The National Program for Research, Development & Inovation – People Program - Reintegration Project (RP) No.2/2008-2010
Project Details
Project Title: Use of endothelial progenitor cells differentiated from human bone marrow and placenta in cellular
therapy
Key words: Endothelial progenitors, stem cells, angiogenesis, ischemia, cell therapy
Domain: Cell Biology
Abstract: Introduction: Transdifferentiation of bone marrow (BM), umbilical cord (UC) blood, and vessel derived stem
cells into endothelial progenitors has been reported both in vitro and in vivo. Encouraging but limited success
with these studies suggested the need for identification of more versatile populations of endothelial progenitors
or signaling pathways to improve their homing to ischemic sites seeking angiogenesis and neovascularization.
Objective: We propose to isolate and characterize in vitro endothelial progenitors derived from BM and UC
blood/matrix and to investigate whether these cells could contribute to vascular regeneration in a novel
experimental model of cardiac ischemia.
Materials and methods: BM and UC blood/matrix derived stem cell subpopulations were assessed in clonal
growth assays under conditions that promote endothelial commitment, and molecularly characterized for the
expression of molecules and genes associated with the endothelial lineage. The capacity of these cells to
participate to vascular regeneration was examined in an in vitro xenotransplntation model. Endothelial
progenitors-induced vasculogenesis was assessed by tracking green fluorescent protein-labeled endothelial
progenitor cells and by immunohistochemistry, using highly specific anti-human antibodies.
Results will be disseminated after acceptance of publication (a manuscript submitted to a high impact-journal is
currently under review process)
Relevance: The proposed study will bring new insights into stem cell biology field, advancing the development
of cell therapies for vascular regeneration relying on angiogenesis and neovascularization.
Project 24 months
duration:
Starting date: May 1st 2008
Ending date: May 1st 2010
Total funding: 510.000 RON
Project Marilena Lupu, PhD
director:
E-mail: marilena.lupu@icbp.ro
Coordinating Institute of Cellular Biology and Pathology "Nicolae Simionescu"
Institution:
Web page: www.icbp.ro
Research The World Health Organization has predicted that by 2020 cardio-vascular disease will be the leading global
background: cause of total disease burden. One out of every ten people who have a myocardial infarction dies within a year.
Although neoangiogenesis within the infarcted bed is an integral component of the remodelling process, the
capillary network is unable to support the great demands of the hypertrophied myocardium, resulting in
progressive loss of viable tissue, infarct extension and fibrous replacement 1. Here we propose to use selected
populations of endothelial progenitors to induce new blood vessel formation in the infarcted bed
(vasculogenesis) and proliferation of the pre-existing vasculature (angiogenesis) after experimental vascular
ischemia, as well as to investigate efficient ways to improve endothelial progenitors homing to ischemic sites.
A large body of evidence indicates that small populations of pluripotent stem cells exist in many tissues,
including bone marrow (BM), umbilical cord (UC) blood (UCB), and blood vessels. During development,
endothelial cells are derived from mesoderm. Commitment of the hemangioblast to the endothelial lineage is
characterized by sequential expression of vascular endothelial (VE)-Cadherin (CD144), PECAM-1 (CD31), and
shortly afterward CD342. In postnatal life, endothelial progenitors express AC133, Flk1, CD34, and MUC18 3-5,
markers that are quickly lost upon differentiation to mature endothelium. Von Willebrand factor (vWF), VECadherin, Weibel-Palade bodies, PECAM-1, and endoglin (CD105) are markers expressed by mature endothelial
cells6.
BM-derived cells have been shown to possess the potential to transdifferentiate into endothelial cells7 and
induce therapeutic angiogenesis of ischemic tissues 8-12. Multipotent adult progenitor CD34-, VE-Cadherin-,
AC133+, and FLk1+13 cells have been identified in the non-hematopoietic BM compartment; when cultured with
VEGF, these cells differentiate into CD34+, VE-Cadherin+, AC133-, and FLk1+ cells, a phenotype characteristic for
angioblasts, and subsequently differentiate into cells that express endothelial markers and contribute to
neoangiogenesis in vivo13. Endothelial progenitors have also been isolated from human peripheral blood 3;9;10;14.
Human BM stem cell compartments are highly complex and perhaps comprising other cell types whose
endothelial fate we propose to define.
The UC is composed of connective tissue called Wharton Jelly, covered by a simple epithelium believed to derive
from amniotic membrane. It has been shown that UC epithelium has the ability to undergo terminal
differentiation when grafted onto the back of nude mice15. Our rationale to use UC matrix as a source of
endothelial progenitors is based on the fact that several groups postulated that the UC matrix is a rich source of
self-renewing, multiple-lineage differentiating cells16;17; however, these cells have not been fully characterized. A
few groups addressed the use of UCB stem cells to generate endothelial progenitors18-23. We propose to identify
and further characterize human UC blood/matrix stem cells with ability to differentiate towards endothelial
lineage and participate to vascular regeneration.
Elevated serum levels of chemokines such as stromal-derived factor-1 (SDF-1), vascular endothelial growth
factor (VEGF), and angiopoietin-1 were able to induce mobilization of endothelial progenitors 24. SDF-1 is
produced within the bone marrow and mediates chemokinesis and chemotaxis on a variety of cell types that
express the CXCR4 receptor. SDF-1-responsive cell types include CD34+ hematopoietic progenitors and stem
cells. High mobility group box 1 (HMGB1), a chromatin protein that acts as a cytokine when released in the
extracellular milieu by the necrotic and inflammatory cells, and its receptor for advanced glycation end products
(RAGE) were shown to induce both migration and proliferation of vessel-associated stem cells25;26. Therefore we
plan to investigate whether such molecules released by the ischemic cardiac tissue are able to induce endothelial
progenitor cells chemotaxis and promote vasculogenesis.
Research Stem cell plasticity studies have a huge potential for both basic and translational research. There is compelling
relevance: evidence from both in vitro and in vivo experiments that BM and UCB derived stem cells can contribute to
endothelial regeneration. This has led to the proposal that humans with serious vascular injuries can potentially
benefit from cellular therapies using the above and perhaps other cell types such as placental vessel and UC
matrix derived stem cells. The proposal seeks to demonstrate the capacity of human BM, placental vessel, and
UC blood/matrix derived endothelial progenitors to contribute to angiogenesis and neovascularization and to
gain insight into the biology of undifferentiated progenitor cells. The selection, characterization, and improved
delivery of autologous stem cell populations endowed with tissue specific plasticity appears to be the obvious
next step towards applying stem cell therapy as a regenerative therapy. We intend to follow this avenue of
research with the goal of developing a feasible therapeutic approach for human patients with vascular diseases.
The regenerative stem cell therapy represents an innovative, less invasive and less expensive treatment
strategy that could address a large spectrum of chronic degenerative disorders. Both morbidity and mortality
associated with these disorders have severe socio-economic consequences. Within the medical community,
studies resembling those proposed here raise hopes for the improvement of population general health status.
The proposed research has a highly significant potential for immediate clinical translation “from bench to
bedside”. Systemic and repeated delivery of autologous endothelial progenitors for the therapeutic management
of vascular diseases would be a simple and safe procedure in human patients. Furthermore, endothelial
progenitor cells isolated at birth from the placental tissues can be stored and used, as needed, for autologous
cell therapy purposes. Another potential advantage of the use of placental-derived stem cells for cell therapy is
that they are less immunogenic and are associated with much lower rates of graft-versus-host disease (GVHD)
after allogeneic transplantation compared to adult stem cells; therefore, placental-derived stem cells could be a
safer source for tissue-targeted allogeneic cell therapy than BM-derived stem cells.
Data protection: Project results will be posted after their publication in scientific journals.
Reference List
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