modeling Colon Stem Cells and Neoplasia Using Primary Explant Cultures Containing an Endogenous Wnt-dependent NicheCalvin Kuo
The intestine undergoes continuous epithelial regeneration that absolutely requires ongoing Wnt signaling. We have developed in vitro culture systems that enable long-term propagation and multi-lineage differentiation of intestinal epithelium and allow sustained intestinal proliferation, multi-lineage differentiation and the support of Lgr5+ and Bmi1+ intestinal stem cells (ISC) over a range of 30 to > 350 d. The defining characteristics of this approach include (1) culture of intestinal epithelium within an air-liquid interface coupled with a 3D culture matrix and (2) the use of explant tissue that include stromal myofibroblasts, neural elements, and recapitulate endogenous peristalsis. Notably, growth does not require the addition of exogenous Wnt agonists, as fetal calf serum alone is sufficient, and growth is ablated by recombinant Dkk1, indicating the presence of endogenous Wnt signaling within the explant cultures. This system recapitulates both the cellular architecture and the rigorous Wnt- and Notch- dependency of the intestinal stem cell niche, and applications to the modeling of colon cancer will be discussed.
Plenary Session 6 – Embryonic to Adult Stem CellsSaturday 5/21/2011
Time: 2:55pm – 4:25pm
Origin of mesenchymal stem cellTakumi Era
Mesenchymal stem cell (MSC) is defined by their ability both to undergo sustained proliferation in vitro and to give rise to multiple mesenchymal cell lineages including bone, cartilage, and fat cells. Although MSCs are a demonstrated reality with applications in regenerative medicine, not much is known about their in vivo characteristics, such as their developmental derivation. Recently we found the new the developmental pathway of MSC during mouse embryogenesis. Using in vitro ES cell culture, we have shown that Sox1+ neuroepithelial cells generate MSCs at the highest efficiency. ES cell-derived day 9 PDGFRa+ precursors induced by RA treatment morphologically exhibit fibroblastic and undergo the self-renew in vitro with maintaining the potential to give rise to multiple lineages including bone, cartilage, and fat cells. Interestingly, the PDGFRa+ cells are derived not from mesoderm cells but from neuroepithelium cells in our ES cell culture. This unexpected result suggests that the neuroepithelium is a candidate of embryonic origin of MSCs. To confirm this result in actual embryo, we are proceeding to search for the origin of MSCs in vivo using genetically fate-trucking method. In E9.5 embryos, we could induce MSCs from Sox1+ cells but not from PDGFRa+ mesoderm. While this type of MSC is found also in neonatal bone marrow at low frequency, most MSCs in postnatal bone marrow are derived from other origins, which are also enriched in the PDGFRa+ population. Thus, we show that MSCs are generated from multiple sources, with those derived from neuroepithelium constituting the earliest wave.
The development of hematopoietic stem cells: Endothelial to hematopoietic transitionElaine Dzierzak
Hematopoietic stem cells (HSC) are the source of all blood cells in the adult. The first HSCs are generated in the aorta-gonad-mesonephros (AGM) region at midgestation in the mouse embryo and at week 4-6 in human gestation. AGM HSCs are generated following the anatomical appearance of clusters of hematopoietic cells closely associated with the lumenal wall of aorta and vitelline/umbilical arteries. The relationship of HSCs to these clusters and the identification of the precursors to HSCs is an area of intense research focus. To begin to understand how clusters are formed we have developed a 3-dimensional whole mount immunostaining and confocal imaging method by which we can temporally map and quantitate all hematopoietic clusters in normal (and hematopoietic defective) mouse embryos (Yokomizo and Dzierzak, Development, 2010). We have localized HSC activity to the hematopoietic clusters. Visual proof that HSCs arise from aortic endothelium comes from time lapse vital imaging of the AGM (Boisset et al., Nature, 2010). Remarkably, HSCs arise directly from endothelial cells of the dorsal aorta in a natural transdifferentiation event. HSC generation is regulated through ventral-derived developmental signals and a group of pivotal (core) transcription factors, including Runx1 and Gata2. Knockout strategies implicate these factors in hematopoietic fate induction and/or expansion. These transcription factors are required for the generation of vascular hematopoietic clusters and HSCs. Developmental signalling pathways triggered by BMP4 and Hedgehog, appear to act upstream of these transcription factors.
Vários dos temas aqui apresentados serão discutidos posteriormente.
Milton Artur Ruiz