Stem cells in molecular and regenerative medicine

Stem Cells are rare cells with the crucial ability to self-renew and to generate mature cells of any tissue through differentiation. Adult stem cells hold great promise for regenerative medicine, tissue repair, and gene therapy. Adult bone marrow cells (BMCs) include two populations of bone marrow stem cells (BMCs): hematopoietic stem cells (HSCs), which give rise to all mature lineages of blood, and mesenchymal stem cells (MSCs), which can differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, tenocytes, and haematopoiesis supporting stromal cells. Under normal condition these stem cells are tightly regulated by both intrinsic and extrinsic signals and malfunctioning in this balance can result in cancer. In this thesis we focused on two different aspects of stem cells: the leukemia stem/initiating cells in acute myeloid leukemia (AML) and the usage of stem cells in regenerative medicine. In the first part we focused on the molecular mechanism of AML-ETO, a results from the t(8:21) translocation which has been associated with leukemic transformation. Acute myeloid leukaemia (AML) is defined as a heterogeneous group of clonal disorders caused by malignant transformation of a bone marrow-derived self-renewing stem or progenitor cell, which demonstrates an enhanced proliferation as well as aberrant differentiation resulting in haematopoietic insufficiency (i.e. granulocytopenia, thrombocytopenia or anaemia). These leukaemias are suggested to result from the acquisition of chromosomal rearrangements and multiple gene mutations in either a hematopoietic multipotent cell or a more differentiated, lineage-restricted progenitor cell that is transformed in a so-called leukaemic stem or initiating cell, which keeps the ability to self-renewal. AML is generally regarded as a stem cell disease and is commonly altered both at the epigenetic as well as the genetic level. AML is the most common acute leukemia affecting adults, and its incidence increases with age. Therapies based on the current knowledge target the bulk leukemic population and spare the leukemic stem cells. It is therefore critical to determine and characterize the exact molecular mechanism involved in leukemic transformation for the development of novel therapeutic targets. AML patients harboring the t(8:21) translocation has intermediate prognosis and the identification of genome wide events in this subset of AML is clinically relevant and would lead to the understanding of molecular mechanism of disease progression. To this end we analyzed the DNA binding pattern of AML1-ETO in AML cell lines and in primary AML blasts. We demonstrate that AML1-ETO preferentially binds regions that contain RUNX1/AML1 and ETS core consensus sequences and that the AML1-ETO binding sites invariably consist of HEB and partially CBFβ, RUNX1/AML1 as well as of ETS factors such as ERG and FLI1. Subsequent analysis in t(8;21) and t(15;17) (another AML associated translocation) cells revealed cell type specific ETS factor binding and preferential AML1-ETO binding to the cell type specific ETS factor binding sites. In addition, we uncovered that binding of the ETS factor ERG correlates with the ‘active’ histone acetylation mark. Together our results suggest that ETS factors demarcate hematopoietic regulatory sites that provide a target for (aberrant) epigenetic regulation by oncofusion proteins. In the second part we attempted to evaluate the possibility to obtain in vitro an implantable tissue-engineered esophagus composed of acellular esophageal matrix and Mesenchymal stem cells (MSCs). Mesenchymal Stem Cells (MSCs) are multipotent precursors to many mesodermal cell lineages in vertebrate animals and are most often obtained from bone marrow. Certain attributes of MSCs, including migration toward sites of inflammation, ease of transduction, and lack of immunogenicity, suggest these cells may be potentially useful for regenerative medicine. Putative therapeutic uses include regeneration of damaged tissue, acting as a vessel for delivering a therapeutic transgene, support of other cell types for tissue repair, and modulating the immune reaction to co-transplanted cells or tissues. The use of MSCs in tissue engineering approaches avoids the moral and technical issues associated with the use of those from embryonic source and MSCs have already demonstrated their efficacy in preliminary tissue engineering application. Artificial materials and autologous tissues used for esophageal reconstruction often induce complications like stenosis and leakage at long-term follow-up. In the present study we attempted to evaluate the adhesion of MSCs on acellular esophageal matrix for esophagus tissue engineering. MSCs were isolated from rabbit bone marrow, characterized, expanded in vitro, and seeded onto rabbit acellular esophageal matrix. Acellular matrices obtained by detergent-enzymatic method did not present any major histocompatibility complex marker. Moreover, they supported cell adhesion, and in as much as just after 24 h from seeding, the scaffold appeared completely covered by MSCs in static as well as in bioreactor. Collectively, these results suggest that patches composed of homologous esophageal acellular matrix and autologous MSCs may represent a promising tissue engineering approach for the repair of esophageal injuries