Download Def-HEP WT - DefiniGEN

Def-HEP WT Wild Type Human Hepatocytes
DefiniGEN has world-leading expertise in the provision of human cell products to the life science and drug discovery sectors. Our hepatocyte
cells display the functional characteristics of primary human hepatocyte cells including albumin production, A1AT secretion, glycogen
storage and CYP450 expression. The cells also have wild-type donor genetics, karotype and metabolism in contrast to immortalized cell
lines. The Def-HEP cells provide customers with a reproducible source of highly functional liver products to enhance their research for key
applications including vaccine development, predictive toxicology and disease modelling. The cells are generated using a ground-breaking
iPS (induced Pluripotent Stem) cell production platform developed at the University of Cambridge. (1)(2)(3)(4)
Hepatocyte cell morphology:
The Def-HEP cell products are highly functional human hepatocytes.
Advantages
When thawed and plated as a monolayer Def-HEP cells form hepatocytes
with cobblestone morphology and tight cell junctions.
- Standardized cell product containing >98%
human hepatocyte cells producing reproducible
and biologically relevant data
- Normal human genetics wild-type donor
genetics and karotype verified
Figure 1. Overview of Def-HEP cell morphology. Def-HEP WT cells exhibit typical
hepatocyte cobblestone morphology and bi-nucleation.
Gene expression profiles:
Def-HEP cells express key hepatocyte markers including albumin, A1AT
and HNF4a at similar levels to Primary Human Hepatocytes. Significantly
the observed AFP (Alfa-fetoprotein levels) are very low in the Def-HEP
cells indicating they have progressed to a mature state comparable to
PHH.
- Human hepatocyte function Def-HEP are
functionally mature hepatocytes which can be
plated and maintain function over a 10 day
window
- Optimized work flow we can deliver industrial
quantities of cryopreserved cell products to
client specification
Hepatocyte key marker analysis
2.500
1.500
1.000
0.500
0.000
Def-HEP WT
PHH
HEP G2
Albumin
+++
+++
++
A1AT
+++
+++
++
Glycogen Storage
+++
+++
++
Urea
++
+++
-
Induction multiple CYP450
++
+++
-
Phase II enzymes
++
+++
-
+++
+++
-
-
-
+++
Functional Characteristics
2.000
Def HEP WT
PHH
AAT
AFP
ALB
HEP G2
HNF4a
Normal WT genetics and karotype
Figure 2. Gene expression analysis demonstrates that Def-HEP express key
hepatocyte markers at similar levels to PHH. AFP levels are extremely low in
Def-HEP indicating the cells have attained a functional mature status.
Crabtree effect
References:
1. Hannan NR, Vallier L et al. Production of hepatocyte-like cells from human pluripotent stem cells.
Nature Protocols. 2013 Feb;8(2):430-7.
2. Yusa K, Rashid ST, Vallier L, et al. Targeted gene correction of a1- antitrypsin deficiency in induced pluripotent stem cells. Nature. 2011; 478; 391-4.
3. Rashid ST, Vallier et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest. 2010;120:3127-36.
4. Takahashi K & Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126; 663-676.
Supported By
Hepatocyte maturation markers:
Def-HEP cells display the functional characteristics of primary human
hepatocytes including albumin secretion, A1AT production, glycogen storage
and LDL uptake (Figure 3). Accordingly they should be handled in a similar
Metabolism - absence of Crabtree effect:
fashion to primary human hepatocytes.
Dosing Def-HEP WT with +/- glucose media does not alter the
hepatotoxic effect of chlorpromazine as assessed via the MTT cell
proliferation assay. Therefore, it is highly unlikely that our cells
exhibit the Crabtree effect, in contrast to immortalized lines.
Figure 3. Functional analysis of Def-HEP WT hepatocytes. (a) Albumin secretion, 10x
magnification (b) Glycogen storage shown by PAS staining (c) LDL cholesterol uptake
shown by fluoresceinated LDL incorporation.
Multiple CYP450 activities:
Def-HEP exhibit multiple drug-metabolizing CYP450 enzyme activities at
levels comparable to PHH. In addition the CYP450 induction levels observed
upon exposure to inducers is similar to PHH (n=3).
Figure 6. Confirmation of absence of the Crabtree effect in Def-HEP WT.
Mitochondrial toxicity:
As assessed by dosing Def-HEP WT with valinomycin (0.3 - 600nM)
and papverine (0.03 - 60 μM) for 48 hr both compounds caused
significant mitochondrial toxicity within concentration ranges
recognised in literature. Cell viability was assessed via the CellTiter
96® Aqueous Non-Radioactive Cell. Both papaverine and
valinomycin caused significant mitochondrial toxicity.
Figure 4. Multiple CYP activites of cryopreserved Def-HEP hepatocyte cells. The results
show Def-HEP cells have comparable CYP activity to PHH and induced activity profiles
that are similar to PHH (CYP1A2 EROD assay inducer Omeprazole), (CYP450 PGlo
assay inducer rifampicin).
Figure 7. Typical Def-HEP WT mitochondrial toxicology drug response to
valinomycin and papverine.
Ordering Information
Product Specification
Hepatitis marker analysis:
There is currently no effective vaccine for hepatitis C and the future
Catalog Number
Def-HEP WT
development of vaccinations and antiviral therapies for this disease relies
Format
Cryopreserved 10 million cells per vial
upon the development of a suitable cell model. HCV entry requires sequential
Viability
> 70%
Applications
Research and Predictive Toxicology
factors including CD81, SR-B1, claudin-1 and occludin. Def-HEP have been
demonstrated to express these key markers at levels similar to PHH.
Usage Statement:
The products are provided for research use only and are not to be
utilized for unauthorised commercial purposes including ex vivo or in
vivo therapeutic uses in humans.
Figure 5. Gene expression analysis demonstrating the presence of key Hepatitis C
markers in Def-HEP including SR-B1, CD81, Claudin-1 and Occludin.
Supported By
Contact Information:
DefiniGEN Ltd, Babraham Research Campus,
Moneta B280, Cambridge, CB22 3AT
Email: enquiries@definigen.com,
Telephone: +44 (0) 1223 497 113
Website: www.definigen.com