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, placental vessels, and
UC blood/matrix and to investigate whether successful in vivo endothelial regeneration, differentiation and maintenance can
be achieved by systemic delivery when a defined demand is placed on the endothelium. Next, we propose to investigate
whether endothelial progenitors homing to ischemic sites can be enhanced by delivery of chemoatractant molecules into the
ischemic bed.
Materials and methods: BM, placental vessel, and UC blood/matrix derived stem cell subpopulations will be 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 in
vascular regeneration will be examined following systemic delivery into immunodeficient NOD/SCID mice with experimentally
induced ischemia. Various chemokines will be investigated for their ability to augment endothelial progenitors homing to
ischemic sites and to promote endothelial regeneration. Endothelial progenitors contribution to vascular reconstitution will be
assessed by histology, immunohistochemistry, electron microscopy and clonal analyses of ischemic tissue samples.
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 duration: 24 months
Starting date: May 1st 2008
Ending date: May 1st 2010
Total funding: 510.000 RON
Project director: Marilena Lupu, PhD
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 cause of total
background: 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
replacement1. 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 MUC183-5, markers that are quickly lost upon differentiation to mature
endothelium. Von Willebrand factor (vWF), VE-Cadherin, 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 cells 7 and induce therapeutic
angiogenesis of ischemic tissues8-12. 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 +, VECadherin+, 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 blood3;9;10;14. Human BM stem cell compartments are highly complex and perhaps comprising other
cell types whose endothelial fate we propose to define.
Vessel-associated progenitor cells, so called pericyte derived cells (in post-natal life) or mesangioblasts (during
embryonic development)15;16, are common progenitors for vascular and extravascular mesodermal derivatives. They express
early endothelial markers such as stem cell antigen-1(Sca-1), Flk-1, CD34, MEF2D, kit and VE-Cadherin15. Mesangioblasts
have been isolated from embryonic mouse aorta15 and the pericyte derived cells have been isolated from vessels of juvenile
dystrophic mice17, outgrowth of small vessel-containing tissue fragments from muscle of adult dogs18, and micro-vasculature
within the BM19. When systemically injected, both mesangioblasts and pericyte derived cells were able to accumulate in the
first capillary filter encountered, transit through the endothelial barrier only in the presence of inflammation15;16, and improve
muscle function both in dystrophic dogs and mice17;18. We propose to investigate whether pericyte derived cells could be
isolated from human placental vessels and contribute to angiogenesis and neovascularization.
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 mice20. 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 cells21;22;
however, these cells have not been fully characterized. A few groups addressed the use of UCB stem cells to generate
endothelial progenitors23-28. 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 progenitors29. 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
cells30;31. Therefore we plan to investigate whether local delivery of such chemotactic molecules within the ischemic sites is
able to improve/accelerate vascular repair and regeneration.
Research Stem cell plasticity studies have a huge potential for both basic and translational research. There is compelling evidence from
relevance: 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.
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