In vivo mesenchymal stem cell tracking with PET using the dopamine type 2 receptor and 18F-fallypride.

UNLABELLED: Human mesenchymal stem cells (hMSCs) represent a promising treatment approach for tissue repair and regeneration. However, little is known about the underlying mechanisms and the fate of the transplanted cells. The objective of the presented work was to determine the feasibility of PET imaging and in vivo monitoring after transplantation of dopamine type 2 receptor-expressing cells. METHODS: An hMSC line constitutively expressing a mutant of the dopamine type 2 receptor (D2R80A) was generated by lentiviral gene transfer. D2R80A messenger RNA expression was confirmed by reverse transcriptase-polymerase chain reaction. Localization of the transmembrane protein was analyzed by confocal fluorescence microscopy. The stem cell character of transduced hMSCs was investigated by adipogenic and osteogenic differentiation. Migration capacity was assessed by scratch assays in time-lapse imaging. In vitro specific binding of ligands was tested by fluorescence-activated cell sorting analysis and by radioligand assay using (18)F-fallypride. Imaging of D2R80A overexpressing hMSC transplanted into athymic rats was performed by PET using (18)F-fallypride. RESULTS: hMSCs showed long-term overexpression of D2R80A. As expected, the fluorescence signal suggested the primary localization of the protein in the membrane of the transduced cells. hMSC and D2R80A retained their stem cell character demonstrated by their osteogenic and adipogenic differentiation capacity and their proliferation and migration behavior. For in vitro hMSCs, at least 90% expressed the D2R80A transgene and hMSC-D2R80A showed specific binding of (18)F-fallypride. In vivo, a specific signal was detected at the transplantation site up to 7 d by PET. CONCLUSION: The mutant of the dopamine type 2 receptor (D2R80A) is a potent reporter to detect hMSCs by PET in vivo.

Evaluation of an automated double-synthesis module: efficiency and reliability of subsequent radiosyntheses of FHBG and FLT.

We optimized the synthesis methods for 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) and 9-(4-[(18)F]fluoro-3-[hydroxymethyl]butyl)guanine) ([(18)F]FHBG) and automated them on an Explora General Nucleophilic double-synthesis module. Furthermore, the synthesis efficiency and reliability and the formation of cross-contaminations of the products when preparing two consecutive batches were evaluated. Whereas the preinstalled FLT synthesis conditions required substantial modification in reaction and neutralization conditions to achieve radiochemical yields of up to 60% within 70±10 min including high-performance liquid chromatography purification, the synthesis of FHBG had to be implemented to the module to obtain competitive radiochemical yields of up to 40% in an overall synthesis time of 60±10 min. The radiochemical purities obtained were ≥99% and ≥96% for the synthesis of [(18)F]FLT and [(18)F]FHBG, respectively. No significant changes in yield or purity could be observed between both batch productions. We found that the yields and purities also did not change when performing FLT after FHBG syntheses and vice versa. Hence, we developed a synthesis setup that offers the opportunity to perform two subsequent syntheses of either [(18)F]FLT, [(18)F]FHBG or [(18)F]FLT after [(18)F]FHBG without decrease in radiochemical yields and purities. Also, no cross-contaminations were observed, which can be attributed to the use of separate product delivery tubes, purification columns and an automated intermediate cleaning program. These results open up the possibility of producing consecutively either two equal (18)F-fluorinated tracers or two different ones in high yields on the same synthesis module.

Towards to hENT1-nucleoside transporter selective imaging agents. Synthesis and in vitro evaluation of the radiolabeled SAENTA analogues.

Three new potential hENT(1) inhibitors suitable for labeling with PET/SPECT radioisotopes were prepared from an advanced intermediate 4. They were tested for their capability to inhibit binding of SAENTA-fluorescein to HL60 leukemia cells in flow cytometry assay and SAENTA-I (5) was determined to be the most active compound. (131)I-5 showed high hENT(1)-specific binding (up to 54% ID) to 6 from 7 tested tumor cell lines and was chosen for further in vivo study.

The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons.

The basic helix-loop-helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.

A function for the calponin family member NP25 in neurite outgrowth.

The neuronal protein 25 (NP25), a member of the calponin (CaP) protein family, has previously been identified as neuron-specific protein in the adult rat brain. Here, we show an early onset of NP25 expression in the chick embryo neural tube. NP25 represents, together with NeuroM, one of the earliest markers for postmitotic neurons. To elucidate its function in the developing nervous system, NP25 was overexpressed in E5 and E9 sensory neurons, E7 sympathetic neurons and PC12 cells that show different endogenous NP25 expression levels. Whereas E5 and E9 sensory neurons and PC12 cells, which express low endogenous levels of NP25, responded by enhanced neurite outgrowth, a reduction of neurite length was observed in sympathetic neurons, which already express high endogenous levels of NP25. Knockdown of NP25 in sensory neurons using NP25 siRNA resulted in shorter neurites, whereas reduction of NP25 expression in sympathetic neurons led to increased neurite length. These results suggest a dynamic function for NP25 in the regulation of neurite growth, with an optimal level of NP25 required for maximal growth.

Target-dependent specification of the neurotransmitter phenotype: cholinergic differentiation of sympathetic neurons is mediated in vivo by gp 130 signaling.

Sympathetic neurons are generated through a succession of differentiation steps that initially lead to noradrenergic neurons innervating different peripheral target tissues. Specific targets, like sweat glands in rodent footpads, induce a change from noradrenergic to cholinergic transmitter phenotype. Here, we show that cytokines acting through the gp 130 receptor are present in sweat glands. Selective elimination of the gp 130 receptor in sympathetic neurons prevents the acquisition of cholinergic and peptidergic features (VAChT, ChT1, VIP) without affecting other properties of sweat gland innervation. The vast majority of cholinergic neurons in the stellate ganglion, generated postnatally, are absent in gp 130-deficient mice. These results demonstrate an essential role of gp 130-signaling in the target-dependent specification of the cholinergic neurotransmitter phenotype.