Differentiation of embryonic stem cells into hepatocytes that coexpress coagulation factors VIII and IX.
OBJECTIVE
To establish an efficient culture system to support embryonic stem (ES) cell differentiation into hepatocytes that coexpress F-VIII and F-IX.


METHODS
Mouse E14 ES cells were cultured in differentiation medium containing sodium butyrate (SB), basic fibroblast growth factor (bFGF), and/or bone morphogenetic protein 4 (BMP4) to induce the differentiation of endoderm cells and hepatic progenitor cells. Hepatocyte growth factor, oncostatin M, and dexamethasone were then used to induce the maturation of ES cell-derived hepatocytes. The mRNA expression levels of endoderm-specific genes and hepatocyte-specific genes, including the levels of F-VIII and F-IX, were detected by RT-PCR and real-time PCR during various stages of differentiation. Protein expression was examined by immunofluorescence and Western blot. At the final stage of differentiation, flow cytometry was performed to determine the percentage of cells coexpressing F-VIII and F-IX, and ELISA was used to detect the levels of F-VIII and F-IX protein secreted into the culture medium.


RESULTS
The expression of endoderm-specific and hepatocyte-specific markers was upregulated to highest level in response to the combination of SB, bFGF, and BMP4. Treatment with the three inducers during hepatic progenitor differentiation significantly enhanced the mRNA and protein levels of F-VIII and F-IX in ES cell-derived hepatocytes. More importantly, F-VIII and F-IX were coexpressed with high efficiency at the final stage of differentiation, and they were also secreted into the culture medium.


CONCLUSIONS
We have established a novel in vitro differentiation protocol for ES-derived hepatocytes that coexpress F-VIII and F-IX that may provide a foundation for stem cell replacement therapy for hemophilia.
Cao
Jun
J
Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
Shang
Chang-zhen
CZ
Lü
Li-hong
LH
Qiu
De-chuan
DC
Ren
Meng
M
Chen
Ya-jin
YJ
Min
Jun
J
D000818:Animals; D015153:Blotting, Western; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002087:Butyrates / Q000494:pharmacology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D005164:Factor IX / Q000096:biosynthesis*; D005169:Factor VIII / Q000096:biosynthesis*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005434:Flow Cytometry; D006467:Hemophilia A / Q000601:surgery; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism* / Q000637:transplantation / Q000648:ultrastructure; D007150:Immunohistochemistry; D051379:Mice; D008854:Microscopy, Electron; D020133:Reverse Transcriptase Polymerase Chain Reaction
D055415:Bone Morphogenetic Protein 4; D002087:Butyrates; D003470:Culture Media; D016222:Fibroblast Growth Factor 2; D005169:Factor VIII; D005164:Factor IX