Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA

Abstract

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

Keywords: cellular reprogramming; direct conversion; neural stem cell; synthetic mRNA; umbilical cord blood.

Figure 1.

Optimization of transfection conditions for effective induction of exogenously transfected mRNA. (a) Human UCB-MSCs were transfected with mRNA-encoding SOX2 at 1, 3, and 5 days post-induction (DPI). Quantitative real-time PCR data demonstrated the expression level of total exogenous and endogenous SOX2 at 5, 10, and 15 DPI. (b) Immunocytochemistry data showed a nuclear localization of SOX2 proteins (red) 48 h post-transfection. Nuclei were counterstained with DAPI. Scale bar = 200 μm. (c) Western blot analysis indicated protein expression of SOX2 48 h post-transfection of unmodified and modified mRNA. Relative expression levels were calculated using the Image J system. (d) Relative gene expression levels of innate immune-related genes (INFA, IFNB, RIG-I, PKR, OAS, and IFIT1) were analyzed at 5 and 10 DPI using quantitative real-time PCR. (e,f) A concentration-dependent transfection test was performed with 0.01, 0.1, 0.5, 1, 2, and 4 µg/ml EGFP mRNA. The GFP-positive cells were counted at 48 h after transfection using flow cytometry. (g,h) A time-dependent transfection test was performed at 24, 48, and 60 hours after EGFP mRNA transfection at a dose of 1 µg/ml, and the GFP-positive cells were counted using flow cytometry. For the control, the cells treated with only transfection reagents (control) and the ESC-derived NSCs were used. Error bars represent the standard deviation of reactions repeated more than three times. ***P < 0.001; **P < 0.01; *P < 0.05. DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; ESC, embryonic stem cell; GFP, green fluorescent protein; NSC, neural stem cell; PCR, polymerase chain reaction; UCB-MSC, umbilical cord blood-derived mesenchymal stem cell.

Figure 2.

Generation of UM-iNSCs from human UCB-MSCs induced by treatment with SOX2 mRNA. (a) A schematic diagram illustrating the procedure for generation of iNSCs from human UCB-MSCs was shown. Arrowheads: SOX2 mRNA transfection. (b) Morphological changes during the reprogramming procedure from UCB-MSCs (i) though NSC-like colonies at 14 DPI (ii) to sub-culture of picked colonies (iii–iv) were observed. (c) The typical morphologies of two UM-iNSC lines (#1 and #2) on an adhesion culture and a floating culture were observed at passage 20. (d) The cumulative CPDL analysis was performed with two UM-iNSC lines to characterize the self-renewal ability. (e) An illustrated schema showed the sphere formation assay procedure. (f,g) the sphere formation assay revealed that there were no significant differences of sizes and numbers between two UM-iNSC lines and ESC-derived NSCs in primary and secondary neurospheres. Error bars represent the standard deviation of reactions repeated more than three times. Scale bar = 200 μm. CPDL, cumulative population-doubling level; DPI, days post-induction; ESC, embryonic stem cell; NSC, neural stem cell; UCB-MSCs, umbilical cord blood-derived mesenchymal stem cell; UM-iNSC, UCB-MSC-derived induced neural stem cell.

Figure 3.

Characterization of the UM-iNSCs by immunocytochemistry. (a) The UM-iNSCs were stained using antibodies for the NSC-enriched markers (SOX2, PAX6, NESTIN) and a cellular proliferation marker (Ki67). Scale bar = 50 μm. (b,c) Immunocytochemistry data were analyzed using the Image J system and showed percentages of SOX2/PAX6 and NESTIN-double-positive cells, Ki67-positive cells, and Ki67 and NESTIN-double-positive cells in UCB-MSCs, UM-iNSCs, and ESC-derived NSCs, respectively. ESC, embryonic stem cell; NSC, neural stem cell; UCB-MSC, UCB-MSCs, umbilical cord blood-derived mesenchymal stem cell; UM-iNSC, UCB-MSC-derived induced neural stem cell.

Figure 4.

Genome-wide transcriptional profiling of UM-iNSCs. (a,b) Relative gene expression levels of NSC-specific genes (endogenous SOX2, PAX6, ASCL1, SLC1A3, NES, OLIG2) and mesenchymal cell- or fibroblast-enriched genes (COL1A2, ACTA2) in UM-iNSCs and ESC-derived NSCs were compared to human UCB-MSCs using quantitative real-time PCR. (c) A pair-wise scatter plot indicated differences of genome-wide transcriptional gene expression in UM-iNSCs and ESC-derived NSCs profiled by microarray analysis. Two-fold change difference boundaries are displayed as black lines. (d) GO enrichment analysis in biological processes are shown. Selected GO categories of two-fold increased (red) and two-fold decreased (blue) genes in UM-iNSCs compared to ESC-derived NSCs are listed. Error bars represent the standard deviation of reactions repeated more than three times. ***P < 0.001; **P < 0.01; *P < 0.05. ESC, embryonic stem cell; GO, gene ontology; NE, no expression; NSC, neural stem cell; PCR, polymerase chain reaction; UCB-MSC, umbilical cord blood-derived mesenchymal stem cell; UM-iNSC, UCB-MSC-derived induced neural stem cell.

Figure 5.

Differentiation capacity of UM-iNSCs into neurons, astrocytes, and oligodendrocytes. Immunocytochemistry results revealed neurons (stained against α-internexin, NF, DCX, MAP2; (a–c)), astrocytes (stained against GFAP; (d)), and oligodendrocytes (stained against MBP; (e)). (f,g) Percentages of NF-, DCX-, and MAP2-positive neuronal cells and GFAP- and MBP-positive neural cells were measured using the Image J system. Error bars represent standard deviation of triplicate reactions. Scale bar = 50 µm. DCX, doublecortin; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; MBP, myelin basic protein; NF, neuro-filament; UM-iNSC, umbilical cord blood-derived mesenchymal stem cell -derived induced neural stem cell.