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STEM CELL

TRACKING
TECHNIQUES
M.Vinoth

MVM09003

ADVISORY COMMITTEE

Chairman:
Dr.A.PALANISAMY,Ph.D
Professor,
Department of Animal Biotechnology,
Members:
Dr.K.KUMANAN, Ph.D
Professor and Head ,
Department of Animal Biotechnology,
Dr.S.BALASUBRAMANIAN,Ph.D
Associate Professor,Department of Animal
Reproduction, Gynaecology &obstetrics

Stem cell
A cell that has the ability to continuously divide
and differentiate (develop) into various other
kind(s) of cells/tissues
Stem cell tracking techniques / in vivo stem cell
imaging
Techniques which has ability to non invasively
monitor stem cell trafficking in vivo

NEEDS OF THIS TECHNOLOGIES


Methods

are needed to non-invasively


monitor the following aspects of stem cell
therapies:
Niche

and engraftment

Where are the cells?

Expansion

and viability

How many cells are there?

Differentiation

Have cells differentiated?

To monitor distribution, density, proliferation,


and transdifferentiation of stem cells after
transplantation ( in vivo)

CHARACTERISTICS OF AN IDEAL
IMAGING TECHNOLOGY
Biocompatible, safe, and nontoxic
No genetic modification or perturbation to the stem
cell
Single-cell detection at any anatomic location
Quantification of cell number
Minimal or no dilution of contrast agent with cell
division
Minimal or no transfer of contrast agent to non
stem cells
Non invasive imaging in the living subject over
months to years
No requirement for injectable contrast agent

(frangioni et al.,2004)

IMAGING TECHNOLOGIES FOR


TRACKING STEM CELLS
Magnetic Resonance Imaging(MRI)
Optical Imaging

Fluorescence

Radionuclear Imaging
Positron

Emission Tomography(PET)
Single Photon Emission Computed Tomography
(SPECT)

X-Ray computed microtomography (microCT)

(zhao et al.,2010)

Bioluminescence

STEM CELL LABELING

Direct Labeling with Exogenous Agent

Using a Reporter Gene

STEM CELL LABELING - STRATEGY #1:


DIRECT LABELING WITH EXOGENOUS
AGENT
Radionuclide tag
SPIONs

radiotracer

fluorescent agent

Fluorescent tag

DIRECT LABELING
Advantages

simple
Can use clinically approved agents

Disadvantages
Signal

per cell dilutes on cell division


Can only follow for a short time (esp.
radiolabeled)
No information on cell viability or
differentiation

(zhao et al.,2010),

Very

STEM CELL LABELING


USING A REPORTER GENE
DNA

promoter

reporter

transcription
mRNA

translation
protein
Fluorescent protein (OPT)
Receptor (NUC)
Enzyme (OPT, NUC, MRI)

LABELING FOR PET,SPECT AND


MRI

[18F]-FHBG

Meral Beksac, 2009

or [124I]-FIAU is radioactive reporter


probe that has specificity for TK enzyme.
The probe is transported into cells and is
phosphorylated by the TK protein only in the
genetically labeled stem cells.
The human dopamine D2 receptor (hD2R)
human somatostatin receptor (hSSTR2)
human transferrin receptor (hTfR) function as
transmembrane receptors
It actively transport their corresponding reporter
probes into the genetically labeled cells

REPORTER GENE LABELING

Advantages

Signal increases if cell population expands


Can follow cells indefinitely as long as reporter gene is
expressed
Can use tissue specific promoters to identify when
differentiation occurs

Disadvantages

Cells must be transduced with reporter plasmid


Use of genetically modified cells more difficult for
translation
For indirect reporters (PET, SPECT, MRI) need to inject
substrate or ligand for reporter protein

Kraitchman et al.,2009

MAGNETIC RESONANCE IMAGING

MAGNETIC RESONANCE IMAGING


It uses a powerful magnetic field to align the
magnetization of some atoms in the body, then
uses radio frequency fields to systematically alter
the alignment of this magnetization.
This causes the nuclei to produce a rotating
magnetic field detectable by the scanner
This information is recorded to construct an
image.

MRI CONTRAST AGENTS


Two main classes of agents:

T1-agents

gadolinium-based
Paramagnetic
loaded via pino/endocytosis into stem cells
permit tracking for up to 6 weeks

T2 agents

Based on superparamagnetic iron oxide (SPION)particles


Superparamagnetic
track extremely small numbers of stem cells for up to several
weeks

BMSCS LABELED SPION; ARROW


INDICATES AN IRON MICROSPHERE

(Nohroudi et al.,2010)

MRI IN VIVO BMSC

MR IMAGING OF MIGRATION
OF STEM CELLS IN RAT BRAIN

From: Modo M, Hoehn M, Bulte JWM: Cellular MR Imaging. Molecular Imaging 4: 143-164, 2005.

ADVANTAGE (MRI)
MRI scan is harmless to the patient. It uses
strong magnetic fields and non-ionizing
radiation, unlike CT scans and traditional X-rays
use ionizing radiation
High spatial resolution
Outstanding anatomic imaging
MRI meets the requirements of penetration
depth
clinical availability
sensitivity 10-3-10-5 M

(Villa et al.,2010), (Nohroudi et al.,2010)

DISADVANTAGE (MRI)
Dilution of contrast with cell division
Difficulty in quantification because of
susceptibility artefact
The potential transfer of contrast to non stem
cells, such as macrophages, after stem cell death.
A significant clinical problem common to all MRI
methods is that certain implantable devices, such
as pacemakers and defibrillators,
Long scan times for large volumes/high
resolution

(Villa et al.,2010), (Nohroudi et al.,2010)

NUCLEAR IMAGING
Positron Emission Tomography (PET)
Single Photon Emission Computed Tomography (SPECT)

POSITRON EMISSION TOMOGRAPHY


(PET)
PET tracer emits positrons which annihilate with
electrons up to a few millimeters away, causing
two gamma photons to be emitted in opposite
directions.
A PET scanner detects these emissions
"coincident" in time, which provides more
radiation event localization information and gives
higher resolution images

gambhir et al .,2000

ADVANTAGE (PET)
The cross-sectional information and threedimensional (3D) reconstruction capability offer
more informative than the optical imaging
techniques
Sensitivity 10-11-10-12 M
Quantification possible

Zhang et al.,2009, gambhir et al .,2000

DISADVANTAGE (PET)
Resolution in PET is less than that which can be
achieved by MRI.
Ionizing radiation
Requires genetic modification of stem cell
Intravenous injection of contrast agent
It is not readily available

Zhang et al.,2009, gambhir et al .,2000

SINGLE PHOTON EMISSION COMPUTED


TOMOGRAPHY (SPECT)
The tracer used in SPECT emits gamma
radiation that is measured directly
SPECT imaging is performed by using a gamma
camera to acquire multiple 2-D images , from
multiple angles. A computer is then used to apply
a tomographic reconstruction algorithm , yielding
a 3-D dataset

ADVANTAGE (SPECT)
Sensitivity 10-10-10-12 M
3D full-body scanning,
No dilution of effect size with cell division
(transgenic approaches)

Blackwood et al 2009,

DISADVANTAGE (SPECT)
Requires genetic modification of stem cell
Intravenous injection of contrast agent
Ionizing radiation
Quantification can be difficult

OPTICAL IMAGING

Bioluminescence imaging (BLI)


Fluorescence imaging

BIOLUMINESCENCE IMAGING
(BLI)
Bioluminescence is the process of light emission
in living organisms.
The DNA encoding the luminescent protein
(luciferase )is incorporated into stem cell via a
viral vector
Bioluminescence utilizes light generated by the
enzyme luciferase to detect cells in vivo.
The reporter probes for these proteins are
substrates that are oxidized and generate light.
Ultra-sensitive CCD camera can image
bioluminescence

IN VIVO BIOLUMINESCENCE IMAGING

CCD

use gene delivery


mechanisms to
introduce luciferase
gene into cells of
interest.
Inject luciferin

image luminescence at
surface of animal

Xenogen Corp.

ADVANTAGE (BLI)
High sensitivity 10-15-10-17 M
No ionizing radiation

DISADVANTAGE (BLI)
Requires genetic modification of stem cell
Intravenous injection of contrast agent,
luciferase genes and substrates described to date
generate only visible (400 to 700 nm) light, which
has very high absorption and scatter in living
tissue.
Limited to small animal use
Even in mice false-negative scanning can occur,
dependent on cell depth

Meral Beksac, 2009

FLUORESCENCE IMAGING
Fluorescence imaging utilizes organic (eg, green
fluorescent protein) as exogenous contrast agents
for in vivo imaging.
Because of high photon absorption and scatter at
visible wavelengths are recorded

(Frangioni et al.,2009)

LABELING FOR FLUORESCENCE


IMAGING

The DNA encoding the GFP is incorporated into


stem cell via a viral vector
Fluorescence imaging detects cells that express
fluorescent proteins - enhanced green fluorescent
protein (eGFP).
The excitation and emission peaks for eGFP
occur well below 600 nm
GFP absorbs blue light and emits green
fluorescence without exogenous substrates or
cofactors and provides a convenient and efficient
way to identify labeled cells.

Meral Beksac, 2009

ADVANTAGE
High sensitivity 10-9-10-12 M
No ionizing radiation,
Fast
Signals from relatively superficial sites, such as
skin and subcutaneous tissues, or from deep sites
after removal of overlying tissues, can offer highresolution images.

DISADVANTAGE
Limited to small animal or intraoperative use
The major problem with NIR fluorescence is that
even with tomographic imaging methods,
detection is limited to only 4 to 10 cm of tissue

X-RAY COMPUTED
MICROTOMOGRAPHY (MICROCT)

X-RAY COMPUTED
MICROTOMOGRAPHY (MICROCT)
Microtomography uses x-rays to create crosssections of a 3D-object
The term micro is used to indicate that the pixel
sizes of the cross-sections are in the micrometer
range
Advanced microCT is capable of achieving a
spatial resolution up to 0.3 m

Cancedda et al 2007

ADVANTAGE (MICROCT)
high definition and resolution human cells after
transplantation
quantification of the number of cells
Readily available,
3D, full-body scanning

DISADVANTAGE (MICROCT)
Requires high molar concentrations of contrast
agent,
Artifacts from bone and cardiac devices,
Ionizing Radiation

SOME FINDINGS THROUGH THESE


TECHNIQUES
Transplantation of predifferentiated rather than
undifferentiated hES cells would be more suited
for avoiding teratoma formation.(Li et al.,2008)
(both MRI/BLI were used)
Labeled NPC are recruited to infarcts with both
parenchymal and cerebrospinal fluid
administration, but higher initial photon counts
suggest that cerebrospinal fluid administration is
more efficient(kim et al.,2004) (BLI was used)
After BMSC transplantation through intravenous
route , 17% of infused cells localized to the marrow
space within 15h(cui et al 2005)(PET was used)

SUMMARY

MR imaging is a better technique for highresolution detection of cell location posttransplantation .


Bioluminescent imaging complementary to other
modalities such as MRI

Zhao et al.,2010

Reporter gene imaging using PET is a better


technique for monitoring long-term cell viability,
death, and proliferation

CONTINUE

Dual- and multimodality imaging technology like


MRI/PET ,MRI/BLI/Ultrasound might improve
the prospects for stem cell tracking
To confirm the fate of stem cells in vivo, it is
crucial to continue the development of stem cell
tracking techniques

Frangioni et al 2010

single contrast agent/detector is not fulfil the


Characteristics of an Ideal Imaging Technology,