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Comparative Study
. 2017 Dec;40(6):1759-1771.
doi: 10.3892/ijmm.2017.3190. Epub 2017 Oct 16.

Hepatic Differentiation of Human iPSCs in Different 3D Models: A Comparative Study

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Free PMC article
Comparative Study

Hepatic Differentiation of Human iPSCs in Different 3D Models: A Comparative Study

Florian Meier et al. Int J Mol Med. .
Free PMC article

Abstract

Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures.

Figures

Figure 1
Figure 1
Cultivation systems used for hepatic differentiation of hiPSCs. (A) In the 2D monolayer culture, hiPSCs were differentiated on Matrigel coated 24-well plates. (B) In the 3D scaffold-free microspheroid culture cells were cultivated in 96-well plates. Within one well of the plate, one microspheroid was generated by self-aggregation of differentiating cells on top of a low attachment surface coated with agarose. (C) In the 3D hollow fiber bioreactor, hiPSCs were differentiated in the extra-capillary space of the bioreactor. The three capillary systems supply medium (blue and red) and oxygen (grey). The capillaries were coated with Matrigel on the extra-capillary-side to allow cell attachment. hiPSCs, human induced pluripotent stem cells.
Figure 2
Figure 2
Microspheroids derived from three different human induced pluripotent stem cell lines at day 18.
Figure 3
Figure 3
Comparative analyses of three cultivation systems for hepatic differentiation of hiPSCs. The cells were differentiated into HLCs by the use of a 2D monolayer culture, a 3D scaffold-free microspheroid culture and a 3D hollow fibre bioreactor. For all systems identical differentiation media were used as indicated. Analyses of glucose consumption/lactate production (Gluc/Lac), mRNA expression (RNA), secretion of proteins (sec. protein), urea production (Urea), intracellular protein expression (protein) and metabolism of substrates by cytochrome P450 isoenzymes (CYP-Metab.) were performed at the indicated days. hiPSCs, human induced pluripotent stem cells; HLCs, hepatocyte-like cells.
Figure 4
Figure 4
Energy metabolism of hiPSCs during hepatic differentiation in 2D cultures (2D, circles), MS (squares) or BR (triangles). (A) Glucose consumption and (B) lactate production are shown. Areas under curve were calculated for each dataset and differences between groups were determined with the unpaired, two-tailed t-test. P-values <0.1 are given in the graphs (2D cultures: n=6; microspheroids and bioreactors: n=3, median of biological replicates ± interquartile range). MS, microspheroids; BR, bioreactors; hiPSCs, human induced pluripotent stem cells.
Figure 5
Figure 5
Gene expression of hiPSC derived hepatocyte-like cells in 2D cultures, microspheroids, bioreactors or in PHH. The mRNA expression of (A) NANOG, (B) SOX17, (C) CXCR4, (D) AFP, (E) SOX9, (F) ALB, (G) HNF4A, (H) CYP3A4, (I) CYP3A7, (J) CYP1A2 and (K) AHR is shown. Samples for expression analysis were taken before (hiPSC day 0) and after hepatic differentiation of hiPSC in 2D cultures (2D day 18), microspheroids (MS day 18) or bioreactors (BR day 18). In addition, samples were taken after definitive endodermal differentiation in 2D cultures (2D day 5). Further, mRNA samples from freshly isolated (0 h) or 2D cultured PHH (24 h) were used for expression analyses. Differences in gene expression between groups were calculated using the Mann-Whitney test. Data from day 0 and 5 were not included in comparative statistics because the aim was to compare the different culture systems among each other and to the current 'gold-standard', the cultured PHH. p<0.1 are given in the graphs [median and data points of 3 resp. 6 (for 2D cultures) independent experiments plus technical replicates (three per experiment) are shown]. hiPSC, human induced pluripotent stem cells; PHH, primary human hepatocytes; MS, microspheroids; BR, bioreactors; NANOG, Nanog homeobox; SOX17, SRY-box 17; CXCR4, C-X-C motif chemokine receptor 4; AFP, α-fetoprotein; SOX9, SRY-box 9; ALB, albumin; HNF4A, hepatocyte nuclear factor 4α; CYP3A4, cytochrome P450 family 3 subfamily A member 4; CYP3A7, cytochrome P450 family 3 subfamily A member 7; CYP1A2, cytochrome P450 family 1 subfamily A member 2; AHR, aryl hydrocarbon receptor; d, day.
Figure 5
Figure 5
Gene expression of hiPSC derived hepatocyte-like cells in 2D cultures, microspheroids, bioreactors or in PHH. The mRNA expression of (A) NANOG, (B) SOX17, (C) CXCR4, (D) AFP, (E) SOX9, (F) ALB, (G) HNF4A, (H) CYP3A4, (I) CYP3A7, (J) CYP1A2 and (K) AHR is shown. Samples for expression analysis were taken before (hiPSC day 0) and after hepatic differentiation of hiPSC in 2D cultures (2D day 18), microspheroids (MS day 18) or bioreactors (BR day 18). In addition, samples were taken after definitive endodermal differentiation in 2D cultures (2D day 5). Further, mRNA samples from freshly isolated (0 h) or 2D cultured PHH (24 h) were used for expression analyses. Differences in gene expression between groups were calculated using the Mann-Whitney test. Data from day 0 and 5 were not included in comparative statistics because the aim was to compare the different culture systems among each other and to the current 'gold-standard', the cultured PHH. p<0.1 are given in the graphs [median and data points of 3 resp. 6 (for 2D cultures) independent experiments plus technical replicates (three per experiment) are shown]. hiPSC, human induced pluripotent stem cells; PHH, primary human hepatocytes; MS, microspheroids; BR, bioreactors; NANOG, Nanog homeobox; SOX17, SRY-box 17; CXCR4, C-X-C motif chemokine receptor 4; AFP, α-fetoprotein; SOX9, SRY-box 9; ALB, albumin; HNF4A, hepatocyte nuclear factor 4α; CYP3A4, cytochrome P450 family 3 subfamily A member 4; CYP3A7, cytochrome P450 family 3 subfamily A member 7; CYP1A2, cytochrome P450 family 1 subfamily A member 2; AHR, aryl hydrocarbon receptor; d, day.
Figure 6
Figure 6
Secretion of specific proteins and metabolites by hiPSCs during hepatic differentiation in 2D cultures (2D, circles), MS (squares) or bioreactors (BR, triangles). (A) Secretion of AFP, (B) albumin (ALB), (C) A1AT and (D) urea is shown. Values are normalized to 106 initial cells on day 0 (2D cultures and bioreactors) or day 11 (microspheroids). Areas under curve were calculated for each dataset and differences between groups were determined with the unpaired, two-tailed t-test (2D cultures: n=6; microspheroids and bioreactors: n=3, median of biological replicates ± interquartile range). MS, microspheroids; BR, bioreactors; AFT, α-fetoprotein; A1AT, α-1-antitrypsin; hiPSCs, human induced pluripotent stem cells.
Figure 7
Figure 7
Expression of liver-specific immunohistochemical markers in human induced pluripotent stem cell-derived hepatocyte-like cells in 2D cultures (2D day 18), MS (day 18) or BR (day 18). (A–C) Bright-field images of the three culture systems at the day of analysis. (D–N) Expression of (D–F and J) HNF4A, (D–F and K) cytokeratin 18, (G–I, L and N) AFP and (G–I, M and N) ALB was determined by quantitative analysis of immunofluorescence pictures. Differences between groups were determined with the unpaired, two-tailed t-test (2D cultures: n=6; MS and BR: n=3, median of biological replicates ± inter-quartile range). p<0.1 are given in the graphs. Scale bars correspond to 100 µm. d, day; MS, microspheroids; BR, bioreactors; HNF4A, hepatocyte nuclear factor 4α; CK18, cytokeratin 18; AFP, α-fetoprotein; ALB, albumin.
Figure 8
Figure 8
Activities of different cytochrome P450 (CYP) isoenzymes in hiPSC-derived hepatocyte-like cells in 2D cultures on day 18 (2D day 18), MS on day 18 (MS day 18), BR on day 18 (BR day 18), in undifferentiated hiPSCs on day 0 (hiPSC day 0) or in PHH 24 h after seeding. CYP activities were determined by measuring the formation of (A) acetaminophen from phenacetin via CYP1A2 and (B) the formation of 1-OH-midazolam from midazolam via CYP3A4/5. Differences in metabolic activity between undifferentiated hiPSCs, 2D cultures, microspheroids, bioreactors and PHH were calculated using the unpaired, two-tailed t-test (hiPSC day 0 cultures and 2D day 18 cultures: n=6; microspheroids day 18, bioreactors day 18 and adult PHH: n=3; median of biological replicates ± interquartile range). p<0.1 are given in the graphs. Values are normalized to the DNA content on day 18. n.d., not detected; d, day; hiPSCs, human induced pluripotent stem cells; PHH, primary human hepatocytes; MS, microspheroids; BR, bioreactors.

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References

    1. Lauschke VM, Ingelman-Sundberg M. The importance of patient-specific factors for hepatic drug response and toxicity. Int J Mol Sci. 2016;17:1714. doi: 10.3390/ijms17101714. - DOI - PMC - PubMed
    1. Mueller SO, Guillouzo A, Hewitt PG, Richert L. Drug biokinetic and toxicity assessments in rat and human primary hepatocytes and HepaRG cells within the EU-funded Predict-IV project. Toxicol In Vitro. 2015;30:19–26. doi: 10.1016/j.tiv.2015.04.014. - DOI - PubMed
    1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. doi: 10.1016/j.cell.2006.07.024. - DOI - PubMed
    1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872. doi: 10.1016/j.cell.2007.11.019. - DOI - PubMed
    1. Rashid ST, Corbineau S, Hannan N, Marciniak SJ, Miranda E, Alexander G, Huang-Doran I, Griffin J, Ahrlund-Richter L, Skepper J, et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest. 2010;120:3127–3136. doi: 10.1172/JCI43122. - DOI - PMC - PubMed

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