Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo

Abstract

Far-red fluorescent reporter genes can be used for tracking cells non-invasively in vivo using fluorescence imaging. Here, we investigate the effectiveness of the far-red fluorescent protein, E2-Crimson (E2C), for tracking mouse embryonic cells (mESCs) in vivo following subcutaneous administration into mice. Using a knock-in strategy, we introduced E2C into the Rosa26 locus of an E14-Bra-GFP mESC line, and after confirming that the E2C had no obvious effect on the phenotype of the mESCs, we injected them into mice and imaged them over nine days. The results showed that fluorescence intensity was weak, and cells could only be detected when injected at high densities. Furthermore, intensity peaked on day 4 and then started to decrease, despite the fact that tumour volume continued to increase beyond day 4. Histopathological analysis showed that although E2C fluorescence could barely be detected in vivo at day 9, analysis of frozen sections indicated that all mESCs within the tumours continued to express E2C. We hypothesise that the decrease in fluorescence intensity in vivo was probably due to the fact that the mESC tumours became more vascular with time, thus leading to increased absorbance of E2C fluorescence by haemoglobin. We conclude that the E2C reporter has limited use for tracking cells in vivo, at least when introduced as a single copy into the Rosa26 locus.

Comparing the differentiation potential ofBrachyury+mesodermal cells generated from 3-D and 2-D culture systems

“…The key differences in the gene expression profile of the Bra-GFP + cells isolated from cavitating EBs 307 (current study) and non-cavitating EBs (previous study) (Rak-Raszewska, 2010) is that in comparison 308 12 of 44 to GFP − cells, the former expressed much higher levels of Foxf1, which is highly expressed in lateral 309 plate mesoderm, and lower levels of the MM genes, Gdnf and Osr1 (Rak-Raszewska, 2010). The high 310 expression levels of Foxf1 might explain why the EB-derived Bra-GFP + cells in the current study had a 311 tendency to generate endothelial cells, because it is known that Foxf1 is essential for vasculogenesis in 312 the developing embryo and is expressed in endothelial cells (Mahlapuu et al, 2001;Ren et al, 2014). 313…”

Functional comparison of distinctBrachyury+ states in a renal differentiation assay

“…Bra-GFP/Rosa26-E2C mESCs ( Zhou et al, 2018 ) were maintained in 0.1% gelatinised six-well tissue culture plates with mitomycin-C (Sigma-Aldrich, M4287) inactivated STO (ATCC, SCRC-1049) feeder cells at 37°C in a humidified incubator with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, D6546) supplemented with 15% fetal bovine serum (FBS) (Sigma-Aldrich, F2442), 1% minimum essential medium (MEM) non-essential amino acid (Sigma-Aldrich, M7145), 2 mmol l −1 L-glutamine (Sigma-Aldrich, G7513), 0.1 mmol l −1 β-mercaptoethanol (Gibco, 31350) and 1000 U ml −1 mouse leukaemia inhibitory factor (mLIF) (Merck Millipore, ESG1107). Cells were passaged every other day and those at passage 13–22 were used for experiments.…”

“…In the present study, we aimed to investigate whether Bra + cells generated using the recently described 2-D culture system, and those derived from cavitating EBs, express similar lineage-specific genes, and have similar developmental potential to those derived from non-cavitating EBs. In order to do this, we have generated a Bra-GFP/Rosa26-E2C mESC reporter line ( Zhou et al, 2018 ) that will allow us to isolate the GFP-expressing mesodermal cells from both systems so that their gene expression can be analysed using RT-PCR and their developmental potential can be assessed by investigating their fate following incorporation into mouse kidney rudiments ex vivo ( Unbekandt and Davies, 2010 ; Kuzma-Kuzniarska et al, 2012 ; Rak-Raszewska et al, 2012 ; Ranghini et al, 2013 ; Dauleh et al, 2016 ).…”

How Non-invasive in vivo Cell Tracking Supports the Development and Translation of Cancer Immunotherapies

“…OPTICAL (bioluminescence): Firefly, Green Click Beetle: D-luciferin; Gaussia, Renilla: coeloenterazine; NanoLuc: imidazopyrazinone. (Lorenz et al, 1991;Loening et al, 2006;Tannous, 2009;Inoue et al, 2011;Hall et al, 2012;Schaub et al, 2015 (Merzlyak et al, 2007;Kremers et al, 2009;Lin et al, 2009;Filonov et al, 2011;Liu et al, 2013;Shcherbakova and Verkhusha, 2013;Deliolanis et al, 2014;Isomura et al, 2017;Zhou et al, 2018;Fukuda et al, 2019) Frequency-selective contrast/other Artificial protein Contrast based on transfer of radiofrequency labeling from the reporter's amide protons to water protons.…”