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First published online May 14, 2008
Experimental Biology and Medicine 233:930-940 (2008)
doi: 10.3181/0709-MR-234
© 2008 by the Society for Experimental Biology and Medicine
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MINIREVIEW

Imaging Stem Cell Implant for Cellular-Based Therapies

Zhenghong Lee*,1, James E. Dennis{dagger} and Stanton L. Gerson{ddagger}

* Departments of Radiology and Biomedical Engineering, National Center for Regenerative Medicine, Case Western Reserve University, Cleveland, Ohio 44106; {dagger} Department of Orthopedics, National Center for Regenerative Medicine, Case Western Reserve University, Cleveland, Ohio 44106; {ddagger} Division of Hematology-Oncology, National Center for Regenerative Medicine, Case Western Reserve University, Cleveland, Ohio 44106

1 To whom requests for reprints should be addressed at Departments of Radiology and Biomedical Engineering, National Center for Regenerative Medicine, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail: Zxl11{at}case.edu


   Abstract
Top
 Abstract
Introduction
 Stem Cell Imaging Techniques
 Issues with Reporter-based...
 New Developments and Future...
 References
 
Stem cell–based cellular therapy represents a promising outlook for regenerative medicine. Imaging techniques provide a means for noninvasive, repeated, and quantitative tracking of stem cell implant or transplant. From initial deposition to the survival, migration and differentiation of the transplant/implanted stem cells, imaging allows monitoring of the infused cells in the same live object over time. The current review briefly summarizes and compares existing imaging methods for cell labeling and imaging in animal models. Several studies performed by our group using different imaging techniques are described, with further discussion on the issues with these current imaging approaches and potential directions for future development in stem cell imaging.

Key Words: stem cell imaging • hematopoietic stem cell • mesenchymal stem cell • bioluminescent imaging • positron emission tomography • cell-based therapy


   Introduction
Top
 Abstract
 Introduction
Stem Cell Imaging Techniques
 Issues with Reporter-based...
 New Developments and Future...
 References
 
There is increasing evidence that adult human tissues harbor stem cells and progenitor cells that can be used for therapeutic purposes. Bone marrow contains pluripotent hematopoietic stem cells (HSCs) that continuously produce blood cells throughout our lifetime with the characteristics of self-renewal and differentiation into progenitor cells, leading to generation of all hematopoietic lineages. HSC transplantation has proven to be a life-saving therapy for diseases refractory to other treatments (1). A total of 27,902 HSC transplantations were performed from 1991–2002 in Europe for solid tumors alone (2). Although effective, this therapy remains toxic with treatment-related mortality of approximately 5% for autologous transplantation and 20–40% for allogeneic transplantation. Interventions to reduce the toxicity and make this therapy available to a larger patient population are urgently needed. HSC-based gene therapy has been an established cellular therapy for patients with hematological diseases, and it has shown great potential for the emerging field of regenerative medicine. However, the efficiency of current oncoretroviral gene transfer is limited. Two of the most problematic reasons are insertional mutagenesis and low transduction efficiency. In vivo drug selection has been shown to be effective in enriching transduced cells, thus enhancing transgene expression. The effective selection using MGMT (P140K) gene has been shown in numerous studies. To monitor this dynamic selection process as well as to follow HSC transplant in general, noninvasive and longitudinal tracking methods are much needed.

In addition to HSCs, bone marrow also contains progenitor cells that can differentiate into multiple mesenchymal phenotypes (3, 4). It has been proposed that the mesenchymal progenitors of bone marrow are part of a mesenchymal lineage (5) analogous to that described for hematopoiesis and that the multipotential progenitors in marrow may be mesenchymal stem cells (MSCs), or multipotent marrow stromal cells (MSCs). MSCs are pluripotent adult stem cells found in adult donor bone marrow that can differentiate into bone, cartilage, and adipose tissue (4–6). Autologous and allogeneic transplantation of hMSCs has been reported to have positive effects for the treatment of several human diseases and conditions. For example, in bone marrow transplant recipients, intravenously (i.v.)-administered MSCs have been reported to promote engraftment of hematopoietic lineages in cancer patients receiving high dose chemotherapy (7) and ameliorate graft versus host disease (GVHD) (8, 9). Furthermore, i.v.-administered hMSCs have been shown to home to neoplasms in vivo, and thus have the potential to serve as vehicles for the delivery of anticancer therapies (10). Finally, hMSCs injected in the setting of myocardial infarction and stroke have been shown to improve postischemic regeneration and function (11, 12). These studies indicate that MSCs can be engineered to secrete functional cytokines and used for hematopoietic support in vivo. Most of the proposed MSC therapies entail the local application of MSCs. However, systemic delivery of MSCs with subsequent organ engraftment would allow clinical use in a variety of settings such as treatment of osteopenia, myocardial infarction, repair of bone metastasis, and facilitation of hematopoietic engraftment. The promise of MSC therapeutics mandates research leading to a better understanding of the long-term fate and trafficking of transplanted MSCs in vivo. Previously, the majority of such research relied on ex vivo labeling MSCs by various methods, performing transplants, and sacrificing animals at serial post-transplant time points to look for histological evidence of MSC fate. This methodology has the obvious drawback of making the understanding of the longitudinal fate of the transplant within a given recipient impossible. A noninvasive, repeated, and quantitative method to monitor allogeneic hMSC transplants in vivo would be highly desirable.

Our imaging work is mostly with HSCs and MSCs (including related multipotent adult progenitor cells or MAPC, etc.) although there are other types stem cells and progenitors, most noticeably, the embryonic stem cells (ESCs) that are pluripotent. We and others realize that high dose anticancer therapy and HSC transplantation is a curative therapy for a number of hematological malignancies and has been investigated in the management of solid tumors as mentioned above. We have focused on improving transplantation outcome by improving hematopoietic engraftment and reducing complications such as GVHD. We have conducted preclinical and clinical studies with a novel marrow-derived stem cell, using the MSCs as facilitator cells to improve HSC engraftment and overcome immune complications of transplantation. We also developed drug resistance gene therapy of HSCs and developed models of bone marrow protection from chemotherapy and selective expansion of donor cells without using high dose therapy. Despite considerable progress in the therapeutic uses of HSCs and MSCs, little is understood about the biology of stem cell engraftment, especially the dynamic processes of homing, tissue migration, engraftment, and expansion. To elucidate in vivo behavior of these cells, we set out to develop sensitive and quantitative imaging technologies, as reported in this review, to measure stem cell distribution, survival, engraftment, and proliferation. The long-term goal is to use in vivo imaging to monitor stem cell engraftment and interaction with the host microenvironment to improve the therapeutic potential of stem cell infusion and organ implantation for the treatment of cancer and other diseases. Quantitative and sensitive in vivo imaging methods for stem cell tracking are also critical to the optimization of therapeutic potential of stem cells through measuring the impact of various cellular and genetic interventions on the in vivo distribution and survival of the stem cells.


   Stem Cell Imaging Techniques
Top
 Abstract
 Introduction
 Stem Cell Imaging Techniques
Issues with Reporter-based...
 New Developments and Future...
 References
 
Overview.
Recently, noninvasive, imaging-based monitoring methods have been developed to track stem cell transplants by labeling the cells. The goal is to track the disposition, distribution, and migration of stem cells once introduced into the model organism. The ability to track the location of cells will allow several questions to be answered: 1) If injected through the vein where do these cells go? 2) If implanted do cells remain at the site of implantation or do they migrate? 3) Once in target locations, how do they interact with their microenvironment? 4) Can transplanted cells differentiate into desired tissues?

Basically, there are two methods to label the cells: direct and indirect. Direct labeling is to introduce a marker into or onto the cells before transplant/implant that is stably incorporated or attached to the cells. Examples include labeling with iron oxide particles such as the super paramagnetic iron oxides (SPIO) or perfluoropolyether or perfluorocarbon nanobeacons for Magnetic Resonance (MR) imaging (13–15), which is extended from cancer cell labeling, or with [18F]-HFB or [18F]-FDG for Positron Emission Tomography (PET) imaging (16, 17), or with [111In]-Oxine or [111In]-Tropolone for gamma scintigraphic imaging (18–20), which is adapted from traditional labeling of blood cells such as leukocytes for use in infection/ inflammation imaging in Nuclear Medicine (21–23). Direct labeling for MR imaging allows the depiction of initial deposition of the implanted stem cells, but the imaging signal diminishes or becomes undetectable with cell division. A confounding factor with this type of labeling and imaging is that dead cells still generate signals for days before macrophage clearance of cellular debris. Recent data confirm that a significant number of MSCs implanted into an infracted heart undergo apoptosis (24), and direct labeling would show the location of the dead or dying cells. Direct labeling of cells with radionuclides can only determine short-term circulation and homing properties of infused stem cells because the imaging signal decreases with radio-decay, or becomes more diffused with cell division and cell dispersion.

Indirect labeling relies on the expression of imaging reporter genes transduced into the cells before transplantation, which are then visualized upon injection of appropriate probes or substrates. Examples of this approach include labeling the cells with firefly luciferase ( fluc) for bioluminescent imaging (BLI), where visible light is emitted from the biochemical reaction of luciferase enzyme with its substrate; thymidine kinase of herpes simplex type one virus (HSV1-tk, or tk) for PET imaging (25–27), where a pair of high energy gamma photons generated from the radio-decay of radiolabeled substrate of HSV1-TK are detected to form an image; and transferrin receptor (TfR) that can be used for nuclear or MR imaging (28). The reporters for use in optical imaging such as fluc (for BLI) or fluorescent proteins (GFP, RFP, etc., for fluorescent imaging) are modified from traditional lab/bench approaches for cell (including stem cell) related research. fluc brings high sensitivity with BLI on small animal models but cannot be easily translated into clinical uses due to constraints of tissue penetration of visible light photons. However, the other reporter genes such as the tk gene allow quantitative PET imaging and have the potential for translation into a clinical setting, because high energy gamma photons from positron decay can penetrate deep tissues and organs. Overall, reporter gene-based imaging offers unique capabilities for noninvasive and longitudinal measurements, which can minimize the number of research animals required by obviating the need to sacrifice animals at multiple time points.

Descriptions and discussions about different imaging techniques and modalities are beyond the scope of this review. However, nonimaging readers can consult several references that cover these different modalities (29–31). In addition, there are recent reviews on using individual imaging modalities such as using MRI for stem cell imaging (32, 33), or specific/disease-based reviews such as imaging stem cell implant for cardiac applications (34). We have conducted imaging studies that include mainly BLI and PET experiments for general purpose stem cell imaging and are reported in the following sections.

Direct Labeling.
As an initial attempt to determine short-term circulatory and homing properties of MSCs, we conducted planar gamma scintigraphic imaging of the rats infused with [111In]-Oxine–labeled MSCs (19). The MSCs were isolated from 3–4-month-old Fisher F-344 rats and cultured as previously described (35). For [111In]-Oxine labeling, the MSCs were transferred to a local pharmacy of Mallinckrodt, Inc. (Independence, OH) approximately 5 hrs before the scheduled infusion. Different routes of infusion were tested. Fifteen planar whole body images were acquired with one-min intervals using a clinical scanner, the E.CAM (Siemens Medical Systems, Inc., Hoffman Estates, IL) immediately after infusion. The initial results of the labeled MSC infusion with and without vasodilator therapy showed pulmonary entrapment (Figure 1Go). In this experiment, vasodilator (sodium nitroprusside, 1.0 mg/kg body mass) was injected prior to MSCs infusion to promote vasodilatation and pulmonary shunts. The image on the right shows the same rat at 48 hrs postinfusion. To validate the imaging data, organs (lungs, liver, kidneys, spleen, and long bones with marrow) of the rats were harvested, weighed, and the radioactivity quantified by a 1282 CompuGamma (Wallac, Inc., Gaithersburg, MD). The results are expressed as a percentage of injected dose (mean and standard deviation) as shown in Table 1Go. The initial distribution of infused MSCs could only be acquired over a limited time period due mainly to the half-life of the radionuclide (2.8 days for 111In), which is the primary limiting factor for most direct labeling. While being able to quantify the number of injected cells found in different tissues is an advantage of this system, there is a need to monitor stem cell transplant over a much longer time span in order to study homing and engraftment, which clearly cannot be done with 111In labeling.


Figure 1
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  		Figure 1. Gamma scintigraphic scans of two rats at 15 minutes and 48 hours after injection of indium111-oxine labeled MSCs (IV) with (D+) or without (D–) vasodilator.

 

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  		Table 1. Biodistribution Data from 48 Hrs After Transplant Used to Validate Image Data
 
BLI with fluc.
The tremendous advantage of BLI has been increasingly recognized over the past few years. More and more researchers are using BLI to monitor and understand dynamic biological processes in real-time. In vitro assays using a luminometer to measure promoter activity, gene expression, or ATP level have existed for a long time. The most studied and widely used luminescent protein is firefly (Photinus pyralis) luciferase ( fluc). fluc is a 61-kDa monomeric protein, which reacts with its substrate D-luciferin in the presence of oxygen, Mg2+, and ATP to release green light with peak wavelength at 562 nm (36). BLI with fluc has become a valuable imaging modality for monitoring gene expression, protein and protein interaction, viral infection, cancer growth and metastasis, treatment progress, and cell trafficking in real-time (37–43). fluc is also very useful as a marker for cells for studying migration in vivo. In transplantation models, bone marrow cells of the donors can be tagged by retroviral vectors or lentiviral vectors containing the fluc gene. After infusion, tagged bone marrow cells can be tracked to provide information on their homing, expansion, and engraftment. Wang et al. used lentiviral vectors to transduce human CD34+ and more primitive CD34+CD38+ cells obtained from umbilical cord blood, and the transduced CD34+ and CD34+CD38+ cells were infused into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Bioluminescent signals were generated after 2 to 3 weeks and up to 108 days, which was consistent with short-term engraftment of CD34+ cells and late-term engraftment of CD34+CD38+ cells (43). Similar experiments were conducted by Cao et al., using fluc transgenic mice with β-actin promoter as donor. KTLS (Lin–Sca-1+c-Kit+Thy-1lo) HSC was purified from the donor and transplanted into the irradiated recipients at different concentrations. BLI signals indicated that initial foci in the spleen and bone marrow have different kinetics, but overall BLI signal was consistent with concentration of purified HSC (44).

We experimented with fluc on HSCs for BLI on mouse models for enrichment of HSC transplant (45). The purpose was to monitor the impact of drug selection on the engraftment of short-term repopulating cells and its potential impact on the long-term repopulating cells. Lentiviral vector containing fluc (pLV-fluc) was generously provided by Dr. D. B. Kohn (Children’s Hospital Los Angeles, CA) with a MND promoter, which was derived from myeloproliferative sarcoma viruses (MPSV) and has been shown to effectively promote gene expression in bone marrow cells (46). The chemo-drug resistant gene P140K MGMT and fluc gene were linked by the ribosomal cleavage site, 2A sequence, a sequence from foot-and-mouth disease virus, which is 19 amino acids long and has been reported to be an intraribosomal cleavage site (47, 48). P140K-MGMT gene and 2A PCR product were cloned together into pLV-fluc with unique restriction sites, and the clones were digested and sequenced to ensure the correct orientation and in frame (Figure 2Go). Bone marrow cells were collected from the femur and tibia of 6–8-week-old female BALB/c mice (Jackson Laboratories) 4 days after treatment with 150 mg/kg 5-fluorouracil (5-FU). Cells were transduced with P140K MGMT-2A-fluc lenti-viruses at MOI of 1.4. Forty-eight hrs after transduction, cells were mixed at different percentage of transduced and untransduced cells (Table 2Go). Congeneric 7–8-week-old female BALB/c mice (Jackson Laboratories) were used as recipient mice and separated into two pre-transplantation conditions. Lethally irradiated recipients received 800 cGy from Cs source 1 day before transplantation, and nonmyeloablated recipients received chemo-drugs BG (30 mg/kg) and BCNU (10 mg/ kg) 48 hrs before transplantation. A mixture of 1 million bone marrow cells labeled and unlabeled with fluc was injected through the tail vein into each recipient mouse. BLI was performed on a cooled-CCD camera IVIS 200 (Xenogen, Palo Alto, CA) at various time points. Each mouse was anesthetized and injected intraperitoneally (i.p.) with 110 mg/kg of D-luciferin (Pierce Biotechnology, Inc.) right before imaging. Image signal from each mouse was compared and pseudo-colored. Lethally irradiated recipients received no further drug treatment. BLI signal was detected in mice with larger number of reporter gene transduced cells (Figure 3BGo) as early as 6–8 days after transplantation. By Day 12–14, the strong signal appeared at the region of the spleen, and at other locations, especially in the bone marrow of the femur and vertebrate. By week 3, signals from the spleen remained while that from the others were reduced and even disappeared. The transgene expression level or clonal expansion from transplanted bone marrow cells appears to be a dynamic process. The progenitor cells may die off or migrate to another location over a couple of days. HSC engraftment in nonmyeloablated recipients appears to be impeded, perhaps because there is no niche for the injected cells to engraft. Introducing a selective gene into transgene and using drug selection have been proven to be a feasible approach (49). We used temozolomide (TMZ), an oral methylating agent that has been very effective against advanced cancer and brain tumor (50), and proven effective for in vivo selection of HSC (51). Three weeks after transplantation, nonmyeloablated recipients were treated with BG (30 mg/kg) and TMZ (80 mg/kg) for 3 consecutive days. Images were taken each week to monitor the clonal expansion of transduced HSC. Three weeks later, a second BG + TMZ treatment was administered. In one non-myeloablated recipient, which received 6 x 105 transduced bone marrow cells without drug treatment, BLI signal was present on Days 8 and 14, but the signal disappeared afterward (Figure 4BGo). In another nonmyeloablated recipient with drug treatment, the signal was detected before the treatment and spread throughout the body after the treatment, indicating the strong expansion and engraftment of transduced HSC (Figure 4AGo). Drug selection could also have induced differentiation of transduced progenitor cells, causing the increased and spread signal. However, the engraftment pattern was more similar to the engraftment of HSC in lethally irradiated recipients. These results are encouraging for using drug selection to enhance engraftment in gene therapy.


Figure 2
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  		Figure 2. P140K-MGMT-2A-fluc lenti-vector with MND promoter and 2A cleavage site.

 

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  		Table 2. Mixture of Transduced and Untransduced Bone Marrow Cells for Transplantation
 

Figure 3
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  		Figure 3. Imaging of P140K-MGMT-2A-fluc lenti-vector transduced BALB/c bone marrow cells infused into lethally irradiated congeneric mice with different cell numbers. (A) 1 x105 transduced cells plus 9 x 105 untransduced cells; (B) 6 x105 transduced cells plus 4 x 105 untransduced cells.

 

Figure 4
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  		Figure 4. Pattern of clonal expansion and engraftment of P140-MGMT-2A-fluc lenti-vector transduced bone marrow cells under selection of BG + TMZ in non-myeloablated condition. (A) 1 x105 transduced plus 9 x105 untransduced cells injected via tail-vein, and the mouse received two sets of BG + TMZ treatments (see text); (B) 6 x105 transduced plus 4 x105 untransduced cells also injected via tail-vein without drug selection.

 
Imaging with NIS Gene.
Na+/I– Symporter (NIS) facilitates the accumulation of iodide by thyroid follicular cells to concentrations 20–40-fold over the plasma levels. Human NIS is expressed in thyroid, salivary, and lactating mammary glands as well as gastric mucosa. NIS is not expressed elsewhere and is thus a good reporter gene for imaging. The radio-probes for NIS are simply the radio-iodide, I-125, I-123, and I-131, or technetium [99mTc]-pertechnetate ([99mTc]-TcO4–). All of these radionuclides are readily available in almost all Nuclear Medicine clinics and no radio synthesis is required. Both radioiodide and [99mTc]-TcO4– are well understood for their metabolism and clearance in the body. The imaging potential of NIS has been shown in vitro (52). Tumor xenografts expressing exogenous NIS have been imaged in vivo (53). Recent mouse scintigraphic studies have thoroughly documented pharmacodynamics of radioiodide and pertechnetate in the target organs and surrounding tissues/organs (54). The NIS reporter gene has been used to monitor neural stem cells (NSCs) (55).

We used NIS as a reporter gene specifically for imaging of MSC homing and engraftment on a mouse model with radioiodide or pertechnetate. A second generation lentiviral vector was used to optimize the transduction of MSCs. A 2.5kb fragment containing human NIS was isolated from 7.9kb pcDNA3 plasmid (gift from Sissy Jhiang, Ohio State University) by restriction digestion and then ligated into the 8.65kb wpt vector using the Fast Link DNA Ligation Kit from Epicenter. The lentiviral vector wpt.hNIS supernatant was added to MSCs and incubated for 6 hrs for 2 consecutive days. After hNIS transduction, radioiodide uptake in NIS-MSCs was determined. The iodide (uptake) counts were proportional to the number of NIS-transduced MSCs (56). The perchlorate inhibition assay was also performed showing complete inhibition of I-123 uptake and validated the specificity of NIS mediated iodide uptake in the transduced cells (56).

Two imaging experiments were conducted using NOD-SCID mice as recipients of human MSC transplant/implant (57). These NOD-SCID mice are deficient in B, T, and NK cells and therefore do not reject human cells, and have been successfully used to test engraftment of human hematopoietic cells (58). All animal procedures were approved by the local IACUC.

In one group, NOD-SCID mice implanted with porous, fibronectin-coated ceramic cubes (Zimmer, Inc., Warsaw, IN) loaded with NIS-transduced human MSC (total of 4 cubes, 2 cubes placed in each flank, 1 cm apart each containing different numbers of NIS-MSCs) were on the same mouse. Ceramic cubes can concentrate a range of MSCs in a physical location to enhance tracer signal and provide a microenvironment for long-term survival of MSCs in NOD-SCID mice. In a second group, NOD-SCID mice were injected subcutaneously (s.c.) with 1 x 106, 2 x 106, and 4 x 106 NIS-MSCs per mouse on different mice. This was to test the mobility of s.c.-injected MSC.

One day after cube implant, [99mTc]-TcO4–, a substrate for NIS, was injected through the tail vein and the NOD-SCID mice were imaged on the X-SPECT (Gamma Medica, Northridge, CA) 25–30 minutes postpertechnetate injection. During SPECT imaging, the animal was kept under isoflurane gas anesthesia maintained by the EZ-anesthesia system (Euthanex, Palmer, PA). The images were reconstructed by using an iterative algorithm available on the scanner. A CT scan was conducted immediately after the SPECT scan while the anesthetized animal remained in the same position, and was easily aligned with SPECT images as shown in Figure 5Go. The top three CT images are axial, sagittal, and coronal cuts of the mouse. The middle three are the same cuts from SPECT scan and bottom three panels are the pseudo-color overlay between the two. Compared with the negative controls (with blank cube implants, images not shown here), the positive controls clearly showed uptake of pertechnetate. Figure 5BGo shows the s.c. injection of 2 x106 NIS-MSC (arrow). The tracer dose and imaging parameters were identical to the cube experiments. While these initial imaging results were very encouraging for identifying the positive control targets, background signals from thyroid and stomach due to endogenous NIS gene expression could potentially interfere with true targeting signals. The other issue was the loss of image signal as encountered by other investigators using the same CMV promoter to drive imaging reporter gene(s) for stem cells imaging (59, 60). We thus moved to another report gene system for labeling as discussed next.


Figure 5
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  		Figure 5. Imaging stem cell implant with NIS gene as the reporter. Overlays of CT and SPECT images with the arrows pointing to the upper left cube (A), which is loaded with NIS-transduced MSCs; to the s.c. injection site (B) with the same NIS-transduced MSCs. The thyroid/salivary glands and the stomach (gastric mucosa) have high uptake in both (A) and (B) due to endogenous NIS expression.

 
Imaging with the Triple Fusion Reporter.
This reporter system allows for longitudinal imaging, both qualitatively and quantitatively, of transplants of transduced human MSCs in vivo in small animal models. The triple reporter gene fluc-mrfp-ttk (a gift from Sam S. Gambhir, Stanford University) encodes a fusion protein containing functional components from fluc, monomeric red fluorescent protein (mrfp), and truncated HSV1 sr39tk (ttk), whose product can thus be visualized using BLI, fluorescent, and PET imaging, respectively (61). The three components or domains were linked by 14-amino acid (LENSHASAGY-QAST) and 8-amino acid (TAGPGSAT) segments, respectively. The ttk domain contains a deletion in the first 135 bp that eliminates the nuclear localization signal. As a result, higher cytosolic versus nuclear concentration brings less cytotoxicity and improved image signal. Enzymatic activity of each domain in this reporter was previously shown to be 54% for fluc, "medium" for mrfp, and 100% for ttk, when compared with their full-length individual counterparts. The choice of this fusion reporter with undiminished tk activity was based on the potential for PET imaging that would be most useful in future clinical translation. Though this and similar reporters have been used previously in the context of stem cell transplants (61–64), our study is unique in its use of a second generation lentiviral vector for reporter gene transduction, in the use of native human MSCs, and in the thorough examination of reporter effects on stem cell phenotype. In particular, the differences between this and the other published MSC tracking study (27) are: 1) this study is a general purpose investigation on imaging-based MSC tracking technique while the other study focused on tumor metastasis tracking; 2) primary human MSCs were used here instead of immortalized MSCs; 3) this study used a triple fusion reporter ( fluc-mrfp-ttk) instead of a dual reporter (egfp-tk), and the addition of fluc allows convenient and sensitive BLI imaging; and 4) a modified myeloprolif-erative sarcoma virus (MPSV) promoter or MND promoter was used to drive triple reporter expression as it has been shown to be more resistant to epigenetic silencing (65) than the promoter of EF1{alpha} or CMV.

Transduction of hMSCs at an MOI of 8 yielded a transduction efficiency of 83% as determined by mrfp FACS analysis. Reporter transduced human MSCs (for imaging) were loaded into the same ceramic cubes used previously as shown in Figure 6AGo and subcutaneously implanted into NOD-SCID mice as before. It was determined that as many as about 50K cells can be loaded into a cube by this procedure (66). To evaluate quantitative correlation of image-based measurement for estimating the number of the cells, cubes were loaded with either reporter transduced MSCs or a diluted mix of reporter transduced MSCs and empty vector-transduced MSCs. Control cubes were loaded with wild-type MSCs, empty vector-transduced MSCs, or no MSCs (empty cubes, yet another control); this allowed for intrasubject positive and negative controls. After receiving hMSCs via cube implants, mice were imaged at multiple time points for up to 3 months by BLI using the Xenogen IVIS 200 System. While still under anesthesia after BLI, the same mice were imaged by a special planar x-ray system developed jointly by Thomas Jefferson National Lab (Newport News, VA) and Case Western Reserve University (Cleveland, OH) to precisely determine cube position. BLI and planar x-ray images were aligned using Pmod (software package from Pmod Technologies, Zurich, Switzerland), which allowed semiquantitative analysis of BLI data in defined regions from the aligned x-ray images. Figure 6BGo shows the BLI signal 4 days after cube implant. There is a remarkable difference in the signal intensity between the diluted and undiluted cells that were transduced with the triple reporter. For the upper two cubes loaded with undiluted human MSC transduced with the reporter, a larger signal region resulted from the extensive diffusion and scattering of the visible light photons. The BLI signals were detectable beyond 3 months postimplantation, during which the BLI imaging signal intensity from the cubes fluctuated (67).


Figure 6
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  		Figure 6. Imaging cube implants using the triple fusion reporter. (A) ceramic cube for cell loading and implant; (B) BLI of 8-cube implants: top row (yellow arrow) loaded with transduced hMSCs, second row (green arrow) loaded with a mixture of wt and transduced hMSCs, and the rest with either vector-transduced or being empty; (C) overlays of PET (hot metal) and CT (grey scale) images: transaxial (left) and coronal (right) views, in which the cubes were also visible on the CT images. While top-row cubes (yellow arrows) had strong PET signals, the second row cubes (green arrow) loaded with a mixture of wt and transduced hMSCs were visible despite of dilution. High and nonspecific uptake of FHBG in the gut is due mainly to FHBG’s lipophilicity.

 
After BLI imaging, selected animals were imaged by CT and PET at different time points up to 2 months postimplantation. The anesthetized animals were scanned by microCT using the CT component of the Gamma Medica’s X-SPECT scanner. After the CT scan, the animal was transported to a nearby R4 microPET scanner (Siemens/Concord, Knoxville, TN) for PET imaging. 9-[4-[18F]fluo-ro-3-(hydroxymethyl)butyl]guanine ([18F]-FHBG), a substrate for herpes thymidine kinase (TK), was injected i.v. by tail vein, and the PET scan was performed 50 min after injection. The PET images were reconstructed using a 2-D ordered subset expectation maximization algorithm. Alignment between PET emission and CT images was done using software developed in-house. Figure 6CGo showed an overlay of PET slice with corresponding CT from the same animal in Figure 5Go (4 days after cube implantation). The top two cubes loaded with undiluted stem cells were clearly visible, and the cube with diluted cells was also visible on the microPET scan. Regions of interest (ROI) were defined centered on cubes seen on the CT images that were aligned with the PET images. Regional data were defined as the sum of the measured radioactivity within a ROI at a specific time point. PET imaging signal from the cubes remained detectable for 2 and half months (67).


   Issues with Reporter-based Imaging
Top
 Abstract
 Introduction
 Stem Cell Imaging Techniques
 Issues with Reporter-based...
New Developments and Future...
 References
 
Stem Cell Properties.
The procedure to transduce reporter into stem cells for imaging would first ensure that the reporter system is working to provide sufficient signal for imaging. An equally important issue is whether such procedures have changed any of the proliferation and differentiation potential of the labeled stem cells. Specifically, is there any alteration to the stem cells after the transduction of the reporter gene system by way of lentiviral vector? To address this crucial issue, transduced human MSCs were compared with wild-type and empty vector-transduced MSCs to examine their ability to differentiate towards adipogenic, chondrogenic, and osteogenic lineages (67).

Stem cells were induced to form adipocytes for adipogenic assay and examined using flow cytometry for the proportion of cells staining with Nile Red, a measure of cellular lipophilic content. By this method, transduced MSCs had an adipocytic phenotype similar to that in hollow lentiviral vector-transduced MSCs (vector-transduced) and untransduced MSCs (wt MSCs or untransduced MSCs) as shown in Figure 7AGo.


Figure 7
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  		Figure 7. Retention of differentiation potentials after lenti-vector transduction of the reporter gene system. (A) adipogenic, (B) osteogenic, (C) chondrogenic assays between wt hMSCs (left) and transduced hMSCs (right).

 
Transduced MSCs, vector-transduced MSCs, and un-transduced MSCs were incubated in osteogenic medium containing 10% fetal bovine serum (FBS), dexamethasone, β-glycerophosphate, and ascorbic acid 2-phosphate for osteogenic assay. Controls were incubated without dexamethasone. Calcium was extracted and measured as a marker for osteoblastic differentiation. The osteogenic potential of transduced MSCs was preserved (Figure 7BGo).

For chondrogenic assay, MSCs were cultured in aggregate form in medium that contains insulin, transferrin, selenous acid, dexamethasone, sodium pyruvate, ascorbic acid 2-phosphate, and TGF-β1. The resulting pellets were fixed and stained with toluidine blue (0.2%) for visual assessment of chondrogenic potential. Figure 7CGo showed no obvious difference between the transduced and wild-type MSCs.

Recently, microarrays have been used to evaluate the changes in the gene expression profiles associated with the stem cell differentiation (68, 69). Although a published study has examined the effects of the transduction of same triple fusion reporter genes on the transcriptional profiles of mouse embryonic stem cells (63), no study has been performed to investigate the transduction effects on human MSCs. Therefore, the transcriptional changes associated with the reporter gene transduction process were evaluated with an oligonucleotide human microarray, and RT-PCR was used to validate the microarray results (70). Significant Analysis of Microarray (SAM) identified 87 upregulated and 69 downregulated genes, respectively, with high accuracy. Annotation analysis showed that genes involved with development (FOXG1B), lipid metabolism (PTGDS), immune response (IL-8), ubiquitin cycle (UBE2D3), cell adhesion (PCDHB10), cytoskeleton organization/biogenesis (COL15A1), etc., were differentially expressed. Furthermore, some anti-apoptosis (SERPINB2, TNFAIP3) and apoptosis (PMAIP1) genes were upregulated. Genes associated with cell growth or cycles are either upregulated (DCBLD2, FGF5, ESM1; E2F7, CKS2, CDC42) or down-regulated (IGF2, FGF7, IGF1; CDKN2A, CDKN2B, CDKN1C). Other genes, such as HMGA2C, which is associated with adiopogenesis, and MSC differentiation were upregulated, while LEPR (a gene related to adipocytes), MGP, PTGER4 (regulation of ossification), COMP (a cartilage-related gene) were downregulated. Despite these changes, the expression of the reporter had no significantly adverse effect on the viability, proliferation, and differentiation of MSCs as the main genes with MSC characteristics pertaining to Adipose (Leptin, Adipose diff-related factor, C/EBP {alpha} and β), Bone (Osteonectin, osf2, Cbfa-1), Cartilage (Jagged 2, Biglycan, Fibromodulin, Decorin, Aggrecan), Endo/Epithelial (VEGF, LDL Recepoter), Muscle (Cardiac Specific ATPase, Actin, gamma 2, Calreticulin, Capping protein, Z-line), Neural (TrkA Receptor, TrkB Receptor, Calretini, Neurotrophin 5), Stromal Support (SCF, SDF, M-CSF, Flt-3 ligand, IL-6), and others (MHC I, MHC II) were not altered by the reporter gene transduction process. Validation assays demonstrated the retention of the potentials of reporter-transduced MSCs for osteogenic, chondrogenic, and adipogenic differentiations.

Quantitative Issues.
Using reporter gene-based approach or direct labeling for imaging, the fundamental questions for quantitative stem cell tracking are how to accurately estimate the number of cells from the image data; how sensitive are the imaging results and what is the smallest number of the cells that can be detected; and how precise the localization of the cells can be in vivo. Usually, it requires a series of calculations and calibrations to convert detected photon counts on the image to the number of the viable stem cells at a site or organ of interest (or an ROI on an image). Previously published data, mostly from using tumor cells, on the sensitivity of similar type of microPET scanner for detecting TK-expressing cells using the same [18F]-FHBG tracer revealed a lower threshold of microPET detection at a cell density of about 1.0 million/mL (27, 71). The cubes loaded with transduced MSCs had the cell density above this lower threshold of microPET detection were clearly imaged as shown in Figure 6CGo, and cell density in the diluted cubes that were also detectable on microPET imaging (Figure 6CGo) was only about one third of the reported lower threshold. Human MSCs are larger than other cell types, such as the tumor cells used to derive the lower limits of detection, which might contribute to the greater detection sensitivity from our study. The absolute number of the transduced human MSCs giving detectable PET imaging signals in one spot (such a cube) was estimated to be around 5,000–10,000 (67).

The factors involved in quantification are equilibrium (quasi steady state) of tracer uptake, concentration of reporter gene tagged the cells, biological half lives of the tracer, etc. We are in the process of setting up a math model to aid quantitative analysis of the sequentially or dynamically acquired image data to account for stem cell proliferation, differentiation, and apoptosis at different stages of the cell life span.

Finally, due to the limitation of spatial resolution and sensitivity of the current BLI, microPET, or microSPECT systems, precise localization of single cells or stem cell niche in animal models remains difficult. We are developing a cryo-macrotome with microscopic fluorescent and potentially β particle (from C-14, H-3 decay) detection capabilities, which would allow single cell detection during whole body slicing of a rodent.


   New Developments and Future Efforts
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 Abstract
 Introduction
 Stem Cell Imaging Techniques
 Issues with Reporter-based...
 New Developments and Future...
References
 
Currently, strong promoters such as CMV, MND, etc., are used to drive constitutive expression of the reporters such as NIS, fluc, GFP, or dual-, tri-fusion reporter genes. Stem cells tagged with such a reporter system will light up like beacons, which would allow longitudinal monitoring of their distribution and dynamics. However, when stem cells differentiate into other lineages, such an event would not be distinguished from the image signal of undifferentiated cells. Our attention has been shifted to event- or location-specific promoters that will turned on when a specific event/ function occurs or the infused stem cells arrive at a specific site or organ. One example was to use cardiac specific promoter {alpha}-Myosin Heavy Chain ({alpha}-MHC) to detect cell differentiation into cardiac myocytes (72). We have constructed vectors with TIE-1 promoter, which is endothelial specific, to drive reporter expression for imaging angiogenesis. Stem cells transduced with such vectors can be tracked for their action in repair/regeneration, or for their role in tumor metastasis. We have also constructed vectors containing collagen {alpha}1 type I promoter (Col2.3) specifically for osteogenic differentiation (73). Stem cells transduced with such vectors can be followed to assess their repair function in a bone fracture model.

So far, all imaging methods are developed to label the cells before transplant or implant. To track endogenous stem cells’ activation, recruitment, homing, and engraftment, or other functions, the logical thinking is to develop specific ligand(s) to target corresponding markers on the surface of these cells. The problem with this kind of approach is that stem cells usually lack specific surface markers (74). An example will be Chemokine (C-X-C motif) receptor 4 (CXCR-4). Homing and engraftment of HSC are directed in part by stromal cell–derived factor-1{alpha} (SDF-1) and CXCR-4 (specific for SDF-1) pair, the so-called SDF-1/CXCR-4 axis (75, 76). SDF-1 and its receptor CXCR-4 were shown to be critical in human stem cell engraftment and repopulation in NOD-SCID mice (77). However, CXCR-4 expression has been documented on mature blood cells, including lymphocytes, monocytes, megakaryocytes, and platelets. It has also been detected on CD34+ progenitors purified from bone marrow, mobilized peripheral blood and cord blood, and is NOT specific for HSCs. In addition, many of the stem cell surface phenotypes are variable depending on the micro-environments that the cells are exposed. Other functional markers are transient in response to the stimuli. For example, the c-Met receptor is induced by HGF and hypoxia (78). Finding targetable marker candidates for imaging endogenous stem cells and their functional behaviors remains a challenge.


   Acknowledgments
 
We thank a group of contributors (listed in alphabetic order) who participated in various experiments or stages of the studies: Amad Awadallah, Yunhui Kim, Dr. Omer Koç, Jeffrey Kolthammer, Yu Kuang, Yuan Lin, Zachary Love, Basabi Maitra, Dr. Stan Majewski, Joseph Molter, Nicolas Salem, Dr. Luis A. Solchaga, Fangjing Wang, and Dr. Andrew Wiesenberger.


   Footnotes
 
This work was supported in part by DOE (DE-FG02-03ER63597 to Z Lee), NIH R21 (EB001847 to Z Lee), NIH R24 (CA110943 to JL Duerk), NIH R01(AR49785 to JE Dennis), NIH R01 (CA073062 to SL Gerson), and JLab sub-contract (SURA-04-Q011).


   References
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 Abstract
 Introduction
 Stem Cell Imaging Techniques
 Issues with Reporter-based...
 New Developments and Future...
 References
 

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