The document describes research differentiating induced pluripotent stem cells (iPSCs) from patients with familial hypercholesterolemia (FH) into hepatocyte-like cells (HLCs). The researchers differentiated FH patient iPSCs into HLCs over five stages. Polymerase chain reaction analysis of gene expression showed that the HLCs expressed markers appropriate for each stage of development. This indicates the iPSCs successfully differentiated into HLCs, which could potentially be used to develop a cell-based treatment for FH. However, further work is needed to restore normal LDL receptor function before the HLCs could provide a therapeutic benefit.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
MilliporeSigma's Jennifer Pratt recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating the utility of HepaRG MRP2 Knockout cells for investigating drug-transporter interactions in the liver involving MRP2.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
MilliporeSigma's Jennifer Pratt recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating the utility of HepaRG MRP2 Knockout cells for investigating drug-transporter interactions in the liver involving MRP2.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
Seminar led by Prof. Thomas Kaufmann. Institute of Pharmacology, University of Bern, Switzerland, at VHIR (15 November 2012).
Content: We are interested to investigate the molecular mechanisms by which pro- and anti-apoptotic members of the BCL-2 family regulate the intrinsic (mitochondrial) apoptotic pathway. The pathway is initiated by members of the BH3-only protein subgroup, which act as sensors in response to a variety of intracellular stress stimuli. Some BH3-only proteins, including Bid and Bim, can be activated downstream of death receptors (e.g. Fas/CD95, TNF-R1) and thus mediate a crosstalk from the extrinsic to the mitochondrial apoptotic pathway. We investigate these processes in mouse liver (and more recently also in mouse granulocytes), as hepatocytes strongly rely on this crosstalk for death receptor-induced apoptosis to be effective. We are further interested in the role of 'X-linked inhibitor of apoptosis protein' (XIAP) in these same cell death pathways.
ABC (ATP‑binding cassette) proteins are one of the largest and most diverse protein superfamilies, whose members can be found in all eukaryotic and prokaryotic organisms studied to date. The most of ABC proteins are transporters that translocate allocrites across biological membranes. Almost half of the 48 human ABC transporter proteins are thought to facilitate the ATP-dependent translocation of lipids or lipid-related compounds. Such substrates include cholesterol, plant sterols, phospholipids, bile acids and sphingolipids. Mutations in a substantial number of the 48 human ABC transporters have been related to human disease.
Intracellular Traffic and Sorting of ProteinsASHIKH SEETHY
Describes intra-cellular trafficking of proteins, protein sorting, clinical aspects of protein targeting, and vesicle transport.
Download and view in slide show mode for better viewing.
ATP-binding cassette or ABC transporters form a large and ubiquitous superfamily of transporters that participate in a wide range of physiological processes. The transport process is energized by ATP.
this is a presentation on gene expression vector that includes what is expression vector, how many types of expression vector and difference between cloning and expression vector
The Beta Amyloid Cleaving Enzymes: From Drug Discovery to Evolution and BackChris Southan
Slides presented at the University of Edinburgh in Jan 2014, based on our paper "A tale of two drug targets: the evolutionary history of BACE1 and BACE2" http://www.ncbi.nlm.nih.gov/pubmed/24381583
The beta amyloid (APP) cleaving enzyme (BACE1) has been a drug target for Alzheimer's Disease (AD) since 1999 with lead inhibitors now entering clinical trials. In 2011, the paralog, BACE2, became a new target for type II diabetes (T2DM) having been identified as a TMEM27 secretase regulating pancreatic β cell function. However, the normal roles of both enzymes are unclear. This study outlines their evolutionary history and new opportunities for functional genomics. We identified 30 homologs (UrBACEs) in basal phyla including Placozoans, Cnidarians, Choanoflagellates, Porifera, Echinoderms, Annelids, Mollusks and Ascidians (but not Ecdysozoans). UrBACEs are predominantly single copy, show 35–45% protein sequence identity with mammalian BACE1, are ~100 residues longer than cathepsin paralogs with an aspartyl protease domain flanked by a signal peptide and a C-terminal transmembrane domain. While multiple paralogs in Trichoplax and Monosiga pre-date the nervous system, duplication of the UrBACE in fish gave rise to BACE1 and BACE2 in the vertebrate lineage. The latter evolved more rapidly as the former maintained the emergent neuronal role. In mammals, Ka/Ks for BACE2 is higher than BACE1 but low ratios for both suggest purifying selection. The 5' exons show higher Ka/Ks than the catalytic section. Model organism genomes show the absence of certain BACE human substrates when the UrBACE is present. Experiments could thus reveal undiscovered substrates and roles. The human protease double-target status means that evolutionary trajectories and functional shifts associated with different substrates will have implications for the development of clinical candidates for both AD and T2DM. A rational basis for inhibition specificity ratios and assessing target-related side effects will be facilitated by a more complete picture of BACE1 and BACE2 functions informed by their evolutionary context.
Seminar led by Prof. Thomas Kaufmann. Institute of Pharmacology, University of Bern, Switzerland, at VHIR (15 November 2012).
Content: We are interested to investigate the molecular mechanisms by which pro- and anti-apoptotic members of the BCL-2 family regulate the intrinsic (mitochondrial) apoptotic pathway. The pathway is initiated by members of the BH3-only protein subgroup, which act as sensors in response to a variety of intracellular stress stimuli. Some BH3-only proteins, including Bid and Bim, can be activated downstream of death receptors (e.g. Fas/CD95, TNF-R1) and thus mediate a crosstalk from the extrinsic to the mitochondrial apoptotic pathway. We investigate these processes in mouse liver (and more recently also in mouse granulocytes), as hepatocytes strongly rely on this crosstalk for death receptor-induced apoptosis to be effective. We are further interested in the role of 'X-linked inhibitor of apoptosis protein' (XIAP) in these same cell death pathways.
ABC (ATP‑binding cassette) proteins are one of the largest and most diverse protein superfamilies, whose members can be found in all eukaryotic and prokaryotic organisms studied to date. The most of ABC proteins are transporters that translocate allocrites across biological membranes. Almost half of the 48 human ABC transporter proteins are thought to facilitate the ATP-dependent translocation of lipids or lipid-related compounds. Such substrates include cholesterol, plant sterols, phospholipids, bile acids and sphingolipids. Mutations in a substantial number of the 48 human ABC transporters have been related to human disease.
Intracellular Traffic and Sorting of ProteinsASHIKH SEETHY
Describes intra-cellular trafficking of proteins, protein sorting, clinical aspects of protein targeting, and vesicle transport.
Download and view in slide show mode for better viewing.
ATP-binding cassette or ABC transporters form a large and ubiquitous superfamily of transporters that participate in a wide range of physiological processes. The transport process is energized by ATP.
this is a presentation on gene expression vector that includes what is expression vector, how many types of expression vector and difference between cloning and expression vector
The Beta Amyloid Cleaving Enzymes: From Drug Discovery to Evolution and BackChris Southan
Slides presented at the University of Edinburgh in Jan 2014, based on our paper "A tale of two drug targets: the evolutionary history of BACE1 and BACE2" http://www.ncbi.nlm.nih.gov/pubmed/24381583
The beta amyloid (APP) cleaving enzyme (BACE1) has been a drug target for Alzheimer's Disease (AD) since 1999 with lead inhibitors now entering clinical trials. In 2011, the paralog, BACE2, became a new target for type II diabetes (T2DM) having been identified as a TMEM27 secretase regulating pancreatic β cell function. However, the normal roles of both enzymes are unclear. This study outlines their evolutionary history and new opportunities for functional genomics. We identified 30 homologs (UrBACEs) in basal phyla including Placozoans, Cnidarians, Choanoflagellates, Porifera, Echinoderms, Annelids, Mollusks and Ascidians (but not Ecdysozoans). UrBACEs are predominantly single copy, show 35–45% protein sequence identity with mammalian BACE1, are ~100 residues longer than cathepsin paralogs with an aspartyl protease domain flanked by a signal peptide and a C-terminal transmembrane domain. While multiple paralogs in Trichoplax and Monosiga pre-date the nervous system, duplication of the UrBACE in fish gave rise to BACE1 and BACE2 in the vertebrate lineage. The latter evolved more rapidly as the former maintained the emergent neuronal role. In mammals, Ka/Ks for BACE2 is higher than BACE1 but low ratios for both suggest purifying selection. The 5' exons show higher Ka/Ks than the catalytic section. Model organism genomes show the absence of certain BACE human substrates when the UrBACE is present. Experiments could thus reveal undiscovered substrates and roles. The human protease double-target status means that evolutionary trajectories and functional shifts associated with different substrates will have implications for the development of clinical candidates for both AD and T2DM. A rational basis for inhibition specificity ratios and assessing target-related side effects will be facilitated by a more complete picture of BACE1 and BACE2 functions informed by their evolutionary context.
Derivation of highly enriched cultures of differentiated cells from human par...Nikolay Turovets
California Institute for Regenerative Medicine (CIRM) & Medical Research Council (MRC)
Human SCNT Workshop.
14 June, 2010, San Francisco, CA
Workshop report: http://www.cirm.ca.gov/files/PDFs/Publications/Human_SCNT_Workshop_Report.pdf
Transfusion Medicine support in live related combined liver and kidney transp...Apollo Hospitals
Combined liver and kidney transplantation (CLKT) is the procedure of choice for patients with dual-organ failure. Transfusion Medicine support in live related combined liver and kidney transplantation (CLKT) not only involves the provision of safest possible blood and histocompatibility testing (HLA typing & CDC Crossmatch) but also ensures better patient care due to availability of various advance immunohematological techniques in a time bound frame. A fully equipped functional and sophisticated blood bank and HLA lab is a must in the hospitals where such surgeries are done.
Flavin-Containing Dimethylaniline Monooxygenase 5 Drives Malignancies in Hepa...semualkaira
Hepatic microsomes play an important role in drug metabolism,
but the potential biological functions of hepatic microsome-containing proteins in Hepatocellular Carcinoma (HCC) remain unclear. Here, we used HCC and corresponding adjacent Non-Tumor
(NT) tissues to isolate hepatic microsomes and then performed
RNA high-throughput sequencing
Flavin-Containing Dimethylaniline Monooxygenase 5 Drives Malignancies in Hepa...semualkaira
Hepatic microsomes play an important role in drug metabolism, but the potential biological functions of hepatic microsome-con- taining proteins in Hepatocellular Carcinoma (HCC) remain un- clear. Here, we used HCC and corresponding adjacent Non-Tumor (NT) tissues to isolate hepatic microsomes and then performed RNA high-throughput sequencing. After screening, flavin-con- taining dimethylaniline monooxygenase (FMO5) showed a significantly high expression level and was associated with poor prognosis in patients with HCC.
Flavin-Containing Dimethylaniline Monooxygenase 5 Drives Malignancies in Hepa...
Poster
1. UofL Design and Print
♦ Abstract
Familial Hypercholesterolemia (FH) is a hereditary
disease resulting in defective Low Density Lipoprotein
Receptor (LDL-R) expression. Due to defective LDL-R,
LDL cholesterol is left to accumulate in the bloodstream,
where it can form atherosclerotic plaques and accelerate
cardiovascular disease. Liver transplant is the only
curative option for FH, but healthy donor livers are in
short supply. Deriving induced pluripotent stem cells
(iPSC) from FH patient fibroblasts and differentiating
them into functional hepatocyte-like cells (HLC) could
offer a significant therapeutic benefit.
We differentiated iPSC into HLC over five stages.
Polymerase Chain Reactions (PCR) performed at the end
of each stage verified the appropriate expression of five
genes (POU5F1, SOX17, HNF4a, AFP, and ALB) used
as markers of cell development and differentiation.
Visualization of PCR products via gel electrophoresis
indicated that the HLC expressed the appropriate makers
for each stage of development.
♦ Introduction & Background
♦ References
Familial Hypercholesterolemia (FH) is an autosomal
dominant disease that results in defective expression of
the Low Density Lipoprotein Receptor (LDL-R). As the
typical FH patient exhibits elevated LDL cholesterol (LDL-
c) levels, cardiovascular diseases (CVD) such as
atherosclerosis or coronary artery disease can result. In
every year since 1900 (except 1918), CVD accounted for
more deaths than any other cause of death in the United
States. It is now the leading cause of death in the world.
Hepatocytes, the parenchymal cells of the liver, have
an especially high concentration of functional LDL-R that
bind circulating LDL-c, which is internalized via receptor-
mediated endocytosis. For this reason, liver transplant is
regarded as the only curative treatment for FH. However,
livers are in short supply, and transplant presents many
challenges (such as immunosuppression, quality of life,
high cost, complication rate, and death).
Induced pluripotent stem cells (iPSC) are embryonic-
like cells that have been reprogrammed from terminally
differentiated adult cells. They express the genes and
factors important for maintaining the defining properties of
embryonic stem cells. Because they are pluripotent (able
to differentiate into the three germ layers – ectoderm,
mesoderm, and endoderm), iPSC can be differentiated
into cells that resemble hepatocytes, termed hepatocyte-
like cells (HLC). We differentiated our iPSC using a
published five-stage protocol (Song, Cell Res, 2009).
♦ Methodology ♦ Conclusions
♦ Acknowledgements
I would like to sincerely thank my mentor Dr. Nolan
Boyd for guiding me through my project and allowing me
to use his lab. I would also like to thank Venkat
Ramakrishnan for supervising me and my progress. I
owe much to my parents, Drs. Huey Tien and Ring Tsai,
for transporting me to and from the lab. Finally, I would
like to acknowledge my science teacher and adult
sponsor, Mr. Robert Baar, who organized the science
fair affairs in my science class.
Development and Characterization of FamilialDevelopment and Characterization of Familial
Hypercholesterolemia Hepatocyte-Like CellsHypercholesterolemia Hepatocyte-Like Cells
Kevin T. Tien
duPont Manual High School, Cardiovascular Innovation Institute, University of Louisville School of Medicine
♦ Purpose
Our lab’s long-term aim is to develop a cell-based
apheresis device that utilizes functionally restored FH
patient-derived HLC (FH-LDLR-HLC). As a first step
towards producing a functional device, the lab
successfully differentiated non-restored FH-iPSC (FH-NT-
iPSC) into HLC (FH-NT-HLC). This experiment sought to
characterize the differentiation of the FH-NT-iPSC into
FH-NT-HLC. The FH-NT-HLC would then serve as a
negative control for functionally restored cells.
♦ Hypothesis
FH-NT-HLC will express the appropriate markers at each
stage of development:
- Stage 0 (undifferentiated): POU5F1
- Stage 1 (early definitive endoderm): SOX17
- Stage 2 (late definitive endoderm/hepatic specification):
HNF4a
- Stage 2 – 5 (hepatic maturation): AFP and ALB
Cells were lysed at the end of each stage of development before being RNA-purified and quantified via QiaShredder
and RNeasy Kits (Qiagen). Isolated RNA was converted into cDNA using SuperScript II Reverse Transcriptase (RT,
Invitrogen). Samples without RT were used as negative controls.
To assess gene expression during HLC development, cell DNA was subjected to Polymerase Chain Reaction (PCR)
at the end of each of five stages. The following genes encode the transcription factors and plasma proteins that were
assessed as markers of HLC development: POU5F1, SOX17, HNF4α, Alpha-fetoprotein (AFP), and Albumin (ALB).
Primers (purchased from Integrated DNA Technologies, IDT) were used to amplify these genes (amplicons). Amplicons
were analyzed via standard gel electrophoresis. GAPDH was used as a loading control.
Gene expression was normalized to GAPDH during densitometry analysis. Densitometry, which indicates the
magnitude of gene expression, was calculated for each band using ImageJ software (NIH). The software measured the
integral of the band for each gene at each stage, allowing us to compare relative expression via a semi-quantitative
means.
We performed these experiments in quadruplicate.
Figure 2: Gene Expression through Various Stages of Hepatocyte-like Cells
OCT4 (POU5F1), a transcription factor, is a marker of pluripotence
that is characteristically expressed in untreated FH-iPSC (Stage 0).
SOX17, another transcription factor, marks the definitive endoderm stage
and is stably expressed at the end of Stage 1, after exposure to
STEMdiff™ Definitive Endoderm media, with decreasing expression
through Stage 5. The HNF4a transcription factor is a late endodermal
marker that is expressed between Stages 2 and 5. Alpha fetoprotein (AFP)
and Albumin are cytoplasmic markers of hepatoblasts/immature
hepatocytes and mature hepatocytes, respectively. They are significantly
up-regulated beginning in Stage 2 and remain stably so through Stage 5.
The GAPDH loading control provided a means for normalization during
densitometry analysis.
Table 1: Densitometry Means of /GAPDH
Table 2: Standard Error Measurements of /GAPDH
Cells exhibited the characteristic genes of
differentiation from iPSC to HLC. Our gel images indicate
that the appropriate genes were expressed at
appropriate times; densitometry measurements
correspond to the expected trends as well.
POU5F1 was expressed during stage 0 as a marker
of pluripotence. SOX17 expression was highest in Stage
1 and gradually decreased in magnitude through Stage
5. HNF4a was first seen in Stage 2 and followed an
expression pattern similar to SOX17. AFP is a marker for
immature hepatocytes while Albumin (ALB) is a marker
associated with mature hepatocytes. Both AFP and ALB
exhibited strong expression starting in Stage 2 (hepatic
specification). AFP expression decreased slightly in
subsequent stages, while ALB was most predominant in
Stage 5 (final stage of differentiation).
The presence of AFP and ALB at Stage 5 indicates that
we (a) have cells that are hepatic in nature and (b) are
potentially of mixed maturity.
♦ Current & Future Directions
The derivation of HLC from FH patient fibroblasts is a
significant milestone in developing a potential therapeutic
for this population. However, as FH is a genetic disease,
it is clear that any cells derived from an FH patient would
be equally as dysfunctional. Therefore, for our iPSC and
HLC to be effective, functional restoration of the LDL-R
activity is warranted. Our lab is working to deliver a
corrective plasmid into iPSC and characterize the
functionally restored HLC.
Further, in order to demonstrate therapeutic potential,
FH-LDLR-HLC will be implanted into FH-model mice and
assessed for their ability to clear LDL cholesterol from
the bloodstream. The ultimate goal would be to utilize
such apheresis devices in human FH patients as a
therapeutic, autologous means of metabolizing their
excess LDL cholesterol and lowering their CVD risk.
Betheseda. (2009). What are Induced Pluripotent Stem Cells? Retrieved October 1, 2013, from National Institutes of
Health: stemcells.nih.gov
Bowen, R. (1998, June 23). Hepatic Histology: Hepatocytes. Retrieved September 26, 2013, from
www.vivo.colostate.edu: http://www.vivo.colostate.edu
Cai, J. (2007). Directed Differentiation of Human Embryonic Stem Cells into Functional Hepatic Cells. Hepatology,
1229-1239.
Cohen, J., Hobbs, H. H., & Rader, D. J. (2003). Monogenic Hypercholesterolemia: New Insights in Pathogenesis
and Treatment. The Journal of Clinical Investigation, 1795-1803.
Hypercholesterolemia. (2007, March). Retrieved September 27, 2013, from Genetics Home Reference:
ghr.nlm.nih.gov
Matsumoto, K., Yoshitomi, H., Rossant, J., & Zaret, K. S. (2001). Liver Organogenesis Promoted by Endothelial
Cells Prior to Vascular Function. www.sciencemag.org, 559-563.
Nunes, S. S., Maijub, J. G., Krishnan, L., Ramakrishnan, V. M., Clayton, L. R., Williams, S. K., . . . Boyd, N. L.
(2013). Generation of a Functional Liver Tissue Mimic Using Adipose Stromal Vascular Fraction Cell-Derived
Vasculatures. Scientific Reports, 1-7.
Song, Z., Cai, J., Liu, Y., Zhao, D., Yong, J., Duo, S., . . . Qin, H. (2009). Efficient Genertation of Hepatocyte-like
Cells from Human Induced Pluripotent Stem Cells. Cell Research.
The University of Utah. (2008). PCR Virtual Lab. Retrieved October 1, 2013, from Learn. Genetics:
learn.genetics.utah.edu
Means S0 S1 S2 S3 S4 S5
POU5F1 0.0715 0.0615 0.0000 0.0000 0.0000 0.0000
SOX17 0.0004 1.4155 0.5915 0.0537 0.1819 0.0247
HNF4A 0.0033 0.1449 0.5626 0.2520 0.4262 0.1932
AFP 0.0064 0.0224 1.6999 1.5441 2.6651 1.2878
ALB 0.0000 0.0260 0.4953 1.3186 2.4401 1.3380
S.E.M. S0 S1 S2 S3 S4 S5
POU5F1 0.0251 0.0341 0.0000 0.0000 0.0000 0.0000
SOX17 0.0004 0.5521 0.1048 0.0146 0.0223 0.0189
HNF4A 0.0033 0.0539 0.1600 0.0965 0.1687 0.0434
AFP 0.0064 0.0087 0.1872 0.1564 1.1704 0.1495
ALB 0.0000 0.0114 0.1329 0.0975 1.0132 0.2479
♦ Results
Figure 4: /GAPDH Means Throughout Differentiation
Day 13730 18 21
iPSC
1. Endoderm
Induction
2. Hepatic
Specification
4. Hepatic
Maturation
3. Hepatoblast
Expansion
5. Mature
HLC
Act A
OSM
Dex
HGF
KGF
FGF4
BMP2
OSM
Dex
N2B27
Figure 1: Flow Chart of Stepwise Differentiation Protocol
Figure 3: Example of Densitometry Curves by ImageJ Software
OCT4 SOX17 HNF4A AFP ALB