Dynamic tracking of human umbilical cord mesenchymal stem cells (hUC-MSCs) following intravenous administration in mice model

Abstract

Introduction: In the past decades, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have sparked interest in cellular therapy due to their immunomodulatory properties. Nevertheless, the fate of hUC-MSCs in the body remains poorly understood. This study aimed to investigate the biodistribution, homing and clearance of systemically administered hUC-MSCs in healthy BALB/c mice model.

Methods: hUC-MSCs were labelled with GFP-Luc2 protein, followed by characterisation with flow cytometry. Upon intravenous infusion of transduced hUC-MSCs into the healthy BALB/c mice, the cells were dynamically monitored through the bioluminescent imaging (BLI) approach.

Results: Transduction of hUC-MSCs with GFP-Luc2 not only preserved the characteristics of MSCs, but also allowed live monitoring of transduced cells in the mice model. Upon systemic administration, BLI showed that transduced hUC-MSCs first localised predominantly in the lungs of healthy BALB/c mice and mainly remained in the lungs for up to 3 days before eventually cleared from the body. At terminal sacrifice, plasma chemistry biomarkers remained unchanged except for C-peptide levels, which were significantly reduced in the hUC-MSCs group. Histopathological findings further revealed that hUC-MSCs infusion did not cause any adverse effects and toxicity to lung, liver and heart tissues.

Conclusions: Collectively, systemically administrated hUC-MSCs was safe and demonstrated dynamic homing capacity before eventually disappearing from the body.

Keywords: Biodistribution; GFP-Luc2; Human umbilical cord mesenchymal stem cell.

Fig. 1

Morphology and GFP-Luc2 expression in parental hUC-MSCs and transduced hUC-MSCs at 24, 48 and 72 h post-transduction. (a) Transduction of GFP-Luc2 proteins in hUC-MSCs did not cause morphological changes. All images taken at 100x magnification. The scale bars represent 50 μm. The GFP signals were taken with 5.5 s exposure time. (b) Mean fluorescence intensities of transduced hUC-MSCs at 24, 48 and 72 h post-transduction (n = 2).

Fig. 2

Time course analysis of GFP expression in transduced hUC-MSCs. (a) Representative histogram showed the percentage of GFP-positive cells at day 0, 7 and 14. (b) Representative microscopic images of transduced hUC-MSCs (GFP-Luc2) at day 0, 7 and 14 in culture. All images taken at 100x magnification. The scale bars represent 50 μm. The GFP signals were taken with 5.5 s exposure time.

Fig. 3

Immunophenotyping of MSCs surface markers in hUC-MSC. Representative histograms of parental hUC-MSCs and transduced hUC-MSCs for the MSCs specific surface markers CD44, CD73, CD90 and CD105, and negative cocktails consisting of CD45, CD34, CD11b, CD19 and HLA-DR. The gating for cells was based on its respective unstained control.

Fig. 4

Linear relationship analysis between the cell number and bioluminescence intensity. (a) Quantitative analysis revealed a strong linear relationship between cell numbers (1, 5 and 10 × 10³ cells) and the bioluminescence signal (R² = 0.9997). (b) Representative images of at least 2 to 3 independent experiments.

Fig. 5

Representative images depicting biodistribution pattern of single i.v. administered transduced hUC-MSCs (18.5 × 10⁶ cells/kg BW) in healthy BALB/c mice at designated time points (1 h, 24 h, 2, 3, 4 days after cell administration). (a) Transduced hUC-MSCs migrated and localised to lungs as early as 1 h post-administration. The signal remained in lungs for up to 3 days before being cleared from the body. (b) Bioluminescence intensity quantification of the whole body of mice at designated time points. (c) Average percentage of signal intensity in lungs at designated time points. Data are presented as mean ± SEM (n = 5).

Fig. 6

Time course of (a) body weight and (b) body temperature changes in mice from before i.v. administration (day 0) until the end of the study (day 7). Data are presented as mean ± SEM (n = 6 for Day 0–1; n = 3 for day 2–7).

Fig. 7

Representatives photomicrographs of the lung, liver and heart as stained by H&E. The lung, liver and heart tissues were found unaffected by the treatment of transduced hUC-MSCs, as evidenced by normal tissues architecture, without any observable lesion. Red arrows indicate pulmonary haemorrhage; black arrows indicate glycogenesis and white arrows indicate vacuolization. All images taken at 100x magnification. The scale bars represent 50 μm.