Use of Ferritin Expression, Regulated by Neural Cell-Specific Promoters in Human Adipose Tissue-Derived Mesenchymal Stem Cells, to Monitor Differentiation with Magnetic Resonance Imaging In Vitro

Abstract

The purpose of this study was to establish a method for monitoring the neural differentiation of stem cells using ferritin transgene expression, under the control of a neural-differentiation-inducible promoter, and magnetic resonance imaging (MRI). Human adipose tissue-derived mesenchymal stem cells (hADMSCs) were transduced with a lentivirus containing the human ferritin heavy chain 1 (FTH1) gene coupled to one of three neural cell-specific promoters: human synapsin 1 promoter (SYN1p, for neurons), human glial fibrillary acidic protein promoter (GFAPp, for astrocytes), and human myelin basic protein promoter (MBPp, for oligodendrocytes). Three groups of neural-differentiation-inducible ferritin-expressing (NDIFE) hADMSCs were established: SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1. The proliferation rate of the NDIFE hADMSCs was evaluated using a Cell Counting Kit-8 assay. Ferritin expression was assessed with western blotting and immunofluorescent staining before and after the induction of differentiation in NDIFE hADMSCs. The intracellular iron content was measured with Prussian blue iron staining and inductively coupled plasma mass spectrometry. R2 relaxation rates were measured with MRI in vitro. The proliferation rates of control and NDIFE hADMSCs did not differ significantly (P > 0.05). SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1 hADMSCs expressed specific markers of neurons, astrocytes, and oligodendrocytes, respectively, after neural differentiation. Neural differentiation increased ferritin expression twofold, the intracellular iron content threefold, and the R2 relaxation rate two- to threefold in NDIFE hADMSCs, resulting in notable hypointensity in T2-weighted images (P < 0.05). These results were cross-validated. Thus, a link between neural differentiation and MRI signals (R2 relaxation rate) was established in hADMSCs. The use of MRI and neural-differentiation-inducible ferritin expression is a viable method for monitoring the neural differentiation of hADMSCs.

Fig 1. Outline of lentiviral vector construction.

Ferritin-expressing lentiviral vectors were constructed in two steps. First, the FTH1 sequence was cloned into the pLVTHM vector between the MluI and ClaI sites, downstream of the H1p promoter, generating the pLVTHM-H1p-FTH1 plasmid vector. Second, one of three neural cell-specific promoter sequences (SYN1p, GFAPp, or MBPp) was cloned into the pLVTHM-H1p-hFTH1 vector between the EcoRI and MluI sites, replacing the H1p sequence, to generate three NDIFE vectors: pLVTHM-SYN1p-FTH1, pLVTHM-GFAPp-FTH1, and pLVTHM-MBPp-FTH1. FTH1: ferritin heavy chain, SYN1: synapsin I, GFAP: glial fibrillary acidic protein, MBP: myelin basic protein.

Fig 2. Effects of the ferritin transgene on human adipose tissue-derived mesenchymal stem cell (hADMSC) proliferation.

There were no significant differences between the control group and the NDIFE hADMSC groups at each time point (one-way analysis of variance, P > 0.05). Error bars represent the mean ± standard deviation (n = 3). The ferritin transgene did not affect the proliferation rate of hADMSCs.

Fig 3. Expression of neural cell-specific markers in differentiating neural-differentiation-inducible ferritin-expressing human adipose tissue-derived mesenchymal stem cells.

After neural differentiation, neural-differentiation-inducible ferritin-expressing (NDIFE) human adipose tissue-derived mesenchymal stem cells (hADMSCs) began to express neural cell-specific markers. The scale bar is 50 μm. SYN1: synapsin I, NSE: neuron-specific enolase, FTH1: ferritin heavy chain 1, GFAP: glial fibrillary acidic protein, MBP: myelin basic protein, DAPI: 4′,6-diamidino-2-phenylindole.

Fig 4. Neural differentiation increases ferritin expression in neural-differentiation-inducible ferritin-expressing human adipose tissue-derived mesenchymal stem cells.

(A) Western blot analysis of ferritin expression in neural-differentiation-inducible ferritin-expressing (NDIFE) and neurally differentiated (ND)-NDIFE human adipose tissue-derived mesenchymal stem cells (hADMSCs). Ferritin expression was significantly higher in ND-NDIFE hADMSCs than in the corresponding control NDIFE hADMSCs. Error bars represent the mean ± standard deviation (n = 3); *P < 0.05. (B) A photomicrograph showing the immunofluorescent staining of ferritin in NDIFE and ND-NDIFE hADMSCs. Ferritin expression (red fluorescence) was significantly stronger in ND-NDIFE hADMSCs than in the corresponding control NDIFE hADMSCs. The scale bar is 50 μm. GFP: green fluorescent protein, DAPI: 4′,6-diamidino-2-phenylindole.

Fig 5. Differentiation increases the intracellular iron level in neural-differentiation-inducible ferritin-expressing human adipose tissue-derived mesenchymal stem cells.

(A) A photomicrograph showing Prussian blue iron staining of intracellular iron in neural-differentiation-inducible ferritin-expressing (NDIFE) and neurally differentiated (ND)-NDIFE human adipose tissue-derived mesenchymal stem cells (hADMSCs). Many disperse cytoplasmic deposits (blue granules) of accumulated intracellular iron were observed in NDIFE hADMSCs, whereas large, dense cytoplasmic deposits appeared in the corresponding control ND-NDIFE hADMSCs. The scale bar is 50 μm. (B) Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the intracellular iron content in NDIFE and ND-NDIFE hADMSCs. The intracellular iron content in ND-NDIFE hADMSCs was significantly higher than that in the corresponding undifferentiated NDIFE hADMSCs. Error bars represent the mean ± standard deviation (n = 3; *P < 0.05).

Fig 6. Differentiation increases the R2 relaxation rates of neural-differentiation-inducible ferritin-expressing human adipose tissue-derived mesenchymal stem cells.

A T2-weighted image from magnetic resonance imaging (MRI) of agarose phantoms of neural-differentiation-inducible ferritin-expressing (NDIFE) and neurally differentiated (ND)-NDIFE human adipose tissue-derived mesenchymal stem cells (hADMSCs). The R2 relaxation rate of ND-NDIFE hADMSCs was significantly higher than that of the corresponding undifferentiated NDIFE hADMSCs, resulting in notable hypointensity in T2-weighted images of ND-NDIFE hADMSCs. Error bars represent the mean ± standard deviation (n = 4; *P < 0.05).