Deana Haralampieva1, Thomas Betzel2, Ivana Dinulovic3, Souzan Salemi4, Meline Stoelting4, Stefanie D Krämer2, Roger Schibli2, Tullio Sulser4, Christoph Handschin3, Daniel Eberli5, Simon M Ametamey6. 1. Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland Laboratory for Tissue Engineering and Stem Cell Therapy, Department of Urology, University Hospital Zurich, University of Zurich, Zurich, Switzerland Zurich Center for Integrative Human Physiology, Zurich, Switzerland; and. 2. Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland. 3. Focal Area of Growth and Development, Biozentrum, University of Basel, Basel, Switzerland. 4. Laboratory for Tissue Engineering and Stem Cell Therapy, Department of Urology, University Hospital Zurich, University of Zurich, Zurich, Switzerland. 5. Laboratory for Tissue Engineering and Stem Cell Therapy, Department of Urology, University Hospital Zurich, University of Zurich, Zurich, Switzerland Zurich Center for Integrative Human Physiology, Zurich, Switzerland; and. 6. Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland Zurich Center for Integrative Human Physiology, Zurich, Switzerland; and simon.ametamey@pharma.ethz.ch.
Abstract
UNLABELLED: Transplantation of human muscle precursor cells (hMPCs) is envisioned for the treatment of various muscle diseases. However, a feasible noninvasive tool to monitor cell survival, migration, and integration into the host tissue is still missing. METHODS: In this study, we designed an adenoviral delivery system to genetically modify hMPCs to express a signaling-deficient form of human dopamine D2 receptor (hD2R). The gene expression levels of the receptor were evaluated by reverse transcriptase polymerase chain reaction, and infection efficiency was evaluated by fluorescent microscopy. The viability, proliferation, and differentiation capacity of the transduced cells, as well as their myogenic phenotype, were determined by flow cytometry analysis and fluorescent microscopy. (18)F-fallypride and (18)F-fluoromisonidazole, two well-established PET radioligands, were assessed for their potential to image engineered hMPCs in a mouse model and their uptakes were evaluated at different time points after cell inoculation in vivo. Biodistribution studies, autoradiography, and PET experiments were performed to determine the extent of signal specificity. To address feasibility for tracking hMPCs in an in vivo model, the safety of the adenoviral gene delivery was evaluated. Finally, the harvested tissues were histologically examined to determine whether survival of the transplanted cells was sustained at different time points. RESULTS: Adenoviral gene delivery was shown to be safe, with no detrimental effects on the primary human cells. The viability, proliferation, and differentiation capacity of the transduced cells were confirmed, and flow cytometry analysis and fluorescent microscopy showed that their myogenic phenotype was sustained. (18)F-fallypride and (18)F-fluoromisonidazole were successfully synthesized. Specific binding of (18)F-fallypride to hD2R hMPCs was demonstrated in vitro and in vivo. Furthermore, the (18)F-fluoromisonidazole signal was high at the early stages. Finally, sustained survival of the transplanted cells at different time points was confirmed histologically, with formation of muscle tissue at the site of injection. CONCLUSION: Our proposed use of a signaling-deficient hD2R as a potent reporter for in vivo hMPC PET tracking by (18)F-fallypride is a significant step toward potential noninvasive tracking of hD2R hMPCs and bioengineered muscle tissues in the clinic.
UNLABELLED: Transplantation of human muscle precursor cells (hMPCs) is envisioned for the treatment of various muscle diseases. However, a feasible noninvasive tool to monitor cell survival, migration, and integration into the host tissue is still missing. METHODS: In this study, we designed an adenoviral delivery system to genetically modify hMPCs to express a signaling-deficient form of human dopamine D2 receptor (hD2R). The gene expression levels of the receptor were evaluated by reverse transcriptase polymerase chain reaction, and infection efficiency was evaluated by fluorescent microscopy. The viability, proliferation, and differentiation capacity of the transduced cells, as well as their myogenic phenotype, were determined by flow cytometry analysis and fluorescent microscopy. (18)F-fallypride and (18)F-fluoromisonidazole, two well-established PET radioligands, were assessed for their potential to image engineered hMPCs in a mouse model and their uptakes were evaluated at different time points after cell inoculation in vivo. Biodistribution studies, autoradiography, and PET experiments were performed to determine the extent of signal specificity. To address feasibility for tracking hMPCs in an in vivo model, the safety of the adenoviral gene delivery was evaluated. Finally, the harvested tissues were histologically examined to determine whether survival of the transplanted cells was sustained at different time points. RESULTS: Adenoviral gene delivery was shown to be safe, with no detrimental effects on the primary human cells. The viability, proliferation, and differentiation capacity of the transduced cells were confirmed, and flow cytometry analysis and fluorescent microscopy showed that their myogenic phenotype was sustained. (18)F-fallypride and (18)F-fluoromisonidazole were successfully synthesized. Specific binding of (18)F-fallypride to hD2R hMPCs was demonstrated in vitro and in vivo. Furthermore, the (18)F-fluoromisonidazole signal was high at the early stages. Finally, sustained survival of the transplanted cells at different time points was confirmed histologically, with formation of muscle tissue at the site of injection. CONCLUSION: Our proposed use of a signaling-deficient hD2R as a potent reporter for in vivo hMPC PET tracking by (18)F-fallypride is a significant step toward potential noninvasive tracking of hD2R hMPCs and bioengineered muscle tissues in the clinic.
Authors: Jinyong Luo; Zhong-Liang Deng; Xiaoji Luo; Ni Tang; Wen-Xin Song; Jin Chen; Katie A Sharff; Hue H Luu; Rex C Haydon; Kenneth W Kinzler; Bert Vogelstein; Tong-Chuan He Journal: Nat Protoc Date: 2007 Impact factor: 13.491
Authors: Deana Haralampieva; Souzan Salemi; Thomas Betzel; Ivana Dinulovic; Stefanie D Krämer; Roger Schibli; Tullio Sulser; Christoph Handschin; Simon M Ametamey; Daniel Eberli Journal: Stem Cells Int Date: 2018-01-21 Impact factor: 5.443