Isolation and Characterization of Quiescent Human Hepatic Stellate Cells For Physiological Modeling ofLiver Metabolism and Function
Mukesh Kumar, Stacy L. Jones, Jessica A. Bonzo, Bina Khaniya and Rafal P. Witek, Cell Biology, Thermo Fisher Scientific, Frederick, MD 21704

Figure 1. Morphological and Immuno-characterization of quiescent humanhepatic stellate Cells

Immuno-characterization of human hepatic stellate cells: Cells wereisolated from human liver tissue and Immunostained for stellate cell markers.
(A) Vitamin A autofluorescence. Inset figure shows magnified view of stellatecells in phase contrast. (B) GFAP (C) Desmin (D) -SMA.

INTRODUCTION

Figure 2. Flow cytometry analysis of quiescent stellate cells

Relative Expression (2‐CT)

18

Co-culture of Hepatic stellate cells with human primary hepatocytes:
Human primary hepatocytes (Life Technologies, HMCPIS) were co-cultured
along with activated hepatic stellate cells at 1:5 ratio of stellate cells/hepatocytesin 6 well plates. Cells were co-cultured either in trans-well or in direct cell to cellcontact.
CYP3A4 Enzymatic Activity: On day 4, cultures were washed with freshWilliam’s E Maintenance Media and incubated for 15 minutes with testosterone
for CYP3A4 activity. After incubation, supernatants were collected and frozen
until analysis. Metabolites were detected using LC/MS/MS or HPLC. Results arenormalized to cell numbers and incubation time.
ELISA for human Albumin and TGF-: Expression of human albumin and TGF was measured using commercially available kits from Abcam (Cambridge, MA)
and Life Technologies (Cat# KAC1688) respectively. The Abcam kit specificallymeasures human albumin.

14LX‐212108620B

DConcentration (pg/ml)

CHGF

PDGFRBvWF9080706050403020100

TGF-β

AHSC

AHSC

Representative flow cytometry analysis of isolated human quiescentstellate cells: Cells were isolated and labeled with anti-GFAP antibody(stellate cell marker) and -SMA (activated stellate cell marker) followed bydetection with alexa fluor 633 labeled secondary antibody.
Activated Stellate Cells

CCL5

CCR5

GLI2

LOX

HHIP

LHX2

TGFB1

IGFBP3 SMAD2

RND2

TLR‐4BAMBI

CCL2Gene expression analysis of activatedhepaticstellatecells(A-HSC)
compared to stellate cell line LX2.
QRT-PCR analysis demonstrated anumber of genes (Nestin, Collagen1A1,PDGFr, LOX, HHIP, IGFBP3) that wereexpressed at higher levels than LX2 cells.
Stellate cell marker Desmin was highly
expressed in A-HSC compared toundetectable levels in LX2.

DesminFunctional
analysisofA-HSC.
Activated stellate cells produce TGF.
Expression of TGF production in cell
culture supernatants of A-HSC and LX2
cells were compared. A-HSC produced
almost twice amount of TGF than LX-2cells.
LX2

LX2AHSCHep Alone
Co‐Culture

BCqPCR - CYP3A41.2
Hep Alone

1Co‐Culture

0.8
0.6
0.40.20
A‐HSC

LX2

LX2LC/MS - CYP3A41800
1600
1400
12001000800600
4002000

Hep Alone
Co‐culture

A‐HSC

LX2

Co-culture of activated stellate cells (A-HSC or LX2) along with humanprimary hepatocytes (A). Albumin expression measured by ELISA at 24 hours
of co-culture demonstrate increased albumin production by co-culturescompared to hepatocyte cultures alone. (B &C). CYP3A4 activity measured atday 4 by qRT-PCR gene expression analysis (B) and by LC/MS (C) . The data
indicates that co-culture of hepatocytes along with activated stellate cells
lowered CYP3A4 activity

LX2

PDGFr

DesminCONCLUSIONS

Vimentin

Nestin

•

GAPDH

GAPDH

Our data indicates that we were able to isolate quiescent hepatic stellatecells from human liver tissue as indicated by positive staining for GFAP andDesmin and negative staining for -SMA .

•Quiescent hepatic stellate cells trans-differentiated to myofibroblast like cells
that expressed -SMA. These cells produced TGF- and had up-regulated
expression of PDGFr indicating their activated state. Comparison of primaryactivated hepatic stellate cell to stellate cell line LX2 indicated enhanced
expression of some of the activation marker in A-HSCs.

•Co-culture of activated primary stellate cells or LX2 cells with hepatocytes
stabilized hepatic function as evident by increased albumin synthesis.
However, a decreased expression of CYP3A4 activity was observed by both
qRT-PCR and LC/MS analysis. Similar decrease in CYP3A4 activity has
been previously shown in NAFLD induced liver fibrosis (Woolsey SJ et al).

Western blot analysis of activated hepatic stellate cells (A-HSC) compared
to stellate cell line LX2. Western blot analysis of A-HSC and LX2 cells
demonstrated that majority of the activation markers e.g. Nestin, PDGFr andmesenchymal cell marker Vimentin were expressed at significantly higher levels
than LX2 cells. Levels of expression of quiescent marker Desmin was unusuallyhigh in AHSC supporting q-PCR data.

Figure 3. Activation of quiescent hepatic stellate cells to A-HSC in vitro

A.

1600
1400
1200
1000
8006004002000A‐HSC

Relative Gene Expression (2‐CT)

NGFR Vimentin NESTIN COL1A1 ACTA1AMATERIALS AND METHODS

Gene Expression analysis: Cells were lysed using Trizol® and RNA waspurified using the Purelink® RNA Mini kit (Life Technologies, 12183018A). Fivehundred nanograms of RNA was reverse transcribed using the High Capacity
cDNA Reverse Transcription kit (Life Technologies, 4368813). Gene expression
was assessed using a custom designed TaqMan® Array card containing either
48 hepatocyte-specific genes of interest to ADME/Tox studies or 28 genes tostudy hepatic stellate cell specific effects.

AA‐HSC164

Hepatic stellate cells being a major non-parenchymal cell (NPC) of the liver are
the essential part of organotypic liver disease models aimed towards
recapitulating the intricate autocrine and paracrine signaling that occurs between
liver cell types.

Immunostaining: Isolated cells were characterized by intracellular staining for
GFAP and -SMA. In brief, cells were fixed, permeabilized, and following
blocking with 10% goat serum incubated overnight in primary antibodies.
Following three washes, bound primary antibody were detected with alexa fluor
labeled secondary antibody. Cells were characterized either by microscopy or byflow cytometry.

Figure 6. Co-culture of activated stellate cells (A-HSC or LX2) stabilizes
albumin production while lowering CYP3A4 activity

20

Hepatic stellate cells (HSCs) are liver non-parenchymal cells located in thespace of Disse between the sinusoidal endothelial cells and hepatocytes, andaccount for 5%–8% of the cells in the liver. In normal uninjured liver they are sitefor vitamin A storage however, when activated by chronic liver injury these cells
acquire a myofibroblastic state secreting ECM matrix, leading to scarring of thetissue and liver fibrosis.

Cell Isolation: Hepatic stellate cells were isolated from resected or whole liverhuman tissue. Briefly, tissue were digested with a two step Pronase andCollagenase/Protease solution followed by purification of non-parenchymal liver
cell using a density gradient.

Figure 4. Comparison of activated hepatic stellate cells (A-HSC) to hepatic
stellate cell line LX2

CYP3A4 (pmol/min*1E6 Cells)

RESULTS

Human hepatic stellate cells (HSC) are liver non-parenchymal cells (NPC’s) thatare present in the peri-sinusoidal tissue space of Disse. In normal liver, stellate
cells store vitamin A in the form of retinol droplets and are deemed quiescent (qHSC). Following hepatic injury, HSCs become activated, proliferate and produce
extracellular matrix (ECM) which accumulates in chronic condition leading to liver
fibrosis. In addition, liver cell-cell interactions, including ones between
hepatocytes and stellate cells, have important functions in stabilization of hepaticfunction. This can be recreated in vitro where the co-culturing of primary
hepatocytes with HSC maintains and stabilizes the differentiated phenotype of
hepatocytes such as albumin secretion and cytochrome P450 expression. To
facilitate these studies we have made attempts to isolate, purify and characterize
quiescent hepatic stellate cells from human liver tissue. Activated HSCs werealso generated from q-HSC and benchmarked against activated HSC cell line
LX2. Cells were characterized for presence of retinol droplets by phase andfluorescent microscopy. Further characterization was carried out by ICC, flowcytometry and microscopic analysis. Gene expression was characterized by qRT PCR for various stellate and mesenchymal cell markers including collagenand vimentin. Activated HSC were co-cultured with primary human hepatocytes
to study their effect on hepatic function and metabolism. Isolated cells werepositive for GFAP and negative for -SMA staining. Flow cytometry analysis
indicated enrichment of approximately 85% or more cells positive for GFAP. As
expected, culturing these cells for more than ten days activated them tomyofibroblasts (A-HSC) indicated by positive staining for -smooth muscle actin.
Activated HSCs (A-HSC) were also positive for Vimentin and CD271 indicating
their mesenchymal origin. Expression of TGF and PDGFr was used todetermine their functionality. Comparative analysis with LX2 indicated that ouractivated stellate cells were found to express all stellate cell markers at
significantly higher levels. When combined with hepatocytes in co-culture HSCs
were able to stabilize the culture of primary human hepatocytes. In this study, we
have generated primary quiescent and activated hepatic stellate cells fromhuman liver tissue with high purity and function. Our data indicates that monoand co-culture of hepatic stellate cells with hepatocytes will mimic physiologically
relevant models of stellate cell activation and their effect on hepatic function.

Albumin Concentration (pg/ml)

ABSTRACT

Figure 5. Co-culture of A-HSC and LX2 with primary human hepatocytes .
Activated Stellate Cells (A-HSC)

Quiescent Stellate Cells

Hepatocytes

Hepatocytes +A-HSC
REFERENCES
1. Friedman, SL. Hepatic stellate cells: protean, multifunctional, and enigmatic
cells of the liver. (2008). 88(1):125-72.

Activation

2. Woolsey SJ, Mansell SE, Kim RB, Tirona RG, Beaton MD. Drug
Metab.Dispos. 2015 Oct;43(10):1484-90.

B.
Control

-SMA

3. Mederacke I, Dapito DH, Affò S, Uchinami H, Schwabe RF. Nat Protoc.
2015 Feb;10(2):305-15.

LX2 Direct Co-culture

GFAP

4. Patil PB, Joshi M, Kuna VK, Xu B, Johannesson L, Olausson M, SumitranHolgersson S. Cytotherapy. 2014 Jul;16(7):990-9.

Hepatocytes +LX2ACKNOWLEDGEMENTS
The authors would like to thank Wendy Bray, Kristen Vaverchak, Patricia
Pawlowski, and Chelsie Fritz for research and program support and Rachel
Witek for Mass Spectrometry analysis.
Generation of activated HSCs from quiescent cells. (A). Quiescent hepatic
stellate cells were grown in uncoated tissue culture plates. After 7-10 days inculture cells turned myofibroblastic and started proliferating. (B). Immunostaining
followed by flow-cytometry analysis demonstrated that more than 90% of cells
were positive of -SMA. Cells were also positive for Vimentin and CD271.

A-HSC or LX2 cells were cocultured with direct cell-cell contact
for five days. Pictures with arrow
indicate myofibroblastic stellate cells
between hepatocytes.

Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com