The Qatar International Conference on Stem Cell Science and Policy

Breast cancer is a major disease leading in both incidence and mortality in women. Although the mortality has been reduced by various therapy approaches, recurrence still occurs in many patients. Evidence indicates that tumor-initiating (cancer stem) cells may contribute to disease relapse, suggesting the importance of effective targeting of this cell population. Recently, it has been shown that the components of the tumor microenvironment, including tumor vascular endothelium, interact with cancer stem cells through intracellular signaling pathways. However, the exact mechanism underlying this interaction is not known. In this study, we intend to investigate how the Akt-activated vascular endothelial cells (E4ORF1) communicate with defined populations of breast tumor. Moreover, we are interested to find out if notch pathway is regulating this crosstalk.

The cancer stem cells were enriched under serum-free conditions in 3D media to obtain mammospheres. To investigate how E4ORF1 maintains breast tumor cells and cancer stem cell in the absence of serum and cytokines, breast cancer MDA-MB231 and MCF-7 cells were co-cultivated with E4ORF1, its conditioned media, or in 3D media. In addition, the expression of pluripotency markers was assessed by real-time PCR in cancer cells after contact with E4ORF1 cells. Besides, the involvement of notch pathway in E4ORF1 interaction with mammospheres was studied by using RNAi against Jagged-1.

Our preliminary results demonstrated that E4ORF1 cells were able to maintain cancer cells under serum-free conditions and in a contact-dependent manner. Also,they were able to enrich mammospheres in contact or after temporary exposure. Moreover, the expression of some pluripotency markers displayed a 1.5-fold increase in cancer cells co-cultured with E4ORF1 cells. Besides, Notch pathway activation seemed to be necessary for E4ORF1 to support mammosphere growth since there was a 3-fold decrease in sphere numbers in Jag-1-/MDA-MB231 cells.

These results suggest that E4ORF1 cells are capable of maintaining cancer stem cell population. Since this interaction is contact dependent, it might indicate the involvement of some intracellular signaling pathways. Our results show that notch might be involved in this crosstalk, which makes it a target for therapy in order to inhibit the cancer stem cell population to prevent breast cancer reappearance.

In vitro neural differentiation of human embryonic stem cells (hESC), or induced pluripotent stem cells (iPSCs) is a powerful system to study early neural development, instrumental for cell replacement therapy and neuropharmacological studies. Here we report the derivation of tyrosine hydroxylase (TH) positive cells from human iPSCs, indicating the differentiation into dopaminergic neurons.

Our research focuses on understanding how pluripotent mammalian stem cells maintain their undifferentiated state and undergo differentiation in culture – this reflecting my enduring interest in the emergence of diversity during mouse gastrulation.

In recent studies we have examined the role of transforming growth factor family members in both pluripotency and differentiation. This involved analysis of the signalling cascade induced by treating hESCs with Activin or Nodal, determining how their response to these growth factors maintains hESC pluripotency. We started by developing chemically defined culture conditions in which the activities of specific growth factors could be identified and studied in a controlled manner. We then carried out a detailed analysis of the roles of Smad proteins (Smad2 and Smad3) as direct regulators of Nanog, which in turn blocks an hESC default differentiation into neuroectoderm. These studies led to our discovery of a novel type of pluripotent epiblast stem cell (EpiSC) from the late epiblast layer of mouse and rat embryos. EpiSCs share many features with hESCs, and subsequent work supports our hypothesis that hESCs are the human counterparts of EpiSCs, with similar responses to growth factors and mechanisms of pluripotency and differentiation.

In our most recent work we have focused on the role of bone morphogenetic protein (BMP)-4 in the cell fate decision between endoderm and mesoderm, demonstrating the similarity of BMP-induced hESC and EpiSC differentiation to mesoderm induction during mouse gastrulation. This work reveals the importance of BRACHYURY and CDX2 genes as key mediators of embryonic and extraembryonic lineage differentiation in hESCs and EpiSCs. Our focus on mesoderm leads us on to molecular pathways for early human cardiomyocyte differentiation, with a goal of understanding the transcriptional networks responsible for cardiomyocyte identity and using this to generate more homogeneous cardiomyocyte populations for therapeutic applications and drug discovery. Taken together, these studies should significantly accelerate the progression from basic stem cell research to clinical applications.

We have previously shown that Endothelial Progenitor Cells (EPCs) bind and inhibit platelet function and impair thrombus formation, but the surrounding mechanisms that regulate this process have not been fully described. Herein, we addressed the mechanistic action of EPCs on platelet-thrombus formation and we highlighted the role of platelet P-selectin in this process.

EPCs were generated from human peripheral blood mononuclear cells after 10 days of culture on fibronectin in conditioned media. The impact of EPCs on platelet aggregation and thrombus formation was investigated in P-selectin deficient (P-sel-/-) and their wild-type counterpart (WT) mice. EPCs significantly impaired, in a concentration dependent-manner, collagen-induced whole blood platelet aggregation in WT mice; whereas in P-sel-/- mice, EPCs had no significant effect. Moreover, in ferric chloride-injured arterial thrombosis model, infusion of EPCs significantly reduced thrombus formation in WT, but not in P-sel-/- mice. Furthermore, the number of EPCs recruited within the thrombi and along the arterial wall was reduced in P-sel-/- mice as compared to WT mice, and the relative mass of thrombi generated in EPC-treated P-sel-/- mice was significantly larger than that in EPC-treated WT mice.

EPCs impair platelet aggregation and reduce thrombus formation by a cellular mechanism implying binding to platelet P-selectin. These findings add new insights into the role of EPCs in the regulation of platelet function and thrombotic reaction during vascular repair.

Heart disease is a leading cause of death in adults and children. We, and others, have described complex signaling, transcriptional and translational networks that guide early differentiation of cardiac progenitors and later morphogenetic events during cardiogenesis. We found that networks of transcription factors and miRNAs function through positive and negative feedback loops to reinforce differentiation and proliferation decisions. Many of the same cues have been leveraged to control differentiation of pluripotent stem cells into cardiac, endothelial and smooth muscle cells that may be useful for regenerative purposes. We have used similar approaches to direct differentiation of disease-specific human induced pluripotent stem cells in order to model human heart disease.

Recently, we utilized a combination of major cardiac regulatory factors, Gata4/Mef2c/Tbx5, to induce direct reprogramming of cardiac fibroblasts into cardiomyocyte-like cells with global gene expression and electrical activity similar to cardiomyocytes. The reprogramming was a stable event at the epigenetic level. We have now used genetic lineage-tracing techniques to demonstrate that resident cardiac fibroblasts can be reprogrammed into induced cardiomyocytes in vivo using a gene therapy approach. The in vivo efficiency of reprogramming into cells that are more fully reprogrammed into beating cardiomyocytes was greater than in vitro and resulted in improved cardiac function after injury. Knowledge regarding the early steps of cardiac differentiation in vivo has led to effective strategies to generate necessary cardiac cell types for disease-modeling and regenerative approaches, and promise to lead to new strategies for human heart disease.

Since the first report on the generation of pluripotent embryonic stem cells (ESCs) from preimplantation embryos, there has been tremendous interest in developing culture systems to produce ESCs from human and other species efficiently to use them in biomedical applications. These cells have provided fascinating possibilities and tools to study human development, genetic diseases, and development of toxicological and pharmaceutical applications as well as in therapeutic transplantation. Developing an efficient method to derive ESCs from various genetic backgrounds should be valuable for establishment of ESCs in various mammalian species, including human. However, it is still encumbered by various technical and ethical objections. This challenge is circumvented by using small molecules that suppress differentiation pathways. Here, we will present our recent studies on efficient and reproducible establishment of ESCs from blastocysts and single blastomere to avoid destruction of the embryo. Additionally, we present our recent researches on testis-derived pluripotent stem cells that share characteristics of ESCs. The application of these small molecules will not only solve the problems of derivation and expansion of pluripotent stem cell in defined conditions, but also clarify new signaling pathways in them.

Human stem cells, particularly embryonic stem cells, have unlimited capacity to develop into different types of cells such as muscle cells, nerve cells, heart cells, blood cells and others. Therefore, human stem cells hold the promise of a renewable source of replacement cells and tissues to treat an ever growing list of human diseases and conditions, including Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury, burns, heart disease, diabetes and arthritis.

On the other hand, stem cell research has led to great controversy. The controversy centers on the ethics of conducting research involving the generation, usage and destruction of human embryonic stem cells. But it should be noted that new approaches and innovative strategies have yielded human stem cells without destruction of embryos via generating pluripotent stem cells from adult cells.

The Supreme Council of Health (SCH) has developed a policy for the ethical use of human stem cells in research. The policy provides guidance and procedures under which proposed research in this area, conducted in Qatar publicly or privately funded, is ethically responsible, scientifically worthy, and conducted in accordance with national and international laws.

The SCH presentation will introduce the research community to what is allowed and not allowed in research involving human embryonic stem cells in Qatar.

The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of MSC from mouse BM (mBM), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of MSC, we hypothesized that a potential reason for the low yield of mMSC from mBM is the flushing of the marrow used to remove single cells suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of MSC physically associated with the abluminal surface of blood vessels.

Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs that yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least two orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal cell populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single cells suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays.

Since the first CB transplant (CBT) was performed by Gluckman et al in 1988, >16,000 patients have received this procedure for a variety of malignant and non-malignant diseases. Despite the lower frequencies of graft versus host disease (GvHD) and event-free survival rates comparable to those achieved after unrelated allogeneic marrow transplantation, a major disadvantage of CBT is the low cell dose, which results in slower engraftment and an elevated risk of engraftment failure by comparison with marrow or PBPC transplantation, even following transplantation with two CB units. A significant shortcoming of the cytokine-driven suspension culture systems initially envisioned for ex vivo expansion of CB cells is their failure to incorporate cues provided by the stromal cell-mediated microenvironment of the bone marrow. Seeking to enhance the efficiency of these culture systems, we have therefore incorporated mesenchymal precursor cells (MPC), a key cellular constituent of the hematopoietic stem cell niche. Optimisation of the expansion culture system and validation of the expanded product in immunodeficient NOD/SCID/IL-2RγNull (NSG) mice led to a clinical trial using haploidentical MSC (from a family member) and more recently using immunoselected allogeneic MPC (Mesoblast Ltd). More than 30 patients with a range of hematological malignancies have now been transplanted with a combination of two CB units, one unmanipulated, the second expanded on MPC. In fulfillment of our hypothesis, a significant enhancement in the rapidity of neutrophil and platelet reconstitution was observed, derived from the expanded unit that was superseded by long-term engraftment from the unmanipulated unit.

One of the fields of medicine that has raised the most expectations in Iran is cell therapy, using somatic or stem cells. The word “cell therapy” refers a series of procedures, which are used to separate and transplant cells in target tissues. However, in the absence of a current consensus on a approved protocol to isolation and identify suitable cells, the most methods in this field are under research yet. Royan Institute, as a leading center in stem cell research, in Iran, simultaneous with global research and increasing the knowledge about stem cells, the first Royan clinical trial in cell therapy field was done in 2004, which was designed and implemented for application of stem cells in patients with recent heart attack. During implementation of this project, at least three other projects were added to the list of Royan institute clinical trials. Two years later, Royan institute started to established GMP facilities for cell culture. By the end of year 2008, increasing number of clinical trials from 4 to 10 studies and termination of investigating stages of using Melanocytes for treatment of vitilligo disease and Limbal stem cells culture for using in patients with Limbal disease, led to establishment of a center for providing medical services to the patients. Now Royan institute in Regenerative department are doing more than 20 NIH registered clinical trials and there are some result in heart, bone, cartilage, limbal, liver and skin field, which will be presented in this lecture.

Haematopoietic stem cell transplantation (HSCT) is the treatment of choice for patients with refractory leukemia. However, HSCT is associated with lethal GVHD and/or disease relapse. The addition of alternative remedies such as adoptive immunotherapy may lead to eradication of minimal residual leukemia and prevention of relapse. Antigen-pulsed ex-vivo dendritic cells (DCs) have been utilized for the initiation of such immunotherapeutic modalities. DCs can successfully be generated from CD34+ haematopoietic stem cells (HPSCs) and DCs generated from patients with chronic myeloid leukemia (CML), displayed clonal heterogeneity with respect to the expression of the bcr/abl fusion gene. One reason could be that, DCs mixtures were originated from diverse sources of normal and leukemia either CD34+ or CD34- HPSC. CD34-HPSCs were reported to possess hematopoisis reconstitution capabilities. So far, the differentiation of DCs from CD34- HPSCs and their expression of bcr/abl have not been explored in CML patients. Consequently, we hypothesized that CD34-HPSCs could be a valuable source for the generation of an effective vaccine which could be stored and used latter to target residual leukemia if the patient encountered any relapses.

Our study showed that DCs generated from CD34-HPSCs were potent and efficient antigen presenting cells and comparable to their counterparts DCs from CD34+ HPSCs and hold promises in immunotherapy modality.

1A. Gaafar, 2H. M. Al-Omar, 1Z. Al-Mokhlafi, 1Manogaran PS, 1A. qniebi1, 1A Al-Mazrou, 2F.Al Mohareb, 1C. Adra, 1K. Al-hussein

1Stem Cells Therapy Program, 2 King Faisal Cancer Center, King Faisal Specialist Hospital & Research Centre.11211Riyadh, Saudi Arabia