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Background:
Longitudinal, noninvasive imaging of hematopoietic stem and progenitor cell (HSPC) engraftment in deep bone marrow remains difficult. Donor-cell persistence after transplantation is still commonly inferred from peripheral blood recovery, chimerism, or invasive marrow sampling, which provide limited spatial information and are not ideal for repeated assessment of marrow-resident engineered cells. Fluorescence imaging is useful for ex vivo validation but is limited in deep tissue, particularly in bone. Chemical exchange saturation transfer (CEST)-MRI offers a radiation-free strategy for reporter gene imaging in deep tissue, but interpretation of in vivo CEST contrast requires orthogonal validation1-4.

 

Hypothesis/Objective:
It was hypothesized that a dual reporter combining CEST-MRI with fluorescence would enable cross-validated detection of engineered hematopoietic cells in vivo and support longitudinal assessment of hematopoietic engraftment in deep bone marrow. The objective was to develop a lentiviral dual-reporter cassette co-expressing the CEST reporter SuperCESTide and the fluorescent protein mScarlet-I, validate reporter performance in vivo, and test feasibility in transplanted HSPCs1-6.

 

Methods:
A lentiviral construct encoding SuperCESTide and mScarlet-I linked by T2A was generated for constitutive co-expression from a single transcript (Fig. A). Reporter performance was first evaluated in CT26 colon carcinoma cells in vitro and in a bilateral flank tumor model, in which reporter-transduced and non-transduced tumors were implanted contralaterally in the same mouse (Fig. B). Imaging included whole-body fluorescence imaging and CEST-MRI, with ROI-based analysis of MTRasym spectra and integrated CEST contrast quantified as area under the curve (AUC) from 1-2 ppm (Fig. C). The reporter was then applied to primary bone marrow-derived lineage-negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) hematopoietic cells following isolation and lentiviral transduction (Fig. D), with expression assessed by fluorescence microscopy, flow cytometry, and imaging flow cytometry. For transplantation studies, recipient mice underwent unilateral half-body irradiation followed by transplantation of reporter-labeled Lin- donor cells and were imaged after 8 weeks by whole-body fluorescence imaging, femoral CEST-MRI, and intravital calvarial microscopy (Fig. E). Ex vivo validation included confocal microscopy, tibial optical imaging, and flow cytometry of marrow compartments (Fig. F,G)7-10.

 

Results:
The dual reporter showed efficient expression in CT26 cells and generated robust in vivo fluorescence and CEST-MRI contrast in bilateral tumors (Fig. B,C). Reporter-positive tumors demonstrated higher fluorescence than contralateral controls and increased reporter-associated MTRasym signal across the relevant frequency range. Integrated CEST contrast (AUC 1-2 ppm) was higher in reporter tumors than in control tumors (Fig. C). In primary hematopoietic cells, reporter expression was detected in Lin- and LSK populations, demonstrating feasibility in sensitive stem and progenitor cells (Fig. D). In the transplantation model, whole-body fluorescence imaging at 8 weeks showed localized signal on the irradiated and reconstituted side, consistent with engraftment and reporter retention (Fig. E). Femoral CEST-MRI demonstrated higher marrow contrast in reporter-transduced recipients than in non-transduced transplanted controls, with increased MTRasym AUC over 1-2 ppm, and intravital calvarial microscopy visualized reporter-positive marrow cells within vascular niches (Fig. E). Ex vivo confocal microscopy, tibial optical imaging, and flow cytometry confirmed reporter retention in reconstituted hematopoietic compartments, including Lin− and LSK populations (Fig. F,G)1-4,7-10.

 

Conclusion:
A dual CEST-fluorescent reporter enabled cross-validated, noninvasive tracking of hematopoietic engraftment in deep bone marrow. By combining deep-tissue CEST-MRI with fluorescence-based validation, intravital microscopy, and ex vivo cellular analyses, this platform links MRI contrast to verified reporter expression in hematopoietic compartments. These findings support the feasibility of imaging engineered hematopoietic cells longitudinally in vivo and establish a framework for future preclinical studies of HSPC transplantation and imaging-enabled cell therapy.

 

Image/Figure:
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Image/Figure Caption:

Figure. Cross-validated multimodal workflow for dual CEST-fluorescent tracking of hematopoietic engraftment in deep bone marrow. (A) Schematic of the lentiviral dual-reporter cassette encoding the CEST reporter SuperCESTide (SC) linked via a T2A sequence to the red fluorescent protein mScarlet-I (hereafter mScarlet), under the indicated promoter and lentiviral backbone elements. (B) Experimental schematic of the bilateral flank tumor model in BALB/c mice, in which reporter-transduced CT26 cells were implanted on one flank and control CT26 cells were implanted contralaterally in the same mouse. Representative in vivo fluorescence imaging (IVIS) is shown, demonstrating reporter and control tumors within the same animal. (C) Representative in vivo MRI of flank tumors showing anatomical T2-weighted images with overlaid CEST-MRI contrast maps at 1.8 ppm (MTRasym overlay; n = 5 per group). (D) Workflow schematic of the hematopoietic cell studies, including bone marrow isolation from female donor mice, enrichment and/or sorting of hematopoietic lineage-negative (Lin-) cells, followed by lentiviral transduction with the mScarlet-SuperCESTide (SC) reporter or control constructs. (E) In vivo detection of reporter-labeled transplanted cells 8 weeks after transplantation. From left to right: representative whole-body IVIS imaging showing localized RFP signal on the irradiated/reconstituted side, consistent with engraftment and reporter retention; representative femoral MRI showing T2-weighted anatomical images with overlaid MTRasym CEST-MRI maps of the bone marrow region; and representative calvarial intravital microscopy images showing mScarlet+ marrow cells within vascular CD31+ bone marrow niches in merged views. (F) Ex vivo validation of reporter retention in reconstituted marrow. Representative maximal-intensity projections (MIPs) of femoral bone marrow from mice transplanted with non-labeled Lin- cells or mScarlet-SC reporter-labeled Lin- cells are shown as whole-region confocal views with DAPI nuclear staining and red fluorescence. Representative ex vivo optical imaging of reconstituted tibias from non-labeled control and reporter-labeled recipients is also shown. (G) Flow cytometric validation of reporter-positive hematopoietic compartments with representative flow cytometry plots of hematopoietic Lin- cells and Lin-Sca-1+c-Kit+ (LSK)cells are shown, together with quantification of mScarlet signal within the Lin− and LSK populations.

 

Author
Katrien Vandoorne
Technion Israel Institute of Technology