Evidence of mesenchymal stromal cell adaptation to local microenvironment following subcutaneous transplantation

Abstract

Subcutaneous transplantation of mesenchymal stromal cells (MSC) emerged as an alternative to intravenous administration because it avoids the pulmonary embolism and prolongs post-transplantation lifetime. The goal of this study was to investigate the mechanisms by which these cells could affect remote organs. To this aim, murine bone marrow-derived MSC were subcutaneously transplanted in different anatomical regions and the survival and behaviour have been followed. The results showed that upon subcutaneous transplantation in mice, MSC formed multicellular aggregates and did not migrate significantly from the site of injection. Our data suggest an important role of hypoxia-inducible signalling pathways in stimulating local angiogenesis and the ensuing modulation of the kinetics of circulating cytokines with putative protective effects at distant sites. These data expand the current understanding of cell behaviour after subcutaneous transplantation and contribute to the development of a non-invasive cell-based therapy for distant organ protection.

Keywords: angiogenesis; hypoxia; mesenchymal stromal cells; remote activity; subcutaneous transplantation.

FIGURE 1

Behaviour of MSC after subcutaneous transplantation in the interscapular region. A, Fluorescent signal of the labelled MSC graft 30 min after the transplantation, as assessed by in vivo imaging. B, representative ex vivo fluorescence imaging of mouse organs at 7d post‐transplantation of CMTPX‐labelled MSC (right) or with vehicle, as control (left); BAT, brown adipose tissue; WAT, white adipose tissue. C, total radiant efficiency values of spectral‐unmixed signal in the MSC aggregate, WAT and lymph node (LN) at 7d after transplantation. D, survival curve for two cell doses (low and high) of grafted cells determined by the in vivo bioluminescent (BLI) signal of Luc‐expressing MSC measured as average radiance. E, representative images of in situ vascularization of MSC aggregates at 7d post‐transplant (after skin excision and graft exposure). F, in vivo assessment of graft‐induced inflammation by quantification of myeloperoxidase‐based bioluminescent signal after i.p. injection of lucigenin in mice receiving low and high MSC doses (**P < .01, Student's t test)

FIGURE 2

HS680 fluorescent imaging agent labels hypoxic MSC in large aggregates. A, Merged confocal images of DIC and fluorescence of U87‐MG cells and MSC incubated with HS680 for 24 h in 2D culture. B, spectral unmixing analysis of 3D multicellular aggregates of U87‐MG cells and MSC cultivated for 3 d in hanging drops in the presence of HS680 dye. This analysis was performed using the IVIS Spectrum system and Living Image 4.5 software, and allowed to separate the autofluorescence signal from the specific signal of HS680 dye. C, The correlation between aggregate diameter and hypoxia level in MSC spheroids formed by hanging‐drop culture of increasing numbers of MSC. The values are mean ± SEM of at least 10 spheroids per experimental condition. D, quantification of hypoxia of in vivo MSC aggregates at 7 d after subcutaneous injection of 1 × 10⁶ or 3.5 × 10⁶ cells (measured as HS680‐specific signal). Data are shown as mean ± SEM of n = 5 mice. (**P < .01, Student's t test)

FIGURE 3

Subcutaneously transplanted MSC activate hypoxia signalling pathways. A, Time‐course evaluation of hypoxia activation in HRE‐Luc‐expressing MSC when 10⁶ cells were transplanted subcutaneously in mice (n = 6). Representative images of one mouse at different time‐points after transplant are illustrated on the right. B, time‐course evaluation of hypoxia activation when five doses of 2 × 10⁵ HRE‐luciferase‐expressing MSC were injected subcutaneously in different adjacent sites in a mouse (n = 4). The representative images of one mouse at different time‐points after transplant are illustrated on the right

FIGURE 4

Dynamic changes in the cytokine abundance in the mouse serum after subcutaneous transplantation of MSC. A, Hierarchically clustered heat map showing the Euclidian distance of serum cytokines differentially expressed at various time‐points after subcutaneous transplantation of MSC in mice. Values represent fold change relative to day 0 and are adjusted to a per row colour scale. The cytokines with first‐day peak level are highlighted in magenta, while cytokines with third‐day peak level are highlighted in violet. B, Graphs illustrating the highly expressed cytokines clustered according to their max peak expression in the serum. At right, the “low expression level” denotes the cytokines whose maximum level of expression at any time‐point was higher than the half but below two thirds of the median value of all spots (highest pixel density between 2000 and 2500). The y‐axis on the left represents the relative expression level as fold change as compared to day 0. The colour‐coded right y‐axis shows the abundance of each cytokine in the serum of mice before transplant (day 0)