1. Jianfei Xie 2015.11.20

2. 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

3. 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.

4. 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)

5. 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)

6. Derivation of Endothelial Cells from human peripheral and umbilical cord blood 1.CD34+ cells 2.CD34- cells 3.Human mononuclear cells (MNCs)

8. 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.

9. Isolation of endothelial progenitor cell colonies derived from adult peripheral and umbilical cord blood.

10. Immunophenotyping of cell monolayers derived from blood EPC-derived ECCs by fluorescence cytometry.

11. C: Endothelial cells incorporate DiI-Ac-LDL D: Endothelial cells expressing EGFP were plated in Matrigel for the formation of capillary-like structures

12. Representative photomicrographs of secondary colonies

13. Growth kinetics of the endothelial cell progeny derived from cord and adult endothelial progenitor cell colonies

14. Quantitation of the clonogenic and proliferative potential of single cord blood and adult endothelial cells derived from EPC colonies

16. Replating potential and long-term culture of the cell progeny derived from a single EPC-derived endothelial cell

18. 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

19. Yi Lin et al. Blood. 2002

20. Yi Lin et al Blood. 2002