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