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animal biotechnology
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STEM CELL TRACKING TECHNIQUESM.VinothMVM09003
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 cellA 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 imagingTechniques 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
Bioluminescence Fluorescence
Radionuclear Imaging Positron Emission Tomography(PET) Single Photon Emission Computed Tomography
(SPECT) X-Ray computed microtomography (microCT)
(zhao et al.,2010)
STEM CELL LABELING Direct Labeling with Exogenous Agent
Using a Reporter Gene
STEM CELL LABELING - STRATEGY #1: DIRECT LABELING WITH EXOGENOUS AGENT
SPIONs
Radionuclide tag
Fluorescent tag
radiotracer
fluorescent agent
DIRECT LABELING Advantages
Very simpleCan use clinically approved agents
DisadvantagesSignal per cell dilutes on cell divisionCan only follow for a short time (esp.
radiolabeled)No information on cell viability or
differentiation
(zhao et al.,2010),
STEM CELL LABELING USING A REPORTER GENE
promoter reporterDNA
mRNA
protein
transcription
translation
Fluorescent protein (OPT)Receptor (NUC)
Enzyme (OPT, NUC, MRI)
LABELING FOR PET,SPECT AND MRI [18F]-FHBG 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
Meral B
eksac, 2009
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
Kraitchm
an 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), (N
ohroudi 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), (N
ohroudi 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 three-
dimensional (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)
Blackw
ood 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
use gene delivery
mechanisms to introduce
luciferase gene into cells of
interest.
Inject luciferinimage luminescence
at surface of animal
CCD
© 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 B
eksac, 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 B
eksac, 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 high-resolution 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
cross-sections 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 µm
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 15 h(cui et al 2005)(PET was used)
SUMMARY Reporter gene imaging using PET is a better
technique for monitoring long-term cell viability, death, and proliferation
MR imaging is a better technique for high-resolution detection of cell location post-transplantation .
Bioluminescent imaging complementary to other modalities such as MRI
Zhao et al.,2010
CONTINUE… single contrast agent/detector is not fulfil
the Characteristics of an Ideal Imaging Technology,
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