By Ning Zhang, Daniel Ansaldi, Stephen Oldfield, and Min Zhu Tuesday, June 1, 2024

Developing sensitive, non-invasive technologies to monitor engraftment in vivo is essential to accelerate the clinical implementation of cell therapies.

Regenerative medicine is a rapidly emerging field using stem and other regenerative cells to repair or restore function to tissues lost or damaged due to the effects of injury, disease, and aging. Medical researchers are applying stem cell therapies to treat a wide variety of diseases including cancer, cardiovascular disease, Parkinson’s disease, spinal cord injuries, multiple sclerosis, and muscle damage. Much work is focused on bringing such advances to patients.

In the research laboratory, this work requires model systems where stem and regenerative cells can be monitored in order to track distribution, measure survival and establish the safety and efficacy of the procedures. At present, most cell therapy protocols require histological analysis to determine viable engraftment of the transplanted cells. The development of sensitive, non-invasive technologies to monitor engraftment in vivo is essential to accelerate the clinical implementation of cell therapies. Optical molecular imaging—particularly using bioluminescent reporters—is an ideal analysis platform for monitoring stem cells in live mouse models because of its exquisite sensitivity, quantitative precision and its high-throughput capability in longitudinal studies.

Labeling strategies
Optical imaging has been used to monitor several varieties of both human and mouse embryonic, mesenchymal, and hematopoietic stem cells. For long-term in vivo studies in animal models three common labeling strategies are used for signal generation:
• Isolation from a transgenic animal ubiquitously expressing luciferase
• Stable transduction of isolated stem cells using a lentiviral vector
• Stable transduction using the Sleeping Beauty transposon or other vectors

In addition, cells are often labeled with fluorescent labels such as DiR for short-term monitoring and immediate localization.

We can explore each strategy with examples in therapeutic areas as diverse as neurology, cardiovascular disease and oncology.

In the first example, Yu-An Cao¹ and a team from Stanford University explored the early dynamics of engraftment by following small numbers of hematopoietic stem cells (HSC) taken from a luciferase transgenic mouse and transferred to an irradiated host. The donor stem cells constitutively expressed firefly luciferase under control of a beta-actin promoter.

Optical imaging using bioluminescence was sensitive enough to detect the first foci of engraftment non-invasively in the recipient and follow the dynamic process of reconstitution quantitatively and in real time. The initial foci in the spleen or bone marrow either expanded locally and seeded other sites, or receded with variable patterns of distribution. The pattern of engraftment was easily followed over time in studies of individual animals—without sacrificing the animals for histology. Optical imaging enabled researchers to track the early stages of the process and measure the signal through to reconstitution after 21 days of monitoring.

Multimodality
Zongjin Li² transduced human embryonic stem cells (hESC) with a lentiviral vector carrying a novel double-fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. The cells were also labeled with iron particles for MRI imaging in the recipient mouse. Optical imaging of the bioluminescent reporter provided the detection of cell viability, showing the kinetics of survival over a four week period. The second optical reporter, GFP, was imaged during post-mortem histology and immunohistochemistry while iron particles in the cells provided precise spatial resolution by MRI. Co-registration of bioluminescence with PET has been shown to provide complementary insights into proliferation and migration³, and optical imaging is frequently used with CT to provide anatomical reference by supplementing the accurate quantitation of a calibrated optical signal with more precise spatial information.

Two optical signals can also be multiplexed to reveal different physiological developments in the same model. As demonstrated by Klopp et al.⁴ firefly luciferase can be used to monitor migration of mesenchymal stem cells while renilla luciferase is used to label 4T1 tumor cells. The multiplexed reporter signals reveal the homing of stem cells to the tumor tissues. Bioluminescent signals are often multiplexed with fluorescence to provide similar signal co-registration and biological insight.

Quantitation
Non-viral vectors can also be used to label stem cells, and the Sleeping Beauty transposon was used by Wilber et al.⁵ to mediate stable gene transfer of luciferase and GFP into human embryonic stem cells. This study demonstrated stable expression of luciferase in stem cells for over five months. Optical imaging can be calibrated to report absolute values so that studies over extended time periods can be quantitatively compared. Many studies have shown the close relationship between bioluminescent signal and cell number,⁶ and the ability to follow an individual animal over time avoids the sampling biases and errors that compromise conventional studies that require groups of animals being sacrificed at different time points for histology purposes. Barberi et al.⁷ monitored stem cell-derived skeletal myoblasts for more than six months in SCID/Beige mice taking advantage of the superior statistics in a non-invasive longitudinal study.

Applications
Non-invasive optical imaging works well through bone and can readily show stem cell proliferation in the brain⁸ and spinal cord.⁹ Stem cells derived from adipose tissue were implanted and imaged in the heart by Bai et al.¹⁰ in vivo results were readily confirmed by ex vivo imaging of bioluminescence and subsequent histological analysis.

Adipose derived regenerative cells
Adipose tissue has been used as cosmetic surgery material. Early experience noted that graft re-absorption could be a significant drawback resulting in 50-90% graft loss and, in 2007, the American Society of Plastic and Reconstructive Surgeons issued a statement to support ongoing research that establishes the safety and efficacy of the procedure.

Adipose derived regenerative cells (ADRCs) encompass a heterogeneous population of mesenchymal stem cells, endothelial progenitor cells, monocytes, macrophages and fibroblasts. In the clinic they are currently used for tissue reconstruction and wound healing procedures because they are abundant and easily harvested by liposuction.

ADRCs secrete a number of growth factors (VEGF, HGF, FGF-2, IGF-1) that improve angiogenesis and vascularity when adipose tissue is grafted into an autologous host. In the laboratory, mouse models are used to monitor the survival of grafted adipose tissue and distribution of ADRCs, and transgenic mice that express luciferase can be used as donors of either grafted adipose tissue or ADRCs for expression in non-transgenic animals. Non-invasive optical imaging can track graft tissue longitudinally and also monitor ADRC distribution.

Mouse model
Transgenic mice with constitutive luciferase expression can be utilized as donors for assessing survival of tissues/cells following implantation to recipient mice. Adult beta actin-luc, GAPDH-luc and Ubc-luc mice show a high level of tissue luciferase expression both in vitro and in vivo and make ideal donors of ADRCs for monitoring cell proliferation and survival.

To track the initial fate of injected cells, ADRCs can be pre-labeled with the membrane dye DiR and tracked in a host animal by fluorescence imaging. 24 hours after injection, ADRCs in an autologous model remain at the site of injection, while cells in an allograft model show fragmentation and dispersal throughout the animal. In the allograft model, secondary staining of phagocytes during the immune responses results in a signal accumulation in the spleen. Using DiR, cells can be monitored for several days after injection although dye is not stably bound and can be dispersed into immune cells and surrounding tissues.

Cells that express luciferase under the control of a constitutive promoter such as beta actin can be tracked for days or weeks so that initial clearance and controlled proliferation can be measured.

Optical imaging is an indispensible tool to investigate both intrinsic and external factors that regulate homing, survival and growth of ADRCs in vivo and can provide insight into the safety and efficacy of procedures for clinical development.

Summary
in vivo optical imaging provides an ideal longitudinal method for monitoring the behavior, survival, and distribution of stem cells in multiple research models. The sensitivity of the technique allows visualization of the early stages of foci formation, and the dynamic range of some imaging systems permits accurate quantitation of the signal through to complete engraftment. Longitudinal studies can monitored from days to months. With multiplexed optical signals, complex biological events can be analyzed in an individual animal, and optical data can also be co-registered with other imaging modalities to provide additional anatomical information.

Because optical imaging is high-throughput, it enables researchers to manage cohorts of animals in a study at the same time. It also provides the ability to follow an individual subject longitudinally, providing improved statistics for even a small sample size. Non-invasive optical methods are becoming routine for all aspects of stem cell work, both in research and in developing a path to the clinic.

References
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2. Li Z, Suzuki Y, Huang M, Cao F, Xie X, Connolly AJ, Yang PC, Wu JC. Comparison of Reporter Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and Differentiated Endothelial Cells in Living Subjects. Stem Cells. January 24, 2008; 26:864-873.

3. Cao F, Lin S, Xie X, Ray P, Patel M, Zhang X, Drukker M, Dylla SJ, Connolly AJ, Chen X, Weissman IL, Gambhir SS, Wu JC. In Vivo Visualization of Embryonic Stem Cell Survival, Proliferation, and Migration After Cardiac Delivery. Circulation. February 21, 2006; 113(7):1005-1014.

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7. Barberi T, Bradbury M, Dincer Z, Panagiotakos G, Socci ND, Studer L. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nature Medicine. May 2007; 13(5):642-648.

8. Bradbury MS, Tomishima M, Panagiotakos G, Chan BK, Zanzonico P, Vider J, Ponomarev V, Studer L, Tabar V. Optical bioluminescence imaging of human ES cell progeny in the rodent CNS. J Neurochem. June 7, 2007.

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10. Bai X, Yan Y, Song YH, Seidensticker M, Rabinovich B, Metzele R, Bankson JA, Vykoukal D, Alt E. Both cultured and freshly isolated adipose tissue-derived stem cells enhance cardiac function after acute myocardial infarction. European heart journal. February 2010; 31(4):489-501.

About the Author
Ning Zhang, Caliper Life Sciences, Daniel Ansaldi, Caliper Life Sciences, Stephen Oldfield, Caliper Life Sciences, and Min Zhu, Cytori Therapeutics Inc. Acknowledgements to Doug Arm and John Fraser of Cytori Therapeutics, Inc. for thoughts and input.

This article was published in Bioscience Technology magazine: Vol. 34, No. 5, May, 2010, pp. 16-19.