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Hematopoietic Stem Cell Protocols 1st Edition Kevin D.
Bunting Digital Instant Download
Author(s): Kevin D. Bunting, Cheng-Kui Qu (eds.)
ISBN(s): 9781493911325, 1493911325
Edition: 1
File Details: PDF, 7.40 MB
Year: 2014
Language: english
Methods in
Molecular Biology 1185
Kevin D. Bunting
Cheng-Kui Qu Editors
Hematopoietic
Stem Cell
Protocols
Third Edition
METHODS IN M O L E C U L A R B I O LO G Y
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Hematopoietic Stem Cell
Protocols
Third Edition
Edited by
Kevin D. Bunting and Cheng-Kui Qu

Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Emory University School of Medicine,
Atlanta, GA, USA
Editors
Kevin D. Bunting Cheng-Kui Qu
Department of Pediatrics Department of Pediatrics
Aflac Cancer and Blood Disorders Center Aflac Cancer and Blood Disorders Center
Emory University School of Medicine Emory University School of Medicine
Atlanta, GA, USA Atlanta, GA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-4939-1132-5 ISBN 978-1-4939-1133-2 (eBook)
DOI 10.1007/978-1-4939-1133-2
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2014942724
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Preface
The hematopoietic stem cell (HSC) field has continued to grow and become more sophis-
ticated with each passing year. Methods for isolation of HSC and progenitor subsets and a
variety of single cell and molecular technologies have led to a greater understanding of the
heterogeneity within the phenotypically defined HSC subsets. The first two editions of
Hematopoietic Stem Cell Protocols are very thorough resources in the user-friendly Methods
in Molecular Medicine format. This is the third edition of Hematopoietic Stem Cell Protocols
and aims to provide timely protocols in the HSC field while continuing the tradition. This
edition teaches major new technologies that have advanced the state of the art in the field
and promise to drive research in many unexpected and exciting ways in the future.
The first chapter is an overview that ties the other chapters together into a larger picture
of the HSC landscape.
The methods chapters include similar categories to the last editions but with entirely
new approaches and innovative insights. The author list is made up of leading researchers
who have made major contributions toward these technical advances and who have pro-
vided a much needed resource for new stem cell investigators.
We thank all of the contributors for their time and effort. It has been a pleasure for both
co-editors to work with the contributing authors on this project. To provide updates in the
fast paced HSC field is an important endeavor. We have specifically focused on the HSC
population and did not focus directly on the stem cell-associated niche which comprises a
new field in itself and deserving of a separate edition. We are delighted to have organized
this information into a comprehensive and essential resource. It is our hope that this
resource will be a critical addition to all laboratories seeking to isolate and characterize
HSCs for research and for therapeutic applications.
Atlanta, GA, USA Kevin D. Bunting, Ph.D.

Cheng-Kui Qu, M.D., Ph.D.
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I OVERVIEW
1 The Hematopoietic Stem Cell Landscape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
PART II STEM CELL ENRICHMENT AND HETEROGENEITY

2 Investigating the Interaction Between Hematopoietic Stem Cells
and Their Niche During Embryonic Development: Optimizing
the Isolation of Fetal and Newborn Stem Cells From Liver, Spleen,
and Bone Marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Huimin Cao, Brenda Williams, and Susan K. Nilsson
3 Single-Cell PCR Profiling of Gene Expression in Hematopoiesis . . . . . . . . . . . . 21
José Teles, Tariq Enver, and Cristina Pina
4 Hematopoietic Stem and Progenitor Cell Mobilization in Mice . . . . . . . . . . . . . 43
Jonathan Hoggatt, Tiffany A. Tate, and Louis M. Pelus
5 Cell Cycle Measurement of Mouse Hematopoietic
Stem/Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Brahmananda Reddy Chitteti and Edward F. Srour
6 Flow Cytometric Analysis of Signaling and Apoptosis
in Hematopoietic Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Lei Dong and Cheng-Kui Qu
PART III MOLECULAR AND CELLULAR CHARACTERIZATION

7 Gene Expression Profiling of Hematopoietic Stem Cells (HSCs) . . . . . . . . . . . . 91
Nalini Raghavachari
8 Measuring MicroRNA Expression in Mouse Hematopoietic Stem Cells. . . . . . . 121
Wenhuo Hu and Christopher Y. Park
9 DNA Methylation Profiling of Hematopoietic Stem Cells . . . . . . . . . . . . . . . . . 141
Amber Hogart Begtrup
10 Metabolic Characterization of Hematopoietic Stem Cells . . . . . . . . . . . . . . . . . 155
Fatih Kocabas, Junke Zheng, Chengcheng Zhang,
and Hesham A. Sadek
11 Nanoproteomic Assays on Hematopoietic Stem Cells . . . . . . . . . . . . . . . . . . . . 165
Heath L. Bradley, Himalee Sabnis, Deborah Pritchett,
and Kevin D. Bunting
vii
viii Contents
PART IV IN VITRO ASSAYS AND DIFFERENTIATION

12 Hematopoietic Differentiation of Pluripotent Stem Cells in Culture . . . . . . . . . 181
Jason A. Mills, Prasuna Paluru, Mitchell J. Weiss,
Paul Gadue, and Deborah L. French
13 Ex Vivo Assays to Study Self-Renewal, Long-Term Expansion,
and Leukemic Transformation of Genetically Modified Human
Hematopoietic and Patient-Derived Leukemic Stem Cells . . . . . . . . . . . . . . . . . 195
Pallavi Sontakke, Marco Carretta, Marta Capala,
Hein Schepers, and Jan Jacob Schuringa
14 Ex Vivo Expansion of Murine and Human Hematopoietic Stem Cells . . . . . . . . 211
Phuong L. Doan and John P. Chute
15 Using Microfluidics to Investigate Hematopoietic Stem Cell
and Microniche Interactions at the Single Cell Level . . . . . . . . . . . . . . . . . . . . . 223
Byungwook Ahn, Zhengqi Wang, David R. Archer,
and Wilbur A. Lam
PART V TRANSPLANTATION ASSAYS AND IMAGING ENGRAFTMENT

16 Five-Lineage Clonal Analysis of Hematopoietic Stem/Progenitor Cells . . . . . . . 237
Ryo Yamamoto, Yohei Morita, and Hiromitsu Nakauchi
17 Intravital Imaging of Hematopoietic Stem Cells in the Mouse Skull. . . . . . . . . . 247
Juwell W. Wu, Judith M. Runnels, and Charles P. Lin
18 Immunodeficient Mouse Model for Human Hematopoietic
Stem Cell Engraftment and Immune System Development . . . . . . . . . . . . . . . . 267
Ken-Edwin Aryee, Leonard D. Shultz, and Michael A. Brehm
19 Homing and Migration Assays of Hematopoietic Stem/Progenitor Cells. . . . . . 279
Xi C. He, Zhenrui Li, Rio Sugimura, Jason Ross, Meng Zhao,
and Linheng Li
PART VI GENETIC MODIFICATION

20 Retroviral Transduction of Murine and Human Hematopoietic
Progenitors and Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Marioara F. Ciuculescu, Christian Brendel, Chad E. Harris,
and David A. Williams
21 Lentiviral Gene Transduction of Mouse and Human Hematopoietic
Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Niek P. van Til and Gerard Wagemaker
22 High-Throughput Genomic Mapping of Vector Integration Sites
in Gene Therapy Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Brian C. Beard, Jennifer E. Adair, Grant D. Trobridge,
and Hans-Peter Kiem
23 Barcoded Vector Libraries and Retroviral or Lentiviral Barcoding
of Hematopoietic Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Leonid V. Bystrykh, Gerald de Haan, and Evgenia Verovskaya
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Contributors
JENNIFER E. ADAIR • Fred Hutchinson Cancer Research Center, University of Washington

School of Medicine, Seattle, WA, USA
BYUNGWOOK AHN • Department of Pediatrics, Aflac Cancer and Blood Disorders Center,
Emory University School of Medicine, Atlanta, GA, USA
DAVID R. ARCHER • Division of Hem/Onc/BMT, Department of Pediatrics, Aflac Cancer
and Blood Disorders Center, Emory University School of Medicine, Atlanta, GA, USA
KEN-EDWIN ARYEE • Program in Molecular Medicine, University of Massachusetts Medical
School, Worcester, MA, USA
BRIAN C. BEARD • Fred Hutchinson Cancer Research Center, Seattle, WA, USA
AMBER H. BEGTRUP • Division of Human Genetics, Department of Pediatrics, College of
Medicine, Cincinnati Children’s Research Hospital Medical Center, Cincinnati, OH, USA
HEATH L. BRADLEY • Division of Hem/Onc/BMT, Department of Pediatrics, Aflac Cancer
MICHAEL A. BREHM • Program of Molecular Medicine, University of Massachusetts Medical
School, Worcester, MA, USA
CHRISTIAN BRENDEL • Boston Children’s Hospital and Dana Farber Cancer Institute,
Harvard Medical School, Boston, MA, USA
KEVIN D. BUNTING • Department of Pediatrics, Aflac Cancer and Blood Disorders Center,
LEONID V. BYSTRYKH • Laboratory of Ageing Biology and Stem Cells, European Research
Institute for the Biology of Ageing, University Medical Centre Groningen, University
of Groningen, Groningen, The Netherlands
HUIMIN CAO • CMSE, Commonwealth Scientific and Industrial Research Organization
(CSIRO), Clayton, Australia
MARTA CAPALA • Department of Experimental Hematology, University Medical Center
Groningen, University of Groningen, Groningen, The Netherlands
MARCO CARRETTA • Department of Experimental Hematology, University Medical Center
BRAHMANANDA REDDY CHITTETI • Indiana University School of Medicine, Indianapolis,
IN, USA
JOHN P. CHUTE • Division of Hem/Onc, Department of Medicine, University of California
Los Angeles, Los Angeles, USA
MARIOARA F. CIUCULESCU • Boston Children’s Hospital and Dana Farber Cancer Institute,
PHUONG L. DOAN • Division of Hematologic Malignancies and Cellular Therapy, Duke
University Medical Center, Durham, NC, USA
LEI DONG • Division of Hem/Onc/BMT, Department of Pediatrics, Aflac Cancer
and Blood Disorders Center, Emory, University School of Medicine, Atlanta, GA, USA
TARIQ ENVER • Stem Cell Laboratory, University College London Cancer Institute,
London, UK
ix
x Contributors
DEBORAH L. FRENCH • Department of Pathology and Laboratory Medicine and Center

for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia,
Philadelphia, PA, USA
PAUL GADUE • Department of Pathology and Laboratory Medicine and Center for
Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia,
GERALDDE HAAN • Laboratory of Ageing Biology and Stem Cells, European Research
CHAD E. HARRIS • Boston Children’s Hospital and Dana Farber Cancer Institute,
XI C. HE • Stowers Institute for Medical Research, Kansas City, MO, USA
JONATHAN HOGGATT • Department of Stem Cell and Regenerative Biology, Harvard
University, Cambridge, MA, USA
WENHUO HU • Human Oncology and Pathogenesis Program, Memorial Sloan Kettering
Cancer Center, New York, NY, USA
HANS-PETER KIEM • Departments of Medicine and Pathology, Program in Transplantation
Biology, Fred Hutchinson Cancer Research Center, University of Washington School
of Medicine, Seattle, WA, USA
FATIH KOCABAS • Department of Education, and Texas Institute of Biotechnology,
Education and Research, North American University, Houston, TX, USA
WILBUR A. LAM • Division of Hem/Onc/BMT, Department of Pediatrics, Aflac Cancer
ZHENRUI LI • Department of Pathology and Laboratory Medicine, University of Kansas
Medical Center, Kansas City, KS, USA
LINHENG LI • Investigator, Stowers Institute for Medical Research, Kansas City, MO, USA;
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center,
Kansas City, KS, USA
CHARLES P. LIN • Center for Systems Biology and Wellman Center for Photomedicine,
Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
JASON A. MILLS • Department of Pathology and Laboratory Medicine and Center for
Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia,
YOHEI MORITA • Division of Stem Cell Therapy, Center for Stem Cell Biology
and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo,
Tokyo, Japan
HIROMITSU NAKAUCHI • Division of Stem Cell Therapy, Center for Stem Cell Biology and
Regenerative Medicine, The Institute of Medical Science, The University of Tokyo,
Tokyo, Japan
SUSAN K. NILSSON • CMSE, Commonwealth Scientific and Industrial Research
Organization (CSIRO), Clayton, Australia
PRASUNA PALURU • Department of Pathology and Laboratory Medicine and Center
for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia,
CHRISTOPHER Y. PARK • Human Oncology and Pathogenesis Program, Departments
of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center,
New York, NY, USA
Contributors xi
LOUIS M. PELUS • Department of Microbiology and Immunology and the Walther Oncology
Center, Indiana University School of Medicine, and the Walther Cancer Institute,
Indianapolis, IN, USA
CRISTINA PINA • Stem Cell Laboratory, University College London Cancer Institute,
London, UK
DEBORAH PRITCHETT • ProteinSimple, Richmond, VA, USA
CHENG-KUI QU • Department of Pediatrics, Aflac Cancer and Blood Disorders Center,
NALINI RAGHAVACHARI • Division of Geriatrics and Clinical Gerontology, National
Institute on Aging, Bethesda, MD, USA
JASON ROSS • Sanford Consortium for Regenerative Medicine, University of California,
San Diego, La Jolla, CA, USA
JUDITH M. RUNNELS • Center for Systems Biology and Wellman Center for Photomedicine,
HIMALEE SABNIS • Division of Hem/Onc/BMT, Department of Pediatrics, Aflac Cancer
HESHAM A. SADEK • Department of Internal Medicine, Division of Cardiology, University
of Texas Southwestern Medical Center, Dallas, TX, USA
HEIN SCHEPERS • Department of Experimental Hematology, University Medical Center
JAN JACOB SCHURINGA • Department of Experimental Hematology, University Medical
Center Groningen, University of Groningen, Groningen, The Netherlands
LEONARD D. SHULTZ • The Jackson Laboratory, Bar Harbor, ME, USA
PALLAVI SONTAKKE • Department of Experimental Hematology, University Medical Center
EDWARD F. SROUR • Indiana University School of Medicine, Indianapolis, IN, USA
RIO SUGIMURA • Stowers Institute for Medical Research, Kansas City, MO, USA
TIFFANY A. TATE • Department of Stem Cell and Regenerative Biology,
Harvard University, Cambridge, MA, USA
JOSÉ TELES • Stem Cell Laboratory, University College London Cancer Institute, London, UK;
Computational Biology and Biological Physics - Department of Astronomy and
Theoretical Physics, Lund University, Lund, Sweden
NIEK P VAN TIL • Department of Hematology, Erasmus University Medical Center,
Rotterdam, The Netherlands
GRANT D. TROBRIDGE • Washington State University, Pullman, WA, USA
EVGENIA VEROVSKAYA • Laboratory of Ageing Biology and Stem Cells, European Research
GERARD WAGEMAKER • Hospital Pharmacy, Erasmus University Medical Center,
Rotterdam, The Netherlands
ZHENGQI WANG • Department of Pediatrics, Aflac Cancer and Blood Disorders Center,
MITCHELL J. WEISS • Department of Pediatrics, The Children’s Hospital of Philadelphia,
BRENDA WILLIAMS • CMSE, Commonwealth Scientific and Industrial Research
Organization (CSIRO), Clayton, Australia
xii Contributors
DAVID A. WILLIAMS • Boston Children’s Hospital and Dana Farber Cancer Institute,
JUWELL W. WU • Center for Systems Biology and Wellman Center for Photomedicine,
RYO YAMAMOTO • Division of Stem Cell Therapy, Center for Stem Cell Biology
and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo,
Tokyo, Japan
CHENGCHENG ZHANG • Departments of Physiology and Developmental Biology,
University of Texas Southwestern Medical Center, Dallas, TX, USA
MENG ZHAO • Stowers Institute for Medical Research, Kansas City, MO, USA
JUNKE ZHENG • Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry
of Education, Shanghai Jiao-Tong University School of Medicine, Shangha, China
Part I
Overview
Chapter 1
The Hematopoietic Stem Cell Landscape

Abstract
Hematopoietic stem cells (HSCs) play critical roles in regulating normal blood cell development. Although
initially these cells were mysterious and difficult to study in isolation, those obstacles have progressively
been rolled away in just a few decades to reveal a heterogeneity of repopulating activity, cell proliferation,
and energy metabolism within defined stem cell populations based on drug transporter and cell surface
marker expression. A wide range of new technologies have driven innovative discovery of the regulators of
HSCs and continued to move the field forward toward a full view of the landscape of single HSCs at the
gene and protein levels. It is the goal of this overview chapter to summarize the array of techniques
included in the third edition of Hematopoietic Stem Cell Protocols which will aid investigators in the field.
Key words Hematopoietic stem cell, Flow cytometry, Embryonic stem cell, Retroviral vector,
Hematology
1 Overview
The hematopoietic stem cell (HSC) has been of great interest for
many decades due to the proven therapeutic potential. Limitations
in the number of HSCs have always been the main drawback to a
wider application of HSC-based therapies. Therefore, methods to
better enrich these cells and to understand their biology have risen
to the forefront of the field in an attempt to increase either the
number in vitro or their repopulating capacity in vivo following
transplantation. It is the goal of this chapter to briefly summarize
the major techniques that are covered in this book. Rapid advances
in our understanding of the biology of HSCs are driven by these
techniques which extend well beyond the basic handling and
manipulation of HSCs and progenitors.
Kevin D. Bunting and Cheng-Kui Qu (eds.), Hematopoietic Stem Cell Protocols, Methods in Molecular Biology,
vol. 1185, DOI 10.1007/978-1-4939-1133-2_1, © Springer Science+Business Media New York 2014
3
4 Kevin D. Bunting and Cheng-Kui Qu
2 Stem Cell Enrichment and Heterogeneity
There are multiple sources of HSCs for experimental study including

fetal liver and adult bone marrow, and it is thus important to be
able to accurately separate the HSCs from their microenvironment
and isolate them for further characterization. This is especially dif-
ficult in embryonic development as the niche(s) are not defined to
the degree that they are in adult bone marrow. Cao et al. (Chapter 2)
summarize their technique for isolating fetal and newborn HSCs
from various tissues. The location from which HSCs are isolated
makes a big difference in the preservation of the self-renewal versus
differentiation profile. Increased lymphoid lineage priming has
been described in fetal liver versus bone marrow HSC using a tech-
nique called single-cell PCR. In the chapter by Teles et al. (Chapter 3),
the single-cell PCR profiling technique is described in great detail
in order to permit the investigator to assess the heterogeneity of
the isolated HSC population.
Another measure of the quality of the isolated HSCs is mea-
surement of the functional characteristics. These are more classi-
cally defined phenotypes including cell cycle status and survival.
Furthermore, the ability to mobilize HSCs back out of the niche is
clinically very important for isolation of normal HSCs for trans-
plantation and may also be very important for stimulating dormant
leukemic stem cells into the circulation where they are more vul-
nerable to chemotherapy. The chapter by Hoggatt et al. (Chapter 4)
describes the techniques for HSC mobilization in mouse models
and offers tips and advice on how to make these experiments more
consistent and reproducible. Likewise, the chapters by Chitteti
et al. (Chapter 5) and Dong et al. (Chapter 6) provide advanced
flow cytometry-based assays for characterizing the response of
HSCs to extracellular cues derived from the niche.
3 Molecular and Cellular Characterization of HSCs
Efforts to better understand HSC biology have been catalyzed by

the advent of technologies capable of analyzing the whole genome.
In the chapter by Raghavachari (Chapter 7) the technique of gene
expression profiling is described, with emphasis on the coding
region-specific mRNAs. In addition, regulation of gene expression
secondary to changes in microRNA (miR) and DNA methylation
changes is highlighted in the chapters by Park (Chapter 8) and
Begtrup (Chapter 9). Once expressed, the critical biology occurs at
the protein level, and thus proteomic based assays are required to
discover changes in protein expression or posttranslational modifi-
cation. Kocabas et al. (Chapter 10) describe how to characterize
metabolic changes in HSCs using flow cytometry-based techniques.
The Hematopoietic Stem Cell Landscape 5
The chapter by Bradley et al. (Chapter 11) describes how the

NanoPro 1000 instrument can be applied to the study of small
numbers of flow cytometry-sorted hematopoietic stem/progenitor
cells in normal and leukemic hematopoiesis.
4 In Vitro Assays and Differentiation
Ultimately it is functional analyses that are critical for defining

newly identified and isolated stem cell subpopulations. The chapter
by Mills et al. (Chapter 12) starts back at the reprogramming stage
in the development of induced pluripotent stem cells and teaches
how to push these cells toward hematopoietic fates. Most commonly,
studies of normal or leukemic stem cell populations start with cells that
are already hematopoietic committed. Sontakke et al. (Chapter 13)
describe how to study leukemic transformation of HSCs by using
patient-derived tissues in long-term expansion culture and also
incorporation of genetic modifications in order to develop useful
animal models of human leukemia. Another major goal in the field
is the ex vivo expansion of normal HSCs for the purpose of trans-
plantation. This goal requires the HSCs to self-renew and maintain
their ability to home and engraft into the appropriate niche.
Endothelium constitutes one of the key niche components that
have been characterized in recent years. Doan et al. (Chapter 14)
describe methods to isolate and expand HSCs based on culture
with an endothelial cell-derived factor called pleiotrophin. Likewise,
Aha et al. (Chapter 15) demonstrate that HSCs can be cultured
with endothelial cells using microfluidic engineering technology as
a novel method for achieving HSC expansion in vitro.
5 Transplantation Assays and Imaging Engraftment
Transplantation is the gold standard assay for normal and leukemic

stem cells. The chapter by Yamamoto et al. (Chapter 16) provides
a new method for clonal analysis of transplanted HSCs in recipient
mice. The localization of HSCs near bone and osteoblasts was one
of the first descriptions of HSC/niche interactions. The chapter by
Wu et al. (Chapter 17) provides key methods for in vivo intravital
imaging of HSC engraftment in the mouse skull. Furthermore,
human HSC engraftment requires an immunodeficient host in
order to achieve engraftment. Aryee et al. (Chapter 18) provide
the method for efficient immune system development following
transplantation of human HSCs into mice. He et al. (Chapter 19)
demonstrate methods for studying the homing and lodgement of
HSCs using in vivo imaging of murine HSCs expressing a Scl-
tTA:H2B-GFP reporter.
6 Kevin D. Bunting and Cheng-Kui Qu
6 Genetic Modification
The final series of chapters in this book highlight the next generation
of methods for genetic modifications of HSCs for further biology
studies and also for therapeutic applications in gene therapy.
Ciuculescu et al. (Chapter 20) highlight the key methods for intro-
ducing retroviral vectors into mouse and human HSCs. Van Til
et al. (Chapter 21) provide a similar focus but rather using the
lentiviral vector system which is more complicated but has a number
of advantages. Beard et al. (Chapter 22) describe high-throughput
genomic mapping of vector integration sites in gene therapy studies.
Finally, barcoded vector libraries using retroviral and lentiviral
systems are described by Bystrykh et al. (Chapter 23) as a novel
tracking system for studies of hematopoiesis.
7 Conclusions
Altogether the techniques described in this book enable a full

characterization of the molecular circuitry controlling HSCs.
Continued understanding of the biology of HSCs is likely to lead
to more rapid advances in therapeutics for a wide range of benign
genetic disorders of hematology and for treatment of hematologic
malignancies.
Acknowledgments
The authors thank the members of the Qu and Bunting labs for
their helpful discussions during the preparation of this summary of
the third edition of Hematopoietic Stem Cell Protocols.
Part II
Stem Cell Enrichment and Heterogeneity

Chapter 2
Investigating the Interaction Between Hematopoietic

Stem Cells and Their Niche During Embryonic
Development: Optimizing the Isolation of Fetal
and Newborn Stem Cells From Liver, Spleen,
and Bone Marrow
Huimin Cao, Brenda Williams, and Susan K. Nilsson
Abstract
Hematopoietic stem cells (HSCs) are maintained in a particular microenvironment termed a “niche.”
Within the niche, a number of critical molecules and supportive cell types have been identified to play key
roles in modulating adult HSC quiescence, proliferation, differentiation, and reconstitution. However,
unlike in the adult bone marrow (BM), the components of stem cell niches, as well as their interactions
with fetal HSC during different stages of embryonic development, are poorly understood. During embryo-
genesis, hematopoietic development migrates through multiple organs, each with different cellular and
molecular components and hence each with a potentially unique HSC niche. As a consequence, isolating
fetal HSC from each organ at the time of hematopoietic colonization is fundamental for assessing and
understanding both HSC function and their interactions with specific microenvironments. Herein, we
describe methodologies for harvesting cells as well as the purification of stem and progenitors from fetal
and newborn liver, spleen, and BM at various developmental stages following the expansion of hematopoiesis
in the fetal liver at E14.5.
Key words Hematopoietic stem cells, Hematopoietic development, Liver, Spleen, Bone marrow
1 Introduction
In the past 30 years since the term “niche” was coined by Schofield
[1] for the adult BM microenvironment in which hematopoietic
stem cells (HSCs) reside, adult HSCs and their niche have been
extensively investigated. To date, this has resulted in a huge body
of literature identifying a number of cell types and extracellular
matrix molecules as components of the niche that play a role in
HSC regulation.
Hematopoiesis develops embryonically in an age-dependent
and microenvironment-controlled process, with fetal HSC
9
10 Huimin Cao et al.
migrating through multiple sites. Initially, in the mouse, primitive

hematopoiesis starts in the yolk sac at E7.5 [2] and gives rise to
erythrocytes. Hematopoiesis then becomes definitive, demonstrat-
ing multi-lineage reconstitution capacity, with evidence suggesting
a contribution from the aorta-gonad-mesonephros (AGM) region,
yolk sac, and placenta [3–5]. HSCs are first detected in the fetal
liver at E11.5 [6], where they expand at E14.5 [7] prior to migrat-
ing to the BM at E16.5, the permanent location for hematopoiesis
throughout adulthood [6]. In addition, the spleen is a temporary
fetal hematopoietic organ from E12 shortly after birth [8]. Besides
inherent intrinsic properties, fetal hematopoiesis receives extrinsic
cues whilst residing within specific microenvironments [9].
However, unlike adult BM, the interaction between fetal HSCs
and their niche during hematopoietic development has not been
thoroughly investigated and hence remains poorly understood.
Herein, we describe methodologies for harvesting cells as well
as the purification of stem and progenitors from fetal and newborn
liver, spleen, and BM at various developmental stages following the
expansion of hematopoiesis in the fetal liver at E14.5. These meth-
odologies have been utilized to identify key interactions between
HSC and specific developmental niches and assess their roles
in HSC regulation.
2 Materials
2.1 Isolation of Liver, 1. Timed mated embryos or newborn pups.

Spleen, and BM 2. Sterile #11 surgical blade and #3 handle.
3. Sterile straight surgical scissors (16 cm Kelly).
4. 30½G needle attached to a 1 ml syringe.
5. Sterile micro-dissecting knife (12 cm knife).
6. Sterile micro-dissecting scissors (100 mm).
7. Sterile fine tweezers.
8. Sterile beveled forceps.
9. Phosphate-buffered saline (PBS): pH 7.2, 310 mOsm
(see Note 1) supplemented with 2 % serum.
10. Sterile 100 mm tissue culture petri dish.
11. Sterile 35 mm tissue culture petri dish.
12. Dissecting microscope.
2.2 Disaggregation 1. PBS (pH 7.2), 310 mOsm.

of Liver, 2. PBS (pH 7.2), 310 mOsm supplemented with 2 % serum.
Spleen, and BM
3. 50 ml Conical polypropylene centrifuge tube.
4. 3 ml Syringe plunger.
Optimizing Isolation of Fetal Hematopoietic Stem Cells 11
5. 40 μm Nylon cell strainer.

6. Sterile straight surgical scissors.
7. 3 mg/ml Collagenase I (we use Clostridium histolyticum) in
PBS made fresh on the day of use.
8. 3 mg/ml Collagenase I/4 mg/ml Dispase II (Bacillus polymyxa,
neutral protease) in PBS made fresh on the day of use.
9. 37 °C Orbital shaker (Eppendorf Thermomixer comfort model
#5355 000.011).
10. 18- and 21-gauge needles attached to 1 ml syringes.
11. Sterile mortar and pestle.
12. Hemocytometer and microscope equipped with phase-contrast
or an automated cell counter (Sysmex model KX-21N).
2.3 Density Gradient 1. 300 ml Density media: Nycoprep Universal: 60 % (w/v) in

Separation water, 1.310 g/cm3, 580 mOsm mixed with 300 ml 20 mM
tricine-NaOH (pH 7.2) and 676.6 ml of 0.65 % NaCl. Confirm
that osmolarity is 265 mOsm and density is 1.077 g/ml at
room temperature. 20 mM Tricine-NaOH is made by diluting
3.584 g of tricine in 900 ml milliQ water and adjusting the
volume to 1 l and pH 7.2. 0.65 % of NaCl is made by adding
6.5 g of NaCl to 900 ml milliQ water and adjusting the
volume to 1 l. Both the 20 mM tricine-NaOH and the 0.65 %
NaCl should be sterile filtered before use.
2. Cannulas attached to 10 or 20 ml syringes.
2.4 Lineage 1. Lineage depletion antibody cocktail: A mixture of optimally

Depletion titrated purified rat anti-mouse antibodies recognizing the cell
surface antigens: B220, Gr-1, and Ter119 (see Note 2) in PBS
(pH 7.2), 310 mOsm supplemented with 2 % serum (antibody
concentrations are all ≤1 μg/ml) (see Note 3).
2. Polypropylene 5 ml round-bottom tubes.
3. Magnetic Dynal beads working buffer: PBS, 310 mOsm sup-
plemented with 2 mM EDTA, and 0.1 % (w/v) fraction
V bovine serum albumen (BSA; pH 7.4).
4. 4.5 µm Diameter sheep anti-rat IgG Dynabeads (Dynal Biotech
ASA, Oslo, Norway).
5. Microtubes.
6. Dynal MPC-S magnet for 2–20 ml samples or MPC-L for
1–8 ml samples.
7. Tube rotator or similar suspension mixer, allowing both tilting
and rotation at 4 °C (we use a MACSmix Tube Rotator placed
in a fridge).
2.5 Hematopoietic 1. HSC antibody cocktail: A mixture of optimally titrated

Stem Cell allophycocyanin-cyanine 7 (APCCy7)-conjugated rat anti-
Fluorescence- mouse B220, Gr-1, CD3, and Ter119 (see Note 4); Pacific
Activated Cell Sorting Blue™ (PB)-conjugated rat anti-mouse stem cell antigen 1
(Sca-1); AlexaFluor®647 (AF647)-conjugated rat anti-mouse
c-Kit; fluorescein isothiocyanate (FITC)-conjugated rat anti-
mouse CD48; and phycoerythrin (PE)-conjugated rat anti-mouse
CD150 in PBS (pH 7.2) and 310 mOsm supplemented with
2 % serum (antibody concentrations are all ≤1 μg/ml).
2. Sterile polypropylene 5 ml round-bottom tubes.
3. Polystyrene, round-bottom tubes: 5 ml with 40 μm cell strainer
cap. The cap is swapped to a 5 ml polypropylene tube for filter-
ing cells.
4. Flow cytometer with sorting capability equipped with five
solid-state lasers (355, 405, 488, 561, and 635 nm). Band-pass
filter settings for the detection of fluorescence for FITC, PE,
AF647, PB, and APCCy7 are 528 ± 19, 605 ± 20, 660 ± 10,
460 ± 25, and 780 ± 30, respectively. We use a 70 mm nozzle,
sort at 30 psi, and drop delay frequency of 61 kHz for HSC
sorting.
3 Methods
3.1 Timed Mating 1. House five sexually mature female mice in one cage (see Note 5)
with mouse chow and acidified water ad libitum.
2. Tease females with bedding from the male for 3 days prior to
mating (see Note 6).
3. Time mate by placing the females into the male’s cage late in
the afternoon for 12 h (see Note 7).
4. Separate the mice, and check each female for the presence of a
vaginal plug. This is designated as 0.5 days (E0.5).
5. Confirm the pregnancy between E12.5 and E14.5.
6. Check pups’ birth twice a day from E18.5. The day when pups
are born is designated as day 0 (D0) and then sequentially as
D1, D2, D3, etc.
3.2 Mice Harvesting 1. Euthanize pregnant mouse by cervical dislocation. Remove

uterus by opening the abdominal cavity and cutting away the
3.2.1 Embryos
connective tissue.
2. Isolate single embryos from the uterus by gently cutting and
dissociating the outer muscular uterine layer and yolk sac.
Remove the placenta, and euthanize each pup by decapitation
(individual institutional animal ethics requirements may vary).
Fig. 1 Fetal organs and bone harvest. (a) Images show fetal liver, spleen, and femur. (b) Comparison of bones
before and after grinding with a mortar and pestle
3.2.2 Newborn Pups 1. Euthanize pups by decapitation with a sharp pair of scissors.
3.3 Isolation 1. In a 100 mm petri dish separate the abdominal and hind
of Liver, Spleen, portions using a surgical blade.
and Long bones 2. Using 30½G needles attached to 1 ml syringes (for embryos)
3.3.1 Liver and Spleen or fine tweezers (for newborn pups) remove other organs
from the posterior end of abdominal portion to expose the
liver and spleen.
3. Under a dissecting microscope, use a pair of 30½G needles to
remove any extra tissue attached to the liver and spleen
(see Note 8) and place in a 35 mm petri dish containing
PBS–2 % serum (Fig. 1a).
3.3.2 Long Bones 1. Lay the hind portion belly down in a 100 mm petri dish, and
remove the skin with scissors to expose the transparently red
long bones (femur and tibia).
2. Under a dissecting microscope, carefully remove muscles from
the bones using a micro knife and/or a pair of micro scissors.
3. Dislocate the femur from the hip and knee, and cut the foot off
the tibia at the ankle. Keep bones in individual groups
(see Note 8) in separate 35 mm petri dishes containing PBS–2 %
serum (see Notes 9 and 10) (Fig. 1a).
3.4 Single-Cell 1. Gently push embryonic livers through a 40 μm strainer on top

Suspensions of Liver, of a 50 ml centrifuge tube with the plunger of a 3 ml syringe.
Spleen, and BM 2. Wash cells in PBS–2 % serum by centrifuging at 400 × g for
3.4.1 Embryonic Livers 5 min at 4 °C.
3. Decant supernatant, and resuspend the cell pellet in 10 ml
PBS–2 % serum.
4. Refilter the cell suspension through a 40 μm strainer into a
new 50 ml conical tube.
5. Perform a cell count.
3.4.2 Newborn Livers 1. Transfer newborn livers into a 50 ml conical tube and chop
finely with a pair of long surgical scissors.
2. Add 1 ml Collagenase I for each minced liver, and agitate for
5 min at 37 °C in an orbital shaker, 750 rpm.
3. Repeatedly flush livers through an 18-gauge needle and then a
21-gauge needle until a single-cell suspension.
4. Add 40 ml PBS–2 % serum, and filter through a 40 μm strainer
into a new 50 ml conical tube.
5. Wash cells twice in PBS–2 % serum by centrifuging cells at
400 × g for 5 min at 4 °C.
6. Decant supernatant, and resuspend the cell pellet in 10 ml
PBS–2 % serum.
7. Perform a cell count.
3.4.3 Spleens Process embryonic and newborn spleens as for embryonic livers
(see Subheading 3.4.1, step 1), except resuspend the cells in 5 ml
PBS–2 % serum.
3.4.4 BM 1. Transfer bones into a sterile mortar.

2. Grind bones with a pestle (see Note 11).
3. Remove the supernatant and filter through a 40 μm strainer
into a 50 ml conical tube.
4. Further grind bones if not dissociated into small fragments
(Fig. 1b). Rinse the crushed bone fragments with PBS–2 %
serum, and filter the supernatant as in step 3, until all bone
fragments become white. Top up the 50 ml tube to 50 ml with
PBS–2 % serum and set aside on ice until step 9.
5. Transfer the crushed bone fragments into a new 50 ml conical
tube containing Collagenase I and Dispase II (1 ml per 12–18
bones) and place at 37 °C in an orbital shaker, 750 rpm, for
5 min (see Note 12).
6. Add 20 ml straight PBS to the digested bone fragments, and
shake vigorously for 20 s.
7. Filter the cell suspension through a 40 μm strainer into a new
50 ml conical tube.
8. Repeat steps 6 and 7, and filter cells into the same 50 ml coni-
cal tube. Top the tube with 10 ml PBS–2 % serum.
9. Centrifuge the cell suspension tubes (from steps 4 and 8) at
400 × g for 5 min at 4 °C.
10. Decant supernatant, resuspend pellets, pool cells in 10 ml
PBS–2 % serum, and perform a cell count.
3.5 Density 1. Top up each cell suspension to 20 ml with PBS–2 % serum

Separation for BM, (see Note 13).
Liver, and Spleen 2. Underlay 10 ml Nycoprep using a cannula attached to a 10 or
a 20 ml syringe.
3. Centrifuge the gradients at 600 × g for 20 min at room tem-
perature with no deceleration.
4. Using a 10 ml pipette, collect mononuclear cells from the
interface between the PBS–2 % serum and the Nycoprep solu-
tion and place in a new 50 ml conical tube.
5. Fill the tube with PBS–2 % serum and centrifuge at 400 × g for
5 min at 4 °C.
6. Decant supernatant, resuspend cells in 10 ml PBS–2 % serum,
and perform a cell count.
3.6 Lineage 1. Pellet cells by centrifuging at 400 × g for 5 min at 4 °C.

Depletion 2. Stain cells at 1 × 107 cells/ml in the cocktail of lineage markers
on ice for 20 min.
3. Wash cells with PBS–2 % serum by centrifuging at 400 × g for
5 min at 4 °C to remove unbound antibodies.
4. Resuspend cells in 2 ml PBS supplemented with 2 mM EDTA
and 0.1 % BSA and transfer into 5 ml sterile polypropylene
tube. Perform a cell count (see Note 14) and set aside on ice
until step 10.
5. Resuspend Dynabeads.
6. Place the required volume of Dynabead suspension, to give
half a bead per cell, into two individual 1.7 ml microtubes. The
optimal number of Dynabeads per cell has previously been
established as half a bead per cell with the depletion repeated
with a second half a bead per cell [10].
7. Remove the azide from the Dynabeads by adding 1 ml of
2 mM EDTA with 0.1 % BSA into each aliquot and mixing
well. Place the tubes in the magnet for 1 min prior to decant-
ing the supernatant and move from the magnet.
8. Repeat step 7.
9. Resuspend the Dynabeads in 500 μl 2 mM EDTA with 0.1 %
BSA.
10. Add the first aliquot of washed Dynabeads to the cells, and mix
well.
11. Incubate the mixture with gentle tilting and rotation for 5 min
at 4 °C.
12. Place the cells in the magnet for 2 min.
13. Transfer the supernatant containing the unbound cells to a
new 5 ml sterile polypropylene tube.
14. Rinse the rosetted bead–cell complexes with 1 ml 2 mM EDTA

with 0.1 % BSA and place in the magnet for 1 min prior to col-
lecting any residual unbound cells to the collection tube used
in step 13.
15. Add the second aliquot of washed Dynabeads into the
unbounded cell suspension.
16. Incubate the mixture with gentle tilting and rotation for
10 min at 4 °C.
17. Place the cells in the magnet for 2 min.
18. Transfer the supernatant containing the unbound cells to a
new 5 ml sterile polypropylene tube.
19. Rinse the rosetted bead–cell complexes with 1 ml 2 mM EDTA
with 0.1 % BSA and place in the magnet for 1 min prior to col-
lecting any residual unbound cells to the collection tube used
in step 18.
20. Measure the total volume of the unbound lineage-negative cell
suspension, and perform a cell count.
3.7 HSC FACS 1. Pellet cells by centrifuging at 400 × g for 5 min at 4 °C.
2. Stain cells at 1 × 107 cells/ml in the HSC antibody cocktail on
ice for 20 min.
3. Add 3 ml PBS 2 % serum, and filter the cell suspension through
a cell strainer into a new 5 ml sterile polypropylene tube
(see Note 15). Centrifuge cells at 400 × g for 5 min at 4 °C to
remove unbound antibodies.
4. Resuspend cells at 25–30 × 106 cells/ml in PBS–2 % serum
(see Note 16) and place on ice until sorted.
5. To set up HSC sorting by flow cytometry, the following sam-
ples are required for each tissue being sorted (see Note 17).
(a) 0.5–1 × 106 Unstained cells to set voltage for forward scat-
ter, side scatter, APCCy7, PB, AF647, PE, and FITC.
(b) Individual tubes containing 0.5–1 × 106 cells stained with
APCCy7, PB AF647, PE, and FITC for compensation
controls.
(c) 0.5–1 × 106 Adult BM cells stained with HSC antibody
cocktail as a positive control.
6. Run the cell samples stained with HSC antibody cocktail and
sequentially gate through FSC-H versus FSC-A, SSC-A versus
FSC-A, SSC-A versus APCCy7, AF647 versus PB, and FITC
versus PE (Fig. 2). Fetal HSCs phenotypically are defined as
lineage+Sca-1+c-Kit+CD150+CD48−.
7. Sort cells at predetermined optimal input speed and collect
into culture medium or PBS–2 % serum depending on the
functional assay requirement.
Fig. 2 HSC gating strategies for flow cytometry. HSCs are sequentially gated through single-cell gate, nucleated
cell gate, and lineage-negative cell gate and then selected as Sca-1+c-Kit+CD150+CD48−
3.8 Summarized Since fetal hematopoiesis is a developing process, maturation, expan-

Optimizing Isolation sion, and migration occur at different time points and in different
of Fetal and Newborn organs. Hence, the strategies for isolating fetal HSC are not always
Liver, Spleen, and BM the same. The optimized time points and methodologies for tissue
at a Variety of harvesting, single-cell preparation, and HSC enrichment are sum-
Developmental Ages marized in Table 1.
4 Notes
1. This osmolarity is appropriate for murine cells and results in

better cell recovery.
2. Mac-1 is excluded from the lineage depletion antibody cocktail
due to its previously described presence on fetal and newborn
HSC.
3. In order to obtain accurate and high-quality fluorescence-
activated cell sorting (FACS) profiles, proper titration is required
for all antibodies. The optimized working concentration
allows the optimal separation of positive cells from negative
Table 1
Optimized methodologies for fetal and newborn HSC isolation
Organ E14.5–16.5 E17.5 E18.5 D0–D4 D5–D8

Liver Observation time HSC isolation –
points
Methods summary Mash with plunger through Mince with scissors –
a 40 μm strainer Digest with Collagenase I
Density separation Flush with 18G and 21G
(see Note 18) needles
Filter through a 40 μm
strainer
Spleen Observation time – HSC isolation
points
Methods summary – Mash with plunger through a
40 μm strainer
Density separation
Lineage depletion
BM Observation time – HSC isolation
points
Methods summary – Grind with a mortar and pestle
Digest with Collagenase I and Dispase II
Density separation
Lineage depletion
cells without causing any shift for isotype control from

unstained.
4. B220, CD3, Gr-1, and Ter119-APCCy7 are included to
exclude residual lineage-committed cell contamination.
5. Co-housing females results in individual estrous cycles being
synchronized.
6. The murine estrous cycle is 4–5 days, with ovulation on the
third day. Therefore teasing females 3 days prior to mating will
produce the maximum number of pregnancies.
7. Only using female mice that are confirmed to be actively in
estrous can increase the success of the timed mating.
8. Due to the size of embryos and newborn pups, a number of
organs need to be pooled to provide sufficient cells. Table 2
shows the number of organs required for harvesting 50 × 106
single cells from different organs at a variety of ages.
9. It is very difficult to clean embryonic and newborn bones as
they are extremely soft and even gentle squeezing will result in
the loss of marrow. As a consequence, holding the muscle
around the bone whilst cleaning works best.
Table 2
The number of organs required for harvesting 50 × 106 cells
Mouse age No. of livers No. of femurs No. of spleens

E14.5 6 _ _
E15.5 2 _ _
E16.5 2 _ _
E17.5 2 _ _
D0 3 220a 40
D1 5 160a 30
a
D2 5 120 20
D3 4 80a 15
D4 4 60 7
D5 _ 30 4
D6 _ 30 3
D7 _ 20 3
D8 _ 15 2
a
Calculated based on observed cellularity
10. As the cellularity of both the femur and tibia is low, the humerus
is also often collected. We have experimentally shown that
there is no significant difference in the cellularity or the HSC
frequency between a femur, tibia, and humerus at E18.5 and
D1–8 bones.
11. The processing of fetal and newborn BM is different from
adult BM [11] in that due to their tiny size they are not sepa-
rated into central and endosteal fractions.
12. 5 min is experimentally determined for ideally digesting bones,
since after 5 min there is a progressive loss of c-Kit and Sca-1
[12], which are important surface markers for isolating HSC.
13. The maximum number of cells per tube for a density separa-
tion is 2 × 108.
14. The maximum number of cells per tube for a Dynal bead sepa-
ration is 3 × 108.
15. As these cells tend to clump easily additional refiltering may be
required immediately prior to sorting.
16. 25–30 × 106 Cells/ml is the ideal concentration for obtaining
optimal yields and purity when sorting cells on our Influx1 cell
sorter.
17. For convenience and saving our enriched cell samples, we use
un-fractionated single cells for the compensation controls.
Furthermore, CD45-conjugated antibodies are used for com-
pensation as CD45 is highly expressed on un-fractionated cells.
18. Fetal liver HSCs are not enriched by lineage depletion, as
E14.5 fetal liver cells do not express lineage antigens at high
levels. Such a population only begins to appear after E16.5,
but still comprises a very low proportion.
References
1. Schofield R (1978) The relationship between maintenance and expansion. Oncogene 23:
the spleen colony-forming cell and the haemo- 7199–7209
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2. Orkin SH, Zon LI (2008) Hematopoiesis: an Traycoff CM, Yoder MC, Srour EF (2002)
evolving paradigm for stem cell biology. Cell Roles of spleen and liver in development of the
132:631–644 murine hematopoietic system. Exp Hematol
3. Kumaravelu P, Hook L, Morrison AM et al 30:1010–1019
(2002) Quantitative developmental anatomy 9. Kiel MJ, Iwashita T, Yilmaz OH, Morrison SJ
of definitive haematopoietic stem cells/long- (2005) Spatial differences in hematopoiesis
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the aorta-gonad-mesonephros (AGM) region patterning in definitive hematopoietic stem
and the yolk sac in colonisation of the cells. Dev Biol 283:29–39
mouse embryonic liver. Development 129: 10. Williams B, Nilsson SK (2009) Investigating
4891–4899 the interactions between haemopoietic stem
4. Gekas C, Dieterlen-Lievre F, Orkin SH, cells and their niche: methods for the analysis
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hematopoietic stem cells. Dev Cell 8:365–375 the marrow following transplantation.
5. Ottersbach K, Dzierzak E (2005) The murine Methods Mol Biol 482:93–107
placenta contains hematopoietic stem cells 11. Grassinger J, Haylock DN, Williams B, Olsen
within the vascular labyrinth region. Dev Cell GH, Nilsson SK (2010) Phenotypically identi-
8:377–387 cal hemopoietic stem cells isolated from differ-
6. Christensen JL, Wright DE, Wagers AJ, ent regions of bone marrow have different
Weissman IL (2004) Circulation and chemo- biologic potential. Blood 116:3185–3196
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Biol 2:E75 (2007) Hemopoietic stem cells with higher
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Chapter 3
Single-Cell PCR Profiling of Gene Expression

in Hematopoiesis
José Teles, Tariq Enver, and Cristina Pina
Abstract
Single-cell analysis of gene expression offers the possibility of exploring cellular and molecular heterogeneity
in stem and developmental cell systems, including cancer, to infer routes of cellular specification and their
respective gene regulatory modules. PCR-based technologies, although limited to the analysis of a
predefined set of genes, afford a cost-effective balance of throughput and biological information and have
become a method of choice in stem cell laboratories. Here we describe an experimental and analytical
protocol based on the Fluidigm microfluidics platform for the simultaneous expression analysis of 48 or 96
genes in multiples of 48 or 96 cells. We detail wet laboratory procedures and describe clustering, principal
component analysis, correlation, and classification tools for the inference of cellular pathways and gene
networks.
Key words Single-cell quantitative RT-PCR, Microfluidics, Hierarchical clustering, Principal compo-
nent analysis, Machine learning, Random forests, Logistic regression, Correlation-based gene networks
1 Introduction
Understanding the gene expression programs that regulate distinct

cell states and the fate transitions between them is central to the
successful delivery of regenerative medicine. An important part of
this challenge lies in the prospective isolation of pure cell compart-
ments [1–3] to reveal the transcriptional signatures of rare popula-
tions [4]. However, it is also increasingly clear that fluctuations in
the molecular composition of individual cells, i.e., molecular
‘noise’, can contribute to cell potential and fate [5–9], and explo-
ration of transcriptional heterogeneity requires single-cell methods
of gene expression analysis.
Early attempts of single-cell transcriptional profiling relied on
end-point multiplex RT-PCR using oligo-dT-based reverse
transcription [10] or target-specific reverse transcription and
amplification with sets of nested primers [11] for a small number
of genes. While limited to qualitative analysis of transcriptional
21
22 José Teles et al.
composition, these approaches were instrumental in advancing the

notion of multi-lineage primed states in hematopoietic stem and
early progenitor cells [11, 12] as well as in supporting alternative
hierarchies of hematopoietic lineage specification [1, 3]. However,
quantitative approaches are required to capture the full spectrum
of fluctuations in gene expression level and, significantly, to begin
to reveal regulatory networks through pairwise correlation analysis
of gene expression.
An early attempt at quantitative analysis of gene expression in
defined hematopoietic populations, namely relatively pure hema-
topoietic stem cells, used global amplification and hybridization to
microarrays [13]. While valuable in demonstrating the ability to
capture biological variation of gene expression above technical
variation, the analysis was limited by the small number of cells sur-
veyed and did not afford a clear separation between cellular and
molecular heterogeneity. Indeed, the cost associated with method-
ologies of global gene expression profiling limits the number of
cells analyzed and compromises full appreciation of the data.
Quantitative RT-PCR is a more cost-effective alternative, par-
ticularly with the development of microfluidic devices that mini-
mize the volume of reagents used and maximize the numbers of
cells and genes inspected per run. While it does not allow
transcriptome-wide analysis of gene expression, and the conclu-
sions are necessarily biased by the identities of the genes studied, it
is possible to analyze tens to hundreds of genes deemed relevant
on the basis of prior knowledge or preliminary experiments to
arrive at biologically meaningful conclusions. Identification of new
cellular compartments [4, 14], inference of molecular mechanisms
of lineage decisions [8, 15], and inference of small regulatory net-
works [14–16] are recent examples of applications in the hemato-
poietic field.
Several platforms—Fluidigm Dynamic Arrays, TaqMan Array
Cards, and NanoString nCounter—are currently available for
single-cell expression analysis, and they all rely on an initial step of
target-specific amplification. Transcript detection and quantifica-
tion are based on qPCR technology in the case of Fluidigm
Dynamic Arrays and TaqMan Array Card methods and on hybrid-
ization of barcoded probes in the case of NanoString nCounter. In
the absence of pre-amplification, nCounter technology allows
direct quantification of the number of copies for each transcript,
but this information is impacted by the efficiency of the pre-
amplification reactions in single-cell applications. Application of
the technology to single cells is very novel [14], and it has recently
been used in hematopoiesis as confirmatory but not as an explor-
atory tool.
Fluidigm and Array Card technologies use quantification by
PCR, which allows for relative quantification of transcripts.
Absolute amounts of RNA and copy numbers can in principle be
Single-Cell PCR Profiling of Gene Expression in Hematopoiesis 23
estimated against pre-quantified standards; however, in the case of

single-cell analysis these can only be indicative rather than defini-
tive, as they are not extracted through in-well cell lysis and have
not been routinely used. Fluidigm Dynamic Arrays allow for the
best high-throughput analysis (96 cells × 96 genes vs. a maximum
of 8 cells in an Array Card) and constitute the most widely used
method. As such, the experimental protocols in this chapter refer
exclusively to the Fluidigm platform. The analytical tools are appli-
cable to other methodologies.
2 Materials
2.1 Reagents 1. DEPC-treated or RNase-free water: Aliquot and store for a

long term at −20 °C.
2. IGEPAL CA-630 (SIGMA): Prepare 10 % solution in DEPC-
treated or RNase-free water; aliquot and store long-term at
−20 °C.
3. RNase inhibitor (e.g., RNase OUT Recombinant Ribonuclease
Inhibitor, Invitrogen).
4. CellsDirect One-Step RT-PCR kit (without ROX) (Invitrogen),
one-step protocol only.
5. Platinum Taq polymerase (Invitrogen), one-step protocol only.
6. Superscript ViLO cDNA synthesis kit (Invitrogen), two-step
protocol only.
7. T4 gene 32 protein, two-step protocol only.
8. TaqMan PreAmp Master Mix, two-step protocol only.
9. Exonuclease I, two-step protocol only.
10. TaqMan Universal PCR Master Mix.
11. 2× Assay Loading Reagent (Fluidigm).
12. 20× Sample Loading Reagent (Fluidigm).
13. 20× TaqMan assays for genes of interest—up to 96 (Applied
Biosystems).
2.2 Microfluidics 1. 0.2 ml Non-skirted 96-well PCR plates.

Chips and Plasticware 2. Optical PCR adhesive films.
3. Disposable 96-well plate plastic lids.
4. 1.5 ml Microfuge clear tubes, RNase and DNase free.
5. Light-touch ergonomic micropipettes (a good choice is the
RAININ LTS system, including an 8-channel 1–10 μl micropi-
pette) and corresponding tips.
6. 48.48 or 96.96 Dynamic Arrays for Gene Expression
(Fluidigm): Note that a 192.24 Dynamic Array format (192
samples × 24 assays) is also available, but requires specific

instrumentation and is not discussed in this chapter.
7. Control Line Fluid (two syringes with 0.3 ml—48.48, or
0.15 ml of fluid—96.96 format (Fluidigm)).
8. Clear (“invisible”) adhesive tape.
2.3 Instrumentation 1. Refrigerated benchtop centrifuge with plate adaptors.

2. Thermal cycler, preferably with multiple block capacity.
3. Integrated Fluid Circuit (IFC) Controller MX (for 48.48
arrays) or HX (for 96.96 arrays) (Fluidigm).
4. Biomark Reader (Fluidigm).
2.4 Software 1. Genesis [17] (http://genome.tugraz.at/genesisclient/

genesisclient_download.shtml).
2. Matlab and Matlab Statistical toolbox (MathWorks).
3. R software for statistical computing (http://www.r-project.org/).
4. Rattle graphical user interface for R [18] (http://rattle.
togaware.com/).
3 Experimental Procedures
Carefully read Notes 1–6 at the end of this chapter, as they high-
light simple procedures that should be adhered to in order to mini-
mize amplicon contamination during pre-amplification and
quantitative PCR setup.
3.1 Single-Cell Although the technicalities of fluorescence-automated cell sorting

Collection do not fall within the scope of this chapter, most experiments will
require prospective isolation of defined hematopoietic cell
3.1.1 Cell Sorting
compartments and single-cell deposition into lysis buffer or alter-
natively directly into the one-step RT/pre-amplification reaction
mix (as detailed in Subheading 3.2.2). Given the low frequency of
highly purified stem and progenitor hematopoietic compartments
as well as the possibility of multiple-way sorting into tubes, but
not plates, we recommend that a maximum number of cell types
be bulk-sorted into tubes containing medium with serum or
serum substitute, with the individual populations subsequently
single-cell deposited onto 96-well PCR plates containing the
appropriate buffer.
Each plate should include at least two wells into which no cells
are deposited as well as three or more serial multiple-cell controls
in duplicate wells for internal validation of the single-cell results.
It is recommended that a maximum of 50 cells are used for any
multiple-cell control, as the PCR can saturate above this cell num-
ber for the most abundant genes.
Prior to cell deposition, it is crucial to verify the alignment of

the plates with the automatic cell deposition unit at two opposite
corners and to ensure that the stream is directed at the center of
the wells by test sorting 25–50 cells at different plate positions
while keeping the plate covered with a clean lid. Sorters should be
set on single-cell mode to prevent doublet deposition. In our expe-
rience, this sorting mode will use a five- to tenfold excess of cells,
and a deposition efficiency of over 85 % is to be expected.
Plates should be covered with optical adhesive film and vor-
texed at maximum speed for 15–30 s to ensure efficient cell lysis
and can be kept on ice for short periods (e.g., while depositing
additional cells). The plates should then be centrifuged in a pre-
cooled centrifuge for 1 min at 2,000 × g and either frozen at −80 °C
for later use or processed immediately.
3.1.2 Cell Lysis Although it is possible to sort cells directly into the reverse-
transcription reaction buffer, we recommend that cells be initially
sorted into a detergent-containing lysis buffer to maximize RNA
extraction. This procedure also adds flexibility to work organiza-
tion, as lysis plates can be frozen long-term at −80 °C prior to
processing.
The lysis buffer can be prepared up to 24 h prior to cell deposi-
tion and stored at −20 °C. If prepared on the day of the experi-
ment, lysis buffer-containing plates can be kept at 4 °C or on ice
until use.
The reagents required for a 96-well plate (including a 10 %
reagent excess) are indicated below:
RNase OUT 40 U/ml 5.3 μl

IGEPAL/NP40 10 % 21.1 μl
DEPC-treated or RNase-free H2O 396.0 μl
Total 422.4 μl—USE 4 μl per well
When using a two-step reverse transcription and pre-amplification

protocol (see Subheading 3.4) use this modified version of the buf-
fer in order to keep reaction volumes as low as possible.
RNase OUT 40 U/ml 5.3 μl

IGEPAL/NP40 10 % 21.1 μl
Superscript VILO RT 5× reaction mix 105.6 μl
Total 422.4 μl—USE 4 μl per well
The buffer mix should be prepared in a PCR hood, with

reagents maintained on ice during preparation; it is possible to
keep the tubes or the plates at room temperature for short periods
for convenience. After aliquoting the lysis buffer, plates should be
covered and centrifuged at 2,000 × g for 1 min in a centrifuge pre-
cooled to 4 °C.
3.2 Multiplex One-step target-specific reverse transcription (RT) and pre-

Reverse Transcription amplification are appropriate for murine primary hematopoietic
and Pre-amplification cells as well as hematopoietic cell lines of human and mouse origin.
It has also been used successfully in the analysis of non-
hematopoietic cell types, including, among others, early stages of
mouse embryonic development, mouse embryonic stem cells, and
induced pluripotent (iPS) cells and normal and cancerous human
epithelia [9, 19–21]. However, we and others have not been able
to apply this method to single human primary hematopoietic cells
[22] and thus describe an alternative two-step random primer-
based RT and target-specific pre-amplification method that allows
amplification of human primary hematopoietic material (Fig. 1).
The two-step method is significantly more expensive, although the
recent introduction by Fluidigm of small cell (5–10 μm)-capture
microfluidics chips for execution of reverse transcription and pre-
amplification steps on their C1 Single-cell Auto-prep System may
allow for cost reduction and potentially for increased sensitivity
and reduced variability of the reactions. However, a comprehensive
comparison of one-step versus two-step reactions on PCR plate
versus microfluidic chip formats is currently still lacking. Potential
downsides of the microfluidic chip format are (1) the inability to
analyze more than one cell type in the same run in the absence of
strong fluorescent reporters than can be localized by microscopy
on the chip and (2) the inability to store single cells in lysis buffer
for later use.
Independently of the method used, we recommend that ‘no-RT’
reactions, where the reverse transcriptase is omitted from the reaction
mix, are included for every gene set or in every plate. This allows
quantification of genomic DNA detection in intron-less genes and,
where primer design is not intron-spanning, attests to the specific-
ity of the detection where exclusive detection of the mRNA species
is expected. Note that ‘no-RT’ reactions can be run simultaneously
with RT reactions on a plate, but not in the C1 chip format.
3.2.1 Assay Mix We recommend the use of TaqMan assays (unlabelled primers +
for Multiplex Analysis fluorescently-labelled probe with quencher moiety) for single-cell
applications, as in our hands, single primer pairs with intercalating
fluorescent dyes such as SYBR or Eva Green often result in nonspe-
cific amplification of multiple products and impair data quantifica-
tion at cell numbers lower than 10 to 30. However, specificity may
be enhanced by the use of nested primers as recently published in
Fig. 1 Single-cell gene expression profiling by quantitative RT-PCR on a microfluidics platform: experimental
and analytical workflow (see [15] for analysis panels)
a comprehensive single-cell RT-qPCR study of surface markers and

regulators in mouse hematopoiesis [14]. Nested primer strategy
was indeed central to early non-quantitative single-cell RT-PCR
strategies and ensured specificity of end-stage products [11].
Prepare 1:96 dilutions of the TaqMan assays of interest
by mixing equal volumes of the assays (commercially provided as
20× or 18 μM each primer and 3 μM probe) with either an equal
total volume of RNase-free water (48.48 format) or no water
added (96.96 format). Make up enough volume for all the chips
planned with the same primer cocktail. Mix can be stored long-
term at −20 °C.
3.2.2 One-Step Reverse Prepare RT/pre-amplification and “no-RT” reaction mixes in a

Transcription PCR hood while keeping reagents on ice.
and Pre-amplification RT + pre-amplification mix—per well:
Cell lysate 4.0 μl

CellsDirect 2× Master Mix 7.5 μl
TaqMan Assay Mix 187.5 nM 2.5 μl
Superscript III/Platinum Taq Mix 1.0 μl
Final volume 15 μl—aliquot 11 μl per well
No-RT control mix—per well:
Cell lysate 4.0 μl

CellsDirect 2× Master Mix 7.5 μl
Platinum Taq 5 U/μl 0.4 μl
Thaw (if working from frozen lysates) and centrifuge plates to

collect contents at the bottom of the wells prior to removing the
film cover. Aliquot 11 μl of the reaction mix into each individual
well, and mix gently by pipetting. Use a different tip in each well
and align the box of pipette tips to mirror the plate layout in order
to prevent cross-contamination of samples. Cover the plate with
adhesive optical PCR film and spin down at 4 °C to collect con-
tents prior to thermal cycler run.
Cycling conditions are as follows:
50 °C, 1 h (Superscript III reverse transcription).
95 °C, 2 min (inactivation of reverse transcriptase and activa-
tion of Platinum Taq polymerase).
22 or 25× (95 °C, 15 s; 60 °C, 4 min): Use a target number of

20–22 cycles for hematopoietic cell lines and 25 cycles for
primary mouse bone marrow cells; refer to discussion in
Subheading 4, Experimental Procedures.
25 °C, 10 s (end step).
Centrifuge plate to collect contents, and keep amplified material
at −20 °C until the quantitative PCR run to prevent evaporation.
3.2.3 Two-Step Protocol: Prepare RT and ‘no-RT’ reaction mixes in a PCR hood while keep-
Random Primer-Based ing reagents on ice.
Reverse Transcription RT mix—per well:
Cell lysate 4.0 μl

T4 Gene 32 Protein 10 mg/ml 0.12 μl
Superscript VILO RT Enzyme Mix 0.15 μl
‘No-RT’ mix—per well:
Cell lysate 4.0 μl

T4 Gene 32 Protein 10 mg/ml 0.12 μl
Thaw (if starting from frozen lysates) and centrifuge plate and
heat at 65 °C for 90 s to denature the RNA molecules; cool plate
rapidly on ice, centrifuge in a precooled centrifuge to collect dena-
tured lysates at the bottom of the wells, and keep on ice until
addition of reaction mix. Only remove film cover at this stage.
Aliquot 1 μl of the reaction mix into each individual well and
mix gently by pipetting. Use a different tip in each well and align
the box of pipette tips to mirror the plate layout in order to prevent
cross-contamination of samples. Cover the plate with adhesive
optical PCR film and spin down at 4 °C to collect contents prior to
thermal cycler run.
Thermal protocol conditions are as follows:
25 °C, 5 min.
50 °C, 30 min.
55 °C, 25 min.
60 °C, 5 min.
70 °C, 10 min.
25 °C, 10 s (end step).
Centrifuge plate in a precooled centrifuge to collect contents

in the bottom of the well and keep on ice until addition of pre-
amplification reaction mix.
3.2.4 Two-Step Protocol: Prepare pre-amplification reaction mix in PCR hood during the
Target-Specific reverse transcription run; keep reagents and reaction mix on ice
Pre-amplification until use.
Pre-amplification mix—per well:
Reverse-transcribed material 5.0 μl

TaqMan PreAmp Master Mix 2× 10.0 μl
Remove film cover after centrifugation and immediately prior

to addition of pre-amplification reaction mix. Aliquot 15 μl of the
reaction mix into each individual well and mix gently by pipetting.
Use a different tip in each well and align the box of pipette tips to
mirror the plate layout in order to prevent cross-contamination of
samples. Cover the plate with adhesive optical PCR film and spin
down at 4 °C to collect contents prior to thermal cycler run.
Cycling conditions are as follows:
95 °C, 10 min (activation of AmpliTaq Gold polymerase).
25× (96 °C, 5 s; 60 °C, 4 min) (cDNA amplification; use 25
cycles as a reference number and refer to Subheading 4,
Experimental Procedures, for additional discussion).
25 °C, 10 s (end step).
Centrifuge plate to collect contents, and keep amplified mate-
rial at −20 °C until the quantitative PCR run to prevent evapora-
tion. Optionally, the pre-amplified material can be treated by
exonuclease I digestion to remove single-stranded DNA, i.e., non-
annealed primers and probes. Addition of exonuclease I mix and all
subsequent steps should be performed on the bench to avoid con-
tamination of the PCR hood with amplified material.
3.2.5 Two-Step Protocol: Exonuclease I mix—per well:

Exonuclease I Treatment
Pre-amplified material 20.0 μl
Exonuclease I reaction buffer 10× 0.5 μl
Exonuclease I 20 U/μl 1.25 μl
Remove film cover after centrifugation of pre-amplified mate-

rial, taking care to do it away from working bench. Aliquot 5 μl of
the exonuclease I reaction mix into each individual well and mix
gently by pipetting. Use a different tip in each well and align the
box of pipette tips to mirror the plate layout in order to prevent
cross-contamination of samples. Cover the plate with adhesive
optical PCR film and spin down to collect contents prior to ther-
mal cycler run.
Thermal protocol conditions are as follows:
37 °C, 30 min.
80 °C, 10 min (inactivation step).
25 °C, 10 s (end step).
Centrifuge plate to collect contents, and keep amplified
material at −20 °C until the quantitative PCR run to prevent
evaporation.
3.3 Gene-Specific The last step in the single-cell analysis of gene expression is a con-
Quantitative PCR ventional quantitative PCR performed in a microfluidics chip that
Analysis allows the partitioning of each pool of multiplexed pre-amplified
cDNAs into 48 or 96 individual reactions.
The Fluidigm Gene Expression Dynamic Arrays give repro-
ducible readings at Ct values between 5 and 27, and the number of
pre-amplification cycles as well as the level of dilution of the pre-
amplified material should be optimized to fit most or all of the
reactions within this range. Fluidigm recommend a minimum 1:5
dilution of the pre-amplified material, and we have indicated the
target numbers of pre-amplification cycles that in our experience
meet these criteria. However, it should be noted that we have ana-
lyzed low-level expression events at the outset of lineage specifica-
tion [8], and it is therefore likely that lower numbers of
pre-amplification cycles be required for the detection and quantifi-
cation of more abundantly expressed gene targets.
Prepare the pre-amplified material and the gene-specific assays
in 96-well plate format that can be used for direct correspondence
with chip locations as shown in Fig. 2. If using a 48.48 Dynamic
Array format, arrange the samples on the right- and the assays on
the left-hand side of the master plate to load directly onto the chip.
In the case of a 96.96 chip format, two 96-well master plates are
necessary, one for assays and the other for samples.
3.3.1 Preparation Centrifuge the plate containing the pre-amplified material to col-
of Pre-amplified Material lect contents at the bottom of the wells, and remove film cover
away from the working bench. Keep plate covered with a clean
disposable plastic lid, either on ice or at room temperature. Due to
well-to-well inequalities in reaction volumes and the possibility of
evaporation during the thermal cycler runs, we recommend
Fig. 2 Fluidigm Dynamic Arrays loading scheme depicting direct correspondences between cell locations on a
96-well plate and on the chip
performing the dilution into a new plate by adding 1 μl of cDNA

to the appropriate volume of DEPC-treated or RNase-free water
(e.g., 4 μl for a 1:5 dilution) while maintaining the original plate
layout. Centrifuge at 2,000 × g for 1 min to collect contents at the
bottom of the wells.
Prepare the sample loading reaction mix (48/96 samples):
20× Sample loading reagent (yellow cap) 156/312 μl

2× TaqMan Universal PCR Master Mix 15.6/31.2 μl
Final volume 171.6/343.2 μl—aliquot
3.3 μl per well
Aliquot 3.3 μl of the sample reaction mix into each well of a

master plate; use a multichannel pipette to add 2.7 μl of the diluted
Exploring the Variety of Random
Documents with Different Content
regular meals. This place where the fish dam is put in is called by
them Cap-pell and is a bar of some twenty or thirty acres, high
enough so the river never over-flows it and yet it is very level. It is a
pretty place, being situated on the south bank of the Klamath river.
There are two villages on this pretty spot, one being Cap-pell which
was very large in the ages gone by and which contained a very large
number of Indians. The other village was called Sy-ah and was very
ancient, being the place where the lodge was situated. The house
they stay in is called Lah-wa-alth and the house where Lock and
Lock-nee sleep is called Ur-girk.
I will say to the white race that my people, or any other Indian
tribes as far as I know them, do not use the name of our Creator
when using profane language, as we would feel it a disgrace to do
so, even to think of such a thing. We never use the sacred name of
God, only in our prayers.
The following are a few expressions sometimes used: Kee-mol-len-
a Ta-ga-ar-a-wah-ma, (bad talk) pointing the right hand, with the
fingers extended, toward a person and at the same time saying:
Woo-saw-ah, means that the person is badly born, and they never
forgive you for this. Another is: Char-reck-quick-cal-lah, and means:
“I wish you were in hell”, and for this also they never forgive.
CHAPTER II.
THE CREATION OF THE WORLD.
In a vision, the Indian through his mysterious eyes

Sees yonder in the distant skies,
A scene sublime of the past ages,
That for aye will enchant bards and sages.
ON His mighty Throne, high in the infinite realms of Heaven, sat the
great ruler of the stars and endless skies, Wah-pec-wah-mow (God).
As he peered down through the darkness of a cheerless and lonely
space, He created a new world, the earth on which we live. He first
made the soil of the earth and placed it in a buck-skin sack. He
opened the sack and shook the soil from it; it fell down into the
chasm of darkness, and Wah-pec-wah-mow could not see anything
but the intense darkness. He commanded that the rays of light
should penetrate the awful darkness, and there should alternately be
night and day. The sun to shine by day and the moon to shine by
night, to break the awful stillness of this once dark and cheerless
world.
Gazing down from His Throne on high, Wah-pec-wah-mow saw
the world he had created was a desolate waste without human life,
or life of any kind. He now began the transformation of the new
world, and lo, the once barren surface of the earth was clothed in
verdure; forests lifted their giant branches sky-ward; tranquil
streams flowed and great rivers wended their way to the ocean.
The first living thing placed upon the earth was the white deer
(Moon-chay-poke). The white deer roamed over the hills, mountains,
in the valleys and on the plains. He was the pride and dignity of the
animal kingdom. This is why the Klamath Indians revere the white
deer that is so sacred to their hearts and use the skin as an emblem
of purity, in one of their greatest festivals, or worships, which is
termed in English as, “The White Deer-skin Dance.” In the Indian
language it is called, “Oh-pure-ah-wah”; which does not mean dance
but means one of their most sacred religious festivals.
The next living creature that Wah-pec-wah-mow placed upon the
earth was the red eagle, Hay-wan-alth, who has ever since ruled as
the monarch of the skies. The Indians prize the feathers of this
eagle very highly, and use them in their great festival. In the
decoration of their head-gear, they take a single feather, fasten it in
the hair at the back of the head, arranging it so that it stands
straight up. They also use the feathers of the bald eagle, Per-gone-
gish, and the gray eagle, Per-gish, sometimes as a substitute for the
feathers of the red eagle.
After the white deer and red eagle was placed upon the earth,
Wah-pec-wah-mow now created all the other animals of the earth.
Some were to roam upon the plains, others in the forests, some to
eat grass and others to devour other animals, etc.
Wah-pec-wah-mow did not give our people any single day during
the week or month, as a day of worship, but gave them a certain
season of the year in which to hold their religious ceremonies. This
season of worshipful ceremonies usually begins in the month of
September, and lasts for several days. It is the season of the year
when the water of the rivers and brooks ebb lowest, and the
summer is almost ready to wane into the glories of Autumn. This
season is called, “Kne-wal-la-taw,” the eighth month of the year,
according to our way of reckoning time.
When Wah-pec-wah-mow had finished creating the plant and
animal life of the earth, He then created the first real man. He made
the first man of the soil of the earth, and placed him in the beautiful
valley of Cheek-cheek-alth. This valley was located in a far off
northern clime. When the first man was created and he became a
living being upon the earth, Wah-pec-wah-mow said to him, “You
are a living man.” God named this man He-quan-neck. Inspired with
the breath of life, He-quan-neck first saw the light of day in this
sweet valley of sunshine, flowers, fruits and herbs. Among the
growing herbs was the herb walth-pay, which has a forked root. God
saw that the man was lonely in this sunny valley, and he was not
pleased with his work. Wah-pec-wah-mow now requested He-quan-
neck to blow his nose, which he did, and immediately the forked
root, or walth-pay turned into a living woman, Kay-y-yourn-nak. Man
now became blessed with a living companion and for a time they
dwelt together in the chaste life of peace and happiness.
Our tradition has been handed down through the long centuries,
the first dwelling place of man and woman was far away in a
northern clime. It would seem a distant land across the waters from
the North American continent that is located in the northern part of
the world, which we call Cheek-cheek-alth.
Man and woman in the valley of Cheek-cheek-alth knew no sin,
two pure souls were they in this valley of perpetual sunshine and
flowers.
The loneliness of two human beings dawned upon Wah-pec-wah-
mow so he decided to have the earth populated with people. He
now caused He-quan-neck and Kay-y-yourn-nah to fall asleep, and
while they slept He caused the snake to crawl across the woman’s
bare abdomen, that awakened the sleepers, and this opened their
eyes to their nudeness and thereafter they knew sin. The finer
senses of the woman awoke, as she became deeply humiliated at
the sight of her naked self, and she began to fasten leaves together
from the herb, Cur-poo-sa-gon, out of which she made an apron to
clothe herself. Thus the first garment that woman wore was from the
leaves of this wonderful plant. This plant grows in abundance along
the lower Klamath river and its surrounding regions, and the little
Indian girls up to this day like to gather these leaves, rub their face
and hands with and wear them upon their heads under their caps.
These leaves have a very strong and unpleasant odor.
Wah-pec-wah-mow commanded the man and woman to go forth
and bring children upon the earth. A curse fell upon the woman, that
she should bear children with pain, therefore every woman after her,
through all the long centuries has had to endure this hardship. The
first children were born some with light hair and fair skin and blue
eyes, and some with black hair, dark skin and black eyes and as they
married they would mate with black hair, the others with light hair
and when they left the old land Cheek-cheek-alth they were not so
dark, many of them were light haired, fair and blue eyed.
Wah-pec-wah-mow put a curse upon the snake that it should
crawl upon its belly as long as the earth should last.
God’s laws were that every man and woman should marry and
bring forth children. These people were taught to obey the laws and
be honest. They increased in number until they became very
numerous, and at that time, they all talked the same language. As
time sped by they became very numerous and Wah-pec-wah-mow
now caused our people, the Indians, to start on their long journey,
away from their native haunts and childhood’s land, Cheek-cheek-
alth. We do not know how long, but they wandered thus in search of
a new land, leaving behind them only a memory of the old land. A
land that claims its own no more in life and like a people in exile
they wandered on.
CHAPTER III.
THE WANDERING TRIBE.
FROM the land of Cheek-cheek-alth, the mystic Eden of long ago,

came our wandering tribe of people who long since inhabited North
and South America; for we are all one people. Among them were our
leaders, the men who possessed in their secret breasts the true
name of God. These men and women in our language we call Talth,
and were the High Priests, and great rulers who ruled our people.
Therefore, we were one of the tribes that was never ruled by a
single chief, but by our Talth, or High Priests. Upon leaving the old
land the Talth carried with them the forked root, Walth-pay, (the root
from which woman was made) and the stalk of this root as a divine
rod of strength, endurance and courage, being used as a saviour of
the tribe. With it the Talth would command food for their famished
members and bring peace and rest to their weary bodies. The
Walth-pay stalk kept perfectly green, and blossomed all the while,
and the High Priests carried it with them on their long journeys and
years of wanderings.
In my infancy, I was taught all that was good, and to make for a
true and noble womanhood; that there was a God in Heaven who
ruled over all, and during my researches throughout I have found
nothing better. When these last two members finish their earthly
reign, with us perishes the true name of God to my people. With it
has perished from the earth our true Indian laws, our sublime
religion, our deeds of chivalry, as rich as the civilized world has ever
beheld. Also our glorious manhood and womanhood; immoral,
corrupt, tottering, down-trodden and debauched by a superior race,
we have perished in that winter night of the transition period. At a
single blow our laws were torn asunder; loathsome diseases we had
never known crushed out the life and beauty of our physical bodies,
and demented our spiritual minds with lowly passions. Poisonous
spiritous drink has set the brain on fire, degrading man and
womanhood, thus as a race we have perished. And this great land,
the richest the world has ever known, the land of our forefathers for
so many thousands of years. Now another race is struggling on
where our reign has ended. Already our great rulers are at rest, and
forever; laureled with the glories of the primeval ages that have
passed away in silence. As a nation, like the ancient Egyptians, we
have grown old and passed away; we have seen a great civilization
rise to the highest of its splendors and pass away to another land
beyond recall. Today we see another civilization endowed with a
splendor of its own, rising over the debris of the eternal years.
We are all one tribe from the source of the Klamath river to its
mouth, and down the coast as far as Trinidad, (Cho-ri) and up the
coast as far as Wilson creek, which we call Ah-man. We are classed
in two divisions and term ourselves as Po-lick-las along the coast and
up the river as far as Weitchpec, designated as the lower division of
our tribe. From Weitchpec on up the river to its source we term as
Petch-ic-la, the upper division of our tribe. We intermarry to a great
extent, having the same marriage laws and religious ceremonies and
all our traditions and teachings are the same. We call God, Wah-pec-
wah-mow, which means in our tongue the father of all and we do
not consider Him as one “which has been so much of the white
man’s allegory, but as an Invisible Omnipotent Being, who rules this
great universe with an all seeing eye, He is everywhere.”
Wah-pec-wah-mow is the common name applied to God, used by
all classes of our tribe, as the real and true name of God is never
spoken. Our high priests, born of the royal marriages, are initiated in
the Holy Lodge and are given the true name of God, but they never
speak it outside of the lodge, it is only spoken inside after they have
gone through a long and secret communion, and then the name is
only whispered in the lowest whisper from mouth to ear. This true
name is only used by the Talth with profound reverence to the Great
Creator, in the sacred lodge and in the hallowed lonely places far
back on the high mountains where they go to worship in the
profound solitudes, away from the gaze of curious people. Our
religion has been too sacred, too sublime an ideal to quarrel over,
hence we have remained silent through the gloom of so many years
and borne patiently the insults on royal society as being heathens.
This true name of God, as great as the universe, will never be
spoken again. If it should be uttered in a loud and harsh tone of
voice, it is said that the earth will tremble, ignite in mighty flames
and pass away forever. Ever thus, since the creation of the world,
the Talth have handed down our religion and traditions from the old
land of Cheek-cheek-alth, from generation to generation. It is the
duty of every Indian child to be pious and worship the Great Creator.
Our sacred religion is O-pure-ah-way (the White Deer-skin dance)
where all the members of the tribes in unison and worship, and
entertain our guests with much hospitality.
In our recollections of the past we left the land of our birth
(Cheek-cheek-alth) many thousands of years ago with our leaders,
the Talth, who were given the true name of God in the old land, and
carried with them the forked root, or Walth-pay. With this divine rod
they commanded food, comfort and peace during their long years of
weary wanderings. After we left the beautiful valley of Cheek-cheek-
alth, for years we wandered down a European land, always moving
toward the south, having our origin in the far north. Over this land
we wandered like exiles, we know not how long, as it might have
been centuries until we reached the rolling waves of the ocean.
Upon reaching this salt water we made boats or canoes, and
paddled over the waves until we reached the opposite shore, having
crossed the straits in safety. Having reached this opposite shore,
upon this new continent we continued our weary years of
wandering, ever on, far on, down this land, always going south as
before. We carried the memory through the long ages, the perils of
the far north, the huge icebergs, the regal monarchs of the North
that floated like ghost-ships at night on dream-land seas, the
splendors of the aurora borealis flickered across the snowy fields and
through this land of the midnight sun came our brave forefathers. In
this land of the frozen North some of our people were left, the
Esquimau; they were given a language as they were separated from
our sturdy band and emigrated over the snowy fields and have long
since from this time on inhabited the land of perpetual ice and snow.
Our tribe would often become weary with travel and become very
dissatisfied and would quarrel much among themselves. The Talth
would stop after hearing so much grumbling and build a lodge
where their members would hold a meeting and offer up worship to
God, that He would guide them aright, endow them with power to
bring peace among their people, comfort them in their wants and
give them food. After the lodge meeting and prayer the Talth would
command with the rod of Walth-pay food for their people. The food
came to them in the form of acorn dough out of which they made
bread or pop-saw. The Indians would never see pop-saw falling to
the ground, but they would find it where the Talth told them to look,
and each one would be compelled to gather up their own, or they
would go hungry. As long as they remained camped in the same
place the pop-saw would come to them but when they would break
up camp and travel on the pop-saw would cease to come and the
tribe would grow very hungry and begin to quarrel again. The Talth
would stop after days of fatigue and hunger, and build another lodge
where their members would worship at the sacred shrine. After the
worship food would come again in the form of the acorn dough,
commanded with rod of Walth-pay. Sometimes the Talth would leave
the camps for several days, during which time the people would
become very restless and discontented and some of the people
would try to perform the duties of the Talth in their absence, and
some of them would pray to the sun, some to the stars and other
idols. The Talth would be very much humiliated upon their return to
find their people so corrupt in their worship, and it would take much
faithful work to assure peace and order among them again. The
Talth would plant the herb, Walth-pay at their stopping places during
their travels, and it would readily take root and grow, at almost
every stopping place some of our people were left and God would
give them a language; they would inhabit the locality permanently
and branch out to other localities, while our part of the people
traveled on until they reached their final earthly home on the
Klamath river, which we call Health-kick-wer-roy, and here we found
the white race, (Wa-gas) which will be told of in another chapter.
Thus we traveled on down a great continent, leaving behind at our
stopping places, a portion of our people, which were given different
languages. Thus were our languages confounded among the tribes
of America, and our tribes became numerous, being scattered over
the land of the midnight sun of perpetual ice and snow, over the
continent of North America to the equator and regions of perpetual
sunshine; and beyond the equator over the continent of South
America to its farthermost southern borders, where we merge into
the regions of ice and snow again, our tribes have been scattered.
Over this great land we are all one people, however some of our
tribes were far superior to others. We know not how many centuries
we wandered, or when we reached our last stopping place on the
Klamath river and where we decided our long journey should end,
and that we would make this our final home. The Wah-teck, Wah-
ker-rah, Cor-tep and Pec-wan villages were among our first camping
grounds on the Klamath river. Here we spread our camps and built
our first houses long ages ago, and have resided in them and kept
them in repair from generation to generation. Some of these
primeval houses yet remain in these old villages, haunted with the
romance of centuries and the inspiring history of past ages. Upon
our first arrival there were a great many of our people and we began
to divide off into different villages and locate along the Klamath river
and down the coast as far as Trinidad, (Cho-ri) and up the coast to
Wilson Creek (Ah-man). The other tribes were placed by Wah-pec-
wah-mow in different localities, that all the people might sustain
themselves with plenty of game and food, and be kept comfortable.
The Talth kept the Walth-pay in commemoration of God’s creation
of woman and their travels, and planted it in a few selected places
back in the lonely mountains. The Talth all know where to find this
wonderful herb growing, but it is also fading with the remote ages
as there are only a few Indians left who know where to find it. With
them passes away the sacred rites and laws of an ancient nation
forever, and the primeval art becomes a thing of the mystic ages.
CHAPTER IV.
TRADITIONS OF THE ANCIENT WHITE PEOPLE.
WHEN the Indians first made their appearance on the Klamath river
it was already inhabited by a white race of people known among us
as the Wa-gas. These white people were found to inhabit the whole
continent, and were a highly moral and civilized race. They heartily
welcomed the Indians to their country and taught us all of their arts
and sciences. The Indians recognized the rights of these ancient
people as the first possessors of the soil and no difficulties ever
arose between the two people. Their hospitality was exceedingly
generous in the welfare of our people and all prospered together in
peace and happiness, in their pursuit of human existence. After a
time there were inter-marriages between the two races, but these
were never promiscuous. For a vast period of time the two races
dwelt together in peace and honored homes, wars and quarrels were
unknown in this golden age of happiness. No depredations were
ever committed upon the property of their people, as the white
people ruled with beacon light of kindness, and our people still
worship the hallowed places where once they trod. Their morals
were far superior to the white people of today, their ideals were high
and inspired our people with greatness. After we had lived with
these ancient people so long, they suddenly called their hosts
together and mysteriously disappeared for a distant land, we know
not where. We have no memory of their reason or cause why they
abandoned their ancient homes where they had dwelt for untold
centuries. Wars did not drive them forth, for we loved them more
than brothers, and difficulties were unknown between the two
people. On leaving they went toward the North from whence we
came, and disappeared from our land beyond the northern seas. It
was a sad farewell when they departed from this land, for our
people mourned their loss, as no more have we found such friends
as they, so true and loyal. In their farewell journey across this land
they left land-marks of stone monuments, on the tops of high
mountains and places commanding a view of the surrounding
country. These land-marks we have kept in repair, down through the
ages in loving remembrance. I have seen many of these land-marks
myself (and often repaired them) that they left as a symbol of the
mystic ages and the grandeur of a mighty nation that passed in a
single season. Oh, how little we know of the depths of the ages
gone, how wide, how profound and deep is the knowledge we seek;
a monument of stone, a stone bowl, a broken symbol, a hallowed
unknown spot, a lodge of ruins, all this makes a golden page
glittering with diamonds that trills the emotions with mysterious
longings for truth and light in the depths unknown.
When the Wag-as left this land they assured my people that they
would return to them at some future time. Perchance thousands of
years have elapsed since then, and they have not returned, we have
waited in vain for it seems that our cherished hopes are fading.
However, some of our people are still looking for the return of the
white man. The traditions handed down lead us to believe that the
Wa-gas returned to the land of their birth, in the far north, the valley
of Cheek-cheek-alth, as their traditions were given to us that their
origin was in this same land of Cheek-cheek-alth, as they came down
from the North when they came to this land. When the Wa-gas first
arrived on this continent they handed down the traditions to us that
it was inhabited by a giant race of people when they first came.
These giants were represented by the Wa-gas as being very swarthy
in complexion, and they used implements so large that no ordinary
man could lift them. It was an age when large animals roamed the
earth, and it seems the birds and fowls were all very large in size. It
appeared to be the first age, and was the age of the giants. The
recollections transmitted by the Wa-gas were that these giants were
very cruel and wicked. It was said that God became displeased with
them and destroyed them and they all perished from the earth. It
was also said that God appeared to the High Priest of the Wa-gas
and told them that he was going to destroy the giant race and that
the Wa-gas themselves would survive upon the earth as a new
people. Smaller birds and animals would appear upon the earth for
the use of man, thus the age of giants perished, but the Wa-gas do
not hand down any tradition of how they perished from the earth, as
my people have no recollections of ever seeing giants. My mother
says that our people in ancient times have seen many relics
belonging to these prehistoric giants, such as huge stone bowls,
stone slabs and other implements so great that our people could not
move them. During the ages of rains and wearing away of the earth,
these implements have been buried so deep and have sunk into the
earth, that is the reason we cannot find them today. The Indian
name for the giant race is Pah-pel-ene, which means people that
have all died and passed away.
When the Wa-gas returned to Cheek-cheek-alth it is supposed
they found a ladder in this beautiful valley which extends from earth
to Heaven, and climbed it to Werse-on-now, (Heaven) where they
dwell with God. All the half castes with the exception of a few went
away with the Wa-gas, and nearly all those that were three quarters
Indian remained with our people. This is said to be the reason why
some of our people are very fair. Some of the Indians are still
looking for their return to the earth, when they come back it is
believed that peace and happiness will reign supreme again over this
great land and all evil will be cast out. When the present race of the
white people made their first appearance upon the American
continent, we believed it was the Wa-gas returning and a hearty
welcome was extended to them and there was great rejoicing
among our tribes. But soon the sad mistake was discovered to our
sorrow, when the men began to debauch our women, give whiskey
to our men and claim our land that our forefathers had inhabited for
so many thousands of years, yet not a single family has ever been
driven from their house on the Klamath river up to this day. We no
longer termed them as Wa-gas, but as Ken-e-yahs, which means
foreigners, who had no right to the land and could never appreciate
our kindness, for they were a very different people from the Wa-gas.
They had corrupt morals that brought dissolution upon our people
and wrought the horrors of untold havoc.
When the Indians first reached the Klamath river there were large
prairies and vast tracts of grassy land, which have since grown up in
timber and under-brush. Many of the prairies were set on fire and
burnt off every year during the dry seasons which kept the timber
from growing up very fast.
The Klamath emptied into the ocean at Wilson creek, about six
miles north of where it now goes into and ocean at Reck-woy. There
were high bluffs of rocks between the river and the ocean all the
way from Reck-woy to Wilson creek, which kept the river in its
course to Ah-man (Wilson creek) where it emptied into the ocean.
The river was said to have kept in this course until our Christ caused
the mighty rocks to split open and the waters of the river rushed
ahead to the ocean at Reck-woy, where it has ever since flowed into
the ocean.
The traditions handed down say that the land, north of Redwood
creek, where it goes into the ocean, extended far out into the sea to
the large rock that is now known to the white people as Redding
rock, has continually washed away leaving this rock jutting up from
the ocean depths and can be seen for many miles over the
surrounding area of land and sea. This rock is located at a distance
of about ten miles from the shore and is called by the Indians Sa-
quan-ow. This name translated into English means an acorn pestle,
a conical shaped stone, carved out of granite and is used to pound
acorns and grass seeds into the finest flour. Long ages ago Redding
rock extended up from the ocean to a great height, and from a
distance appeared to be a huge Sa-quan, or pestle, hence its name.
After ages of erosion the massive rock became surrounded by water
and the receding bluffs left it alone out in the ocean where its
greater portion has crumbled and fallen beneath the waves as it is
seen today. The Indians still call it Sa-quan-ow.
There has been but little change in the channel of the Klamath
river, except at its mouth since our arrival in this land. In olden times
the channel of the river was very deep and clear and much narrower
than it is now and large bars of alluvial soil composed its banks,
where luxuriant grasses grew, and upon these lowlands during the
winter months great herds of deer and elk would graze, coming
down from the snow covered mountains. The channels of the large
creeks and tributaries of the river, such as Blue creek, (Ur-ner) Tec-
tah and Pec-wan have practically never changed as they still flow
into the river in the same places. Where the Trinity river flows into
the Klamath river it has made but little or no change during the
passing ages as has been handed down to us.
We have no word of severe earthquakes in our regions, but have
had slight shocks from time to time throughout the centuries. We
have no tales of any great damage ever done by earthquakes and
our people never held any fear of tremors of the earth. But my
people tell of great tidal waves that have swept our country. They
say a long time ago one swept up the Klamath river to the mouth of
the Trinity river, a distance of over forty miles, and did great
damage, as it swept away houses and thousands of our people were
drowned and carried away by the rolling waves of the ocean, so few
of our tribe were left that they were well nigh exterminated. Many
smaller tidal waves have swept over the coast where the destruction
was not so great.
They tell of epidemics that came up the river and laid us low in
the devastation of life, thousands of our people would pass away in
a single season; they would die so fast that they could not be buried
and many of the bodies would be thrown into the river. The only way
we could keep the whole tribe from complete devastation by the
ravages of these dreadful diseases was to abandon the dead and
leave the river and go back into the high mountains and there we
built bark houses and remain until the snow and cold would compel
us to retreat to the lowlands again. In our mountain home we
subsisted on wild game, berries, pine nuts, roots and herbs. Some of
our people would have such a terror of the fatal diseases that they
would refuse to return to their homes and would brave the fierce
storms of the cold winter until they were convinced that all dangers
had ceased. In our traditions of the passing centuries many of these
epidemics have almost devastated the land of human life. During
one of these contagions it was said that the children would go down
to the river to swim and would lie down in rows from six to twelve in
number upon the sand, as if they were alive and had been placed
there by careful hands; but they would be in their eternal sleep,
contagion having overtaken them.
CHAPTER V.
TIME AND NAMES.
WE have ten months for one year, and four seasons, as follows:—
1st month: Caw-cha-witch.
2nd month: Nan-ah-wetch.
3rd month: Nachk-sa-witch.
4th month: Chaw-na-ah-wertch.
5th month: Mere-i-yaw.
6th month: Cauh-chow.
7th month: Chere-wer-sere.
8th month: Cana-wal-a-ture.
9th month: Cher-mick.
10th month: Wealth-ah-wah.
Spring: Key-atch-ker.
Summer: Kis-sa-no.
Autumn: Ka-yock-ka-muck.
Winter: Cah-mah.
We lose time in our count each year, so we throw in or stop
counting until the time comes around to start again. The Klamath
Indians are good in counting and can count up into the thousands.
We count ten, and ten hundreds for one thousand. All of our
counting is done by whole numbers; we have no fractions. All the
women have to count and count closely in weaving baskets in order
to make the designs come out correctly. We have astronomers,
called Haw-getch-neens, and they keep close observation of the sun,
which we call Ca-chine-wan-now-slay. Day we call Ca-chine; the
moon, Nas-cha-wan-now-sloy, this means the night sun.
English names. Klamath Indian.
An old woman Ca-par-a
Young women Way-yun
Little girl Wer-yes
English names. Klamath Indian.
Baby Oaks
Boat or canoe Yacht
House Och-lum-ilth
Come in the house Och-la-may
How do you do my friend I-ya-quay Nec-tor-mer
Me or I Neck
Yes A
Fire Metch
Mother Calk
Father Tat, or Tatus
Grandfather Peach
Grandmother Gooch
Old man Ma-we-mer
Young man Pay-girk
Large boy Che-na-mouse
Small boy May-wah
Mother-in-law Cha-win
Father-in-law Par-ah
Sister-in-law Netch-nah
Brother-in-law Weitch-tay, or Tay
Uncle Jim
Aunt Tool
Klamath river Health-kick-wer-roy
Redwood timber Keilth
Mermaids Squer-tuck
Silver Salmon Nep-puoy
Steelhead Salmon Squalth
King Salmon Ah-pus
Hook-bill Salmon Cha-goon
Grizzly Bear Nick-witch
Sea or Ocean Pis-calth
The Bald Hills we call Cho-lu, contains many hundreds of acres of
open land, high up where one can see as far as the eye can reach in
all directions.
There is another species of the Salmon caught in the Klamath
river, the English name of which I do not know but we call it Ra-
gawk.
In the year 1850 my people had never heard of the present white
race and we were then making our fires with two pieces of wood,
one the willow and the other of hardwood.
My mother and father never learned to talk English, so I talk to
them only in our own language.
CHAPTER VI.
DEATH AND THE SPIRIT LAND.
THERE is a large and silent river that flows through the shadowy
vale of death. On the banks of this awful and mysterious river dwells
an old woman, called Sye-elth, and she keeps at her side a large
dog, Chish-yah, (the common name for dog).
When an Indian dies, if he has led a dishonorable and wicked life,
a broad path leads his soul down to the banks of the river to the
very door where the old woman lives in her house. When the
wandering soul reaches her door, the Chish-yah tries to drive it back
to the dead body, but the old woman fights the dog off and if she is
successful in her efforts she takes charge of the miserable soul and
sends it on to the opposite side of the river, in the shadowy land of
endless anguish. If the dog is successful in fighting the soul back it
returns to the dead body where life is regained and the person lives
again. This seldom occurs, and only where the body lives in a state
of coma and is supposed to be dead, but after a few hours comes
out of that state and revives into life again. The Chish-yah is seldom
successful, as a case rarely occurs. This is why the Indian never likes
to scold or treat the dog badly.
The old Indians do not like to look at a photograph or to have
their photographs taken, because they say it is a reflection or a
shadowy image of the departed spirit, O-quirlth. They do not like to
see spirits, but they say they have often seen them. This is the
reason they turn their backs on the camera and object so strongly to
having their pictures taken. Often have my people been ridiculed for
their strange actions, but they have a reason for every one of them.
If the civilized man could only respect the reasons and simple ways
of the highest type of primitive man, as much as primitive man
venerates his civilization.
When the spirit comes back to the tired and weary body, and that
body lives again, that person is said to meet a very unfortunate
existence. It is said he is never satisfied with earthly things again.
He is very restless and unhappy as nothing can satisfy his longing
soul, and always meets death suddenly.
On the shore of this mysterious River of Death awaits a young
man, Pa-ga-rick, in his canoe; he is always ready to receive the soul
from the old woman as she hands it into his care. His canoe is
similar in shape and size to the earthly Indian canoes, with the
exception that if one may note carefully that all the canoes contain
in the bow a knob in the center, some three feet back from the bow,
which is the heart, and they say it is the life of the boat. Also the
canoe the Indians use is burned inside and out, and polished
smooth. The canoe that Pa-ga-rick uses for the crossing of the souls
is neither burned or polished and has no heart, therefore it is called
the dead boat, merm-ma. In olden times no Indian would venture
out in a boat upon the water that did not contain a heart, as they
said it was lifeless and would be sure to sink or some disaster befall
it. We call our canoe here on earth, Yatch.
Sye-elth [TN: lives?] just on the bank of this dark River of Death,
Char-reck-quick-werroy, where she gets the souls away from the
dog. She takes it to the water’s edge and gives it to the man in the
dead boat. He takes the soul into his canoe, paddles it across those
silent waters, the awful stillness, the awful fear of death. When the
canoe, Merm-mo or Nee-girk, either name, touches the opposite
shore, Po-ga-rick, takes the soul, o-quirlth, and banishes it into exile,
exile without an end or example in story, and leaves it in a
wilderness. In this wilderness it is damp, a constant gloom is cast,
dark and fearful clouds forever flit, cold winds forever howl and
shriek the agonies of hell.
In this terrible wildness, the souls of the condemned men and
women sustain their misery up on bitter berries, bitter grasses and
roots, and cannot die. They had never lived but a wasted life upon
earth, therefore they can wait to die, as souls never die. These
wretched souls since Time began, and I think the time is sad and
heavy through all the weary ages, since they go wandering,
hallowing, moaning, weeping and wailing, grieving grief without an
end and suffering pain, intense pain that knows no ending. Thus,
Wah-pec-wah-mow, the Great God has seen fit to punish his
disreputable children until the judgment day.
Sye-elth, this old woman, is the satan of my people, Chish-yah,
the dog, is our Guardian Angel. This old woman is our evil doer who
is always trying to influence the Indians away from the path of
rectitude. She hovers about them in life unseen, seeking out their
weak points, that she may lead them evil ways and vindicate her
cruel wants upon their death by taking their souls down the broad
path to the wilderness of anguish. Fearing her powers, fearing the
Unhappy Land, the Indians struggle to live simple and peaceful lives
and never quarrel over their religion.
The wretched souls banished into the wilderness of anguish do not
quarrel with one another, as they are too wretched in their own
agony to concern themselves about others.
The Indian seeing a vision of the unhappy land tries to live the
simple and honest life, near to nature, and their nature’s God.
However, there is not a tribe however well guarded but some and
sometimes many stray afar from the path of rectitude and are lead
into the wilderness of anguish by their cruel Satan, Sye-elth.
My people believe that there will sometime come a chance for
them to become regenerated, or reborn, so that many of them will
be given the opportunity to recompensate for the wickedness of
their former lives and given a chance to live good clean lives in their
second birth. Thus given the opportunity by God when they die
again, they will be rewarded in going to Heaven, Werse-on-now.
However, if the ones given the opportunity of being saved, do not
live lives of integrity after their second birth, they are cast off and
destroyed forever.
The Indians who had always lived the life of integrity on earth
when they die their soul or spirit travels a narrow and winding trail
which takes the soul to north, to a land far away from their native
haunts. This far northern clime is said to be the old land of Cheek-
cheek-alth, where the spirit finds a ladder that reaches from earth
into Heaven. As the spirit climbs the ladder to Heaven it reaches God
on that infinite shore where it dwells forever in flowery fields of light,
straying together with the Master in peace and love, and joining the
spirits of those that have gone before them.
Can you of the Christian faith comprehend why we take so kindly
to your own belief? Yet we think that ours is the most perfect and
yet you call us savage. We love our God almost akin to sadness and
are always ready with a prayer-offering, be it midday hour or in the
hours of the silent night. The Indian in all his savagery, could never
blaspheme the sacred name of his Creator in man’s builded houses,
or in his daily life as he is a child of nature, akin to nature’s God,
that the Divine Being is the beacon light of his soul, showing him life
beyond the grave and into the flowery fields of light and love, on
that infinite shore, into the glories of Heaven.
The Indian through his long centuries of barbarism battled with
the environments of barbaric man. In his child-like nature he taught
his sons and daughters to be kind, courageous, self-denying,
industrious and above all have integrity that could not be
questioned. Fathers, brothers and cousins guarded the mothers,
daughters and sisters, that not one of them may stray into a life of
shame by the passions of designing men. Woman was manifestly the
upholder of her race, loved as the unassuming creature, who gave to
the race clean limbed and vigorous men. But ah, the sad knell, the
approach of civilized man, and his crushing hand of debauchery to
the sorrow of our race, and our laws have long since been
demolished, and with it our true religion, our life blood, our all. Out
of the gloom of saddened years, rising in scattered remnants, who
like the children of Israel that have lived without a country for many
weary centuries, we are struggling to gain our own once more.
Freedom to worship God in our own way and to be allowed to
become citizens of this our own glorious country.
When a illegitimate child was born, mother and child lived in
disgrace and after death could never reach the kingdom of Heaven,
but traveled that broad road which leads to the wilderness, being
forever lost. During their life the mother is always addressed as Caw-
haw, a name that reminds her always of her disgrace every time she
is spoken to, and the child is always reminded of its unwedded
mother. Sometimes the unfortunate mother may marry, but she is
always known as Caw-haw as long as she lives and can not take the
name of the man she marries.
Those who sought unscrupulous brawls were low and disgraced,
all traveled after death the broad road to Satan and are never given
an opportunity to go to Werse-on-now. There are many of the
miserable souls who lived a wasted life on earth, only to enter in the
Spirit Land, the wilderness of anguish.
In marriage the wife takes the husband’s name and the husband
takes the wife’s name, just as an exchange of names and the family
names are handed down from one generation to another. This is
done by giving the name to a daughter, son, cousin, etc., either the
mother or father’s name on both sides of the family. Sometimes the
generation dies out and there are none left of a near kindred, in this
case they sometimes give the name to a close friend and this
custom is followed more by the high families. As an example, some
years ago an old man lived in the Pec-wan village, his name was Ta-
poo-sen. He died some thirty years ago, and at this writing a middle
aged man is living in the Cor-tep village who adopted his name after
his death, and he is known to every one as Ta-poo-sen. There are
quite a number of Indians living at the present time who have taken
the names of deceased relatives or friends. The deceased has been
laid at rest for at least one year before any one takes his or her
name.
The Klamath Indians are very much prejudiced against one taking
their own life. They look down on the act, and if one should take his

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Hematopoietic Stem Cell Protocols Methods in Molecular

Genetic modification of hematopoietic stem cells methods

Stem Cell Protocols 1st Edition Ivan N. Rich (Eds.)

Stem Cell Niche Methods and Protocols 1st Edition Lichao

Human cell culture protocols 3rd Edition Robin D Hugues

Neural Stem Cell Assays 1st Edition Mohan Vemuri

Stem Cell Regulators Volume 87 1st Edition Gerald Litwack

Stem Cell Culture 1st Edition Dr. Jennie P. Mather (Eds.)

For further volumes:

Kevin D. Bunting and Cheng-Kui Qu

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Library of Congress Control Number: 2014942724

© Springer Science+Business Media New York 2014

Printed on acid-free paper

Humana Press is a brand of Springer

Atlanta, GA, USA Kevin D. Bunting, Ph.D.

PART II STEM CELL ENRICHMENT AND HETEROGENEITY

PART III MOLECULAR AND CELLULAR CHARACTERIZATION

PART IV IN VITRO ASSAYS AND DIFFERENTIATION

PART V TRANSPLANTATION ASSAYS AND IMAGING ENGRAFTMENT

PART VI GENETIC MODIFICATION

JENNIFER E. ADAIR • Fred Hutchinson Cancer Research Center, University of Washington

DEBORAH L. FRENCH • Department of Pathology and Laboratory Medicine and Center

The Hematopoietic Stem Cell Landscape

2 Stem Cell Enrichment and Heterogeneity

There are multiple sources of HSCs for experimental study including

3 Molecular and Cellular Characterization of HSCs

Efforts to better understand HSC biology have been catalyzed by

The chapter by Bradley et al. (Chapter 11) describes how the

4 In Vitro Assays and Differentiation

Ultimately it is functional analyses that are critical for defining

5 Transplantation Assays and Imaging Engraftment

Transplantation is the gold standard assay for normal and leukemic

Altogether the techniques described in this book enable a full

Stem Cell Enrichment and Heterogeneity

Investigating the Interaction Between Hematopoietic

migrating through multiple sites. Initially, in the mouse, primitive

2.1 Isolation of Liver, 1. Timed mated embryos or newborn pups.

2.2 Disaggregation 1. PBS (pH 7.2), 310 mOsm.

5. 40 μm Nylon cell strainer.

2.3 Density Gradient 1. 300 ml Density media: Nycoprep Universal: 60 % (w/v) in

2.4 Lineage 1. Lineage depletion antibody cocktail: A mixture of optimally

2.5 Hematopoietic 1. HSC antibody cocktail: A mixture of optimally titrated

3.2 Mice Harvesting 1. Euthanize pregnant mouse by cervical dislocation. Remove

3.4 Single-Cell 1. Gently push embryonic livers through a 40 μm strainer on top

3.4.4 BM 1. Transfer bones into a sterile mortar.

3.5 Density 1. Top up each cell suspension to 20 ml with PBS–2 % serum

3.6 Lineage 1. Pellet cells by centrifuging at 400 × g for 5 min at 4 °C.

14. Rinse the rosetted bead–cell complexes with 1 ml 2 mM EDTA

3.8 Summarized Since fetal hematopoiesis is a developing process, maturation, expan-

1. This osmolarity is appropriate for murine cells and results in

Organ E14.5–16.5 E17.5 E18.5 D0–D4 D5–D8

cells without causing any shift for isotype control from

Mouse age No. of livers No. of femurs No. of spleens

Single-Cell PCR Profiling of Gene Expression

Understanding the gene expression programs that regulate distinct

composition, these approaches were instrumental in advancing the