Molecular imaging of human embryonic stem cells.
ABSTRACT Human embryonic stem cells (hESCs) are a renewable source of differentiated cell types that may be employed in various tissue regeneration strategies. However, clinical implementation of cell transplantation therapy is hindered by legitimate concerns regarding the in vivo teratoma formation of undifferentiated hESCs and host immune reactions to allogenic cells. Investigating in vivo hESC behaviour and the ultimate feasibility of cell transplantation therapy necessitates the development of novel molecular imaging techniques to longitudinally monitor hESC localization, proliferation, and viability in living subjects. An innovative approach to harness the respective strengths of various imaging platforms is the creation and use of a fusion reporter construct composed of red fluorescent protein (RFP), firefly luciferase (fluc), and herpes simplex virus thymidine kinase (HSV-tk). The imaging modalities made available by use of this construct, including optical fluorescence, bioluminescence, and positron emission tomography (PET), mat be adapted to investigate a variety of physiological phenomena, including the spatio-temporal kinetics of hESC engraftment and proliferation in living subjects. This chapter describes the applications of reporter gene imaging to accelerate basic science research and clinical studies involving hESCs through (1) isolation of a homogenous hESC population, (2) noninvasive, longitudinal tracking of the location and proliferation of hESCs administered to a living subject, and (3) ablation of the hESC graft in the event of cellular misbehavior.
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ABSTRACT: Coccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively. We found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (T(EM)) and IFN-γ release. In conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.BMC Immunology 01/2010; 11:60. · 2.25 Impact Factor
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ABSTRACT: Molecular imaging is revolutionizing the way we study the inner workings of the human body, diagnose diseases, approach drug design, and assess therapies. The field as a whole is making possible the visualization of complex biochemical processes involved in normal physiology and disease states, in real time, in living cells, tissues, and intact subjects. In this review, we focus specifically on molecular imaging of intact living subjects. We provide a basic primer for those who are new to molecular imaging, and a resource for those involved in the field. We begin by describing classical molecular imaging techniques together with their key strengths and limitations, after which we introduce some of the latest emerging imaging modalities. We provide an overview of the main classes of molecular imaging agents (i.e., small molecules, peptides, aptamers, engineered proteins, and nanoparticles) and cite examples of how molecular imaging is being applied in oncology, neuroscience, cardiology, gene therapy, cell tracking, and theranostics (therapy combined with diagnostics). A step-by-step guide to answering biological and/or clinical questions using the tools of molecular imaging is also provided. We conclude by discussing the grand challenges of the field, its future directions, and enormous potential for further impacting how we approach research and medicine.Physiological Reviews 04/2012; 92(2):897-965. · 29.04 Impact Factor
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ABSTRACT: During the past two decades, stem cells have created enthusiasm as a regenerative therapy for ischemic heart disease. Transplantation of bone marrow stem cells, skeletal myoblasts, and endothelial progenitor cells has shown to improve myocardial function after infarction. Recently, attention has focused on the potential use of embryonic stem cells and induced pluripotent stem cells because they possess the capacity to differentiate into various cell types, including cardiac and endothelial cells. Clinical trials have shown positive effects on the functional recovery of heart after myocardial infarction and have answered questions on timing, dosage, and cell delivery route of stem cells such as those derived from bone marrow. Despite the current advances in stem cell research, one main hurdle remains the lack of reliable information about the fate of cell engraftment, survival, and proliferation after transplantation. This review discusses the different cell types used in cardiac cell therapy as well as molecular imaging modalities relevant to survival issues.Trends in cardiovascular medicine 08/2010; 20(6):183-8. · 4.37 Impact Factor
©2006 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
Molecular Imaging of Human Embryonic Stem Cells
Keeping an Eye on Differentiation, Tumorigenicity and Immunogenicity
Koen E.A. van der Bogt1-3
Joseph C. Wu1,4,*
1Molecular Imaging Program at Stanford; 2Laboratory of Cardiothoracic
Transplantation; 4Department of Medicine, Division of Cardiology; Stanford
University School of Medicine; Stanford, California USA
3Department of Surgery, Leiden University School of Medicine; Leiden, The
*Correspondence to: Joseph C. Wu; Department of Medicine and Radiology;
Edwards Building, Room R-354; Stanford, California 94305-5324 USA; Tel.:
650.736.0234; Fax: 650.736.0234; Email: firstname.lastname@example.org
Original manuscript submitted: 10/13/06
Revised manuscript submitted: 10/20/06
Manuscript accepted: 10/22/06
Previously published online as a Cell Cycle E-publication:
embryonic? stem? cell,? molecular? imaging,?
immunogenicity,? tumorigenicity,? teratoma,?
[18F]-FHBG? 9-(4-[18F]-fluoro-? ?
Human embryonic stem cells (hESCs) are capable of differentiation into every cell
type of the human being. They are under extensive investigation for their regenerative
potential in a variety of debilitating diseases. However, the field of hESC research is still
in its infancy, as there are several critical issues that need to be resolved before clinical
translation. Two major concerns are the ability of undifferentiated hESCs to form teratomas
and the possibility of a provoked immune reaction after transplantation of hESCs into a
new host. Therefore, it is imperative to develop noninvasive imaging modalities that
allow for longitudinal, repetitive, and quantitative assessment of transplanted cell survival,
proliferation, and migration in vivo. Reporter gene‑based molecular imaging offers these
characteristics and has great potential in the field of stem cell therapy. Moreover, it has
recently been shown that reporter gene imaging can be combined with therapeutic
strategies. Here, we provide an outline of the current status of hESC research and discuss
the concerns of tumorigenicity and immunogenicity. Furthermore, we describe how
molecular imaging can be utilized to follow and resolve these issues.
“NIH Human Embryonic Stem Cell Registry”.5
dERivAtion, MAintEnAnCE And diFFEREntiAtion
feeder? layer? for? the? undifferentiated? growth? of? hESCs? because?
cross-species? retroviral? infection,? however,? this? is? an? unattractive?
option? in? the? long? term.? Recently,? several? reports? have? described?
proteoglycans?TRA-1-60/81? and?TRA-2-54;? and? enzyme? alkaline?
differentiated? cultures? is? indispensable? for? developing? cell? based?
diFFEREntiAtion into MESodERM LinEAgES
diFFEREntiAtion into ECtodERM LinEAgES
Using? different? growth? factors? and? stimulating? environments,?
of? neurodegenerative? disorders? is? under? intensive? investigation.?
the? EB,20-22? factor-induced? neuronal? differentiation? seems? to? be?
diFFEREntiAtion into EndodERM LinEAgES
a? variety? of? growth? factors.15? The? creation? of? insulin-secreting?
pancreatic? cell? populations? has? generated? much? interest,? as? this?
transplanted? in? immunodeficient? mice,? hESCs? form? teratomas?
One? factor? influencing? hESC-based? teratoma? formation? is? the?
differentiate? at? the? subcutaneous? site? but? remain? undifferentiated?
Another? factor? influencing? teratoma? formation? is? the? number?
discussed? earlier,? transplantation? of? pure? undifferentiated? hESC?
Recently,? Muotri? and? colleagues? have? studied? undifferentiated?
for? cell? incompatibility? is? the? major? histocompatibility? complex?
cells.? Thus,? the? transplanted? cells? may? not? ?
priming? of? alloreactive? CD4+? T? cells? through?
ferentiated? mESCs? for? their? ability? to? trigger?
alloimmune? response? in? a? murine? model? of?
myocardial? infarction.36? We? found? progressive?
intra-graft? infiltration? of? inflammatory? cells? ?
mediating? both? adaptive? (T? cells,? B? cells,? and?
dendritic? cells)? and? innate? (macrophages? and?
granulocytes)? immunity.? Cellular? infiltration?
progressed? from? mild? infiltration? at? two? weeks?
rejection? of? the? mESC? allograft.? Moreover,? we?
found? an? accelerated? immune? response? against?
mESCs? that? had? differentiated? in? vivo? for? two?
weeks,? suggesting? that? mESC? immunogenicity?
Although? it? was? previously? reported? that?
immunocompetent?mice,37 a?recent report?using?
eliminated? at? 1? month? post-transplantation.38?
response? to? transplanted? allogeneic? hESCs? are?
strategies? include:? (1)? forming? HLA? isotyped?
hESC-line? banks;? (2)? creating? a? universal? donor? cell? by? genetic?
monitor? cell? survival? and? differentiation.? However,? these? reporter?
genes? cannot? be? used? to? reliably? track? in? vivo? characteristics? of?
of? background? signal.? Instead,? GFP-labeled? cells? are? typically?
Figure 1. Schematic overview of molecular imaging. Outline of a vector containing a DNA report‑
er construct with the reporter gene(s) driven by a promoter of choice. Transcription and translation
lead to production of mRNA and reporter protein, respectively. After administration of a reporter
probe systemically, the reporter probe will be catalyzed by specific cells that have the reporter
proteins. This amplification process can be detected by a sensitive imaging device. Examples of
reporter genes and their specific reporter probes are listed per imaging modality. Abbreviations:
Fluc, Firefly luciferase; PET, positron emission tomography; HSV‑ttk, herpes simplex virus truncated
thymidine kinase; [18F]‑FHBG, 9‑(4‑[18F]‑fluoro‑3hydroxymethylbutyl) guanine; SPECT, single
photon emission computed tomography; hNIS, human sodium/iodide symporter; MRI, magnetic
resonance imaging; CCD, charged coupled device; BLI, bioluminescence imaging.
or? tissue? specific.? The? construct? can? be?
ular? biology? techniques? using? either? viral?
or? nonviral? techniques.? Transcription? of?
protein? reacts? with? the? reporter? probe,?
photon? emission? computed? tomography?
iMAging ESC tRAnSpLAntAtion,
A? major? advantage? of? reporter? gene?
construct? into? the? cellular? DNA.? This?
ensures? that? the? reporter? gene? will? only?
be? expressed? by? living? cells? and? will? be?
passed? on? equally? to? the? cell’s? progeny.?
Thus,? this? imaging? modality? can? provide?
significant? insight? into? cell? viability? and?
proliferation.? As? discussed? earlier,? moni-
to? assess? immunogenicity,? as? a? provoked?
Monitoring? cell? proliferation? is? another?
carrying? triple-fusion? (TF)? construct? containing? firefly? luciferase?
(Fluc),? monomeric? red? fluorescent? protein? (mRFP),? and? herpes?
able? to? ablate? teratoma? formation? and? follow? this? progress? non-?
injection? (Fig.? 2).?Whether? lower? cell? numbers? (e.g.,? 1,? 10,? 50),?
whether? they? will? affect? ESC? differentiation? and? hamper?efforts? ?
that? the? TF? reporter? genes? affect? <2%? of? total? genes? of? mESC?
differences? between? control? mESCs? versus? mESCs? with? reporter?
Figure 2. In vivo bioluminescence imaging of teratoma formation after transplantation of 100 hESCs.
(A) Bioluminescence image showing longitudinal follow up after transplantation of 100 hESCs stably
expressing a double fusion reporter gene (Fluc‑GFP). Faint imaging signals were seen as early as 2
hrs after transplant, which became progressively stronger over two weeks. Histology at eight weeks
confirmed teratoma formation. Note one of the hESC transplanted sites did not successfully engraft
(arrow) as there were no detectable signals by two weeks. (B) Histology from a representative explanted
teratoma showing hESCs that have differentiated into derivates from different germ layers. (I) squamous
cell differentiation with keratin pearl; (II) respiratory epithelium with ciliated columnar and mucin produc‑
ing goblet cells; (III) osteoid (nonmineralized bone) formation; (IV) cartilage formation; (V) gland cells;
and (VI) rosette consistent with neuroectodermal differentiation (400x magnification).
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human? cell? types? highlights? their? promising? role? in? regenerative? ?
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