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PSRC 60th Annual Meeting

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Adipose Derived Stem Cells Express von Willebrand Factor and Factor VIII
Maricel Miguelino, MD, David Sahar, MD, Jerry Powell, MD.
University of California, Davis, Sacramento, CA, USA.

Adipose Derived Stem Cells Express von Willebrand Factor and Factor VIII
Authors: Maricel Miguelino, MD1, Brittany Busse MD2, Christopher Pivetti, MS2, David Sahar, MD2, Jerry Powell MD1
1Department of Internal Medicine Division of Hematology Oncology
2Department of Surgery
University of California, Davis
PURPOSE: Hemophilia A is an x-linked recessive bleeding disorder due to a mutation of the coagulation Factor VIII (FVIII) gene that manifests as a deficiency or complete absence of FVIII. Patients with severe HA (FVIII levels <1%) experience severe bleeding in the joints that may result in debilitating hemarthroses, oropharygneal bleeds or fatal intracranial hemorrhage. Monogenic disorders such as HA is highly attractive for stem cell based therapy due to its broad therapeutic window were an increase in plasma clotting FVIII levels above 5% converts disease phenotype from severe HA to moderate HA reducing episodes of spontaneous bleeds, improving the patient’s quality of life and may provide a permanent cure for the disease. Putative adipose derived stem cells (ASCs) represent an abundant and readily available source of stem cells that possess the ability to undergo multi-lineage differentiation analogous to mesenchymal stem cells (MSCs) from the bone marrow [1]. MSCs from bone marrow have been reported to contribute to endogenous FVIII production and to phenotypically correct hemophilia in murine and sheep[2].The aim of this study is to demonstrate that ASCs may contribute to endogenous production of FVIII and after ex vivo transduction, ectopically express sustained Factor VIII.
METHODOLOGY: ASCs were isolated from murine stromal vascular fraction (n=6), and characterized via flow cytometry. Osteogenic, adipogenic, chondrogenic and endothelial differentiation was assessed in lineage-specific induction media after passage three. Identification of von Willebrand factor (vWF) and FVIII was conducted through immunocytochemistry. Lentiviral vector transduction of the ASCs with B-domain deleted FVIII tagged with green fluorescent protein (GFP) was performed. 72 hours after transduction, transgene FVIII expression was examined via immunofluorescence imaging and flow cytometry.
RESULTS: Isolated ASCs showed spindle shaped morphology after 5-7 days in culture.
Flow cytometry showed that majority of ASCs were from mesenchymal origin. Adipogenic differentiation was characterized by microscopic observation of intracellular lipid droplets by Oil Red O staining. Osteogenic differentiation potential was confirmed by Alizarin red staining. Intense Alcian blue staining confirmed presence of proteoglycans in chondrogenic differentiation. Immunocytochemistry is positive for both vWF and Factor VIII. About 70% of the cells were GFP positive.
CONCLUSION: ASCs exhibit similar differentiation and multi-functional abilities as bone marrow MSCs. After endothelial differentiation and transduction with FVIII, ASCs express FVIII and vWF which stabilizes FVIII in circulation. ASCs may provide a novel approach and a valuable cell source for establishing cell-based gene therapy for hemophilia A.
1.
Zhu, Y., et al., Adipose-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct, 2008. 26(6): p. 664-75.
2.
Porada, C.D. and G. Almeida-Porada, Treatment of Hemophilia A in Utero and Postnatally using Sheep as a Model for Cell and Gene Delivery. J Genet Syndr Gene Ther, 2012. S1.


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