Generation of hTERT-immortalized human mesenchymal stromal cells with optical and magnetic labels for in vivo transplantation and tracking

Abstract

Background: Mesenchymal stem/stromal cells (MSCs) are the focus of increasing research as a potential therapeutic agent for a range of nervous system diseases, due to their unique capacity for self-renewal and differentiation. The subsequent tracking of cells post-transplantation into the organism is of pivotal significance, as it elucidates their fate, distribution, and enables the timely monitoring of any adverse effects. In the context of cell monitoring, the utilization of a non-toxic label that exhibits long-term stability is of paramount importance.

Methods: A human immortalized MSCs cell line was engineered to express a green fluorescent protein (GFP) and bacterial nanocompartments (high-molecular-weight icosahedral capsid-like protein complexes) via lentiviral transduction. The obtained cells were characterized by inductively coupled plasma mass spectrometry (ICP-MS) and Perls staining as well as using the nonlinear magnetization method, confocal microscopy and flow cytometry. An animal study was conducted in Sprague-Dawley rats.

Results: In this study, an immortalized human MSCs cell line with stable expression of a novel magnetic resonance (MR) reporter label was established for the first time. GFP was genetically produced for utilization as an optical tag. A nanocompartment of the bacterium Quasibacillus thermotolerans (Qt) was used as a carrier of the magnetic label. The Qt nanocompartment consists of a shell and a ferroxidase cargo protein. Ferroxidase provides the biomineralization of iron ions within the nanocompartment shell. As a result, ferric oxide nanoparticles are formed inside the encapsulin nanocompartments, which have T2-contrast properties and serve as genetically encoded labels for magnetic resonance imaging (MRI) and for quantification by the nonlinear magnetization method.

Conclusions: The experimental results indicate that the use of two complementary labels allows for the multimodal visualization of the derived cells post-transplantation into the rat brain striatum, which is promising for monitoring MSCs-based therapy.

Keywords: Cell tracking in vivo; Encapsulins; MRI visualization; Magnetic particle quantification; Multimodal imaging; Stem cells.

Fig. 1

Generation and characterization of ASC52telo-Qt cell line. a Schematic representation of the genetic construct encoding the shell (QtEnc^FLAG) and cargo (QtIMEF) proteins, and the assembled Qt encapsulin shell with QtIMEF payload. Color code: Qt encapsulin shell in light orange, QtIMEF in green, FLAG-tag in blue, CMV (cytomegalovirus promoter) in grey; (b) RT-PCR analysis of ASC52telo-Qt cells; (c) Western blot analysis against FLAG-tag on QtEnc protomer proteins; (d) GFP expression in ASC52telo-Qt cells assessed by flow cytometry; (e) Immunofluorescence staining of ASC52telo-Qt cells with primary mouse anti-FLAG-tag monoclonal antibodies and goat anti-mouse AlexaFluor647 labeled secondary antibodies. White arrows indicate Qt encapsulins in ASC52telo-Qt cell cytoplasm, scale bar 20 μm; (f) Flow cytometry of ASC52telo-Qt cells stained with primary anti-FLAG-tag antibodies and secondary AlexaFluor647 labeled antibodies; (g) Resazurin assay of various FAS concentrations in the growth medium for ASC52telo-Qt and ASC52telo-RFP cells; (h) Intracellular iron content in ASC52telo-Qt and ASC52telo-RFP cells after incubation with 0–2 mM FAS for 24 h quantified by ICP-MS; (i) The magnetic signals of ASC52telo-Qt cells and ASC52telo-Qt cells preincubated with 2 mM FAS measured using the MPQ technique. All data are shown as the mean ± S.D. of three independent experiments (* indicate p-value < 0.05, ** indicate p‐value < 0.001). Full-length gels are presented in Fig. S7 and S8

Fig. 2

Ferrous iron uptake in ASC52telo-Qt and ASC52telo-GFP cells. a Schematic representation of the fluorescence response of HMRhoNox-M for Fe²⁺. Laser scanning confocal microscopy of ASC52telo-Qt (b) and ASC52telo-GFP (c) cells after pretreatment with 2 mM FAS for 30 min followed by incubation with HMRhoNox-M and LysoTracker Deep Red

Fig. 3

Bivalent iron uptake in ASC52telo-Qt and ASC52telo-GFP cells reveald via Fe²⁺ selective sensor. a In the absence of a source of divalent iron, ASC52telo-GFP cells have been demonstrated to possess the capacity to capture iron contained within the growth medium and serum. In this case, the addition of the HMRhoNox-M sensor to the cells results in the observation of a fluorescent signal with an average intensity ranging from 10² to 10³ RFU; (b) In ASC52telo-Qt cells cultured under identical conditions, an increase in the fluorescence intensity of the sensor is observed, reaching a peak intensity of 10³ RFU; (c) Following a 24-hour incubation of ASC52telo-GFP cells with FAS, a robust HMRhoNox-M fluorescent signal is detected in lysosomes, exhibiting a substantially elevated signal intensity ranging from 10⁴ to 10⁵ RFU; (d) The IMEF enzyme within Qt nanocompartments facilitates the conversion of Fe²⁺ to Fe³⁺.This process results in a reduction of the divalent iron content in cells, consequently leading to a decline in the fluorescent signal of the sensor to 10³–10⁴ RFU. The inserts provide the intensity of the GFP fluorescent signals in the cells

Fig. 4

Histological and MR images obtained 3 days after the transplantation of ASC52telo-Qt cells into the rat brain. a Perls stained histological section of the brain, red arrow indicates the injection site, blue arrows point at the areas of iron accumulation, scale bars are 1000 μm and 100 μm in high magnification images; (b) Panoramic image of the right brain hemisphere obtained by confocal microscopy, green fluorescent signal – GFP in transplanted cells, nuclei are stained with DAPI, scale bar – 500 μm; (c) In vivo MR image of a rat brain, black arrows indicate cells distributed in the corpus striatum and corpus callosum

Fig. 5

Histological and MR images obtained 10 days after the transplantation of ASC52telo-Qt cells into the rat brain. a Perls stained histological section of the brain, red arrow indicates the injection site, blue arrows point at the areas of iron accumulation, scale bars are 1000 μm and 100 μm in the regions of interest; (b) Panoramic confocal image of the right brain hemisphere, green fluorescent signal – GFP in transplanted cells, green arrows point to individual cells at the injection site, nuclei are stained with DAPI, scale bar – 500 μm. In vivo MR image of a rat brain, obtained 3 days (c) and 10 days (d) after ASC52telo-Qt cells injection. Black arrows indicate cells distributed in the cortex, corpus callosum and striatum

Fig. 6

Histological and MR images obtained 3 and 10 days after intracerebral transplantation of ASC52telo-RFP cells. Panoramic confocal images of the right brain hemisphere acquired 3 days (a) and 10 days (c) after injection, red fluorescent signal – RFP in transplanted cells, nuclei are stained with DAPI, scale bar – 500 μm. Perls stained histological sections of the brain collected 3 days (b) and 10 days (d) after cell transplantation, scale bars are 1000 μm and 100 μm in the regions of interest. MR image of live rat brain 10 days after ASC52telo-RFP cells injection (e)

Fig. 7

Ex vivo analysis of rat brain sections by the MPQ method. Preparation of rat brain sections (a); detection of grafted cells by GFP/RFP fluorescence (b) (source: bioart.niaid.nih.gov/bioart/370, red arrow indicates the cell injection site); brain sections in the MPQ detection coil (c); magnetic signals measured by the MPQ method 3 and 10 days after transplantation of ASC52telo-Qt cells/ASC52telo-RFP cells (n = 3, * indicate p-value < 0.05) (d)