Transcript C: Endothelial cells incorporate DiI-Ac

Jianfei Xie
2015.11.20
Introduction
Hemophilia A is characterized by inability to clot blood because
of FVIII gene mutations and deficiency of this coagulation factor
(Follenzi et al. 2012).
3D structure of FVIII, PDB ID: 2R7E
Cell therapy
The goal of cell therapy is to introduce long-term expression
of therapeutic levels of FVIII in vivo resulting in a cure of the
disease .
Where is factor VIII synthesized?
Controversial !
Hepatic and Extrahepatic
Hepatic
1. Liver sinusoidal endothelial cells (LSEC): Main source
(Everett LA et al, 2014; Fahs SA et al, 2014; Diego Zanolini et al, 2015)
2. Hepatocytes and Küpffer cells: Low level
(Diego Zanolini et al 2015)
Extrahepatic
1. Peripheral or cord blood derived cells, including:
(1) Endothelial progenitor/endothelial cells.
(2) Monocytes, macrophages and megakaryocytes from
hematopoietic stem cells.
2. Bone marrow derived cells, including
Monocytes, macrophages and endothelial cells.
These extrahepatic cells could synthesize factor VIII in sufficient
amount to ameliorate the bleeding phenotype in hemophilic
mice (Diego Zanolini et al 2015)
Derivation of Endothelial Cells from human
peripheral and umbilical cord blood
1. CD34+ cells
2. CD34- cells
3. Human mononuclear cells (MNCs)
1. Human mononuclear cells (MNCs)
2. Blood (20-100 mL)
3. Histopaque 1077
MNCs were resuspended in 12 mL complete EGM-2 medium.
Cells were seeded onto 3 separate wells of a 6-well tissue
culture plate precoated with type 1 rat tail collagen After 24
hours of culture, nonadherent cells and debris were aspirated.
Medium was changed daily for 7 days and then every other
day until the first passage.
Isolation of endothelial progenitor cell colonies derived
from adult peripheral and umbilical cord blood.
Immunophenotyping of cell monolayers derived from
blood EPC-derived ECCs by fluorescence cytometry.
C: Endothelial cells incorporate DiI-Ac-LDL
D: Endothelial cells expressing EGFP were plated in
Matrigel for the formation of capillary-like structures
Representative photomicrographs of secondary colonies
Growth kinetics of the endothelial cell progeny derived
from cord and adult endothelial progenitor cell colonies
Quantitation of the clonogenic and proliferative potential of single
cord blood and adult endothelial cells derived from EPC colonies
Quantitation of the clonogenic and proliferative potential of single
cord blood and adult endothelial cells derived from EPC colonies
Replating potential and long-term culture of the cell
progeny derived from a single EPC-derived endothelial cell
Replating potential and long-term culture of the cell
progeny derived from a single EPC-derived endothelial cell
Use of blood outgrowth endothelial cells for gene therapy
for hemophilia A. Yi Lin et al. Blood. 2002
Human venous blood  Mononuclear  Endothelial cells
 Transduced with GFP-FVIII expression vector
 NOD/SCID mice model
Yi Lin et al. Blood. 2002
Yi Lin et al
Blood. 2002