Subproject 11
1. IdentificationSubproject title: “Investigation of angiogenic process and molecular interactions between mesenchymal
stem cells or pericytes with endothelial cells using developed in vitro and in vivo systems”
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.
Blood vessels form in response to angiogenic factor stimulation by two main processes: vasculogenesis and angiogenesis. Vasculogenesis is the de novo formation of vessels from progenitor endothelial cells (angioblasts), whereas angiogenesis refers to the sprouting of neovessels form a preexisting vasculature. Angiogenic sprouting in the adult was initially considered an exclusive feature of terminally differentiated endothelial cells, which have the capacity to form vascular tubes and recruit pericytes.
Previous studies conducted by our group gave us the hypothesis that immature mesenchymal stem cells reside in several peripheral tissues where they participate in the angiogenic response to injury (Covas et al., 2008). Moreover, we conducted studies of generation mature endothelial cells from endothelial progenitor cells (EPC) derived from bone marrow (BM) and cord blood (CB) after selection of specific CD133 marker expression. These CD133 positive cells from BM and CB were induced to differentiate into endothelial cells using three different protocols. At the end of differentiation protocol the endothelial cells were characterized by performing Leishman staining, immunophenotypical staining, real time RT-PCR, AcLDL uptake as well as neovessels formed in the ex-vivo matrigel system. Altogether, these studies demonstrated that vasculogenesis can be reproduced ex-vivo by culturing BM and UCV progenitor endothelial cells with specific cytokines and in matrigel system. Also, several transcriptional factors involved in this process could be elucidated (Covas, personal communication).
In order to continue these studies the propose of present sub-project consists on investigation the molecular interactions between bone marrow derived-mesenchymal stem cells (BM-MSC), umbilical cord vein derived-MSC (UCV-MSC) and central nervous system microvascular pericytes (CNS-P) after co-culture with human umbilical vein endothelial cell (HUVEC) and ex-vivo derived endothelial cells from BM and CB. The study of these interactions will provide relevant information undergoing physiological or pathological angiogenic and vasculogenic responses as well as will provide important information about vascularization process which still remains one of the major challenges in cell therapy protocols.
The aim objective of the present work is to determine the molecular interactions of cross-talk between FACS-sorted BM-MSC, UCV-MSC and CNS-P with CD31 positive endothelial cells. To perform and optimal way to stimulate angiogenesis in vitro and in vivo we will develop three models using BM-MSC, UCV-MSC or CNS-P with endothelial cells: 1) in vitro three dimensional co-culture model of BM-MSC, UCV-MSC and CNS-P with endothelial cells (HUVEC and BM and UCV-derived endothelial cells); 2) the matrigel plug assay co-infused with MSC or pericytes plus endothelial cells followed by subcutaneous injection in NODSCID mice and 3) the treatment of acute ischemic injury in a mouse hind limb model with endothelial progenitor or mature endothelial cells, as well as, BM-MSC, UCV-MSC and CNS-P.
For in vitro analyses of three dimensional co-culture model all cultures will be grown for 5 to 15 days and then cells will be harvested for: a) the morphology characterization by conventional microscopy; b) the immunophenotypic profile by flow cytometry; c) the capacity to take up DiI-labeled acetylated low-density lipoprotein (ac-LDL); d) gene expression analysis by real time PCR; e) assessment of in vitro angiogenesis by the formation of vascular-like structure on a matrigel system and f) characterization of new blood vessel formed by confocal microscopy as well as by conventional microscopy after staining the tissue formed with HE to investigate the presence of erythrocytes indicating functional capillaries.
For in vivo analysis two angiogenesis models will be performed: 1) the Matrigel plug assay containing mesenchymal stem cells or pericytes plus endothelial cells followed by subcutaneous injection in NODSCID mice and 3) NODSCID mouse hindlimb ischemic injury produced by produced by femoral artery removal. For these in vivo models MSC or pericytes will be genetically modified with fluorescent and bioluminescent proteins using transposon system which will allow the characterization of new capillaries by confocal microscopy and the analysis of new blood vessel formation in live animals using Xenogen IVIS system.
Moreover, the interactions between these two cell types during angiogenesis process will be investigate using MSC and endothelial cells from large animal models such as canine model in collaboration with Professor Maria Angelica Miglino from Faculty of Veterinary Medicine and Zootecnia from University of São Paulo. In this study, it will be performed the isolation and characterization of MSC from yolk sac canine which consists the first site of blood-cell production during embryogenesis.
Recent studies conducted by Maria Angelica’ group demonstrated that the canine yolk sac contains three preferential sites of MSC and endothelial cells (Miglino et al 2008). Therefore, after isolation, culture and characterization of MSC and endothelial cells from canine yolk sac it will be perform the three dimensional co-culture model for assess the cross-talk between these two cell types which will provide molecular insights into vasculogenesis and angiogenesis process during development and tissue repair. Similarly, the cultures will be grown for 5 to 15 days and then cells will be harvested to perform the same analysis as mentioned above.
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 determine the molecular interactions of cross-talk between FACS-sorted bone marrow or umbilical cord vein mesenchymal stem cells, as well as with pericytes with CD31 positive endothelial cells and contribute for understanding the blood vessels formation by two main processes: vasculogenesis and angiogenesis.
Specific Objectives
1. Phenotypic characterization of mesenchymal stem cells from bone marrow (BM-MSC) and umbilical cord vein (UCV-MSC) and central nervous system microvascular pericytes (CNS-P) using 6 CD markers: CD271, CD140B, 3G5, NG2, Stro-1 and CD146. Among of them select the surface antigen which is more expressed in MSC and CNS-P;
2. Clonogenic assays and morphological characterization of FACS-sorted BM-MSC, UCV-MSC and CNS-P using a specific marker previously selected;
3. Morphological and phenotypical characterization of FACS-sorted cell populations;
4. Isolation, morphologic and phenotypic characterization of mesenchymal stem cells from canine yolk sac (cYS-MSC);
for osteogenic, chondrogenic and adipogenic differentiation;
6. Genetic modification of human and canine MSC with firefly luciferase (luc) and and DsRed2 fluorescent protein using transposon-mediated gene transfer;
7. Morphological and phenotypical characterization of FACS-sorted cell population and DsRed2 fluorescent protein positive cells;
8. Isolation and culture of human umbilical vein endothelial cell (HUVEC) and canine yolk sac endothelial cells (cYS-EC) FACS-sorted according to CD31 high level expression;
9. Generation of mature endothelial cells from human bone marrow - and cord blood–derived endothelial progenitor cells (CD133+KDR+);
10. Morphological and phenotypical characterization of FACS-sorted CD31+ cell population;
11. The influence of endothelial soluble factors, as well as, the requirement of direct cell-cell contact between endothelial cells and BM-MSC, UCV-MSC or NS-P using co-culture in vitro systems;
12. Morphological, phenotypical and gene expression analysis of both cell populations before and after co-culture systems, as well as, the formation of vascular like structure on matrigel for the evaluation of in vitro angiogenesis;
13. Assessment of the role of BM-MSC,UCV-MSC, CNS-P and cYS-MSC in angiogenesis after co-infusion with HUVEC or cYS-EC using the Matrigel plug assay followed by subcutaneous injection in NODSCID mice. Image of new blood vessels in live animals with IVIS Xenogen system will be performed;
14. Characterization of new blood vessel formation by confocal microscopy;
15. Analyze the contribution of mesenchymal stem cells or pericyte together with endothelial cells to the recovery of the hind limb ischemic injury in NODSCID mice by confocal microscopy.
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 com a equipe do Hemocentro Ribeirão Preto Coordenador MD, PhD Aparecida Maria Fontes 13113802806 Responsável pela elaboração do Pesquisadora Pós-doc em terapia gênica PhD
protocolo experimental Ricardo B.
Silva
Executará os ensaios de expressão gênica dos ensaios de co-cultivo, seguida da caracterização em modelo animal
bolsista Execução dos ensaios pilotos em camundongos Bacharel Danilo Almeida Responsável pelos ensaios de co-infusão em matrigel bolsista Cultura e expansão de CTM e com ensaios animais Mestrando Maristela Delgado Orellana Responsável pelo isolamento e cultivo das MSC, pericitos e endoteliais Responsável pelo laboratório de cultura celular Isolamento e cultivo de MSC e endoteliais humanas Mestre Estudante DTI
Auxiliará nos ensaios de lesão isquêmica Patrícia VB
Palma
Responsável pelo “sorting” por citometria
Responsável pelo laboratório de citometria de fluxo Análise imunofenotípica por Citometria de Fluxo Bióloga Virgínia Picanço-Castro Auxiliará nas discussões dos resultados Bolsista pós-doc Modificação Gênica células-tronco PhD Danielle AR Magalhães
Auxiliará nas análises de expressão gênica e modificação gênica Tec. Pesq. Lab. Transf. Gênica Análise de expressão gênica em microarray PhD Angélica Miglino Avaliação semanal do andamento do projeto com a equipe da Fac. Medicina Veterinária e Zootecnia da USP Coordenadora do projeto com a equipe da Fac. Medicina Veterinária e Zootecnia da USP Coordenadoras de Projeto temático, auxílios à pesquisa e auxílios viagens Profa. Titular do Departamento de Cirurgia
Irina Kerkis 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 Responsável pelo isolamento e cultivo das CTM e endoteliais 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 Desenvolvimento da gestação conceptos bovinos clonados e Profa. Dra. da Faculdade Estadual Paulista – unidade
pelo L@mpe, Unesp Dracena transgênicos GFP+ 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.
