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Methods in
Molecular Biology 1905

Naoki Tanimizu Editor

Hepatic
Stem Cells
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
Hepatic Stem Cells

Methods and Protocols

Edited by

Naoki Tanimizu
Sapporo Medical University, Sapporo, Hokkaido, Japan
Editor
Naoki Tanimizu
Sapporo Medical University
Sapporo, Hokkaido, Japan

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8960-7 ISBN 978-1-4939-8961-4 (eBook)
https://doi.org/10.1007/978-1-4939-8961-4

Library of Congress Control Number: 2018962407

© Springer Science+Business Media, LLC, part of Springer Nature 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
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Cover Illustration: Biliary epithelial cells (BECs) labeled with tdTomato proliferate to expand the CK19+ biliary epithelial
tissue structure upon injury, indicating that pre-existing BECs contribute to the “ductular reaction”. (The image related
to Chapter 7 has been provided by Dr. Kenji Kamimoto).

This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of
Springer Nature.
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface

The liver contains two types of epithelial cells, namely, hepatocytes and cholangiocytes
(biliary epithelial cells). Therefore, liver stem/progenitor cells (LPCs) are defined as bipo-
tential cells that can differentiate into hepatocytes and cholangiocytes. Hepatoblasts, fetal
LPCs, split into hepatocytes and cholangiocytes in mid-gestation. Bipotential LPCs also
have been isolated from neonatal liver. On the other hand, it is still controversial whether
bipotential LPCs contribute to maintenance of cellular homeostasis in healthy adult liver.
Recent works demonstrated that both hepatocytes and cholangiocytes are heterogeneous
cell populations and may contain committed progenitors. This book first contains methods
for isolation and expansion of bipotential LPCs from fetal and neonatal liver, for adult
clonogenic cholangiocytes and for hepatocyte progenitors.
The liver has the strong regenerative capability: it has been generally considered that
self-duplication of hepatocytes or cholangiocytes compensates the lost tissue after acute
injuries, whereas LPCs are activated and supply new hepatocytes after chronic injuries.
However, recent works demonstrated that dedifferentiation and lineage conversion of
liver epithelial cells also contribute to liver regeneration. These results highlight that liver
regeneration can be achieved by multiple modes of cellular responses. In the second part,
this book contains experimental methods for identifying and characterizing progenitor cells
involved in liver regeneration in vivo as well as those for investigating molecular mechanisms
regulating progenitor cell-driven liver regeneration.
It is crucial to expand functional hepatocytes and cholangiocytes ex vivo for implement-
ing regenerative medicine such as cell therapy and for establishing drug screening systems.
Recently, novel culture methods have been established to supply hepatocytes and/or
cholangiocytes from pluripotent stem cells as well as somatic terminally differentiated
cells. In addition, it was demonstrated that in vivo conversion of fibrogenic stellate cells
into hepatocytes could be useful to ameliorate liver fibrosis. In the third part, this book
contains culture methods for generating hepatocytes and cholangiocytes from multiple
cellular sources and a technique converting fibroblasts into hepatocytes in vivo.
Liver epithelial cells form three-dimensional (3D) tissue structures, which are indispens-
able for the liver to perform their physiological functions. Facing various types of injuries,
3D hepatic tissue structures are dynamically rearranged to avoid fatal liver damages. Thus,
this book also contains experimental methods for the reconstitution of 3D tissue structures
ex vivo as well as for the investigation of the dynamic structural changes induced after
injuries and during regeneration.
Fibrosis is the major pathology of the liver. Another issue is liver cancers. It is important
to establish a novel strategy to resolve liver fibrosis and to understand etiologies of hepato-
cellular (HCC) and cholangiocellular carcinomas (CC). Therefore, the last part of this book
includes methods resolving hepatic fibrosis by bone marrow transplantation as well as two
novel mouse models of HCCs and CCs.

v
vi Preface

In addition to experimental protocols, each author provides a great introduction sum-

marizing the background of each topic. This book would help researchers understand the
current concept about hepatic stem/progenitor cells and perform basic and translational
works related to liver biology and pathophysiology.

Sapporo, Hokkaido, Japan Naoki Tanimizu

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I ISOLATION OF PROGENITOR CELLS

1 Long-Term Culture of Mouse Fetal Hepatic Stem/
Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Atsunori Tsuchiya and Shuji Terai
2 Isolation of Bipotential Liver Progenitor Cells from Neonatal
Mouse Liver. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Naoki Tanimizu
3 Identification and Isolation of Clonogenic Cholangiocyte in Mouse. . . . . . . . . . . 19
Bin Li, Craig Dorrell, Pamela S. Canady, and Leslie Wakefield
4 Isolation and Expansion of Rat Hepatocytic Progenitor Cells. . . . . . . . . . . . . . . . . 29
Junichi Kino, Norihisa Ichinohe, Masayuki Ishii, and Toshihiro Mitaka

PART II CHARACTERIZATION OF LIVER PROGENITORS IN VIVO

5 Genetic Lineage Tracing of Biliary Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Teresa Rubio-Tomás, Beatriz Aguilar-Bravo, and Pau Sancho-Bru
6 Specific Labeling and Lineage Tracing of Periportal Hepatocytes
Using Two-Step Genetic Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Nicola de Prisco, Eleanor Stout, and Joan Font-Burgada
7 Analysis for the Heterogeneity of Liver Progenitor Cells . . . . . . . . . . . . . . . . . . . . . 71
Kenji Kamimoto
8 Chemical Screening Using a Zebrafish Model for Liver Progenitor
Cell-Driven Liver Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Sungjin Ko and Donghun Shin

PART III GENERATION OF HEPATOCYTES, CHOLANGIOCYTES,

AND THEIR PROGENITORS

9 Conversion of Fibroblasts to Hepatocytes In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Pengyu Huang, Lulu Sun, Ludi Zhang, and Lijian Hui
10 Conversion of Fibroblasts to Hepatocyte-Like Cells In Vivo . . . . . . . . . . . . . . . . . 103
Guangqi Song, Qinggong Yuan, Zhen Dai, Hsin-Chieh Tsay,
Xizhong Shen, Michael Ott, and Amar Deep Sharma
11 Chemically Induced Liver Progenitors (CLiPs): A Novel Cell Source
for Hepatocytes and Biliary Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Takeshi Katsuda and Takahiro Ochiya

vii
viii Contents

12 Induction of Functional Hepatocytes from Human iPSCs . . . . . . . . . . . . . . . . . . . 131

Taketomo Kido and Yuta Koui
13 Culture System of Bile Duct-Like Cystic Structures Derived from
Human-Inducible Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Akihide Kamiya, Kazuya Anzai, Kota Tsuruya, and Hiromi Chikada

PART IV RECONSTITUTION OF LIVER TISSUE STRUCTURES

14 Generation of Hepatic Tissue Structures Using Multicellular

Spheroid Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Fumiya Tao, Hirotaka Mihara, and Nobuhiko Kojima
15 Reconstruction of Hepatic Tissue Structures Using Interstitial
Flow in a Microfluidic Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Ryo Sudo
16 Generation of Hepatic Organoids with Biliary Structures . . . . . . . . . . . . . . . . . . . . 175
Takeshi Katsuda, Takahiro Ochiya, and Yasuyuki Sakai
17 Analysis for Remodeling of Hepatic Tissue Structures in 3D
During Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Kota Kaneko

PART V LIVER INJURY MODELS

18 Canine Liver Fibrosis Model to Assess the Functions of Infused

Autologous Bone Marrow-Derived Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Taro Takami, Kenji Tani, Yasuho Taura, and Isao Sakaida
19 A Rodent Model for Cell Transplantation of Hepatic Progenitor Cells . . . . . . . . 211
Sei Kakinuma and Akihide Kamiya
20 Mouse Model for Hepatocellular Carcinoma and Cholangiocarcinoma
Originated from Mature Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Masahiro Yamamoto, Bing Xin, and Yuji Nishikawa
21 Mouse Model for Cholangiocarcinoma from Peribiliary Glands. . . . . . . . . . . . . . . 237
Hayato Nakagawa, Nobumi Suzuki, and Kazuhiko Koike

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Contributors

BEATRIZ AGUILAR-BRAVO  Institut d’Investigacions Biomèdiques August Pi i Sunyer

(IDIBAPS), Barcelona, Spain
KAZUYA ANZAI  Department of Gastroenterology, Tokai University School of Medicine,
Isehara, Kanagawa, Japan
PAMELA S. CANADY  Oregon Stem Cell Center, Oregon Health and Science University,
Portland, OR, USA
HIROMI CHIKADA  Department of Molecular Life Sciences, Tokai University School of
Medicine, Isehara, Kanagawa, Japan
ZHEN DAI  Department of Gastroenterology, Hepatology and Endocrinology, Hannover
Medical School, Hannover, Germany
NICOLA DE PRISCO  Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA,
USA
CRAIG DORRELL  Oregon Stem Cell Center, Oregon Health and Science University,
Portland, OR, USA
JOAN FONT-BURGADA  Cancer Biology Program, Fox Chase Cancer Center, Philadelphia,
PA, USA
PENGYU HUANG  School of Life Science and Technology, Shanghai Tech University, Shanghai,
China
LIJIAN HUI  School of Life Science and Technology, Shanghai Tech University, Shanghai,
China; State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell
Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences,
University of Chinese Academy of Sciences, Shanghai, China
NORIHISA ICHINOHE  Department of Tissue Development and Regeneration, Research
Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo,
Japan
MASAYUKI ISHII  Department of Tissue Development and Regeneration, Research Institute
for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan;
Department of Surgery, Surgical Oncology and Science, Sapporo Medical University School
of Medicine, Sapporo, Japan
SEI KAKINUMA  Department of Gastroenterology and Hepatology, Tokyo Medical and Dental
University, Tokyo, Japan; Department of Liver Disease Control, Tokyo Medical and Dental
University, Tokyo, Japan
KENJI KAMIMOTO  Department of Developmental Biology, Washington University School of
Medicine in St. Louis, St. Louis, MO, USA
AKIHIDE KAMIYA  Department of Molecular Life Sciences, Tokai University School of
Medicine, Isehara, Kanagawa, Japan; Center for Matrix Biology and Medicine, Graduate
School of Medicine, Tokai University, Isehara, Kanagawa, Japan
KOTA KANEKO  Department of Pathology, School of Medicine, University of California, San
Diego, CA, USA
TAKESHI KATSUDA  Division of Molecular and Cellular Medicine, National Cancer Center
Research Institute, Tokyo, Japan
TAKETOMO KIDO  Laboratory of Cell Growth and Differentiation, Institute of Molecular
and Cellular Biosciences, The University of Tokyo, Tokyo, Japan

ix
x Contributors

JUNICHI KINO  Department of Tissue Development and Regeneration, Research Institute for
Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan;
Tokushima Research Institute, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan
SUNGJIN KO  Department of Developmental Biology, McGowan Institute for Regenerative
Medicine, Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
KAZUHIKO KOIKE  Department of Gastroenterology, The University of Tokyo, Tokyo, Japan
NOBUHIKO KOJIMA  Department of Life and Environmental System Science, Graduate
School of Nanobioscience, Yokohama City University, Yokohama, Japan
YUTA KOUI  Laboratory of Cell Growth and Differentiation, Institute of Molecular and
Cellular Biosciences, The University of Tokyo, Tokyo, Japan
BIN LI  Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR,
USA
HIROTAKA MIHARA  Department of Life and Environmental System Science, Graduate
School of Nanobioscience, Yokohama City University, Yokohama, Japan
TOSHIHIRO MITAKA  Department of Tissue Development and Regeneration, Research
Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo,
Japan
HAYATO NAKAGAWA  Department of Gastroenterology, The University of Tokyo, Tokyo, Japan
YUJI NISHIKAWA  Division of Tumor Pathology, Department of Pathology, Asahikawa
Medical University, Asahikawa, Hokkaido, Japan
TAKAHIRO OCHIYA  Division of Molecular and Cellular Medicine, National Cancer Center
Research Institute, Tokyo, Japan
MICHAEL OTT  Department of Gastroenterology, Hepatology and Endocrinology, Hannover
Medical School, Hannover, Germany
TERESA RUBIO-TOMÁS  Institut d’Investigacions Biomèdiques August Pi i Sunyer
(IDIBAPS), Barcelona, Spain
YASUYUKI SAKAI  Institute of Industrial Science, The University of Tokyo, Tokyo, Japan
ISAO SAKAIDA  Department of Gastroenterology and Hepatology, Yamaguchi University
Graduate School of Medicine, Yamaguchi University, Ube, Yamaguchi, Japan
PAU SANCHO-BRU  Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS),
Barcelona, Spain; Centro de Investigacion Biomédica en Red de Enfermedades Hepáticas y
Digestivas (CIBERehd), Barcelona, Spain
AMAR DEEP SHARMA  Department of Gastroenterology, Hepatology and Endocrinology,
Hannover Medical School, Hannover, Germany
XIZHONG SHEN  Department of Gastroenterology, Zhongshan Hospital, Fudan University,
Shanghai, China
DONGHUN SHIN  Department of Developmental Biology, McGowan Institute for
Regenerative Medicine, Pittsburgh Liver Research Center, University of Pittsburgh,
Pittsburgh, PA, USA
GUANGQI SONG  Department of Gastroenterology, Zhongshan Hospital, Fudan University,
Shanghai, China
ELEANOR STOUT  Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA,
USA
RYO SUDO  Department of System Design Engineering, Keio University, Yokohama, Japan
LULU SUN  State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular
Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences, University of Chinese Academy of Sciences, Shanghai, China
NOBUMI SUZUKI  Department of Gastroenterology, The University of Tokyo, Tokyo, Japan
 Contributors xi

TARO TAKAMI  Department of Gastroenterology and Hepatology, Yamaguchi University

Graduate School of Medicine, Yamaguchi University, Ube, Yamaguchi, Japan
KENJI TANI  Department of Veterinary Surgery, Joint Faculty of Veterinary Medicine,
Yamaguchi University, Yamaguchi, Yamaguchi, Japan
NAOKI TANIMIZU  Department of Tissue Development and Regeneration, Research Institute
for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Hokkaido,
Japan
FUMIYA TAO  Department of Life and Environmental System Science, Graduate School of
Nanobioscience, Yokohama City University, Yokohama, Japan
YASUHO TAURA  Department of Veterinary Surgery, Joint Faculty of Veterinary Medicine,
Yamaguchi University, Yamaguchi, Yamaguchi, Japan
SHUJI TERAI  Division of Gastroenterology and Hepatology, Graduate School of Medical and
Dental Science, Niigata University, Niigata, Japan
HSIN-CHIEH TSAY  Department of Gastroenterology, Hepatology and Endocrinology,
Hannover Medical School, Hannover, Germany
ATSUNORI TSUCHIYA  Division of Gastroenterology and Hepatology, Graduate School of
Medical and Dental Science, Niigata University, Niigata, Japan
KOTA TSURUYA  Department of Gastroenterology, Tokai University School of Medicine,
Isehara, Kanagawa, Japan
LESLIE WAKEFIELD  Oregon Stem Cell Center, Oregon Health and Science University,
Portland, OR, USA
BING XIN  Division of Tumor Pathology, Department of Pathology, Asahikawa Medical
University, Asahikawa, Hokkaido, Japan
MASAHIRO YAMAMOTO  Division of Tumor Pathology, Department of Pathology, Asahikawa
Medical University, Asahikawa, Hokkaido, Japan
QINGGONG YUAN  Department of Gastroenterology, Hepatology and Endocrinology,
Hannover Medical School, Hannover, Germany
LUDI ZHANG  State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular
Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences, University of Chinese Academy of Sciences, Shanghai, China
 Part I

Isolation of Progenitor Cells

 Chapter 1

Long-Term Culture of Mouse Fetal Hepatic

Stem/Progenitor Cells
Atsunori Tsuchiya and Shuji Terai

Abstract
Mouse fetal liver includes abundant hepatic stem/progenitor cells (HSPCs). Easy expansion with passage of
HSPCs is necessary to obtain steady data. However, it is often difficult to enrich only HSPCs, and HSPCs
can die when usual trypsin is used for replating. Here, we introduce serum-free long-term culture with
passage of HSPCs using fetal mouse liver without a cell sorter.

Key words Hepatic progenitor cell, Spheroid, Serum-free culture, Colony, Mouse

1 Introduction

Mouse fetal liver includes abundant hepatic stem/progenitor cells

(HSPCs) surrounding hematopoietic cells, endothelial cells, mes-
enchymal cells, and other cells. Owing to the high expansion ability
of HSPCs, it is very convenient to use these cells after expansion for
in vitro and in vivo studies. There are two main problems in long-
term culture of HSPCs. First, while HSPCs have a high expansion
ability, other cells, such as hematopoietic cells, endothelial cells, and
mesenchymal cells, also have high expansion ability. Sometimes
when fetal liver cells are cultured without enrichment with serum,
mesenchymal cells occupy most of the culture dish. Adopting a cell
sorter is a very good approach to isolate only HSPCs by using the
markers CD45 Ter119 c-kit CD29+CD49f+/low [1, 2], epithelial
cell adhesion antigen (EpCAM) [3], CD13 [4, 5], and delta-like
1 homolog (DLK1) [6, 7]. However, culture from single cells is not
easy, and not all laboratories can apply this approach. Second,
HSPCs can die when usual trypsin is used for replating. To over-
come these problems and achieve long-term culture of HSPCs, we
developed an approach involving spheroid culture followed by
two-dimensional (2D) culture with replating using diluted trypsin
[8] (Fig. 1).

Naoki Tanimizu (ed.), Hepatic Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1905,
https://doi.org/10.1007/978-1-4939-8961-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

3
4 Atsunori Tsuchiya and Shuji Terai

Culture Scheme Medium

Dissociated E13.5 fetal liver cells DMEM/F12 (1:1)
+B27+ITS+
For 6 days 20 ng/ml EGF+
20 ng/ml bFGF+
Floating culture 20 ng/ml HGF
Hepatic spheroid formation

Plating culture Monolayer colony formation

DMEM/F12 (1:1)
+B27+ITS+
20 ng/ml EGF+
Subpassage with maintaining With trypsin diluted 20 ng/ml bFGF+
colony formation by KSR and CaC12 10 ng/ml HGF

E13.5 fetal liver cell

Expansion of fetal hepatic stem/progenitor cells Hepatic spheroid

Flow cytometry Ultra-low attachment plate

Immunocytochemistry +OSM etc.
RT-PCR Expanding monolayer colony
Transplantation

Analysis Differentiation Type-1 collagen coated

dish

Fig. 1 Flow diagram presenting our culture system

2 Materials

2.1 Isolation of 1. Mice: A pregnant C57BL/6 mouse at day 13.5 of gestation

HSPCs was used as a source of fetal liver. Other stains were not
attempted for culture.
2. Tweezers.
3. Tissue culture dishes (100 mm, polystyrene).
4. Phosphate-buffered saline (PBS).
5. 1 mL pipette tips.

2.2 For Spheroid 1. Medium: Dulbecco’s Modified Eagle Medium/F12

Culture (Floating (DMEM/F12).
Culture) 2. Supplement: B27 supplement (50), ITS mixture (100), and
HEPES (final concentration: 10 nmol/L) (see Note 1).
3. Antibiotics: Penicillin-streptomycin liquid (see Note 1).
4. Growth factors: Epidermal growth factor (EGF; final concen-
tration 20 ng/mL), hepatocyte growth factor (HGF; final
concentration 20 ng/mL), and basic fibroblast growth factor
(bFGF; final concentration 20 ng/mL).
5. Dish: Six-well ultralow attachment plates (see Note 2).
 Culture of Mouse Fetal HSPCs 5

2.3 For 2D Culture 1. Medium: Same as that in the spheroid culture.

(Plating Culture) 2. Supplement: Same as that in the spheroid culture.
3. Antibiotics: Same as that in the spheroid culture.
4. Growth factors: Same as that in the spheroid culture. It is
possible to reduce the concentration of each growth factor
(20–10 ng/mL).
5. Dish: Six-well collagen type I-coated plates (see Note 2).

2.4 Diluted Trypsin 1. Trypsin (0.25%) 100 mL (final concentration 0.2%).

2. Knockout serum replacement (KSR) 25 mL.
3. CaCl2 (final concentration 1 mmol/L) (see Note 3).
4. Diluted trypsin: Mix 1–3 to make diluted trypsin. Aliquot
15 mL to a Falcon tube, and freeze at 30  C until use.
Warm the Falcon tube at 37  C before use.

3 Methods

3.1 Spheroid Culture 1. Before performing 2D culture, create spheroids using an ultra-
low attachment plate.
2. Kill a pregnant C57BL/6 mouse at day 13.5 of gestation using
isoflurane, and perform cesarean delivery.
3. Remove and dissect fetal livers using tweezers in cold PBS.
4. Dissociate the dissected fetal livers using a 1 mL pipette tip, and
collect the fragments with centrifugation at 500 rpm for 3 min
(see Note 4).
5. Count and plate the collected cells (see Note 5) at a density of
5  105 cells/mL in the medium with growth factors men-
tioned in Subheading 2.2 on a 6-well ultralow attachment plate
(2 mL/well).
6. Add 2 mL of medium and growth factors at days 2 and
4. Hepatic spheroids will form and increase in size for 6 days
(Fig. 2) (see Note 6).

3.2 2D Culture 1. Collect the hepatic spheroids formed in Subheading 3.1 by

centrifugation at 300 rpm for 2 min.
2. Plate the spheroids on a type I collagen-coated plate with the
medium and growth factors mentioned in Subheading 2.2.
Each spheroid will form a colony (Fig. 3a) in the 6-well colla-
gen-coated plate (see Note 7). Hepatic progenitor cells will be
highly enriched in this serum-free condition (see Note 8).
3. Change the medium and growth factors every 3 days.
4. After reaching approximately 80% of the confluency, collect
cells according to the method mentioned below.
6 Atsunori Tsuchiya and Shuji Terai

Fig. 2 Spheroids formed for 6 days by spheroid (floating) culture. The size of
spheroids was approximately 50–250 μm, comprising approximately 20–150
cells

3.3 Replating 1. Remove the medium and wash with PBS.

2. Add 1 mL of diluted trypsin warmed at 37  C to the
6-well collagen-coated plate, and warm the plate at 37  C for
2–5 min.
3. Check the status of the colonies by microscopy every 2–3 min.
If the colonies are partially removed from the plates while
maintaining cell-cell contact, add medium and stop the enzy-
matic digestion/reaction (see Note 9).
4. Press a 2 mL or 5 mL pipette to the plates, and remove
colonies with the strong hydraulic pressure of the pipette
(Fig. 3b).
5. Collect the removed colonies with low-speed centrifugation
(500 rpm for 3 min).
6. Divide the colonies into 2 or 3 aliquots, and replate on newly
prepared collagen-coated plates (Fig. 3c). This procedure can
be repeated to expand the mouse fetal HSPCs for more than
ten passages. A scheme of the culture is shown in Fig. 1 (see
Note 10).
7. Collected colonies can be preserved at 80  C for several
weeks or in liquid nitrogen for years by using
STEM-CELLBANKER®. CELLBANKER® 2 can also be
used (see Note 11).
 Culture of Mouse Fetal HSPCs 7

Fig. 3 Images of the 2D (plating) culture and replating. Colonies are formed from each spheroid (a). Collected
colonies by diluted trypsin (b) are replated onto newly prepared collagen-coated plates (c). During passage,
cell-cell contact was maintained

4 Notes

1. B27 supplement (50), ITS mixture (100), and penicillin-

streptomycin liquid (100) can be purchased from Thermo-
Fisher Scientific.
2. Any type of plate may be appropriate if cells cannot attach to
the plate. We purchased plates from Corning.
3. We made 100 mM CaCl2 solution (100) by dissolving CaCl2
in distilled water and then filtering the solution. The solution
was added to the mixture of trypsin and KSR.
4. Faster centrifugation might be better. However, 500 rpm for
3 min is sufficient to collect dissociated fetal liver cells.
5. During counting, red blood cells obviously distinguished by
size were excluded.
8 Atsunori Tsuchiya and Shuji Terai

6. We examined the efficacy to form spheroids through growth

factor matching. Matching with EGF, HGF, and bFGF was the
most appropriate to obtain many spheroids.
7. Spheroids from each 6-well plate were plated on each well of
6-well collagen type I-coated plates.
8. Hematopoietic cells and mesenchymal cells were included in
the culture. However, most of the cells disappeared after 2–3
passages (confirmed by cell morphology and flow cytometry).
9. If colonies are difficult to remove from the plates, the time for
trypsin treatment can be extended to 10–15 min. Maintaining
cell-cell contact is the most important point for successful
replating.
10. The replating method using diluted trypsin can be employed
for adult mouse hepatic progenitor cells [9, 10].
11. The cell bankers can be purchased from ZENOAQ.

References
1. Suzuki A, Zheng Y, Kondo R et al (2000)
Flow-cytometric separation and enrichment of
hepatic progenitor cells in the developing
mouse liver. Hepatology 32:1230–1239
2. Suzuki A, Zheng YW, Kaneko S et al (2002)
Clonal identification and characterization of
self-renewing pluripotent stem cells in the
developing liver. J Cell Biol 156:173–184
3. Schmelzer E, Zhang L, Bruce A et al (2007)
Human hepatic stem cells from fetal and post-
natal donors. J Exp Med 204:1973–1987
4. Kamiya A, Kakinuma S, Yamazaki Y et al
(2009) Enrichment and clonal culture of pro-
genitor cells during mouse postnatal liver
development in mice. Gastroenterology
137:1114–1126 1126 e1-14
5. Kakinuma S, Ohta H, Kamiya A et al (2009)
Analyses of cell surface molecules on hepatic
stem/progenitor cells in mouse fetal liver. J
Hepatol 51:127–138
6. Tanimizu N, Nishikawa M, Saito H et al (2003)
Isolation of hepatoblasts based on the expres-
sion of Dlk/Pref-1. J Cell Sci 116:1775–1786
7. Tanimizu N, Tsujimura T, Takahide K et al
(2004) Expression of Dlk/Pref-1 defines a sub-
population in the oval cell compartment of rat
liver. Gene Expr Patterns 5:209–218
8. Tsuchiya A, Heike T, Fujino H et al (2005)
Long-term extensive expansion of mouse
hepatic stem/progenitor cells in a novel
serum-free culture system. Gastroenterology
128:2089–2104
9. Tsuchiya A, Heike T, Baba S et al (2007) Long-
term culture of postnatal mouse hepatic stem/
progenitor cells and their relative developmen-
tal hierarchy. Stem Cells 25:895–902
10. Lu WY, Bird TG, Boulter L et al (2015)
Hepatic progenitor cells of biliary origin with
liver repopulation capacity. Nat Cell Biol
17:971–983
 Chapter 2

Isolation of Bipotential Liver Progenitor Cells from Neonatal

Mouse Liver
Naoki Tanimizu

Abstract
Liver stem/progenitor cells (LPCs) are defined as bipotential cells differentiating into both hepatocytes and
cholangiocytes. The Notch, TGFβ, and Hippo pathways have been implicated in lineage determination of
LPCs during development and regeneration. However, the molecular mechanisms governing the lineage
specification have not been fully elucidated, yet. Epithelial adhesion molecule (EpCAM) is a marker of
cholangiocytes and of LPCs. We found that EpCAM+ cells isolated from neonatal liver contain LPCs that
clonally proliferate and are bipotential in vitro and in vivo. Furthermore, EpCAM+ progenies keep the
capacity of bidirectional differentiation even after long-term culture. These cells are useful to investigate the
molecular mechanisms regulating lineage commitment and epithelial differentiation of LPCs.

Key words Tissue stem cell, Liver, Neonate, Bipotential, Hepatocyte, Cholangiocyte, Liver stem/
progenitor cell, Fluorescence-activated cell sorting, Colony assay, Clonal culture

1 Introduction

Liver stem/progenitor cells (LPCs) are defined as bipotential cells

differentiating into two types of liver epithelial cells, hepatocytes
and cholangiocytes. LPCs have been isolated from normal and
injured livers based on expression of surface antigens including
EpCAM (epithelial adhesion molecule), CD13, and CD133
[1]. However, the genetic lineage tracing done in the past 5 years
suggests that LPCs significantly contribute neither to cellular turn-
over nor regeneration [2, 3]. In contrast, recent results showed that
cholangiocytes supply new hepatocytes when hepatocytes are
almost completely depleted or the proliferative capability of hepa-
tocytes is impaired [4, 5], suggesting that part of cholangiocytes
may have plasticity to act as facultative LPCs. Although the hetero-
geneity of cholangiocytes has been demonstrated [6, 7], it is still
difficult to label those cells in vivo and to prospectively isolate the
sauce of facultative LPCs in normal and injured adult liver.

Naoki Tanimizu (ed.), Hepatic Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1905,
https://doi.org/10.1007/978-1-4939-8961-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019

9
10 Naoki Tanimizu

We isolated EpCAM+ cells from the liver between E17 and

12 W after birth and showed that colony-forming cells are enriched
in this fraction [8]. We further found that neonatal EpCAM+ cells
differentiate to hepatocytes and cholangiocytes in vitro and in vivo,
though they gradually lose such differentiation capability during
the subsequent developmental process. It is also possible to estab-
lish bipotential liver progenitor cell lines from neonatal EpCAM+
cells. This chapter includes the protocols for isolating EpCAM+
cells from 1 W mouse liver, expanding them in vitro and inducing
their differentiation to either hepatocyte or cholangiocytes.

2 Materials

2.1 Cell Isolation 1. Isoflurane.

2. Hanks’ balanced salt solution (HBSS).
3. Pre-perfusion solution: 0.5 mM EGTA in HBSS.
4. Twenty-seven-gauge needle.
5. Ten milliliter syringe.
6. Collagenase: 1 mg/mL in HBSS.
7. 6 cm plastic dish.
8. Liver perfusion medium: Liver perfusion medium (Thermo
Fisher Scientific, Cat. No. 17701038) or HBSS
containing EGTA.
9. PBS (+/+):100 mg/mL MgCl2 and 100 mg/mL CaCl2 in
phosphate buffer saline (PBS).
10. Liberase TM (Roche Diagnostics, Basel, Switzerland): It is the
blendzyme of collagenase and thermolysin. Dissolve in PBS,
make aliquots, and store at 80  C. Dilute the stock solution
50 times in PBS (+/+) before use.
11. 70 μm cell strainer.
12. Hemolysis buffer: 10 mM Tris-HCl, 50 mM NH4Cl. Pass
through a 0.22 μm pore filter to sterilize.

2.2 Cell Sorting 1. Antibodies: FITC-conjugated anti-mouse CD329/EpCAM,

APC-conjugated anti-mouse CD31, and APC-conjugated
anti-CD45 and APC-Cy7-conjugated anti-mouse TER119.
2. Wash buffer: Add 2% FBS to PBS. Store at 4  C.
3. Propidium iodide (PI) (1 mg/mL).

2.3 Cell Culture 1. Growth factor-reduced Matrigel® (MG) (BD Biosciences, Bed-
ford, MA): Thaw the bottle of MG on ice, and make aliquots in
1.5 mL tubes. Freeze those tubes in liquid nitrogen, and store
 Isolation of Neonatal Bipotential Liver Progenitors 11

them at 30  C. Thaw MG in a tube on ice before use (see

Note 1).
2. Collagen type IPC (Koken Co., Ltd., Tokyo, Japan).
3. Collagen reconstitution buffer: 2.2% NaHCO3, 0.2 M
HEPES, and 50 mM NaOH. Pass through a 0.22 μm pore
filter to sterilize.
4. 10 Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s
F-12 Nutrient Mixture (1:1) (DMEM/F-12) medium.
5. Laminin solution: Thaw laminin-111 in a glass bottle at 4  C.
Transfer it to a 1.5 mL tube, and store at 4  C. Dilute it in PBS
at 10 μg/mL. Add 1 mL diluted solution to 35 mm plate. Wait
about 1 h and remove the solution before plating cells (see
Note 2).
6. Gelatin solution: Autoclave PBS containing 0.1% gelatin. Add
300 μL gelatin solution to each well of 24-well plate. Aspirate
gelatin solution and wash wells with PBS once.
7. Basal medium: Prepare DMEM/F-12 medium supplemented
with 10% fetal bovine serum, 10 mM nicotinamide, and 10 μg/
mL gentamicin.
8. Growth medium: Add 1 insulin/transferrin/selenium (ITS),
1  10 7 M dexamethasone (DEX), 10 ng/mL epidermal
growth factor (EGF), and 10 ng/mL hepatocyte growth factor
(HGF) to the basal medium.
9. Differentiation medium: Add 1 ITS, 1  10 7 M DEX, and
1% dimethyl sulfoxide (DMSO) to the basal medium. Add
10 ng/mL oncostatin M (OSM) or 5% MG before use.
10. Lysis solution for RNA extraction.
11. Cloning ring: Autoclave glass or stainless rings. Apply auto-
claved Vaseline on the bottom of the ring before surrounding a
colony.
12. Trypsin/EDTA: Dissolve 0.25% trypsin in PBS, make aliquots,
and store at 30  C. Dilute the stock solution 5 times with
PBS and add with 1 mM EDTA before use.
13. Blocking solution: Dissolve Block Ace in PBS.
14. Bovine serum albumin (BSA).
15. PFS: Dissolve 0.7% fish gelatin, 0.07% saponin, and 0.02%
sodium azide. Store at 4  C.
16. Paraformaldehyde (PFA) solution: 4% PFA in PBS.
17. Permeabilization buffer: 0.2% Triton X-100 in PBS. Make 10%
Triton X-100 and keep it at room temperature. Dilute the
stock solution by 50 times with PBS.
12 Naoki Tanimizu

3 Methods

3.1 Cell Isolation 1. Anesthetize a mouse by isoflurane. Open the abdomen, and
then cut the diagram and rib to expose the heart.
3.1.1 Collagenase
Perfusion 2. Attach a needle to a 10 mL syringe filled with pre-perfusion
medium that is pre-warmed at 37  C. Insert the needle into the
left ventricle, and inject pre-perfusion medium. Cut the inferior
vena cava after confirming the liver tissue becomes enlarged
due to pressure (Fig. 1). Change the syringe to one filled with
perfusion medium. Inject the perfusion solution slowly.
3. Isolate the liver and place it on a 6 cm plastic dish. Add 5 mL of
HBSS. Hold the liver with a forceps, and scrape off hepatocytes
from undigested liver tissue using another forceps. (Use undi-
gested tissue for further enzymatic digestion. See the next
paragraph.) Pass the cell suspension through a 70 μm cell
strainer.
4. Centrifuge the cell suspension at 50  g for 1 min. Collect the
supernatant and centrifuge it at 350  g for 4 min (see Note 1).
Resuspend the pellet in 1 mL of basal medium.

3.1.2 Enzymatic
Digestion of the Remaining
Tissue After Collagenase
Perfusion
1. Mince the undigested tissue after collagenase perfusion. Sus-
pend tissue pieces in 3 mL of liver perfusion medium, and
collect them in a 15 mL tube. Centrifuge at 150  g for 3 min.
2. Resuspend the pellet in 1 mL of Liberase TM solution (see
Note 3). Incubate the cell suspension at 37  C for 15 min.
Dissolve the tissue by pipetting (see Note 4). Add 1–2 mL of

Fig. 1 Schematic view of perfusion for a neonatal mouse. A mouse is fixed on a

board with tapes. After exposing the heart and identifying the left ventricle, a
needle is attached to a 10 mL syringe filled with pre-perfusion medium
 Isolation of Neonatal Bipotential Liver Progenitors 13

basal medium to stop enzymatic digestion. Pass the cell sus-

pension through a 70 μm cell strainer.
3. Centrifugation at 50  g for 1 min. Then, centrifuge the
supernatant at 350  g for 4 min to collect dissociated cells.
Resuspend cells in 1 mL of basal medium.

3.1.3 Hemolysis 1. Combine the cell suspensions from Subheading 3.1.1, step 4,
and Subheading 3.1.2, step 3, into a new 15 mL tube. Centri-
fuge at 350  g for 4 min.
2. Resuspend pellet in 1 mL of hemolysis buffer. Incubate at 4  C
for 5 min. Then, add 1–2 mL of basal medium, and pass
through a 70 μm cell strainer. Centrifuge at 350  g for 4 min.
3. Resuspend the pellet in 200 μL of basal medium, and count the
number of cells.

3.2 Cell Sorting 1. Centrifuge the cell suspension at 350  g for 4 min. Resuspend
cells in 100 μL of basal medium.
2. Add 1 μL of anti-CD16/CD32 antibody, and incubate at 4  C
for 20 min to avoid non-specific binding of antibodies by
masking Fcγ receptors. Add ice-cold wash buffer and centrifuge
at 350  g for 4 min.
3. Resuspend cells in 100 μL of basal medium, and then add 1 μL
each of FITC-conjugated anti-mouse EpCAM,
APC-conjugated anti-CD31, APC-conjugated anti-CD45,
and APC-Cy7-conjugated anti-TER119 (see Note 5).
4. For an isotype control, add FITC-conjugated rat IgG,
APC-conjugated rat IgG, and APC-Cy7-conjugated rat IgG.
5. Incubate cells with antibodies at 4  C for 20 min.
6. Add 2 mL of ice-cold wash buffer and centrifuge at 350  g for
4 min. Resuspend cells in 300 μL of basal medium containing
1 μg/mL PI. Pass through 40 μm cell strainer before analysis
on FACS Aria II (BD Biosciences).
7. Select CD31 CD45 TER119 cells in PI singlet cells by
gating on FACS Aria II. Identify and collect EpCAM + cells
in CD31 CD45 fraction into a 5 mL tube (Fig. 2).
8. Transfer the collected cells into a 15 mL tube. Centrifuge at
350  g for 8 min. Resuspend cells and add 7 mL of basal
medium and centrifuge at 350  g for 8 min. Resuspend cells in
1 mL of basal medium.
14 Naoki Tanimizu

Fig. 2 Isolation of EpCAM+ cells by FACS. After gating live singlet CD31 CD45 cells, EpCAM+ cells can be
identified and isolated

3.3 Culture 1. Resuspend cells in growth medium. Plate 5000 cells in 35 mm

dish coated with laminin.
3.3.1 Colony Assay
2. At day 7, fix cells in 4% PFA at 4  C for 15 min. Wash 1 mL of
PBS for 3 times. Permeabilize with 0.7 mL of 0.02% Triton
X-100, and incubate at room temperature for 5 min. Wash cells
with 1 mL of PBS for 2 times. Add 1 mL of Block Ace and
incubate at room temperature for 30 min.
3. Replace blocking solution to 1 mL of PBS containing 1% BSA
and 1 μL each of rabbit anti-mouse cytokeratin 19 (CK19) and
anti-mouse albumin (ALB) antibody. Incubate at 4  C for 4 h.
Wash cells with PBS three times. Add PBS containing 1% BSA,
0.5 μL each of AlexaFluor488-conjugated anti-rabbit IgG and
AlexaFluor555-conjugated anti-goat IgG and 1 μL each of
Hoechst 33342. Incubate at 4  C for 2 h. Wash cells with
PBS three times before examination of the number of cells
and expression of ALB and CK19 under a fluorescence micro-
scope. A typical bipotential colony is shown in Fig. 3a.

3.3.2 Hepatocyte 1. Plate 5000 EpCAM+ cells in a well of 24-well plate coated with
Differentiation gelatin.
2. Replace basal medium to the differentiation medium contain-
ing OSM and incubate for 2 days. Replace the medium to one
containing 5% MG. Keep the culture for additional 3 days.
3. To examine expression of hepatocyte marker genes, dissolve
cells in a lysis solution and extract total RNA. Synthesize cDNA
and perform PCR with gene-specific primers.
4. To examine expression of hepatocyte marker proteins, treat
cells with Subheading 3.3.1, step 2. Incubate cells with rabbit
anti-hepatocyte nuclear factor 4 α (HNF4α) and goat anti-ALB
or with rabbit anti-CCAAT enhancer binding protein α
(C/EBPα) and goat anti-carbamoyl phosphate synthetase I
(CPSI) antibody. Typical images before and after the treatment
of OSM and MG are shown in Fig. 3b.
 Isolation of Neonatal Bipotential Liver Progenitors 15

Fig. 3 Proliferation and hepatocyte differentiation of neonatal EpCAM+ cells. A EpCAM+ cell clonally
proliferates and forms a bipotential colony containing ALB+ hepatocytes and CK19+ cholangiocytes. (a) Part
of EpCAM+ cells expresses ALB and HNF4α before inducing hepatocyte differentiation, whereas they become
HNF4α+ALB+ hepatocytes in the presence of OSM and MG. (b) Bars represent 100 μm

3.3.3 Cholangiocyte 1. Add PBS, collagen reconstitution buffer, 10 DF medium,

Differentiation and collagen IPC at 4:3:3:20 (v:v:v:v) ratio in a 15 mL tube
on ice. Pipette up and down gently to mix thoroughly.
2. Mix collagen gel with MG at 1:1 ratio. Add 80 μL of the mixed
gel to each well of 8-well cover glass chamber. Incubate it at
37  C for 30 min.
3. Plate 1  104 cells in 150 μL of growth medium, and incubate
for 5 min. Add 150 μL of growth medium containing 10% MG.
16 Naoki Tanimizu

4. To examine expression of cholangiocyte marker genes, dissolve

cells in a lysis solution, and extract total RNA. Synthesize
cDNA and perform PCR with gene-specific primers.
To examine expression of cholangiocyte marker proteins, fix
cells in 4% PFA and permeabilize in 0.02% Triton X-100. Wash
cells with PBS once, add PFS, and incubate at room tempera-
ture for 30 min. Incubate cells with PFS containing primary
antibodies such as anti-CK19, anti-integrin beta4, anti-protein
kinase C zeta, and anti-aquaporin 1 antibody followed by Alexa
Fluor dye-conjugated antibodies and Hoechst 33342. Examine
the localization of cholangiocyte markers under a confocal laser
scanning microscope.

3.3.4 Isolation of 1. Keep clonal culture for about 4 weeks.

Bipotential Clones 2. Wash cells with PBS once, and then surround each colony with
a cloning ring. Add 100 μL of trypsin/EDTA, and incubate for
5–10 min at 37  C. Pipette up and down for 3–4 times, and
transfer the cell suspension into 1 mL of basal medium in
15 mL tube. Centrifuge at 350  g for 3 min.
3. Resuspend cells in growth medium, and plate them onto a well
of 24-well plate coated with laminin-111 (see Note 6).

4 Notes

1. It takes about 2 h to dissolve an aliquot of 500 μL MG on ice.

2. The diluted laminin solution can be reused up to three times to
coat tissue culture dishes. We usually recover the laminin solu-
tion and store it in a 15 mL tube at 4  C.
3. For scale-up, undigested tissues can be pooled at this step. The
tissue pieces derived from 2 to 3 mice are collected in a 15 mL
tube and then added with 2 mL of Liberase TM. For digesting
tissue pieces from more than three mice, make another tube for
Liberase digestion and then pooled cell suspensions into one
tube after Liberase digestion.
4. Pipette up and down for 20–30 times to dissociate the most of
cells from connective tissue.
5. Count the number of cells. Use 1 μL of each antibody to label
cells less than 1  107 cells. Increase the quantity of antibodies
according to the amount of cells.
6. The size of culture is gradually increased to 12-well plate and
then 35 mm dish. EpCAM expression is examined by FACS to
acquire EpCAM+ clones. EpCAM+ clones are replated every
4 days by plating 5  104 cells in 35 mm dish coated with
laminin. Hepatocyte and cholangiocyte differentiation can be
 Isolation of Neonatal Bipotential Liver Progenitors 17

induced using the same protocol as the primary EpCAM+ cells,

which are described in Subheadings 3.3.2 and 3.3.3.

Acknowledgments

I thank Dr. Toshihiro Mitaka and Dr. Norihisa Ichinohe for helpful
discussion and Ms. Yumiko Tsukamoto and Ms. Minako Kuwano
for technical assistance. This work is supported by the Ministry of
Education, Culture, Sports, Science and Technology, Japan,
Grants-in-Aid for Scientific Research (C) (25460271,
16 K08716), and Grants-in-Aid for Scientific Research on Innova-
tive Areas “Stem Cell Aging and Disease” (17H05653).

References
1. Miyajima A, Tanaka M, Itoh T (2014) Stem
Progenitor cell sin liver development, homeosta-
sis, regeneration, and reprogramming. Cell Stem
Cell 14:261–274
2. Espanol-Suner R, Carpentier R, Van HN et al
(2012) Liver progenitor cells yield functional
hepatocytes in response to chronic liver injury
in mice. Gastroenterology 143:1564–1575
e1567
3. Malato Y, Naqvi S, Schurmann N et al (2011)
Fate tracing of mature hepatocytes in mouse
liver homeostasis and regeneration. J Clin Invest
121:4850–4860
4. Choi TY, Ninov N, Stainer DY et al (2014)
Extensive conversion of hepatic biliary epithelial
cells to hepatocytes after near total loss of hepa-
tocytes in zebrafish. Gastroenterology
146:776–788
5. Raven A, Lu WY, Man TY et al (2017) Cholan-
giocytes act as facultative liver stem cells during
impaired hepatocyte regeneration. Nature
547:350–354
6. Kamimoto K, Kaneko K, Kok CY et al (2016)
Heterogeneity and stochastic growth regulation
of biliary epithelial cells dictate dynamic epithe-
lial tissue remodeling. Elife 5:e15034
7. Li B, DorrellC CPS et al (2017) Adult mouse
liver contains two distinct populations of cho-
langiocytes. Stem Cell Reports 9:478–489
8. Tanimizu N, Kobayashi S, Ichinohe N et al
(2014) Downregulation of miR122 by
grainyhead-like 2 restricts the hepatocytic differ-
entiation potential of adult liver progenitor cells.
Development 141:4448–4456
 Chapter 3

Identification and Isolation of Clonogenic Cholangiocyte

in Mouse
Bin Li, Craig Dorrell, Pamela S. Canady, and Leslie Wakefield

Abstract
Cholangiocytes are proliferative and are one of the sources for liver progenitor cells. Clonogenic cholan-
giocytes are defined as cells capable of clonally proliferating and differentiating cholangiocytes both in vitro
and in vivo. In this protocol, we describe the method for isolation of primary cholangiocytes from mouse.
To study the heterogeneity of cholangiocytes, we used flow cytometry-based cell sorting to isolate different
subsets of cholangiocytes. Organoid-forming efficiencies from sorted single cells are compared within
different cholangiocyte populations to identify clonogenic cholangiocytes.

Key words Duct cells, ST14, FACS, Organoid

1 Introduction

Liver duct cells, so-called cholangiocytes, are highly proliferative

in vitro and in vivo. In our previous report found in the normal and
injured mouse liver, MIC1-1C3+ liver duct cells are proliferative:
they formed colony in vitro, and gene expression showed MIC1-
1C3+ cells that are expressing Sox9, a progenitor marker [1]. More-
over, MIC1-1C3+ colonies could engraft in the Fah
/
mouse, a
transgenic mouse model for liver injury, and the transplanted liver
cells could replace the damaged host liver [2], indicating the stem/
progenitor capabilities of donor cells. Thus, MIC1-1C3 is used as
an oval cell marker. Lgr5, a member of Wnt signal pathway, has
been previously found as a liver stem cell marker. In contrast to
MIC1-1C3, normal liver doesn’t express Lgr5 [3], whereas, under
chronic liver injury, liver duct cells in mouse become Lgr5+. Those
Lgr5+ cells are proliferative and form liver organoid in vitro. More-
over, transplantation of Lgr5+ organoids could engraft into the
host liver, indicating the stem cell capability [4]. Further report
demonstrated that MIC1-1C3+ duct cells in both normal liver and
pancreas form organoids in vitro [5]. ST14 (suppressor of tumori-
genicity 14 protein) is a marker for pancreatic cancer; however, not

Naoki Tanimizu (ed.), Hepatic Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1905,
https://doi.org/10.1007/978-1-4939-8961-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019

19
20 Bin Li et al.

any research has studied ST14 in the liver. In our previous report,
with the cell surface markers MIC1-1C3 [5] and ST14 [3], we
demonstrated that normal mouse cholangiocytes are heteroge-
neous: ST14hiMIC1-1C3+ cells are highly proliferative; they form
single-cell-derived organoid more efficiently than ST14loMIC1-
1C3+ cholangiocytes [3]. Here we will describe the method for
isolating cholangiocytes via liver perfusion. ST14hiMIC1-1C3+
cells in primary liver non-parenchymal cells (NPCs) are directly
sorted into 96-well plate by fluorescence-activated cell sorting
(FACS). In this format of colony-forming assay, clonogenic cho-
langiocytes form single-cell-derived organoids. In addition, orga-
noids cultured for ~3 months are used for the differentiation assay
examining their bidirectional differentiation potential.

2 Materials

2.1 Liver Perfusion 1. Solution 1: 10 mM HEPES and 0.5 mM EGTA in Earle’s

Balanced Salt Solution (EBSS) without Ca2+/Mg2+, pH 7.4
(~30 mL per mouse).
2. Solution 2: 10 mM HEPES in EBSS with Ca2+/Mg2+, pH 7.4
(~20 mL per mouse).
3. Solution 3: 0.1% collagenase II in solution 2 (~50 mL per
mouse) see Note 1.
4. NPC digestion solution: 2.5 mg/mL collagenase IV and
0.1 mg/mL DNase I in solution 2 (~5 mL per mouse).
5. Wash medium: 10% FCS and 0.1 mg/mL DNase in DMEM.
6. 0.05% trypsin.
7. Phosphate-buffered saline (PBS).
8. 70% ethanol.
9. Cotton applicators (sterile).
10. Intravenous (IV) infusion set.
11. 24-gauge catheters.
12. Peristaltic pump.
13. Forceps, scissors (sterile).
14. Tape.
15. 100 μm cell strainer.
16. 60 mm petri dish.
17. Anesthetic solution.
18. Centrifuge, set at 10  C.
19. Mouse: 8-week-old C57B/L6 male mouse. All animal experi-
mentation was conducted in accordance with protocol
IP00445 of the Institutional Review Committee at Oregon
Health and Science University.
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no related content on Scribd:
 Fig. 220. Fig. 221.

131. Pine.—Fig. 221. Very variable, very light and soft in “soft” pine, such as
white pine; of medium weight to heavy and quite hard in “hard” pine, of which
longleaf or Georgia pine is the extreme form. Usually it is stiff, quite strong, of even
texture and more or less resinous. The sapwood is yellowish white; the heartwood,
orange brown. Pine shrinks moderately, seasons rapidly and without much injury; it
works easily; is never too hard to nail (unlike oak or hickory); it is mostly quite
durable, and if well seasoned is not subject to the attacks of boring insects. The
heavier the wood, the darker, stronger and harder it is, and the more it shrinks and
checks. Pine is used more extensively than any other kind of wood. It is the
principal wood in common carpentry, as well as in all heavy construction, bridges,
trestles, etc. It is used also in almost every other wood industry, for spars, masts,
planks, and timbers in ship building, in car and wagon construction, in cooperage,
for crates and boxes, in furniture work, for toys and patterns, railway ties, water
pipes, excelsior, etc. Pines are usually large trees with few branches, the straight,
cylindrical, useful stem forming by far the greatest part of the tree.
132. Spruce.—Fig. 222. Resembles soft pine, is light, very soft, stiff, moderately
strong, less resinous than pine; has no distinct heartwood, and is of whitish color.
Used like soft pine, but also employed as resonance wood and preferred for paper
pulp. Spruces, like pines, form extensive forests; they are more frugal, thrive on
thinner soils, and bear more shade, but usually require a more humid climate.
“Black” and “white” spruce as applied by lumbermen, usually refer to narrow and
wide ringed forms of black spruce.
 Fig. 222. Fig. 223.

Broad-Leaved Woods.
133. Ash.—Fig. 223. Wood heavy, hard, strong, stiff, quite tough, not durable in
contact with soil, straight grained, rough on the split surface and coarse in texture.
The wood shrinks moderately, seasons with little injury, stands well and takes a
good polish. In carpentry ash is used for finishing lumber, stairways, panels, etc.; it
is used in shipbuilding, in the construction of cars, wagons, carriages, etc., in the
manufacture of farm implements, machinery, and especially of furniture of all
kinds, and also for harness work; for barrels, baskets, oars, tool handles, hoops,
clothespins, and toys. The trees of the several species of ash are rapid growers, of
small to medium height with stout trunks; they form no forests, but occur scattered
in almost all broad-leaved forests.
134. Basswood.—Fig. 224. (Lime tree, American linden, lin, bee tree): Wood
light, soft, stiff but not strong, of fine texture, and white to light brown color. The
wood shrinks considerably in drying, works and stands well; it is used in carpentry,
in the manufacture of furniture and woodenware, both turned and carved, in
cooperage, for toys, also for paneling of car and carriage bodies. Medium to large
sized trees, common in all Northern broad-leaved forests; found throughout the
Eastern United States.
Fig. 224.
 Fig. 225. Fig. 226.

135. Birch.—Fig. 225. Wood heavy, hard, strong, of fine texture; sapwood
whitish, heartwood in shades of brown with red and yellow; very handsome, with
satiny luster, equaling cherry. The wood shrinks considerably in drying, works and
stands well and takes a good polish, but is not durable if exposed. Birch is used for
finishing lumber in building, in the manufacture of furniture, in woodturnery for
spools, boxes, wooden shoes, etc., for shoe lasts and pegs, for wagon hubs, ox
yokes, etc., also in wood carving. The birches are medium sized trees, form
extensive forests northward and occur scattered in all broad-leaved forests of the
Eastern United States.
136. Butternut.—Fig. 226. (White Walnut.) Wood very similar to black walnut,
but light, quite soft, not strong and of light brown color. Used chiefly for finishing
lumber, cabinet work and cooperage. Medium sized tree, largest and most
common in the Ohio basin; Maine to Minnesota and southward to Georgia and
Alabama.
 Fig. 227.

137. Cherry.—Fig. 227. Wood heavy, hard, strong, of fine texture: sapwood
yellowish white, heartwood reddish to brown. The wood shrinks considerably in
drying, works and stands well, takes a good polish, and is much esteemed for its
beauty. Cherry is used chiefly as a decorative finishing lumber for buildings, cars
and boats, also for furniture and for turnery. It is becoming too costly for many
purposes for which it is naturally suited. The lumber-furnishing cherry of this
country, the wild black cherry, is a small to medium sized tree, scattered through
many of the broad-leaved woods of the western slope of the Alleghanies, but
found from Michigan to Florida and west to Texas.
138. Chestnut.—Fig. 228. Wood light, moderately soft, stiff, not strong, of
coarse texture; the sapwood light, the heartwood darker brown. It shrinks and
checks considerably in drying, works easily, stands well, and is very durable. Used
in cabinet work, cooperage, for railway ties, telegraph poles, and locally in heavy
construction. Medium sized tree very common in the Alleghanies, occurs from
Maine to Michigan and southward to Alabama.
 Fig. 228. Fig. 229.

139. Elm.—Fig. 229. Wood heavy, hard, strong, very tough; moderately durable
in contact with the soil; commonly cross-grained, difficult to split and shape, warps
and checks considerably in drying, but stands well if properly handled. The broad
sapwood whitish, heart brown, both shades of gray and red; on split surface rough,
texture coarse to fine, capable of high polish. Elm is used in the construction of
cars, wagons, etc., in boat and ship building, for agricultural implements and
machinery; in rough cooperage, saddlery, and harness work, but particularly in the
manufacture of all kinds of furniture, where the beautiful figures, especially of the
tangential or bastard section, are just beginning to be duly appreciated. The elms
are medium to large sized trees, of fairly rapid growth, with stout trunk, form no
forests of pure growth, but are found scattered in all the broad-leaved woods of our
country.
140. Gum.—This general term refers to two kinds of wood usually distinguished
as sweet or red gum, and sour, black, or tupelo gum, the former being a relative of
the witch-hazel, the latter belonging to the dogwood family.
Sweet Gum. Fig. 230. (red gum, liquidambar); Wood rather heavy, rather soft,
quite stiff and strong, tough, commonly cross-grained, of fine texture; the broad
sapwood whitish, the heartwood reddish brown; the wood warps and shrinks
considerably, but does not check badly, stands well when fully seasoned, and
takes good polish. Sweet gum is used in carpentry, in the manufacture of furniture,
for cut veneer, for wooden plates, plaques, baskets, etc., also for wagon hubs, hat
blocks, etc. A large sized tree, very abundant, often the principal tree in the
swampy parts of the bottoms of the Lower Mississippi Valley; occurs from New
York to Texas and from Indiana to Florida.
 Fig. 230. Fig. 231.

141. Hickory.—Fig. 231. Wood very heavy, hard and strong, proverbially tough,
of rather coarse texture, smooth and of straight grain. The broad sapwood white,
the heart reddish nut brown. It dries slowly, shrinks and checks considerably, is not
durable in the ground, or if exposed, and, especially the sapwood, is always
subject to the inroads of boring insects. Hickory excels as carriage and wagon
stock, but is also extensively used in the manufacture of implements and
machinery, for tool handles, timber pins, for harness work and cooperage. The
hickories are tall trees with slender stems, never form forests, occasionally small
groves, but usually occur scattered among other broad-leaved trees in suitable
localities.
Hickory excels as carriage and wagon stock, but is also extensively used in the
manufacture of implements and machinery, for tool handles, timber pins, for
harness work and cooperage. The hickories are tall trees with slender stems,
never form forests, occasionally small groves, but usually occur scattered among
other broad-leaved trees in suitable localities.
142. Maple.—Fig. 232. Wood heavy, hard, strong, stiff, and tough, of fine
texture, frequently wavy grained, thus giving rise to “curly” and blister” figures; not
durable in the ground or otherwise exposed. Maple is creamy white, with shades of
light brown in the heart; shrinks moderately, seasons, works and stands well,
wears smoothly and takes fine polish. The wood is used for ceiling, flooring,
paneling, stairway and other finishing lumber in house, ship and car construction; it
is used for the keels of boats and ships, in the manufacture of implements and
machinery, but especially for furniture, where entire chamber sets of maple rival
those of oak. Maple is also used for shoe lasts and other form blocks, for shoe
pegs, for piano actions, school apparatus, for wood type in show bill printing, tool
handles, wood carving, turnery and scroll work.

Fig. 232.

The maples are medium sized trees, of fairly rapid growth; sometimes form
forests and frequently constitute a large proportion of the arborescent growth.
143. Oak.—Fig. 233. Wood very variable, usually very heavy and hard, very
strong and tough, porous, and of coarse texture; the sapwood whitish, the heart
“oak” brown to reddish brown. It shrinks and checks badly, giving trouble in
seasoning, but stands well, is durable and little subject to attacks of insects. Oak is
used for many purposes; in shipbuilding, for heavy construction, in common
carpentry, in furniture, car and wagon work, cooperage, turnery, and even in wood
carving; also in the manufacture of all kinds of farm implements, wooden mill
machinery, for piles and wharves, railway ties, etc. The oaks are medium to large
sized trees, forming the predominant part of a large portion of our broad-leaved
forests, so that these are generally “oak forests” though they always contain a
considerable proportion of other kinds of trees. Three well marked kinds, white,
red, and live oak are distinguished and kept separate in the market. Of the two
principal kinds, white oak is the stronger, tougher, less porous, and more durable.
Red oak is usually of coarser texture, more porous, often brittle, less durable, and
even more troublesome in seasoning than white oak. In carpentry and furniture
work, red oak brings about the same price at present as white oak. The red oaks
everywhere accompany the white oaks, and like the latter, are usually represented
by several species in any given locality. Live oak, once largely employed in
shipbuilding, possesses all the good qualities (except that of size) of the white oak,
even to a greater degree. It is one of the heaviest, hardest and most durable
building timbers of this country; in structure it resembles the red oak but is much
less porous.

Fig. 233.

144. Sycamore.—Fig. 234 (button wood, button-ball tree, water beech): Wood
moderately heavy, quite hard, stiff, strong, tough, usually crossgrained, of coarse
texture, and white to light brown color; the wood is hard to split and work, shrinks
moderately, warps and checks considerably but stands well. It is used extensively
for drawers, backs, bottoms, etc., in cabinetwork, for tobacco boxes, in cooperage,
and also for finishing lumber, where it has too long been underrated. A large tree,
of rapid growth, common and largest in the Ohio and Mississippi valleys, at home
in nearly all parts of the eastern United States.

Fig. 234. Fig. 235.

145. Tulip Wood.—Fig. 235. Tulip tree. (yellow poplar, white wood): Wood quite
variable in weight, usually light, soft, stiff but not strong, of fine texture, and
yellowish color; the wood shrinks considerably, but seasons without much injury;
works and stands remarkably well. Used for siding, for paneling, and finishing
lumber in house, car and shipbuilding, for sideboards and panels of wagons and
carriages; also in the manufacture of furniture, implements and machinery, for
pump logs, and almost every kind of common woodenware, boxes, shelving,
drawers, etc. An ideal wood for the carver and toy man. A large tree, does not form
forests, but is quite common, especially in the Ohio basin; occurs from New
England to Missouri and southward to Florida.
146. Walnut.—Fig. 236. Black Walnut. Wood heavy, hard, strong, of coarse
texture; the narrow sapwood whitish, the heartwood chocolate brown. The wood
shrinks moderately in drying, works and stands well, takes a good polish, is quite
handsome, and has been for a long time the favorite cabinet wood in this country.
Walnut formerly used, even for fencing, has become too costly for ordinary uses,
and is to-day employed largely as a veneer, for inside finish and cabinet work, also
for turnery, for gunstocks, etc. Black walnut is a large tree, with stout trunk, of rapid
growth, and was formerly quite abundant throughout the Alleghany region,
occurring from New England to Texas, and from Michigan to Florida.
Fig. 236.
 CHAPTER XIII.
Wood Finishing.

147. Wood Finishes.—Finishes are applied to wood surfaces (1)

that the wood may be preserved, (2) that the
appearance may be enhanced.
Finishing materials may be classed under one or the other of the
following: Filler, stain, wax, varnish, oil, paint. These materials may
be used singly upon a piece of wood or they may be combined in
various ways to produce results desired.
148. Brushes.—Good brushes are made of bristles of the wild
boar of Russia and China. These bristles are set in
cement and are firmly bound by being wrapped with wire in round
brushes or enclosed in metal in flat brushes. Fig. 237.
 Fig. 237. Fig. 238.

A large brush, called a duster, is used for removing dust or loose

dirt from the wood, Fig. 238. Small brushes, used for tracing, usually
have chiseled edges, Fig. 239.
Bristle brushes are expensive and should be well cared for.
Brushes that have been used in shellac and are not soon to be used
again should be cleaned by rinsing them thoroughly in a cup of
alcohol. This alcohol may be used later for thinning shellac.
 Fig. 239.

Varnish and paint brushes should be cleaned in turpentine. If they

are to be laid away for some time, a strong soap suds, or lather
made from some of the soap powders, should be well worked into
the brush, after the preliminary cleansing. It should then be carefully
pressed into proper shape and laid away flat on a shelf. When the
brush is to be used again, it should first be washed out, to get rid of
all the soap.
Brushes that are used from day to day should be kept suspended,
when not in use, as in Fig. 240, so that their bristles shall be kept
moist, without their touching the bottom of the bucket or can.
 Fig. 240. Fig. 241.

Since alcohol evaporates rapidly, shellac cans with cone tops

should be used or, better, a can in which the brush handle may
extend through the top.
Fig. 241 shows a can which is made double. Varnish is kept in the
inner portion and water in the outer ring. The cover fits over the inner
can and into the water space, thus sealing the varnish air-tight but
removing all danger of the cover’s sticking to the sides of the can.
The brush is suspended from the “cleaning wire” so that its bristles
rest in the liquid.
If delicate woods are to be varnished, stone or glass jars would
better be used to hold the liquid, for metal discolors it slightly.
 Fig. 242. Fig. 243.

149. General Directions for Using Brush.—(1) Hold the brush

as in Fig. 242. (2) Dip
the end of the brush in the liquid to about one-third the length of the
bristles. (3) Wipe off the surplus liquid on the edge of the can, wiping
both sides of the brush no more than is necessary to keep the liquid
from dripping. A wire stretched across the can as in Fig. 243
provides a better wiping place for the dripping brush. In wiping the
brush on the edge of the can, some of liquid is likely to “run” down
the outside. (4) Using the end of the brush, apply the liquid near one
end of the surface to be covered. (5) “Brush” in the direction of the
grain. (6) Work towards and out over the end of the board, leveling
the liquid to a smooth film of uniform thinness. The strokes should be
“feathered,” that is, the brush should be lowered gradually at the
beginning of the sweep and raised gradually at the close, otherwise,
ugly “laps” will result. The reason for working out over the ends
rather than from them will appear with a little thought. (7) Now work
toward the second end. The arrows, Fig. 244, show the general
directions of the final or feathering strokes.

Fig. 244.

Edges are usually covered first and adjoining surfaces afterward.

It frequently happens that surplus liquid runs over a finished
surface, especially when working near the arrises. This surplus can
be “picked up” by wiping the brush upon the wire of the bucket until
the bristles are quite free of liquid, and giving the part affected a
feathering sweep.
If the object has an internal corner, work from that out over the
neighboring surfaces.
Panels and sunk places should be covered first. Afterward, the
raised places, such as stiles, rails, etc., may be attended to.
Wherever possible the work should be laid flat so that the liquid may
be flowed on horizontally. This is of especial advantage in
varnishing. Vertical work should always be begun at the top and
carried downward.
 Tracing consists in working a liquid up to a given line but not over
it, such as painting the sash of a window. Tracing requires a steady
hand and some practice. A small brush is generally used and the
stroke is made as nearly continuous as the flow of the liquid will
allow. Fig. 245.

Fig. 245.

150. Fillers.—Fillers are of two kinds, paste and liquid. They are
used to fill up the wood pores and thus give a smooth,
level, non-absorbent surface, upon which other coverings may be
placed. Paste fillers are for use upon coarse grained woods such as
oak and chestnut, while liquid fillers are for close grained woods
such as Georgia pine.
 Fillers are not a necessity, especially the liquid, but the saving
affected by their use is considerable. Not only are they cheaper than
varnish but one or two coats of filler will take the place and permit a
saving of two or three coats of the more expensive material.
Liquid filler should be applied evenly with a brush and allowed to
dry twenty-four hours, after which it may be sanded smooth with No.
00 paper. It is used mainly upon large work such as porch ceilings
and interior finish, like Georgia pine. On fine cabinet work, one or
two coats of thin white shellac is used as a filler upon close grained
wood. Shellac forms a surface which after twenty-four hours, can be
sandpapered so as to make a very smooth surface. Varnish applied
to the bare wood has a tendency to darken and discolor it. Filling
with shellac preserves the natural color.
Paste filler is sold by the pound in cans of various sizes. The best
fillers are made of ground rock crystal mixed with raw linseed oil,
japan and turpentine.
For preserving the natural color of the wood, filler is left white; for
Flemish, it is colored brown; for antique and weathered finishes, it is
dark. Fillers can be purchased ready colored.
151. Filling with Paste Filler.—(1) Thin the filler with turpentine
until it makes a thin paste. (2) With a
stiff-bristled brush, force the filler into the pores of the wood and
leave the surface covered with a thin coating. (3) Allow this to stand
until the filler has “flatted,” that is, until the “gloss” has disappeared
and the filler becomes dull and chalkish. The time required for this to
take place varies. Twenty minutes is not unusual. (4) Rub the filler off
just as soon as it has flatted—do not let it stand longer, for the longer
it stands the harder it is to remove. Rub across the grain as much as
is possible, using a wad of excelsior. Finish fine work by going over it
a second time with a cloth, rubbing with the grain as well as across,
that the “high lights” may be clear of filler.
On fine work use a felt pad to rub the filler into the pores, and rub
off with a cloth only.
Twenty-four hours should be allowed the filler to harden. One
filling is sufficient for ordinary work; on fine work the above process
is sometimes repeated after the first filling has hardened.
 The striking contrasts in the grain of wood such as oak and
chestnut, obtained by the use of colored fillers, are due to the dark
filler’s remaining in the open grain but being wiped off of the close
grain—the “high lights.”
On quarter-sawed oak, each flake is sometimes sanded with fine
paper, No. 00, to remove the stain that the contrast may be sharper.
Excelsior and rags used in cleaning off filler must not be allowed to
lie around but must be burned for they are subject to spontaneous
combustion and are dangerous.
152. Stains.—Stains are used to darken the high lights of wood
preparatory to the application of a relatively darker
filler. By varying the intensity of the stain different results may be
obtained with the same color of filler. Stains are also used without
fillers.
There are three kinds of stains: (1) water, (2) oil, (3) spirit. Each
kind has its advantages and its disadvantages.
Wood stains are cheap, penetrate the wood deeply, and are
transparent. They cause the grain of the wood to “rough up,”
however, and for this reason are used mainly upon hard woods
which require darkening before the application of a filler. The wood is
sanded before the filler is applied. Where water stain is not to be
followed by filler, it is customary to thoroughly moisten the surface to
be covered with water alone. After this has dried, the surface is
sanded with fine paper and the stain applied. The stain does not
raise the grain as it otherwise would.
Water stains may be applied with a brush or a sponge. They are
sometimes heated that they may enter the wood more deeply. Any
coloring matter that can be dissolved in water will make a wood dye
or stain.
Oil stains, like water stains, are often used to stain wood before
filling. They are more generally used where no filling is desired. They
are easier to apply evenly than water or spirit stains. They do not
raise the grain of the wood like the other stains. On the other hand,
they do not penetrate and therefore cannot color hard woods dark.
Neither do they give the clear effects.
Most oil stains are applied with a brush, after which the surface of
the wood is immediately wiped clean with a cloth.