Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture
Abstract
:1. Introduction
2. Results
2.1. Effect of Culture Media Variations on Mono-Cultures of Human Umbilical Vein Endothelial Cells (HUVEC)
2.2. Hepatic Differentiation of hiPSC-Derived Definitive Endoderm (DE) Cells with or without HUVEC Co-Cultivation Using Different Co-Culture Media
2.2.1. Morphological Characteristics of hiPSC-Derived Hepatocyte-like Cells (HLC) and Co-Cultured HUVEC
2.2.2. Gene Expression of Stage-Specific and Endothelial Cell Markers
2.2.3. Immunocytochemical Analysis of Stage-Specific and Endothelial Cell Markers
2.2.4. Secretion of α-Fetoprotein (AFP), Albumin and Urea
2.2.5. Functional Analysis of Different Cytochrome P450 (CYP) Isoenzymes
3. Discussion
4. Materials and Methods
4.1. Culture of HUVEC
4.2. Culture and Hepatic Differentiation of hiPSC
4.3. Culture Medium Testing in Mono-Cultures of HUVEC
4.4. Co-Culture of hiPSC-Derived DE cells with HUVEC
4.5. Analyses of Biochemical Parameters
4.6. Gene Expression Analysis
4.7. Immunocytochemical Staining
4.8. Measurement of Cytochrome P450 (CYP) Isoenzyme Activities
4.9. Statistical Evaluation
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
AFP | α-fetoprotein |
ALB | Albumin |
bFGF | Basic fibroblast growth factor |
BMP | Bone morphogenetic proteins |
CYP | Cytochrome P450 |
DE | Definitive endoderm |
DMEM | Dulbecco’s modified eagle’s medium |
DMSO | Dimethyl sulfoxide |
ECGF | Endothelial cell growth factor |
EGF | Epidermal growth factor |
EGM | Endothelial cell growth medium |
FBS | Fetal bovine serum |
FGF | Fibroblast growth factor |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
HCM | Hepatocyte culture medium |
hESC | Human embryonic stem cells |
HGF | hepatocyte growth factor |
hiPSC | Human induced pluripotent stem cells |
HLC | Hepatocyte-like cells |
HNF4A | Hepatocyte nuclear factor 4 α |
HUVEC | Human umbilical vein endothelial cells |
KRT18 | Cytokeratin 18 |
L15 | Leibovitz’s |
LDH | Lactate dehydrogenase |
OSM | Oncostatin M |
PECAM1 | Platelet and endothelial cell adhesion molecule 1 |
POU5F1 | POU domain, class 5, transcription factor 1 |
RPMI | Roswell Park Memorial Institute |
VWF | Von Willebrand factor |
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Cell Type Used for Co-Culture with hiPSC/hESC | Ratio (hiPSC/hESC-Derived Cells:Co-Cultured Cell Type(s)) | Co-Culture Method | Culture Medium Applied | Ref. |
---|---|---|---|---|
Murine hepatic stromal cell line MLSgt20 | 1:1 | Self-aggregation in microwells | DMEM + FBS + differentiation factors | [58] |
Murine embryonic fibroblasts (3T3-J2) | 0.04:1 | Seeding of DE cells onto mitomycin-treated 3T3-J2 feeder cells | Hepatocyte culture medium + FBS | [60] |
Murine embryonic fibroblasts (swiss 3T3) | Not specified | Hepatoblast monolayer covered with 3T3 cell sheet | L15 medium + differentiation factors | [61] |
HUVEC and mesenchymal stem cells | 10:7:2 | Spontaneous formation of 3D liver buds | HCM (without EGF) + EGM, 1:1 | [25,67] |
hiPSC-derived endothelial cells | 2 : 1 | Multicomponent hydrogel fibers containing galactose for HLC and collagen for endothelial cells | Not specified | [55] |
Hepatic stellate cell line TWNT-1 | Not specified | Cell inserts | DMEM-F12 + knockout serum replacer + DMSO | [56] |
Murine embryonic fibroblasts (3T3-J2) | 2:1 or 2.5:1 for cryopreserved HLC | Micropatterned co-culture containing collagen coating and matrigel overlay | RPMI + B27 supplement + differentiation factors | [57] |
Murine embryonic fibroblasts (3T3-J2) | 2:1 | Self-aggregation in microwells | RPMI + B27 supplement + differentiation factors | [59] |
Primary rat hepatocytes | 1:2 | Microfluidic co-culture | DMEM + FBS + maintenance factors and IMDM + FBS + DMSO + differentiation factors | [66] |
HUVEC and adipose derived stem cells | 1:1:0.02 | 3D bioprinting of in vivo like liver lobule structures | HCM (without EGF) + EGM-2, 1:1 | [52] |
HUVEC | 2:1 | HUVEC grow in free spaces of hiPSC-derived DE cell monolayers | HCM + EGM complete, 1:1 and HCM + EGM Supplements | Present study |
Component | EGM complete | HCM-I | HCM-II | HCM-I/II + EGM Complete | HCM-I/II + EGM Supplements |
---|---|---|---|---|---|
Hepatocyte culture medium (HCM) Bullet Kit | - | 100% (v/v) | 50% (v/v) | 97.5% (v/v) | |
Endothelial cell growth medium (EGM) | 97.5% (v/v) | - | 48.75% (v/v) | - | |
EGM Supplements | 2.5% (v/v) | - | 1.25% (v/v) | 2.5% (v/v) | |
Human hepatocyte growth factor, recombinant | - | 10 ng/mL | 10 ng/mL | 10 ng/mL | 10 ng/mL |
Human oncostatin M, recombinant | - | - | 10 ng/mL | 10 ng/mL 1 | 10 ng/mL 1 |
Gentamycin | 0.05 mg/mL | 0.05 mg/mL | 0.05 mg/mL | 0.05 mg/mL | 0.05 mg/mL |
Gene Symbol | Gene Name | Assay ID |
---|---|---|
AFP | α fetoprotein | HS00173490_m1 |
ALB | albumin | HS00910225_m1 |
GAPDH | glyceraldehyde-3-phosphate dehydrogenase | HS03929097_g1 |
HNF4A | Hepatocyte nuclear factor 4, α | Hs00230853_m1 |
KRT18 | Keratin 18 | Hs02827483_g1 |
PECAM1 | platelet and endothelial cell adhesion molecule 1 | Hs00169777_m1 |
POU5F1 | POU domain, class 5, transcription factor 1 | HS00999632_g1 |
VWF | von Willebrand factor | Hs00169795_m1 |
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---|---|---|---|---|---|
Primary Antibody | |||||
Cytokeratin 18 | CK18 | mouse | Santa Cruz | Sc-6259 | 2 |
Hepatocyte nuclear factor 4 α | HNF4A | rabbit | Santa Cruz | Sc-8987 | 4 |
Platelet endothelial cell adhesion molecule 1 | PECAM1 | mouse | Abcam | ab24590 | 5 |
POU domain, class 5, transcription factor 1 | OCT3 | rabbit | Santa Cruz | Sc-9081 | 2 |
Von Willebrand factor | VWF | rabbit | Abcam | ab6994 | 35.5 |
Secondary antibody | |||||
Alexa Fluor® 488 anti-mouse | goat | Life Technologies | A-11029 | 2 | |
Alexa Fluor® 594 anti-rabbit | goat | Life Technologies | A-11037 | 2 |
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Freyer, N.; Greuel, S.; Knöspel, F.; Strahl, N.; Amini, L.; Jacobs, F.; Monshouwer, M.; Zeilinger, K. Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture. Int. J. Mol. Sci. 2017, 18, 1724. https://doi.org/10.3390/ijms18081724
Freyer N, Greuel S, Knöspel F, Strahl N, Amini L, Jacobs F, Monshouwer M, Zeilinger K. Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture. International Journal of Molecular Sciences. 2017; 18(8):1724. https://doi.org/10.3390/ijms18081724
Chicago/Turabian StyleFreyer, Nora, Selina Greuel, Fanny Knöspel, Nadja Strahl, Leila Amini, Frank Jacobs, Mario Monshouwer, and Katrin Zeilinger. 2017. "Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture" International Journal of Molecular Sciences 18, no. 8: 1724. https://doi.org/10.3390/ijms18081724