Sage Journals: Discover world-class research

Abstract

Noninvasive positron emission tomography (PET) provides a potential method for in vivo tracking of radiolabeled cells. The goal of this study was to assess the potential toxicity of ⁶⁴Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (PTSM) on rhesus monkey CD34⁺ hematopoietic and mesenchymal stem cells in vitro in preparation for developing imaging protocols posttransplantation. CD34⁺ hematopoietic cells were radiolabeled with 0 to 40 μCi/mL ⁶⁴Cu-PTSM and viability and colony formation were assessed. Rhesus monkey mesenchymal stem cells (rhMSCs) were placed in culture postradiolabeling for assessments of growth and differentiation toward adipogenic, osteogenic, and chondrogenic lineages. The results indicated that CD34⁺ cells radiolabeled with 20 μCi/mL and rhMSCs radiolabeled with 10 μCi/mL ⁶⁴Cu-PTSM did not result in adverse effects on growth or differentiation. Nonradioactive copper was also evaluated and showed that the presence of copper was not harmful to the cells. CD34⁺ cells and rhMSCs radiolabeled with the optimized concentrations of 20 and 10 μCi/mL, respectively, were also assessed using the microPET scanner. Studies showed that a minimum of 2.50 × 10⁴ CD34⁺ cells (1.1 pCi/cell) and 6.25 × 10³ rhMSCs (4.4 pCi/cell) could be detected. These studies indicate that CD34⁺ hematopoietic cells and rhMSCs can be safely radiolabeled with ⁶⁴Cu-PTSM without adverse cellular effects.

STEM AND PROGENITOR CELL THERAPY focuses on using the unique characteristics of stem and progenitor cells to regenerate damaged tissues.^1–3 However, understanding the fate of transplanted cells requires in vivo imaging technologies that can safely and effectively monitor the cells. Traditionally, cell trafficking has been performed using laborious tissue analysis,⁴ but with the development of noninvasive imaging technologies, studying the trafficking of cells in vivo is now feasible. With the wide array of imaging techniques currently in use, it is crucial to consider an imaging modality that is relatively noninvasive, is sufficiently sensitive to detect the cells in vivo, provides a method for quantitation, and uses labeling techniques for imaging that will not alter the structure or function of the cells.

Three common imaging technologies used experimentally for cellular tracking are optical imaging, magnetic resonance imaging (MRI), and nuclear imaging. Optical imaging techniques are based on fluorescence (eg, enhanced green fluorescent protein) or bioluminescence (eg, luciferase).⁵ These techniques have the advantages of being relatively noninvasive and inexpensive and typically do not involve hazardous materials. However, they have some limitations when compared with MRI and positron emission tomography (PET) owing to high scattering and absorption of light in tissue, resulting in limited depth penetration. MRI, using cells labeled with iron oxide nanoparticles, provides another approach.^6,7 Signals are readily obtained at a depth from within tissue, and detection of small numbers of cells has been demonstrated in mouse models.^8,9 However, the labeled cells produce negative contrast and therefore can be readily detected only in tissues that exhibit high MRI signal levels. This makes whole-body imaging studies to determine the biodistribution of cells difficult. The signal generation mechanism, and the fact that the signal void produced by labeled cells can “bleed” into surrounding tissues, also makes quantification problematic.

Nuclear imaging consists of two common modalities, single-photon emission computed tomography (SPECT) and PET,^10,11 which have exceptional sensitivity, reaching nano- to picomolar levels, with the gamma radiation produced having a good penetration depth in tissues. However, PET and SPECT are limited by their low spatial resolution. Recent developments in instrumentation for small animal PET and SPECT has resulted in improved resolution performance (typically 1 to 2 mm for in vivo studies) while still maintaining high sensitivity.¹² The images are also readily quantified, allowing numbers of cells to be determined in vivo, and the whole body can be studied, allowing the biodistribution of cells to be followed over time.

For these studies, the positron-emitting radionuclide ⁶⁴Cu was chosen in conjunction with PET.¹¹ ⁶⁴Cu has a half-life of 12.7 hours and is one of the longer-lived PET radionuclides, allowing labeled cells to be tracked for 2 to 3 days. It also has a relatively low positron emission energy (0.65 MeV),¹¹ which reduces positron range effects in PET imaging. However, in addition to positron emission, ⁶⁴Cu also decays by electron capture and β⁻ decay, making radiation dosimetry somewhat complex. ⁶⁴Cu can be passively delivered into the cells via the lipophilic redoxactive carrier molecule pyruvaldehyde-bis(N4-methylthiosemicarbazone) (PTSM),^13,14 allowing for rapid radiolabeling of cells. PTSM has higher binding affinity for divalent [Cu(II)] rather than monovalent Cu [Cu(I)]. The stable Cu(II)-PTSM is reduced to the more labile Cu(I)-PTSM complex. The charged Cu(I) ion dissociates and is captured by intracellular macromolecules, whereas the neutral PTSM diffuses back out of the cell.^13,14 This passive diffusion process results in the uptake of Cu-PTSM, which is driven by the concentration gradient across the cellular membrane (Figure 1). As previously reported,¹³ the influx of ⁶⁴Cu-PTSM into glioma cells has been shown to equilibrate after about 3 hours.

Figure 1.

Schematic of ⁶⁴Cu-PTSM uptake and retention by cells. PTSM binds with the divalent form of copper to form a complex that can passively diffuse across the cell membrane. Once inside the cell, Cu(II)-PTSM is reduced to the monovalent Cu(I)-PTSM complex and dissociates. Copper is captured by the intracellular macromolecules, whereas the neutral PTSM diffuses back out of the cell. Adapted from Adonai et al.¹³

Although ⁶⁴Cu has good imaging characteristics, very little is known about the effects of ⁶⁴Cu-PTSM on the growth and function of radiolabeled cells. Thus, the studies described focused on assessing the effects of ⁶⁴Cu radiolabeling on the viability, growth, and differentiation of rhesus monkey CD34⁺ hematopoietic cells¹⁵ and mesenchymal stem cells (MSCs)^16,17 at different ⁶⁴Cu-PTSM concentrations. The overall goal was to determine the feasibility of using ⁶⁴Cu to radiolabel stem and progenitor cells for in vivo imaging and to assess cell migration and trafficking. These studies have shown that although the toxic effects on cell growth and differentiation can be observed at the highest concentrations studied, radiolabeling CD34⁺ hematopoietic cells and rhesus monkey mesenchymal stem cells (rhMSCs) is feasible and permits adequately sensitive imaging while ensuring that the toxic effects of the labeling are kept to a minimum.

Materials and Methods

Cell Isolation and Culture

All animal procedures conformed to the requirements of the Animal Welfare Act, and protocols were approved prior to implementation by the Institutional Animal Care and Use Committee at the University of California, Davis.

Preliminary studies were performed using peripheral blood mononuclear cells (PBMCs) obtained from two healthy adult rhesus monkeys (10 mL) following protocols previously described.¹⁸ Briefly, PBMCs were isolated by density gradient centrifugation using Histopaque (1.077 g/cm³; Sigma-Aldrich, St. Louis, MO) at 400g for 30 minutes at room temperature. The mononuclear cell layer was collected, washed with RPMI-1640 medium (Invitrogen, Carlsbad, CA), and centrifuged for 20 minutes at room temperature using established protocols.^15,18

CD34⁺ hematopoietic cells were isolated from bone marrow aspirates (≈10 mL) collected from the iliac crest of 11 rhesus monkeys (4 infants, 4 juveniles, 3 adults) using standardized protocols.¹⁵ The collected cells were filtered through Falcon 40 μm nylon cell strainers (Fisher Scientific, Pittsburgh, PA) and isolated with density gradient centrifugation. Cells were then immunoselected using MiniMACS separation columns using an antibody for CD34⁺ cells (Clone 563 PE antihuman CD34, BD Biosciences, San Jose, CA), as previously described.¹⁵ The CD34⁺ cells were cryopreserved using a controlled rate cryopreservation protocol using 10% dimethylsulfoxide (DMSO) (Sigma) and 10% fetal bovine serum (Invitrogen) and then stored in liquid nitrogen. Cells were thawed immediately before radiolabeling using established techniques.

The rhMSCs were isolated and expanded from early third-trimester fetal bone marrow (120 days gestation [term 165 ± 10 days], N = 3) collected after tissue harvest by flushing the long bones with medium, as previously described.^16,17 The isolated cells were plated at 1 × 10⁵ cells/cm² in polystyrene culture dishes (Falcon, Franklin Lakes, NJ) in Dulbecco's Modified Eagle Medium (Invitrogen) consisting of 20% fetal bovine serum, 1% penicillin-streptomycin (Invitrogen), and 1% L-glutamine (Invitrogen). After 48 hours of incubation at 37°C and 5% CO₂, the cells were washed with phosphate-buffered saline (PBS) (Invitrogen) three times to remove the nonadherent cells, as previously described.^16,17 Medium was replaced every other day until the cells reached ≈80% confluence. The collected cells were then plated at 1 × 10³ cells/cm² in MSC culture medium, incubated at 37°C, 5% CO₂, and passaged when the cells reached ≈80% confluence. The second passage was used for radiolabeling.

⁶⁴Cu-PTSM Production and Labeling

⁶⁴Cu (half-life = 12.7 hours; β⁺: 0.653 MeV, 17.4%; β⁻: 0.578 MeV, 39%)¹⁹ was produced by cyclotron irradiation of ⁶⁴Ni at the Washington University at St. Louis School of Medicine, Department of Radiological Science, using established methods.²⁰ The ⁶⁴Cu chloride solution was buffered in 0.25 mM ammonium acetate. Appropriate amounts of ⁶⁴Cu-ammonium acetate solution was added to 2 μL (10 mg/mL, 82 nM) of PTSM in DMSO and incubated at room temperature for 10 minutes. The crude ⁶⁴Cu-PTSM solution was purified using mini C-18 cartridges (ORTG, Oakdale, TN). The product was loaded onto the C-18 cartridge, washed with water, and subsequently eluted with ethanol (0.1 mL). The ethanol was removed from the product using an Eppendorf Vacufuge 5301 Concentrator (Brinkmann Instruments, Westbury, NY) at room temperature. The remaining radioactive residue was resuspended in PBS and filtered (0.22 μm) for sterility. The product was analyzed using silica thin-layer chromatography (TLC) plates (EMD Chemicals, Gibbstown, NJ), eluted with ethyl acetate, and the plate scanned using an AR-2000 TLC plate reader (Bioscan, Washington, DC).

PBMCs were radiolabeled in suspensions of 0.5 ×10⁶ cells/tube at ⁶⁴Cu-PTSM concentrations of0 to 40 μCi/mL in different volumes of RPMI-1640 medium (250, 500, 1,000 μL). Suspensions of CD34⁺ hematopoietic cells (1.0 × 10⁶ cells/tube) were radiolabeled with ⁶⁴Cu-PTSM at concentrations of 0 to 40 μCi/mL in 1 mL of culture medium consisting of X-Vivo 15 serum-free medium (Cambrex Bio Science, Baltimore, MD), Flt3/Flk2 ligand (R&D Systems, Minneapolis, MN), stem cell factor (R&D Systems), and thrombopoietin (R&D Systems). rhMSCs were plated in six-well plates at cell concentrations of 0.5 × 10⁶ cells/well and allowed to adhere overnight. The attached rhMSCs were incubated with 0 to 40 μCi/mL ⁶⁴Cu-PTSM in MSC culture medium (1 mL). All cell labelings were performed at 37°C, 5% CO₂ conditions for 3 hours. After incubation, all free ⁶⁴Cu-PTSM was removed by washing three times with PBS. The pellet and washes were counted using the Wallac Wizard 1470 gamma counter (Perkin Elmer, Shelton, CT).

Nonradioactive copper labeling was performed using the same methods described above. The labeling concentrations used were 0 to 1,000 pmol of Cu/mL for CD34⁺ hematopoietic cells and 0 to 230 pmol of Cu/mL for rhMSCs.

T-Cell Proliferation Assay

Radiolabeled PBMCs were evaluated for their proliferation capability using the colorimetric 5-bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (Roche, Penzberg, Germany) according to the manufacturer's instructions and established protocols.^21,22 Briefly, the cells were plated onto 96-well plates at 5.0 × 10⁴ cells/well with 50 μL 10× ConA in triplicate and incubated at 37°C, 5% CO₂ conditions for 4 days. After incubation, 10 μL/well of 100 μM BrdU labeling reagent was added and incubated for 24 hours. Plates were then centrifuged for 10 minutes at 1,500 rpm at room temperature and denatured with FixDenat solution (Roche) for 30 minutes. With the addition of 100 μL/well anti-BrdU conjugated to peroxidase, the cells were then incubated at room temperature for another 2 hours. After removing the antibody conjugate, substrate solution was added for 15 minutes, and then the reaction was stopped by the addition of 25 μL/well of 1 M H₂SO₄. The absorbance was immediately measured at 450 nm using a microplate reader (Model 680, Bio-Rad, Hercules, CA).

Hematopoietic Colony-Forming Unit Assay

Colony-forming unit (CFU) assays were performed on the radiolabeled CD34⁺ hematopoietic cells as previously described.¹⁵ Briefly, the cells were suspended in MethoCult GF⁺ H4435 containing erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, stem cell factor, interleukin-3, and interleukin-6 (Stem Cell Technologies, Inc., Vancouver, BC) and plated in fibroblast-free 35 mm plates at 5.0 × 10³ cells/cm² in duplicate. The plates were incubated at 37°C, 5% CO₂ for a standard incubation period of approximately 10 days.¹⁵ At the end of the culture period, the individual erythroid and myeloid progenitors were counted and collected.

rhMSC Viability and Proliferation

⁶⁴Cu-PTSM-labeled rhMSCs were cultured overnight in fresh medium and then trypsinized, and the viability was assessed using trypan blue exclusion dye. The quantity of viable cells was determined using a hemacytometer and light microscopy (Olympus America Inc., Center Valley, PA). Cells were replated in six-well plates at 1 × 10³ cells/cm². Proliferation was checked every other day for approximately 14 days. For each time point, the number of viable cells was counted.

In Vitro rhMSC Differentiation

rhMSCs were differentiated toward adipogenic, osteogenic, and chondrogenic lineages using Adipogenic, Osteogenic, and Chondrogenic Differentiation Medium, respectively (Cambrex Bio Science) and following the manufacturer's recommendations.¹⁶ For adipogenic differentiation, ⁶⁴Cu-PTSM-labeled rhMSCs were plated at 2.1 × 10⁴ cells/cm² in duplicate and cultured in MSC culture medium until confluent with fresh medium changes every 2 to 3 days. Once confluence was achieved, cells were induced with Adipogenic Induction Medium. Cells were stimulated toward adipogenic differentiation under three cycles of induction and maintenance culture conditions at 37°C, 5% CO₂. The cells were then cultured for approximately 7 days in the Maintenance medium, with medium changes every 3 days. Adipogenic differentiation was noted by observation of lipid vacuoles in the culture plates. For analysis, cells were washed with PBS, fixed with 10% formalin, and stained with Oil Red O dye. The dye was then extracted with isopropanol and measured for optical density (OD) at 510 nm using a spectrophotometer.

For osteogenic differentiation, ⁶⁴Cu-PTSM-labeled rhMSCs were plated at 3.1 × 10³ cells/cm² in duplicate at 37°C, 5% CO₂ overnight. Cells were induced with Osteogenic Induction Medium, which was changed every 3 to 4 days for 2 to 3 weeks. Cells were then assessed for calcium production by suspension in 0.5 N HCl for 4 hours. The supernatant was mixed with Calcium Coloring Reagent (Teco Diagnostics, Anaheim, CA), and OD was measured at 570 nm. Using a standard curve, the OD values were converted into calcium concentration.

For chondrogenic differentiation, approximately 2.5 × 10⁵ labeled rhMSCs were pelleted by centrifugation at 150g for 5 minutes at room temperature. Cells were induced to differentiate with Chondrogenic Induction Medium. The pellets were incubated at 37°C, 5% CO₂ for 3 to 4 weeks while replacing the induction medium every 2 to 3 days. At the end of induction, pellets were collected and frozen in OCT embedding compound (Sakura Finetek, Inc., Torrance, CA). The pellets were then sectioned at 5 μm and stained for glycosaminoglycans using the Safranin O stain. Ten random sections were chosen using a random generator and counted to estimate the volume of the chondrogenic fraction of the pellet using the Cavalieri method and CAST Grid Stereology System (Visiopharm, Hørsholm, Denmark).²³

In Vitro Imaging

In vitro microPET of the radiolabeled CD34⁺ cells and rhMSCs was performed in 96-well plates. Cells were plated at eight different cell densities: 0, 6.25 × 10³, 1.25 × 10⁴, 2.50 × 10⁴, 5.00 × 10⁴, 1.00 × 10⁵, 2.00 × 10⁵, and 4.00 × 10⁵ cells/well, which approximately corresponded to a total radioactivity of 0, 0.007, 0.014, 0.03, 0.06, 0.11, 0.22, and 0.45 μCi, respectively, for CD34⁺ cells and 0.03, 0.06, 0.11, 0.22, 0.44, 0.89, and 1.78 μCi, respectively, for rhMSCs. The final volume of each well was 200 μL. One plate contained the four higher cell densities (5.00 × 10⁴ to 4.00 × 10⁵ cells/well) and the second plate the lower cell densities (0 to 2.50 × 10⁴ cells/well). The plates were placed on the scanning bed and imaged for 30 minutes using a microPET P4 scanner (Siemens Preclinical Solutions, Knoxville, TN).²⁴ The images were reconstructed using a filtered back-projection algorithm to yield a pixel size of approximately 1.9 × 1.9 mm (matrix = 128 × 128). The slice thickness (z dimension) was 1.2 mm (with 53 slices total).

Statistical Analysis

The results were reported as the mean ± standard error of the mean, and calculations were performed using Microsoft Excel (Microsoft, Redmond, WA). The statistical significance for group comparisons was determined by analysis of variance and a two-sided Student t-test, where p < .05 was considered statistically significant.

Results

Preliminary Studies with PBMCs

Preliminary studies were performed with PBMCs and focused on viability and proliferation when cells were radiolabeled with differing ⁶⁴Cu-PTSM concentrations (0, 5, 10, 20, and 40 μCi) in different media incubation volumes (250, 500, 1,000 μL RPMI). With ≥ 20 μCi, viability was found to decrease approximately 10-fold when compared with control, unlabeled cells (p < .05). Using an established lymphocyte proliferation assay,^21,22 significant differences were observed with concentrations ≥ 20 μCi (250 μL volume) (data not shown). At ⁶⁴Cu-PTSM levels < 20 μCi, no adverse effects were observed. Since the effects on viability and proliferation were observed in these preliminary studies with the 20 μCi/mL concentration, the same volume range of ⁶⁴Cu-PTSM (0–40 μL/mL) was used for studies with CD34⁺ hematopoietic cells and rhMSCs. In addition, because the 1 mL medium volume with 3 hours of incubation showed good labeling efficiency, cell viability, and cell proliferation, this volume and incubation time were chosen for the study.

Labeling Efficiency and Cell Viability

CD34⁺ hematopoietic cells from 11 rhesus monkeys and rhMSCs from three fetal rhesus monkeys were radiolabeled with 0 to 40 μCi/mL ⁶⁴Cu-PTSM, and the labeling efficiency was measured using a gamma counter with an energy window (400–600 keV) around the 511 keV photopeak. The results of these studies are shown in Figure 2 and suggest differences in the quantity of radioactivity retained in each of the cell types assessed. For example, CD34⁺ hematopoietic cells had a labeling efficiency of up to 0.8 pCi/cell (at 40 μCi/mL), whereas rhMSCs radiolabeled with the same quantity of ⁶⁴Cu-PTSM showed a labeling efficiency of up to 8.2 pCi/cell.

Figure 2.

⁶⁴Cu uptake per cell for CD34⁺ hematopoietic cells and rhesus monkey mesenchymal stem cells (rhMSCs). Uptake of ⁶⁴Cu for rhMSCs was 3 to 10 times higher when compared with CD34⁺ hematopoietic cells. The amount of ⁶⁴Cu retained at 10 μCi/mL with rhMSCs was also higher when compared with CD34⁺ cells at 40 μCi/mL.

Viability was assessed 24 hours postlabeling using trypan blue dye exclusion. The percentage of viable CD34⁺ cells (14 to 30% range for all of the radiolabeling concentrations, including the unlabeled control) showed a decreasing trend as the ⁶⁴Cu-PTSM concentration increased. In contrast, there was no evidence of effects on viability when rhMSCs were radiolabeled with ≤ 20 μCi/mL ⁶⁴Cu-PTSM. At 40 μCi/mL, the viability of rhMSCs was found to be 88%, which was not statistically significant when compared with the lower ⁶⁴Cu-PTSM concentrations (p > .05).

CD34+ Hematopoietic Cells

CD34⁺ hematopoietic cells were assessed for effects on hematopoietic colony formation (CFUs) by plating 5.0 × 10⁴ radiolabeled CD34⁺ hematopoietic cells in MethoCult, as previously described, and the resulting erythroid (CFU-E) and myeloid (CFU-GM) progenitors were assessed.¹⁵ CD34⁺ cells radiolabeled with 10 and 20 μCi/mL resulted in 64 ± 15 and 45 ± 12 myeloid (CFU-GM) colonies, respectively, which were not significantly different when compared with colony formation of unlabeled, control CD34⁺ cells (83 ± 19 colonies). However, at 40 μCi/mL, a significantly lower number of CFU-GM (22 ± 6, p < .05) (Figure 3) was found. Erythroid progenitors (CFU-E) ranged from 16 ± 7 to 31 ± 15, which was not significantly different when compared with the unlabeled, control CFU-E (29 ± 10). These findings suggest that radiolabeling of CD34⁺ hematopoietic cells with ⁶⁴Cu-PTSM at 40 μCi/mL had an adverse effect on CFU-GM colony formation when assessed in vitro.

Figure 3.

Effects of radiation on CD34⁺ hematopoietic cells. ⁶⁴Cu-labeled CD34⁺ hematopoietic cells (10–40 μCi/mL) were assessed in a colony-forming unit (CFU) assay, and BFU-E and CFU-GM colonies were evaluated. No effects were observed on BFU-E, whereas CFU-GM colonies were significantly decreased at 40 μCi when compared with unlabeled, control cells.

rhMSC Growth and Differentiation

The initial functional assay performed on ⁶⁴Cu-PTSM-labeled rhMSCs was to assess proliferation potential when cultured at 1 × 10³ cells/cm², using established protocols.¹⁶ When rhMSCs were radiolabeled with 5 or 10 μCi/mL ⁶⁴Cu-PTSM, no effects on growth were observed (Figure 4A). Cells were found to enter the exponential growth phase at approximately the second day in culture with a plateau at approximately 9 days in culture. By day 7, the total population doubling times of cells radiolabeled with 5 to 10 μCi/mL ⁶⁴Cu-PTSM (4.6 ± 0.2) were not significantly different when compared with the unlabeled, control cells (4.7 ± 0.2). However, when cells were radiolabeled with ≥ 20 μCi/mL, significant effects on growth were observed (2.1 ± 0.5 population doublings by day 7) when compared with the lower ⁶⁴Cu-PTSM concentrations (p < .01). Cells labeled at the higher concentrations showed a growth lag and slower entry into the exponential growth phase at 4 to 7 days. After 13 days in culture, rhMSCs radiolabeled with 20 μCi/mL ⁶⁴Cu-PTSM showed cell counts (6.0 × 10⁵ ± 2.0 × 10⁵ cells) similar to those cells labeled with 0 to 10 μCi/mL (7.7 × 10⁵ ± 2.4 × 10⁵ cells), whereas cells radiolabeled with 40 μCi/mL were about half (3.8 × 10⁵ ± 1.3 × 10⁵ cells). No significant differences were found in the total population doubling times after 13 days of culture when comparing cells labeled with 5 to 40 μCi/mL (5.7 ± 0.3) and the unlabeled, control cells (6.1 ± 0.5). In addition, once cells radiolabeled with 5 to 40 μCi/mL entered the exponential growth phase, the population doubling times (29.3 ± 1.6 to 37.4 ± 6.9 hours) were shown to be similar to unlabeled, control cells (27.8 ± 4.5 hours) (p > .05).

Figure 4.

Effects of radiation on rhesus monkey mesenchymal stem cell (rhMSC) growth and differentiation. Fetal rhMSCs radiolabeled with ⁶⁴Cu (5–40 μCi/mL) were evaluated for growth and differentiation potential (B-D). Growth (A) decreased when cells were exposed to ≥ 20 μCi/mL. The labeling concentrations did not have any effects on adipogenic differentiation when compared with control, unlabeled cells (B). Osteogenic differentiation (C) was significantly reduced with 40 μCi/mL (p < .01). Chondrogenic differentiation (D) was affected at all concentrations studied, although the most significant effects were observed at 20 to 40 μCi/mL (p < .05).

⁶⁴Cu-PTSM-radiolabeled rhMSCs were differentiated toward the adipogenic lineage, and the results were quantified with Oil Red O stain.¹⁶ The results for cells radiolabeled with 5 to 40 μCi/mL ⁶⁴Cu-PTSM (0.42 ± 0.29 to 0.48 ± 0.25) did not vary significantly when compared with the unlabeled, control cells (0.49 ± 0.21) (p > .05) (Figure 4B).

Osteogenic differentiation was also performed on ⁶⁴Cu-PTSM-labeled rhMSCs, and the results were analyzed using a colorimetric assay for the amount of calcium produced. Using a standard curve, results were converted to the corresponding calcium concentration. The results for 5 to 20 μCi/mL (range of 18.4 ± 11.4 mg/dL to 22.8 ± 11.0 mg/dL) did not show any significant differences when compared with the unlabeled, control cells (22.1 ± 10.7 mg/dL) (p > .05) (Figure 4C). However, at 40 μCi/mL ⁶⁴Cu-PTSM, osteogenic differentiation (0.8 ± 0.8 mg/dL) was shown to decrease significantly when compared with the lower ⁶⁴Cu-PTSM concentrations (p < .01).

Chondrogenic differentiation was also quantified using the Cavalieri method to estimate the chondrogenic pellet volumes. The pellets were compared by the percentage of differentiated chondrogenic volumes to the total pellet volumes. The percentage of chondrogenic differentiation was shown to decrease with increasing ⁶⁴Cu-PTSM concentrations (Figure 4D). At 5 and 10 μCi/mL, the chondrogenic percentages (26.8 ± 0.6% to 28.6 ± 1.2%) were significantly lower (p < .05) than the unlabeled, control cells (48.8 ± 5.6%). At 20 and 40 μCi/mL, the percentages (8.5 ± 6.5% to 19.0 ± 3.0%) also decreased significantly when compared with 5 and 10 μCi/mL ⁶⁴Cu-PTSM (p < .01).

Nonradioactive Copper Labeling

The final ⁶⁴Cu-PTSM solution used for radiolabeling of cells included ⁶⁴Cu-PTSM (0.005–0.05%), nonradioactive Cu-PTSM (1.0–55.0%), and free PTSM (45–99%). Each ⁶⁴Cu-PTSM production had varying specific activity, and the amount of actual copper ions retained inside the cells (CD34⁺ and rhMSCs) differed between the studies. To assess the effects of copper toxicity, various nonradioactive copper concentrations were chosen depending on the quantity of copper used for the ⁶⁴Cu-PTSM radiolabeling studies. CD34⁺ cells from infant, juvenile, and adult rhesus monkeys were labeled with a range of 0 to 1,000 pmol Cu/mL and did not show any significant differences in cell viability or in the CFU assay. rhMSCs from fetal monkeys labeled with 0 to 230 pmol Cu/mL also showed no significant differences in cell viability, growth, or differentiation. Thus, these studies supported the hypothesis that the effects on the cells resulted from the radioactivity and not the presence of copper.

Radiolabeled Cell Quantity versus MicroPET Signal Intensity

To assess the imaging potential of the cells, different cell densities (0–4 × 10⁵ cells/well) radiolabeled under the optimized conditions of 20 μCi/mL for CD34⁺ hematopoietic cells and 10 μCi/mL for rhMSCs were plated in 96-well plates in triplicate and then imaged on the microPET scanning bed 1 hour postlabeling (Figure 5). The relationship between cell number and the signal intensity was found to be linear (see Figure 5A). For rhMSCs, the cell signal was sufficiently strong to be visible at 6.25 × 10³ cells (Figure 5C), whereas the CD34⁺ hematopoietic cells were visible at 2.5 × 10⁴ cells (Figure 5E). The background noise in the absence of cells was negligible.

Figure 5.

Cell density and microPET. The relationship between cell density and signal intensity was found to be linear for both CD34⁺ hematopoietic cells radiolabeled at 20 μCi/mL and rhesus monkey mesenchymal stem cells (rhMSCs) radiolabeled at 10 μCi/mL (A). rhMSCs (B) and CD34⁺ cells (C) were plated at higher cell densities of 5 × 10⁴ to 4 × 10⁵ cells/well. Lower densities of rhMSCs (D) and CD34⁺ cells (E) were plated at 0 to 2.5 × 10⁴ cells/well. Signals were detected with minimal background noise at ≈0.03 μCi for both cell types. Artificial color ranges were assigned to the signal intensities and were not the same for all images.

Discussion

The purpose of this study was to determine the feasibility of radiolabeling CD34⁺ hematopoietic cells and rhMSCs with ⁶⁴Cu-PTSM for imaging and tracking posttransplantation. These studies have shown that the radiolabeling efficiency with ⁶⁴Cu-PTSM differed depending on the cell type investigated. For CD34⁺ hematopoietic cells, significant effects were observed on colony formation at 40 μCi/mL, whereas the effects on rhMSC growth and differentiation were evident at 20 μCi/mL. The microPET images showed that the cells were readily detected when radiolabeled with a total radioactivity of ≈0.03 μCi. These studies were performed in vitro; thus, it is possible that in vivo detection of the cells will be somewhat less sensitive because of attenuation and scatter, although preliminary results in vivo in nonhuman primates using 1 to 5 × 10⁶ CD34⁺ autologous cells have suggested that the radiolabeling techniques described are highly efficient and provide a method for readily imaging the cells posttransplantation. These studies are in progress, and the results will be reported separately.

Copper exists in two oxidation states (Cu(I) and Cu(II)) and is an essential element for many key physiologic and biochemical functions owing to its redox properties.^25–27 In copper homeostasis, Cu(I) is transported into the cells by a copper transporter and Cu(II) is transported by a divalent metal transporter.^25,28 However, once complexed with the lipophilic copper chelator PTSM, copper can be transported into the cells by passive diffusion, thus bypassing the normal cellular metabolic and excretory pathways. In normal, noncancerous cells, Cu(II)-PTSM has been shown to be reduced specifically in the mitochondria.^29,30 The copper molecule subsequently enters the pathway of copper metabolism remaining in the cytosol bound to metallothioneins and other proteins.^27,31 Although copper is very important physiologically, it can be toxic when available in excess.²⁶ Cellularly, free copper ions participate in the formation of reactive oxygen species,³² which can induce deoxyribonucleic acid (DNA) strand breaks and oxidation of bases.³³

When using cyclotron-produced ⁶⁴Cu, the radioisotopic purity of the ⁶⁴Cu is very high, but the amount of copper contaminants prior to processing in the cyclotron can result in a significant difference in the specific activity.²⁰ Thus, the ⁶⁴Cu to Cu ratio inconsistency can result in very different copper concentrations. However, the results of these studies have shown that the variations in the copper concentrations studied did not result in any detectable effects on the cells. Studies have shown that at 5 μM, copper may stimulate respiration and collapse of the membrane potential in the mitochondria.³⁴ Given that all of the ⁶⁴Cu labeling experiments used copper concentrations below the nanomolar range, the copper amounts were not sufficiently high to cause toxic cellular effects. Thus, all of the effects observed in these studies were determined to be from radiation (⁶⁴Cu) and not from the copper per se.

Considerable experimental evidence has shown that radiation primarily targets DNA, leading to mitotic death.^35,36 Thus, radiation can result in single- and double-strand breaks, DNA-protein cross-links, and oxidized bases.^36,37 Cells may attempt repair; however, this can lead to chromosomal aberrations, cell death, mutagenesis, and carcinogenesis.^38,39 Irradiation has also been shown to induce delays in the cell cycle.^40,41 Given that these are phases of DNA checkpoints and DNA replication, DNA damage could cause repair delay. This could explain the initial rhMSC growth delays that were observed at 20 and 40 μCi/mL in this study. Owing to the possible radiation damage to the DNA strands, it may take some time to repair the cells before dividing. It is also possible that at 20 and 40 μCi/mL, some of the cells may no longer be viable; therefore, the time for the remaining viable cells to expand to the quantity of cells grown at the lower concentrations could result in longer population doubling times. This suggests that once cells reach the exponential growth phase, or when the initial radiation damage has been repaired, the radiation may not have any further adverse effects on the cells. This could explain the results observed with rhMSC viability. Although no significant differences in cell viability were shown when comparing the concentrations studied, there was a slight decline in cell viability with increasing concentrations. In addition, approximately 24 hours after radiolabeling, there were more viable cells present than at the initial plating, suggesting that the cells proliferated.

Studies that suggest that radiation impedes cell growth may also explain the outcomes in the rhMSC differentiation studies. No significant effects were observed on adipogenic differentiation, but a significant decline was observed at 40 μCi/mL for osteogenic differentiation. With adipogenic differentiation, substantial proliferation of the cells is not required prior to differentiation; with osteogenic differentiation, the quantity of cells plated was approximately one-tenth of those required for adipogenesis. Thus, more time was necessary for the cells to proliferate and reach confluence before differentiation was induced. Consequently, the effects on cell growth may have played a role in osteogenic differentiation, with effects on differentiation owing to the quantity of cells available. For chondrogenic differentiation, the cells were differentiated in a pellet and are concentrated in one small volume. Thus, the amount of radiation absorbed by the cells is more significant than for those cells grown in monolayers.

Different cell types may respond differently to ⁶⁴Cu radiolabeling. This was shown in the uptake of ⁶⁴Cu when comparing CD34⁺ hematopoietic cells with rhMSCs. CD34⁺ cells are smaller than rhMSCs and thus may retain less ⁶⁴Cu. Alternatively, CD34⁺ cells may be less able to reduce Cu(II)-PTSM to Cu(I)-PTSM, thus allowing more of the activity to efflux from the cells and lowering the amount of radioactivity trapped within the cells. Although the CD34⁺ cells were shown to have lower radioactivity per cell, the radiolabeling of the cells indicated some alterations in proliferation capabilities, as evidenced in the CFU assay. This was primarily related to CFU-GM colony growth at 40 μCi/mL. Studies have suggested variation in sensitivity to radiation and that erythroid progenitors may be less sensitive to radiation than GM progenitors.^42–44

Radiolabeled CD34⁺ hematopoietic cells showed significant adverse effects when 40 μCi/mL ⁶⁴Cu-PTSM was used, and an optimized concentration was found to be 20 μCi/mL. For rhMSCs, proliferation and differentiation were altered at 20 μCi/mL, and an optimized radiolabeling concentration was determined to be 10 μCi/mL. CD34⁺ hematopoietic cells were also shown to retain less ⁶⁴Cu than rhMSCs. Owing to this difference in retained radioactivity, a higher quantity of CD34⁺ cells was found to be necessary for efficient microPET imaging. It was noted that the signal activity increased linearly with an increase in the cell number and that this trend did not vary significantly between the two cell types studied.

Footnotes

Acknowledgments

The authors acknowledge David Kukis, Steve Rendig, and other members of the Center for Molecular and Genomic Imaging for assistance with the microPET imaging.

References

Bruder

Kurth

Shea

. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. J Orthop Res 1998;16:155–62.

Sierra

Storer

Hansen

. Transplantation of marrow cells from unrelated donors for treatment of high-risk acute leukemia: the effect of leukemic burden, donor HLA-matching, and marrow cell dose. Blood 1997;89:4226–35.

Zhao

Liu

Ahmad

. Differentiation of embryonic stem cells to retinal cells in vitro. Methods Mol Biol 2006;330:401–16.

Reinhardt

Khoruts

Merica

. Visualizing the generation of memory CD4 T cells in the whole body. Nature 2001;410:101–5.

Tarantal

Lee

Jimenez

. Fetal gene transfer using lentiviral vectors: in vivo detection of gene expression by microPET and optical imaging in fetal and infant monkeys. Hum Gene Ther 2006;17:1254–61.

Bulte

Kraitchman

. Monitoring cell therapy using iron oxide MR contrast agents. Curr Pharm Biotechnol 2004;5:567–84.

Frank

Anderson

Kalsih

. Methods for magnetically labeling stem and other cells for detection by in vivo magnetic resonance imaging. Cytotherapy 2004;6:621–5.

Gao

Kar

Zhang

. MRI of intravenously injected bone marrow cells homing to the site of injured arteries. NMR Biomed 2007. [In press].

Smirnov

Lavergne

Gazeau

. In vivo cellular imaging of lymphocyte trafficking by MRI: a tumor model approach to cell-based anticancer therapy. Magn Reson Med 2006;56:498–508.

10.

Cherry

Gambhir

. Use of positron emission tomography in animal research. ILAR J 2001;42:219–32.

11.

Smith

. Molecular imaging with copper-64. J Inorg Biochem 2004;98:1874–901.

12.

Chatziioannou

. Molecular imaging of small animals with dedicated PET tomographs. Eur J Nucl Med Mol Imaging 2002;29:98–114.

13.

Adonai

Nguyen

Walsh

. Ex vivo cell labeling with ⁶⁴Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc Natl Acad Sci U S A 2002;99:3030–5.

14.

Blower

Lewis

Zweit

. Copper radionuclides and radiopharmaceuticals in nuclear medicine. Nucl Med Biol 1996;23:957–80.

15.

Lee

Fletcher

Tarantal

. Effect of age on the frequency, cell cycle, and lineage maturation of rhesus monkey (Macaca mulatta) CD34⁺ and hematopoietic progenitor cells. Pediatr Res 2005;58:315–22.

16.

Lee

Tarantal

. Comparison of growth and differentiation of fetal and adult rhesus monkey mesenchymal stem cells. Stem Cells Dev 2006;15:209–20.

17.

Lee

Kohn

Tarantal

. Morphological analysis and lentiviral transduction of fetal monkey bone marrow-derived mesenchymal stem cells. Mol Ther 2004;9:112–23.

18.

Jimenez

Leapley

Lee

. Fetal CD34⁺ cells in the maternal circulation and long-term microchimerism in rhesus monkeys (Macaca mulatta). Transplantation 2005;79:142–6.

19.

Sun

Kim

Martell

. In vivo evaluation of copper-64-labeled monooxo-tetraazamacrocyclic ligands. Nucl Med Biol 2004;31:1051–9.

20.

McCarthy

Shefer

Klinkowstein

. Efficient production of high specific activity ⁶⁴Cu using a biomedical cyclotron. Nucl Med Biol 1997;24:35–43.

21.

DeMatteo

Raper

Ahn

. Gene transfer to the thymus. A means of abrogating the immune response to recombinant adenovirus. Ann Surg 1995;222:229–39.

22.

Huong

PLT

Kolk

AHJ

Eggelte

. Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation. An easy and low cost alternative to radioactive thymidine incorporation. J Immunol Methods 1991;140:243–8.

23.

Gundersen

Jensen

. The efficiency of systematic sampling in stereology and its prediction. J Microsc 1987;147:229–63.

24.

Tai

Chatziioannou

Siegel

. Performance evaluation of the microPET P4: a PET system dedicated to animal imaging. Phys Med Biol 2001;46:1845–62.

25.

Harris . Basic and clinical aspects of copper. Crit Rev Clin Lab Sci 2003;40:547–86.

26.

Sharp

. Ctr1 and its role in body copper homeostasis. Int J Biochem Cell Bio 2003;35:288–91.

27.

Arredondo

Nunez

. Iron and copper metabolism. Mol Aspects Med 2005;26:313–27.

28.

Eisses

Kaplan

. The mechanism of copper uptake mediated by human CTR1: a mutational analysis. J Biol Chem 2005;280:37159–68.

29.

Fujibayashi

Wada

Taniuchi

. Mitochondria-selective reduction of ⁶²Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (⁶²Cu-PTSM) in the murine brain; a novel radiopharmaceutical for brain positron emission tomography (PET) imaging. Biol Pharm Bull 1993;16:146–9.

30.

Shibuya

Fujibayashi

Yoshimi

. Cytosolic/microsomal redox pathway: a reductive retention mechanism of a PET-oncology tracer, Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM). Ann Nucl Med 1999;13:287–92.

31.

Baerga

Maickel

Green

. Subcellular distribution of tissue radiocopper following intravenous administration of ⁶⁷Cu-labeled Cu-PTSM. Int J Radiat Appl Instrum B 1992;19:697–701.

32.

Gaetke

Chow

. Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology 2003;189:147–63.

33.

Kawanishi

Inoue

Yamamoto

. Hydroxyl radical and singlet oxygen production and DNA damage induced by carcinogenic metal compounds and hydrogen peroxide. Biol Trace Elem Res 1989;21:367–72.

34.

Saris

Skulskii

. Interaction of Cu⁺ with mitochondria. Acta Chem Scand 1991;45:1042–6.

35.

Hutchinson

. The molecular basis for radiation effects on cells. Cancer Res 1966;26:2045–52.

36.

Stalnacke

Sundell-Bergman

Halldin

. Radiotoxicity of ¹¹C-methionine measured by the accumulation of DNA strand breaks in mammalian cells. Eur J Nucl Med 1985;11:166–70.

37.

Cadet

Delatour

Douki

. Hydroxyl radicals and DNA base damage. Mutat Res 1999;424:9–21.

38.

Frankenberg-Schwager

. Induction, repair and biological relevance of radiation-induced DNA lesions in eukaryotic cells. Radiat Environ Biophys 1990;29:273–92.

39.

Goodhead

Thacker

Cox

. Effects of radiations of different qualities on cells: molecular mechanisms of damage and repair. Int J Radiat Biol 1993;63:543–56.

40.

Painter

Robertson

. Effect of irradiation and theory of role of mitotic delay on the time course of labeling of HeLa S3 cells with tritiated thymidine. Radiat Res 1959;11:206–17.

41.

Yamada

Puck

. Action of radiation on mammalian cells. IV. Reversible mitotic lag in the S3 HeLa cell produced by low doses of x-rays. Proc Natl Acad Sci U S A 1961;47:1181–91.

42.

Laver

Ebell

Castro-Malaspina

. Radiobiological properties of the human hematopoietic microenvironment: contrasting sensitivities of proliferative capacity and hematopoietic function to in vitro irradiation. Blood 1986;67:1090–7.

43.

Goff

Shields

Boggs

. Effects of recombinant cytokines on colony formation by irradiated human cord blood CD34⁺ hematopoietic progenitor cells. Radiat Res 1997;147:61–9.

44.

Ziegler

Sandor

Plappert

. Short-term effects of early-acting and multilineage hematopoietic growth factors on the repair and proliferation of irradiated pure cord blood (CB) CD34⁺ hematopoietic progenitor cells. Int J Radiat Oncol Biol Phys 1998;40:1193–203.

Radiolabeling Rhesus Monkey CD34 + Hematopoietic and Mesenchymal Stem Cells with 64 Cu-Pyruvaldehyde-Bis(N4-Methylthiosemicarbazone) for MicroPET Imaging

Abstract

Materials and Methods

Cell Isolation and Culture

64Cu-PTSM Production and Labeling

T-Cell Proliferation Assay

Hematopoietic Colony-Forming Unit Assay

rhMSC Viability and Proliferation

In Vitro rhMSC Differentiation

In Vitro Imaging

Statistical Analysis

Results

Preliminary Studies with PBMCs

Labeling Efficiency and Cell Viability

CD34+ Hematopoietic Cells

rhMSC Growth and Differentiation

Nonradioactive Copper Labeling

Radiolabeled Cell Quantity versus MicroPET Signal Intensity

Discussion

Footnotes

Acknowledgments

References

⁶⁴Cu-PTSM Production and Labeling