Human SOX2 mRNA

Neural stem cells (NSCs) are an important cell source for understanding neuropathogenesis and developing therapeutic applications for neurodegenerative diseases due to their regenerative capacity and multipotency. Reprogramming using in vitro transcribed (IVT) mRNA is a genetically safe reprogramming method because the exogenous mRNA is transiently present in the cell and does not integrate into chromosomes. Here, researchers successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) by transfecting in vitro transcribed (IVT) mRNA encoding SOX2 (SOX2 mRNA) under appropriately optimized conditions. They demonstrated that the generated human UCB-MSC-derived iNSCs (UM-iNSCs) possessed NSC characteristics, including multipotency and self-renewal. In addition, researchers transfected SOX2 mRNA into human dermal fibroblasts (HDFs). Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited similar neural reprogramming morphology and NSC-enriched mRNA levels, but their proliferation capacity was limited. These findings suggest that human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) can be directly reprogrammed into NSCs by transfection with IVT mRNA encoding individual factors, which provides an integration-free reprogramming tool for future therapeutic applications.

Here, researchers developed a protocol for direct conversion of neural stem cells (NSCs), as shown in Figure 1a). Human umbilical cord mesenchymal stem cells (UCB-MSCs) were used as the source for NSC generation (Figure 1b). These cells were transfected with SOX2 mRNA three times every other day. After 14 days, NSC-like clones were formed with an efficiency of 0.015% (Figure 2(b)). Subsequently, the researchers transferred these clones to coated cell culture dishes that promote adhesion, and then passaged these cells to uncoated spheroid culture dishes to obtain a homogeneous population of human umbilical cord mesenchymal stem cell-derived neural stem cells (UM-iNSCs). In both culture dishes, the cells maintained a monolayer or neurosphere morphology, which was similar to human embryonic stem cell-derived NSCs (Figure 1c). As expected, there was no significant difference between UM-iNSCs and ESC-derived NSCs at passage numbers 20 to 22 (Figure 1d). Next, the researchers evaluated the formation of secondary neurospheres by comparing the size and number of secondary neurospheres between UM-iNSCs and ESC-derived NSCs. To measure these results, cells were formed into primary neurospheres by transferring from coated dishes to uncoated dishes and then dissociated and re-seeded into uncoated dishes to form secondary neurospheres (Figure 1e). The results showed that there was no significant difference in the size and number of neurospheres between the UM-iNSC line and ESC-derived NSCs (Figure 1f,g). These results indicate that overexpression of SOX2 by IVT mRNA transfection is sufficient to directly reprogram human umbilical cord blood mesenchymal stem cells (UCB-MSCs) into iNSCs.

Figure 1. Generation of UM-iNSCs from human UCB-MSCs induced by treatment with SOX2 mRNA. (Kim B E, et al., 2018)