This document summarizes research characterizing the differentiation of human embryonic stem cells into hepatocyte-like cells using gene expression analysis. Key findings include: 1) Gene expression signatures of differentiated cells showed downregulation of pluripotency markers and upregulation of hepatocyte-specific genes over time, indicating loss of stemness and gain of liver cell function. 2) The differentiated cells expressed proteins and exhibited metabolic functions characteristic of mature hepatocytes such as glycogen storage, albumin production, and drug metabolism enzyme activity. 3) Mitochondrial DNA content increased in differentiated cells compared to stem cells, reflecting increased energy demands of hepatocytes. 4) Gene expression profiling provided a fast method to

1. 1336
Characterization of Stem Cell Differentiation Using Gene Expression Signatures
Jason C. Poole1, Aleksandra J. Poole2, Craig T. Fredrickson2, Tony L. Gaige1, Gabriel Nistor2, Robert Kovelman1, Adrian Vilalta1
1 EMD Millipore Bioscience, 2 California Stem Cell Inc.
Jason.Poole@merckgroup.com
Abstract hESC Derived Hepatocytes Express Loss of Pluripotency Biomarkers in Metabolic Activity of Mature hESC
Stem cell-based therapies look promising for the treatment Functional Markers of Mature Differentiating Hepatocytes Hepatocytes
of currently intractable medical conditions. These therapies rely on
Hepatocytes We selected 13 human stem cell pluripotency markers for use in Expression of CYPs in hESC-derived hepatocytes was examined by
transplantation of differentiated stem cells that are functionally
qPCR gene expression assays. Markers were tested during the immunocytochemistry (ICC) for CYP2A6, CYP3A4 and CYP2C9. All
analogous to the differentiated tissues they replace. A major Cells derived using this method possess the morphological,
course of a 20 day hepatocyte differentiation protocol. RNA was show strong expression in the differentiated cells.
bottleneck in efforts to efficiently differentiate stem cells is derived biochemical and functional characteristics of mature
isolated and tested every 4th day. Nearly all the markers are down
from the inability to quickly and accurately monitor the hepatocytes, such as:
regulated indicating the cells are losing their pluripotency as
CYP3A4
effectiveness of a protocol and thereby determine if a functional
CYP2A6
Glycogen storage Indocyanine green dye expected during differentiation.
cell type has been obtained. Ultimately the final product may
require functional testing to satisfy this requirement, but this is (PAS assay) uptake Human Pluripotency Marker Gene Expression in
Differentiating Hepatocytes (Time Course)
impractical when differentiation protocols are under development
and evolving quickly. Therefore, a fast and accurate method to
monitor the efficacy of differentiation protocols is needed using
1.0
biomarkers that are specific and predictive for the differentiated
cell type.
0.0
Relative Quantity Log2
CYP2C9
D0 (hESC)
-1.0 D4
D8
Approach -2.0
D12
D16
We use a series of qPCR panels specifically designed to • Albumin production D20
-3.0
identify molecular profiles that accurately monitor functional • Expression of mature hepatocytes-specific markers,
hepatocyte differentiation (NovaQUANT gene expression panels). Albumin, A1AT, and CYP1A2 below. -4.0
We demonstrate that the use of a small set of lineage-specific Induction of CYP mRNAs During
markers can effectively track the differentiation of human
CK8/CYP1A2
embryonic stem cells into mature hepatocytes. We show that
-5.0
Gene Target Carbamazepine (CBZ) Treatment in Stem Cell
Albumin
these cells express the appropriate immunocytochemical markers Derived Hepatocytes
and possess functional activities of mature hepatocytes.
Hepatocyte Specific Biomarkers are CBZ induces the expression and activity of the hepatic microsomal enzyme
system including CYP3A4, which in turn metabolizes CBZ. Stem cells
. Upregulated in a Time Dependent Manner undergoing a procedure of hepatocyte differentiation were tested for mRNA
Performed as above but using hepatocyte specific genes. induction of a cross section of CYP subunits (left). A large induction of all the
Critical markers of hepatocyte function were upregulated at the level CYPs is seen in this trial. At right, A 48 hour treatment with CBZ induces the
of transcription for part or all of the differentiation period. Several
CK18/A1AT
metabolism of the CYP3A4 substrate erythromycin in hepatocyte
Elevated mtDNA Content in Differentiated genes showed onset of regulation by day four and remained differentiated cells but not hESCs.
upregulated throughout the protocol including CYP2E1, DPP4, and
Hepatocytes G6PC. Other genes showed a specific onset of regulation CBZ induced HepDiff HepG2 hESC 0.35
We tested stem cell derived hepatocytes for their indicative of a more mature marker of hepatocyte function. Genes
mitochondrial DNA content compared to pluripotent stem cells using 15
Relative Enzyme Activity
such as AFP and Albumin expressed relatively early whereas 0.30
Relative Quentity Log2
our NovaQUANT Mitochondrial to Nuclear DNA ratio kit. If the 13
others such as A1AT and APOH did not express until day 16. 11
differentiation protocol was effective we reasoned the mtDNA copy 0.25
Human Hepatocyte Markers Expressed in Differentiating 9
number would increase in maturing hepatocytes due to the increased BASAL
Hepatocytes (Time Course) 7
energy needs of this cell type. In fact, these cells showed a greater
than 50% increase in mitochondrial copy number as compared to
Oxidative Stress Targets are Down 5 0.20 CBZ
3
their undifferentiated counterparts indicating these cells are Regulated During hepatocyte 1 0.15
expanding the metabolic machinery required in mature hepatocytes. 6
Differentiation -1
-3
We tested differentiated hepatocytes for their Oxidative 4 0.10
Relative Quantity Log2
D0 (hESC) CYP2C9 CYP3A4 CYP2C19 hESC HepDiff
mtDNA Content in Hepatocyte Differentiated Stem stress signature using the NovaQUANT Oxidative Stress gene D4 CYP2C8 CYP1A2 CYP2D6
2
Cells expression panel. Surprisingly many of the genes were D8
1.75 downregulated compared to hESC, perhaps due to resources D12
0
1.65
being redirected to differentiation efforts. D16
D20
Summary
-2 By validating our qPCR panels with functional testing, we
Relative mtDNA copy number
1.55 D20 Hepatocytes
2.00 show that gene expression signatures can be a useful
1.45
Relative Expression Levels of hESC Derived Hepatocyte -4
Compared to hESC method for monitoring differentiation protocols with high
1.00 Gene Target
1.35 -6 functional relevance. The mRNA expression signals show
1.25 0.00
good correlation with traditional hepatocyte markers such as
While the expression of hESC early and definitive endoderm glycogen storage, albumin production and indocyanine
Relative Quantity Log2
1.15
-1.00 markers decreased, the expression of late hepatic cell markers green uptake. In addition, key genes in drug metabolism are
1.05 increased. Likewise, Albumin secretion from these cells induced at the level of transcription including CYP3A4,
-2.00 increases with time CYP2D6 and CYP2C9. mRNA Levels of mature hepatocytes
0.95 25
-3.00 Albumin Secretion markers such as Albumin and Alpha-1 antitrypsin are found
0.85 hNQO1 hHMOX1 hGPX1(b) hGCLM 20
to closely follow that of their respective proteins. The data
% Increase
hGSTP1 hTP53 hATF4 hSOD1
0.75 -4.00 hSOD2 hCAT hHIF1a hTALDO1 15 between mRNA and protein expression corroborate well with
Mito1 Mito2 Mito1 Mito2 Mito1 Mito2
10
one another during differentiation and qPCR is an effective
HepDiff HepDiff hESC1 hESC1 hESC2 hESC2 -5.00
Gene Target / Sample method to quickly screen cells during the optimization of
5 differentiation protocols.
-6.00
0
-7.00 Hepato Media HepDiff D13 HepDiff D20 HepDiff D27
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