Establishment of embryonic stem cells secreting human factor VIII for cell‐based treatment of hemophilia A

Summary.  Background: Hemophilia A is an X‐chromosome‐linked recessive bleeding disorder resulting from an F8 gene abnormality. Although various gene therapies have been attempted with the aim of eliminating the need for factor VIII replacement therapy, obstacles to their clinical application remain. Objectives: We evaluated whether embryonic stem (ES) cells with a tetracycline‐inducible system could secrete human FVIII. Methods and results: We found that embryoid bodies (EBs) developed under conditions promoting liver differentiation efficiently secreted human FVIII after doxycycline induction. Moreover, use of a B‐domain variant F8 cDNA (226aa/N6) dramatically enhanced FVIII secretion. Sorting based on green fluorescent protein (GFP)–brachyury (Bry) and c‐kit revealed that GFP–Bry+/c‐kit+ cells during EB differentiation with serum contain an endoderm progenitor population. When GFP–Bry+/c‐kit+ cells were cultured under the liver cell‐promoting conditions, these cells secreted FVIII more efficiently than other populations tested. Conclusion: Our findings suggest the potential for future development of an effective ES cell‐based approach to treating hemophilia A.

[1]  R. Montgomery,et al.  Lentivirus‐mediated platelet‐derived factor VIII gene therapy in murine haemophilia A , 2007, Journal of thrombosis and haemostasis : JTH.

[2]  Gordon Keller,et al.  BMP-4 is required for hepatic specification of mouse embryonic stem cell–derived definitive endoderm , 2006, Nature Biotechnology.

[3]  Ralf Kettenhofen,et al.  Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium , 2006, The Journal of experimental medicine.

[4]  K. High,et al.  Cellular and genetic therapies for haemophilia , 2006, Haemophilia : the official journal of the World Federation of Hemophilia.

[5]  J. Sommer,et al.  Inadvertent germline transmission of AAV2 vector: findings in a rabbit model correlate with those in a human clinical trial. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[6]  Chikara Furusawa,et al.  Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture , 2005, Development.

[7]  R. Sarkar,et al.  Transplantation of endothelial cells corrects the phenotype in hemophilia A mice , 2005, Journal of thrombosis and haemostasis : JTH.

[8]  G. Daley,et al.  The homeobox gene HEX regulates proliferation and differentiation of hemangioblasts and endothelial cells during ES cell differentiation. , 2005, Blood.

[9]  Takahisa Fujikawa,et al.  Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells. , 2005, The American journal of pathology.

[10]  A. Giles,et al.  Preclinical experiment of auxiliary partial orthotopic liver transplantation as a curative treatment for hemophilia , 2005, Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society.

[11]  M. Manco‐Johnson.  Hemophilia management: optimizing treatment based on patient needs , 2005, Current opinion in pediatrics.

[12]  M. Dake,et al.  Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases , 2005, Hepatology.

[13]  S. Pipe,et al.  New high‐technology products for the treatment of haemophilia , 2004, Haemophilia : the official journal of the World Federation of Hemophilia.

[14]  J. Kaiser.  Side Effects Sideline Hemophilia Trial , 2004, Science.

[15]  S. Pipe,et al.  1 Bioengineering of Coagulation Factor VIII for Improved Secretion , 2004, Pediatric Research.

[16]  Gordon Keller,et al.  Development of definitive endoderm from embryonic stem cells in culture , 2004, Development.

[17]  Lili Wang,et al.  Total correction of hemophilia A mice with canine FVIII using an AAV 8 serotype. , 2004, Blood.

[18]  V. Kouskoff,et al.  Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation , 2003, Development.

[19]  G. Daley,et al.  HoxB4 Confers Definitive Lymphoid-Myeloid Engraftment Potential on Embryonic Stem Cell and Yolk Sac Hematopoietic Progenitors , 2002, Cell.

[20]  A. Hubbard,et al.  Potency estimation of recombinant factor VIII: effect of assay method and standard , 2001, British journal of haematology.

[21]  K. Nogami,et al.  An Alloantibody Recognizing the FVIII A1 Domain in a Patient with CRM Reduced Haemophilia A due to Deletion of a Large Portion of the A1 Domain DNA Sequence , 2000, Thrombosis and Haemostasis.

[22]  D. Kioussis,et al.  The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation. , 2000, Development.

[23]  S. Antonarakis.  Molecular genetics of coagulation factor VIII gene and haemophilia A , 1998, Haemophilia : the official journal of the World Federation of Hemophilia.

[24]  R. Kaufman,et al.  Differential Interaction of Coagulation Factor VIII and Factor V with Protein Chaperones Calnexin and Calreticulin* , 1998, The Journal of Biological Chemistry.

[25]  R. Kaufman,et al.  Role of the B domain for factor VIII and factor V expression and function. , 1994, Blood.

[26]  A. Giles,et al.  Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII. , 1993, Blood.

[27]  A. Dorner,et al.  Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. , 1988, The Journal of biological chemistry.

[28]  R. Kaufman,et al.  A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[29]  J. Kaiser.  Gene therapy. Side effects sideline hemophilia trial. , 2004, Science.