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LABORATORY OF: Stem Cell Biology
CONTACT PERSON: Prof.Rodolfo Quarto (University of Genova)
Phone +39 010 5737240 E-mail: rodolfo.quarto@unige.it

Description of Laboratory and Expertise:

The main research activities of the laboratory are focused on the cellular biology of progenitor cells/adult stem cells for basic research and regenerative medicine applications. The repair of skeletal tissues represent the endpoint of cell-mediated approaches that take into account the properties of the progenitor cells selected from different sources and the influence of the microenvironment on cell delivery and differentiation. In this respect the laboratory is also strongly involved in the technological improvement of cell culturing conditions. The laboratory is presently involved in several projects funded by MUR and EU related to the area of tissue engineering.

Abstract of Activities:

In our laboratory, we investigate the prospect of using stem/ progenitor cells in the cell therapy of bone, cartilage and other mature tissues taking into consideration the properties of osteoprogenitor cells from different sources and the influence of the microenvironment on cell delivery and differentiation. Cell phenotype, gene expression and regulation, extracellular matrix production and its components during culture and differentiation conditions are evaluated by using cytochemical, immunochemical and molecular biology approaches. The research activity focuses also on the generation of bone grafts/substitutes, based on autologous cells and 3D scaffolds. To do this, we aim at identifying appropriate 3D scaffolds (in terms of chemical compositions and internal architecture) where cells may growth. In vitro and in vivo approaches are currently applied to test the scaffolds in terms of biocompatibility, cell seeding requirements, osteoconductivity and osteoinductivity. Results obtained so far have led to the identification of some osteoconductive biomaterials (polymers, ceramics, composites) supporting the expansion and differentiation of mesenchymal progenitor stem cells (MSC) from the bone marrow into bone-like tissues. Different bioreactor systems for an efficient seeding and culture of cells into 3D scaffolds under automated and controlled conditions have been also tested and used to engineer bone grafts.

Detailed Research Activities:

Stem and progenitor cells from adult tissues represent an important promise in the therapy of several pathological conditions. Stem cells have the ability to self-replicate for long periods or,in the case of adult stem cells, maintain their differentiation potential throughout the life of the organism. Progenitor cells are derived from stem cells; they retain the differentiation potential and high proliferation capability, but they have lost the self-replication property. Adult stem cells were first identified in tissues characterized by a high rate of cell turnover, such as bone marrow. Recently, stem cells also have been isolated from adult organs previously thought to be capable of limited self-repair,offering the prospect of new therapeutic strategies for repair of damaged tissues.
From a classical view, adult stem cells are long-lived cells restricted to differentiating along the lineage pathways of their own tissue. Similarly, progenitor cells have been considered committed to the cell phenotypes of their tissues of origin.This concept of lineage restriction has been challenged by experimental evidences over the past few years. Indeed, most stem and progenitor cell types display an amazing plasticity, which is the property of cells to differentiate into phenotypes not restricted tothe tissues and, in some cases, to the germ layers from which they are derived. Bone marrow (BM) transplantation made it possible to follow the fates of transplanted cells in vivo by designing experiments in which donor cells were distinguishable from those of the recipient. In a similar experimental setting, although often at a very low frequency, BM-derived cells of adult donors were found integrated in the parenchyma of a variety of non-hemopoietic tissues, such as liver, brain, vascular wall, skeletal, and cardiac muscle, and even in skin, lung, and digestive tract epithelium. In our laboratory, we investigate the prospect of using stem/ progenitor cells in the cell therapy of bone, cartilage and other mature tissues, taking into consideration the properties of osteoprogenitor cells from different sources and the influence of the microenvironment on cell delivery and differentiation. For instance, multipotent cells obtained by an autologous harvest may be processed and expanded in vitro to amplify their number, seeded into suitable three-dimensional (3D) resorbable biomaterials, and then elaborated in vitro to induce their differentiation and neo-tissue formation, before their implant. The factors regulating the growth and development of mesenchymal precursor cells toward mature tissue are not well identified. Thus, part of our efforts are devoted to the formulation of chemically defined cell culture media; in this respect, assays are performed to determine the medium components effects on the cell morphology, phenotype and metabolism, via proteomic or genomic approaches and via differentiation assays, in vivo and in vitro. FACS, microarray and ELISA kits for chemokine assays kits are also feasible and applied. All these assays are used to integrate physiological parameters of the bone marrow, its internal architecture, the composition of the matrix, the cell population subsets, the cell-cell interactions, and the hemo-dynamics conditions in a comprehensive approach to tissue engineering. Within this light, the most challenging of our goals is the maintenance/amplification of the stemness of bone marrow derived cells in our defined 3D culture conditions, once more to be verified through the characterization of the morphology, phenotype and clonogenicity of cells. In this respect, we also aim at modifying the clinical practice in the field of regenerative medicine, through a cell therapy approach that reconstitutes the BM niches in which stroma and hemopoietic stem cells coexist. Main goals are (i) the identification of the best cellular sources and bio-active molecules, (ii) planning and development of 3D biomaterials with internal structures and chemical compositions favouring the in vivo tissue formation, (iii) the development of specific bioreactor systems for the multipotent cells culture within three-dimensional biomaterials under specific physical stimuli and automatic experimental conditions.
Our laboratory has been also focusing its attention on generating osteoinductive constructs exclusively within a 3D culture environment (biomaterials and bioreactor systems). In particular, we tested the hypothesis that BMSC can be isolated and expanded within 3D ceramic scaffolds by direct loading and culture of bone marrow nucleated cells into the scaffold pores. Moreover, based on the demonstrated efficacy of 3D perfusion systems to improve the efficiency and uniformity of cell seeding within porous 3D scaffolds, we are investigating whether dynamic perfusion seeding of bone marrow nucleated cells within 3D ceramics could improve the amount and uniformity of bone formed in vivo as compared to static cell loading.

Applications and Developments:

The Stem Cells Biology Laboratory research activities may provide future applications and comebacks in the fields of (i) stem cell sources, cultures and microenvironment (ii) planning and development of 3D biomaterials with internal structures and chemical compositions favouring the in vivo mature tissue formation, (iii) development of specific bioreactor systems for the stem cells culture within three-dimensional biomaterials under specific physical stimuli and automatic experimental conditions. These outcomes may be relevant for the field of cell-mediated tissue repair and for medical devices productions.

Managed core facilities:

1. optical microscope
2. cell culture equipment
3. molecular biology equipment

Ongoing collaborations:

Our research in the field of Tissue engineering is going on in collaboration with the Laboratory for Tissue Engineering at the University Hospital of Basel (Prof. I. Martin), Finceramica Faenza s.r.l, the University of Genova and the Laboratory of Stem Cell Bioengineering (Dr. Silvia Scaglione).

Most recent and significant publications:

Marcacci, M., E. Kon, V. Moukhachev, A. Lavroukov, S. Kutepov, R. Quarto, M. Mastrogiacomo, and R. Cancedda Stem cells associated with macroporous bioceramics for long bone repair: 6- to 7-year outcome of a pilot clinical study. Tissue Eng. 2007 May;13(5):947-55.

Li Pira, G., F. Ivaldi, L. Bottone, R. Quarto, and F. Manca. Human bone marrow stromal cells hamper specific interactions of CD4 and CD8 T lymphocytes with antigen-presenting cells. Hum Immunol. 2006 Dec;67(12):976-85

Braccini, A., D. Wendt, C. Jaquiery, M. Jakob, M. Heberer, L. Kenins, A. Wodnar-Filipowicz, R. Quarto, and I. Martin. Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts. Stem Cells. 2005 Sep;23(8):1066-72.

Monticone, M., Y. Liu, L. Tonachini, M. Mastrogiacomo, S. Parodi, R. Quarto, R. Cancedda, and P. Castagnola. Gene expression profile of human bone marrow stromal cells determined by restriction fragment differential display analysis. J Cell Biochem. 2004 Jul 1;92(4):733-44.

Garofalo, S., and R. Quarto. Knocking out the bad allele. Gene Ther. 2004 Sep;11(17):1301-2

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