BIOL-UH 3122 Stem Cell Biology

Week 1_2: Stem cell identification, isolation,

and characterization
Ken-ichiro Kamei
Associate Professor of Biology and Bioengineering
Divisions of Science and Engineering
New York University Abu Dhabi

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Stem cell identification, isolation, and
characterization
2.1 Identification of stem cells

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Stem cells express surface molecules to be used for their
isolation.

Antigen combinations define specific cells.

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Fluorochromes

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Microscope techniques: Confocal Microscopy

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Super resolution microscopy

L. DONG ET AL., SCIENCE 349, 944 (2015) 6

 Relative Benefits and Limitations of
Different Super-resolution Techniques

J. A.Thorley et al., Fluorescence Microscopy (2014), 199-212 7

The Nobel Prize in Chemistry 2014

Single-molecule microscopy STED Single-molecule microscopy 8

 Flow cytometry for multiparameter analysis of
stem cell populations

: Shape and Internal

complexicity
: Size

PMT: Photomultiplier tube 9

Cytoplasmic stem cell features can be detected using
Fluorescence-based approaches.

Red: ABCG2 (ATP-binding cassette (ABC) transporter G2)

Stem cells often express ABCG2 to export Hoechst to outside of cells.

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Determination of frequency of cell division in a population

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Fluorescent proteins

A reporter for gene expression and cell location

Fusion of proteins of interests to track protein location, stability, etc.
A biosensor to investigate molecular functions 12
The Nobel Prize in Chemistry 2008

Discover GFP Use GFP as a reporter Develop GFP variants 13

Lineage tracing by Cre-activated reporters

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Cre-loxP system

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Lineage tracing by Cre-activated reporters

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Lineage tracing by Confetti mouse

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Stem cell identification, isolation, and
characterization
2.2 Isolation of stem cells

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Fluorescence-activated cell sorting (FACS)
A method used to separate different types of cells
from a mixed group.

1. Marking Cells: First, we attach fluorescent tags

to the cells. These tags glow under a special light
and are designed to stick to certain types of cells.
2. Flowing Through a Tube: The cells are put in a
liquid and passed through a tube one by one.
3. Shining a Light: As each cell goes through the
tube, a laser light shines on it. The fluorescent
tags on the cells glow when the laser hits them.
4. Detecting the Glow: A detector measures how
much each cell glows and uses this information
to figure out what type of cell it is.
5. Sorting the Cells: Based on how they glow, the
machine can sort the cells into different groups. It
uses an electric charge to push each cell into a
separate container.

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Magnetic-activated cell sorting (MACS)

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Magnetic-activated cell sorting (MACS)

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Stem cell identification, isolation, and
characterization
2.3 Characterization of stem cells

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“Gold standard” tests: in vivo assays
Xenograft mouse model

SCID: Severe combined immunodeficiency (Deficient of B- and T-cell mediated immunity)

NOD: Non-obese diabetic mutant
NOD/SCID: Lack of B-/T-lymphocytes and low level of natural killer cells 23
Transplantation assay for hematopoietic stem cell activity

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Scheme of teratoma formation assay

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Teratoma formation assay

(A) Photograph of an explanted

teratoma. Images in panels
(B–F) are sections of a teratoma
stained with hematoxylin and eosin to
identify embryonic tissues that
represent all three embryonic germ
layers. Mesoderm is depicted in (B)
and (C) as nascent renal tubules and
glomeruli within a bed of primitive renal
epithelium and as cartilage surrounded
by condensing mesenchyme,
respectively. Endoderm is shown in (D)
as glandular intestinal tissue. Ectoderm
is shown in (E) and (F) as nascent
neural tube and primitive squamous
epithelium, respectively. Bars = 100 µm.

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Cancer stem cells

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Assay for cancer stem cells

Patient-derived xenograft (PDX) model

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Advanced in vivo imaging

BLI: Bioluminecent imaging

FLI: Fluorescent imaging
PET:Positoron emission tomography
SPECT: Single-photon emission
computerized tomography
CT: Computed tomography
US: Ultrasound
MRI: Magnetic resonance imaging

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Pros/Cons of in vivo imaging

Ultrashort echo time

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Principle of positron emission tomography (PET) imaging

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PET tracers

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in vitro stem cell assays: in vitro colony formation assay

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in vitro stem cell assays: organoid culture

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Molecular analysis: Chromatin immunoprecipitation (ChIP)

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Transcriptomic analyses
Reverse-transpription quantitative
real time polymerase chain reaction Microarray RNA sequencing (RNA-seq)

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Single-cell transcriptomics

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10X Genomics

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 Metabolites as cellular functional markers

Metabolites are the outputs of cellular

changes upon stimuli and environmental
changes, and the players for affecting the
other cells.

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Steuer A. E., et al., Front. Chem. 7. Article 319 (2019)
Metabolomics

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Summary

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Quiz and Discussion

If you have a gene-of-interest, how do you investigate its role

during tissue development?

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