Subproject 14
1. IdentificationSubproject title: "Isolation and functional characterization of pluripotent stem cells – side population” Country: Brazil
Knowledge area, according to CNPq table http://www.cnpq.br/areasconhecimento Area: Hematology Code: 4.01.01.05-3
2. Subproject Coordinator’s Data
Full name: Aparecida Maria Fontes
Date of birth: 26/11/1966 Sex: Male Female Nationality: Brazilian
e-mail: fontesam@hemocentro.fmrp.usp.br
3. Data concerning the subproject coordinator’s institution
Institution (university, center, company, etc):Fundation Hemocenter of Ribeirão Preto Acronym: FUNDHERP
Organ (institute, college, etc): Regional Blood Center of Hemotherapy, HC-FMRP/USP Unit (department, laboratory, etc): Transferência Gênica
Position/Function: Research Scientific Technologic II
4. Subproject summary
Summarize, in a maximum of 1 page, the subproject.
The stem cell compartment is heterogeneous and there is the identification of more primitive cells is of great interest because they have greater therapeutic potential compared, for example, with mesenchymal stem cells which have been used in some cell therapy protocols based on their plastic adherence property. Among these more primitive we can cite the side population (SP) stem cells, which is the objective of the actual project
.
Originally, side population phenotype was described in murine bonemarrow preparations and are present with a frequency of 0,05%. The SP isolation is based on the property to exclude lipofilic staining Hoechst and acquire the profile of low side scatter and bright Hoechst staining cells, which is termed side population (Goodell et al. ,1996). Moreover, the SP cells contain higher ability to reconstituted hematopoietic system compared to HSC-CD34+ obtaining from bone marrow. Next, the presence of SP cells was demonstrated at human bone marrow, as well as, other tissue such as, liver, brain, mammary gland, kidney, skin, testis and retina (reviewed in Challen e Little, 2006). These data suggest that SP might be widely distributed into several tissues, although functional role and characterization is not well understood.
Previous studies conducted by our group demonstrated the presence of SP in human bone marrow which express high level of pluripotent gene markers, such as, Nanog, Oct4, c-myc e b-catenin (Picanço-Castro, personal comm.). Moreover, considering that embryonic stem cells present two important application limitations: a) immunological rejection and b) tumorigenic potential, the use of SP cells can constitute an alternative safe cell source for autologous clinical applications. The group of researchers from Center for Cell-based Therapy develops research projects with mesenchymal stem cells since eight years ago. Also, we were the first one who demonstrated the presence of MSC in umbilical cord vein (Covas et al., 2003) and recently we shown that MSC are widely distributed into the organism and present high similarities with pericytes (Covas et al., 2008).
Considering the importance to identify and characterize more primitive stem cells followed by the evaluation of their therapeutic potential, the aim objective is to isolate side population from two human tissues (umbilical cord vein and central nervous system blood capillaries) and to compare with SP isolated from bone marrow. Also, the isolation and characterization of human SP obtained from mesenchymal stem cells expanded in culture and isolated from bone marrow, umbilical cord vein, as well as, brain pericytes expanded in culture is the second aim objective of the present project. In parallel, SP isolated from human bone marrow, umbilical cord vein and brain capillaries at day 0 will be submitted to expansion in culture using four different medium conditions: a) culture medium for MSC expansion (MSC-M); b) culture medium for expansion of hematopoietic stem cell (HSC-M); c) culture medium for expansion of endothelial progenitor cells (EPC-M) and d) culture medium for expansion of human embryonic stem cells. After expansion SP will be characterized by morphological, immunophenotypical and multipotentiality features.
Also, gene expression profile by RT-PCR will be performed for functional characterization of these different sources of SP. Finally, the third aim objective consists on investigate the therapeutic potential cell-derived SP using murine model for hepatic endothelial injury, as well as, for hind limb ischemia injury.
In collaboration with the group of Professor Dr. Maria Angélica Miglino the fourth aim objective of the Project consists on isolating SP from ovine bone marrow and brain capillaries, as well as, from canine yolk sac. The frequency of SP will be analyzed by flow cytometry and clonogenic assay. Next, these SP will be grown and expanded under three specific medium cultures: a) MSC-M; b) HSC-M and c) EPC-M and the therapeutic potential of some of these SP will be investigated in a canine bone marrow aplasia model and ovine brain ischemia injury.
A second collaboration with the group of Professor Dr. Flávio Vieira Meirelles to isolate SP from two bovine/GFP+ fetal tissues consists on the fifth aim objective of this Project. The bovine fetal GFP tissues are the bone marrow (BM) and umbilical cord vein (UCV) with collection in utero. After the isolation, the SP cells will be grown and expanded under three specific medium cultures: a) MSC-M; b) HSC-M and c) EPC-M and the therapeutic potential of some of these SP will be investigated in a NODSCID murine bone marrow transplantation model. Additionally, will be performed nuclear transfer experiments in bovine enucleated oocytes using BM or UCV-SP/GFP+ cells in order to evaluate the genetic reprogramming ability of SP cells. Also, in collaboration with Prof Dr. Flávio the nucleus of human SP cells will be used for genetic reprogrammed studies using bovine cytoplast as receptor, once the project get approved by the Research Committee.
5. Objectives
List the general and the specific objectives of the subproject.
General Objectives
1. Integration with people from other institutions and ensure contribution with different researchers to give support for in vitro studies as well as pre-clinical trials in the field of cell therapy;
2. To identify and characterize more primitive stem cells termed side population, which expresses pluripotent genes, from two human tissues and to compare with side population cells from bone marrow. Also, the isolation and characterization of side population stem cells from three sheep tissues, one canine tissues and two bovine tissues are the second aim objective of this project.
Specific Objectives
1- Isolation and characterization of human side population stem cells in bone marrow, umbilical cord vein and in blood capillaries from central nervous system (day 0);
2- Isolation and characterization of human side population stem cells obtained from cultured mesenchymal stem cells (MSC-SP) isolated from bone marrow, umbilical cord vein, as well as, cultured pericytes isolated from central nervous system blood capillaries using the plastic adherence classical protocol;
3- Isolation and characterization of ovine side population stem cells in bone marrow and in blood capillaries from central nervous system (day 0);
4- Isolation and characterization of canine side population stem cells in yolk sac (day 0);
5- Isolation and characterization of side population stem cells in bone marrow and umbilical cord vein from transgenic fetal bovine, which express GFP (day 0);
6- Analysis of side population frequency from human, dog, sheep and bovine tissues at day 0 by flow cytometry and in vitro clonogenic assay;
7- Analysis of side population frequency from human, dog, sheep and bovine cultured mesenchymal stem cells tissues obtained after plastic adherence classical protocol;
8- Culture and expansion of “side-population” stem cells isolated at day 0, as well as, SP cells isolated from MSC cultures using four different culture media: a) culture medium for MSC expansion; b) culture medium
for expansion of hematopoietic stem cell (HSC-M); c) culture medium for expansion of endothelial progenitor cells and d) culture medium for expansion of human embryonic stem cells;
9- Analysis of karyotype and cytogenetic characteristics of human SP;
10- Analysis of gene expression profile of human SP culture and expanded under MSC conditions by microarray; 11- Culture and expansion of “side-population” stem cells isolated sheep, canine and bovine tissues using
medium for MSC expansion;
12- Morphologic and immunophenotypic characterization of side population stem cells undergoing expansion using different conditions;
13- Multipotentiality potential of mesenchymal stem cells derived from human and animal SP;
14- Analysis of gene expression profile of human SP cultured under other culture media (EPC, HSC and hES), as well as, bovine, canine and ovine SP cells under several media by real time PCR;
6. Work plan
Describe the stages of the execution of the subproject.
7. Methodology
Indicate the methodology which will be applied in the research intended.
8. Executive Team - Activities of the researchers who belong to the project
Detailed explanation of the proposing team, explaining the qualifications of the researchers.
Specify the activities to be performed by the team members, informing their previous experiences in research and development activities.
Name CPF Objective/Justification of the activity Function in the project (researcher or responsible for the laboratory Previous experiences Titles Dimas Tadeu Covas Avaliação semanal do andamento do projeto Coordenador MD, PhD Aparecida Maria Fontes 13113802806 Responsável pela elaboração do protocolo experimental Pesquisadora Pós-doc em terapia gênica e experiência com cultivo e expansão de hES PhD Maristela Delgado Orellana Responsável pelo cultivo e expansão das células-tronco “side population” Responsável pelo laboratório de cultura celular Isolamento e cultivo de MSC e endoteliais humanas Mestre Patrícia VB Palma Responsável pelo isolamento de células “side population” por citometria de fluxo Responsável pelo laboratório de citometria de fluxo Análise imunofenotípica por Citometria de Fluxo Bióloga Kamilla Swiech
Auxiliará no cultivo das células-tronco nos diferentes meios Pós-doutoranda Produção de células em larga escala PhD
DTI de modelo animal murino
Danielle AR Magalhães
Auxiliará nas análises de expressão gênica por RT-PCR em tempo real Tec. Pesq. laboratório de Transf. Gênica Análise de expressão gênica por microarray PhD Rodrigo A. Panepucci Responsável pelas análises de expressão gênica por micro-array
Pesquisador Análise de expressão gênica por SAGE PhD Fábio M. Oliveira Responsável pela análise citogenética das células SP humanas Pós-doutorando Citogenética clássica de células somáticas normasi e tumorais PhD Danielle AR Magalhães
Auxiliará nas análises de expressão gênica por RT-PCR em tempo real Tec. Pesq. laboratório de Transf. Gênica Análise de expressão gênica por microarray PhD Lindolfo da Silva Meirelles Auxiliará no cultivo e expansão das células-tronco mesenquimais humanas Pós-doutorando Cultivo e expansão de MSC murinas PhD Angélica Miglino 00542335859 Avaliação semanal do andamento do projeto com a equipe da Fac. Medicina Veterinária e Zootecnia da USP Coordenadora do projeto Coordenadoras de Projeto temático, auxílios à pesquisa e auxílios viagens Profa. Titular do Departamento de Cirurgia
Irina Kerkis 05218440701 Responsável pela elaboração do protocolo experimental com a equipe da Fac. Medicina Veterinária e Zootecnia da USP Pesquisador Pesquisadora com mestrado e doutorado na Rússia, desenvolveu no Brasil a patente da extração das células de polpa de dente Profa. Dra. Pesquisador PcQ IV do Instituto Butantan Daniele dos Santos Martins 294226528-04 Responsável pela caracterização das células tronco Pesquisadora Células-tronco germinativas de origem fetal canina PhD Carlos Eduardo Ambrosio 255397158-37 Responsável pelo laboratório de extração de células tronco do liquido amniótico e vitelino. Pesquisador Pesquisador com experiência na área de placentação em carnívoros e cultivo celular Jovem Pesquisador da Faculdade de Medicina Veterinária e Zootecnia Flávia Thomaz Verechia Pereira 191.637.838-27
Obtenção das células bovinas GFP+ Pós-doc do LMMD, FZEA-USP, Pirassununga e responsável pelo L@mpe, Unesp Dracena Desenvolvimento da gestação conceptos bovinos clonados e transgênicos GFP+ Profa. Dra. da Faculdade Estadual Paulista – unidade Dracena/SP
9. Funding Sources
List of projects funded in the last 5 years (current or completed)
Funding Source Title Term Value Relation to the present
request
10. Contribution of the participant institutions
Detail, in national currency, the contribution of the Brazilian and French institutions, participants of the thematic subproject, under the form of: infrastructure, financial resources, human resources (working hours), consumables, daily payments and traveling tickets.
11.Budget, justified and adequate to the proposal Attached Form
12. Scholarships
These resources cannot overcome 15% of the total value requested for the project Scholarship Modality: (IC, ITI, DTI, AT, PDJ, BEV)
Modality Duration Quantity
13. Establishment of the post-graduation courses or subjects:
Detail high-level technological training or establishment of new laboratorial methodologies – indicate hours/class and the program. Indicate CAPES classification.
