Quantification of early adipose-derived stem cell survival: comparison between sodium iodide symporter and enhanced green fluorescence protein imaging

Abstract

Objective

Strategies to overcome the problem of extensive early stem cell loss following transplantation requires a method to quantitatively assess their efficacy. This study compared the ability of sodium/iodide symporter (NIS) and enhanced green fluorescent protein (EGFP) imaging to monitor the effectiveness of treatments to enhance early stem cell survival.

Methods

Human adipose-derived stem cells (ADSCs) transduced with an adenoviral vector to express both NIS and EGFP were mixed with culture media (control), matrigel (matrigel group) or pro-survival cocktail (PSC group), and 5 × 10⁶ cells were injected into thigh muscles of C57BL/6 mice. Animals underwent serial optical imaging and ^99mTcO₄^- scintigraphy. Image-based EGFP fluorescence and ^99mTcO₄^- uptake was measured by region-of-interest analysis, and extracted tissues were measured for ^99mTc activity. Fluorescent intensity measured from homogenized muscle tissue was used as reference for actual amount of viable ADSCs.

Results

ADSCs were efficiently transduced to express EGFP and NIS without affecting proliferative capacity. The absence of significant apoptosis was confirmed by annexin V FACS analysis and Western blots for activated caspase-3. Both fluorescence optical imaging and ^99mTcO₄^- scintigraphy visualized implanted cells in living mice for up to 5 days. However, optical imaging displayed large variations in fluorescence intensity, and thus failed to detect difference in cell survival between groups or its change over time. In comparison, ^99mTcO₄^- scintigraphy provided more reliable assessment of within-in group donor cell content as well as its temporal change. As a result, NIS imaging was able to discern beneficial effects of matrigel and pro-survival cocktail treatment on early ADSC survival, and provided quantitative measurements that correlated to actual donor cell content within implanted tissue.

Conclusion

NIS reporter imaging may be useful for noninvasively assessing the efficacies of strategies designed to improve early survival of transplanted stem cells.

Introduction

Stem cells have the capacity to self-renew and differentiate into multiple cell types, and are thus a promising therapeutic option for repair and regeneration of ischemic tissue [1]. However, before these cells are widely applied in the clinics, it is crucial to better understand their behavior and fate in vivo. A major obstacle limiting the efficacy of stem cell therapy is an extensive loss of donor cells early after transplantation. A large number of studies extending across various cell types and animal models have revealed that only a small fraction of therapeutic cells remain viable within days of implantation [2], [3], [4], [5], [6]. Indeed, donor cell loss rates as high as 40% within the first 1 h [3], and over 80% by 24 h have been reported [4].

Accordingly, there is much effort in developing strategies to enhance early survival of transplanted donor cells [7], [8], [9], [10], [11]. The efficacy of these strategies is determined by the increase in number of surviving donor cells when treated in a certain way compared to untreated controls. Histological examination for donor cells is not only highly subject to sampling error, but its invasiveness precludes evaluation of temporal change. Molecular imaging techniques provide a means to noninvasively and longitudinally track therapeutic cells in living subjects. Direct cell labeling has drawbacks of label leakage and false positive signals from macrophage engulfed debris [12], [13]. As such, reporter gene imaging that produces signals only in viable cells is preferable for monitoring transplanted cell survival [12], [14], [15].

The sodium/iodide symporter (NIS), a transmembrane carrier that selectively transports iodide, offers several advantages for in vivo reporter gene imaging [12], [16]. NIS expression is limited to only a few tissues; the protein does not significantly influence underlying cell biochemistry; and the use of species-specific NIS sequences can avoid immune responses that are present with foreign reporter proteins [12]. Furthermore, NIS imaging tracers do not require radiochemical synthesis, and multiple radioisotope types with a wide range of half-lives can be selected for either positron emission tomography (PET) or γ-camera imaging [16]. Indeed, NIS gene imaging has been shown to allow in vivo stem cell tracking [13], [17], [18], as well as monitoring of stem cell-mediated therapeutic gene delivery [19], [20]. However, several characteristics of the NIS system, including susceptibility to damage during cell harvesting [21] and influence of transport activity by intracellular signaling [22], [23], [24], cause concern over the accuracy of this technique for monitoring viable cell content. Thus, a critical question that remains for the role of NIS imaging in stem cell therapy is whether it has the ability to accurately quantify surviving donor cells and discern beneficial effects of strategies to enhance early survival following implantation.

In this study, we thus investigated the accuracy of NIS imaging for evaluating the efficacy of strategies to enhance early survival of adipose derived stem cells (ADSCs) transplanted into mouse thigh muscles. These cells are gaining wide attention for their high accessibility, and ability to differentiate into various cell types [25], [26], [27]. To transiently express enhanced green fluorescent protein (EGFP) and NIS for a few days, cells were infected with an adenoviral vector. Scintigraphic ^99mTcO₄^- images, optical images, and ex vivo radiouptake measurements were compared to fluorescent intensities of homogenated tissue as reference for actual cell content.