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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. 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. Because direct reprogramming bypasses the pluripotent state, it can prevent the risk of teratoma formation 2 — 4. Moreover, reports have shown that it is possible to directly reprogram mouse or human somatic cells by transducing cells with the single-factor SOX2 using viral methods 8 They demonstrated that overexpression of a single-factor, SOX2, via a viral method is sufficient to convert human somatic cells into self-renewing and multipotent neural stem cells NSCs.

However, virus-mediated reprogramming entails a high risk of genetic insertion leading to tumor formation in vivo. To replace the virus-mediated method, a of transgene-free reprogramming technologies — including non-integrating adenoviral vectors, DNA plasmid-based vectors, and recombinant proteins incorporating cell-penetrating peptides CPPs — for transduction have recently been developed. While adenoviral vectors are non-integrating vectors, they can trigger multiple components of the immune response, such as cytotoxic T lymphocyte activation 11 Although transfection using DNA plasmid-based vectors is safer than using viral vectors, there are concerns about insertional mutagenesis, and it is difficult to completely eliminate the risk of genomic insertion It is also difficult to directly introduce reprogramming factors as proteins and peptides into cells because penetrating the lipid bilayer of the cell membrane to enter the intracellular space while maintaining protein tertiary structure is difficult, and direct introduction of proteins causes instability in the extracellular space Importantly, these DNA- or protein-based methods depend on repeated administration of transient vectors and thus have shown very low reprogramming efficiency 15 — In addition to integration-free gene delivery systems, it has been shown that direct transfection of in vitro transcribed IVT mRNA-encoding transcription factors can reprogram human somatic cells into pluripotent stem cells, which could be redifferentiated into myogenic cells 20 and a retinal lineage Importantly, it is reported that human fibroblasts can be directly reprogrammed into hepatocyte-like cells by IVT mRNAs Moreover, IVT mRNA-encoding transcription factors can efficiently overexpress the target gene without risk of insertional mutagenesis.

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Because exogenously transfected mRNA is translated in the cells and only temporally expressed, it is a genetically safe method compared to the other approaches 15 Moreover, the mRNA-based method does not leave a genetic footprint or have a risk of genome integration, suggesting the potential safety advance of the mRNA-mediated method 1523 Therefore, thus far, mRNA-based methodologies are the most suitable for cell therapy and clinical approaches due to the safety aspects 13 However, it has a low reprogramming success rate because the influx of exogenous mRNA exists only temporarily.

Therefore, reports have suggested that daily transfection of mRNA is needed to retain gene expression for cellular reprogramming 1320 By interacting with pattern-recognition receptors such as RIG-I receptor family, these proteins inhibit translation initiation and global protein expression from both endogenous and exogenous mRNA, and lead to pro-inflammatory cytokine responses 25 — To conduct an effective reprogramming process, optimal conditions are needed to maintain gene expression and to minimize the innate immune response.

Non-integrative direct reprogramming into induced NSCs iNSCs and induced neurons is promising for neurodegenerative disease therapy. Unlike terminally differentiated induced neurons, iNSCs are more potent for transplantation therapies and investigation of pathology for neurodegenerative disease because of their self-renewal ability and multipotency 928 — As a further study of our reports, we used the transcription factor SOX2 as a master direct neural reprogramming factor via a non-integrative gene delivery system.

In this study, we hypothesized that a SOX2 mRNA-mediated method facilitates overexpression of the SOX2 protein in nuclei, and it is sufficient to reprogram the human umbilical cord blood-derived mesenchymal stem cells UCB-MSCs into iNSCs available for various clinical approaches without concerns about uncontrolled genetic integrations. Briefly, to remove red blood cells in human cord blood samples, HetaSep solution Stem Cell Technologies, Vancouver, British Columbia, Canada was incubated with the samples at a ratio of at room temperature.

The supernatant was collected, and mononuclear cells were harvested using Ficoll Sigma Aldrich, St. The cells were washed twice in phosphate-buffered saline PBS. We purchased pcDNA3. The following day, the medium was exchanged with the same fresh medium. Rohidas Arote. After a few days, the cells were dissociated with Accutase Gibco BRL and subcultured on non-coated dishes for suspension culture. To produce a homogeneous population, the cells were passed into non-coated dishes and coated dishes by turn. In brief,cells of each NSC line were seeded in six-well plates in triplicate.

The cells were passaged every 3 days three or four passagesand the same population of cells were seeded as before and counted using trypan blue to detect live cells. The population-doubling level was calculated by adding to the passages. The colony-forming assay procedure has been ly described After 3 days, the primary neurospheres were dissociated and re-plated at the same density for formation of secondary neurospheres.

After 24 h, the medium was changed to specific medium to induce differentiation into three lineages neurons, astrocytes, and oligodendrocytes. The differentiation medium was prepared as ly described All primers are listed in Supplementary Table 1. Next day, the cells were incubated with secondary Alexa or Alexa conjugated antibodies ; Invitrogen for 1 h at room temperature diluted in PBS. We performed three washes with PBS in the intervals. Cells were washed in PBS and harvested in a tube. Fold-changes in genes in all samples were normalized and selected using GeneSpringGX 7.

Then, we observed that the transfected SOX2 mRNA was not only translated into protein but was also localized in the nucleus Figure 1 b. Optimization of transfection conditions for effective induction of exogenously transfected mRNA. Nuclei were counterstained with DAPI. Relative expression levels were calculated using the Image J system.

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The GFP-positive cells were counted at 48 h after transfection using flow cytometry. Error bars represent the standard deviation of reactions repeated more than three times. In prominent studies about modification of IVT mRNA transcripts, modified nucleotides with 5-methylcytidine substituted for cytidine and pseudouridine for uridine showed ificant improvement in protein expression, accompanied by reduced activation of the antiviral innate immune system. Therefore, we investigated whether the expression levels of SOX2 and antiviral aling-related proteins changed depending on the modification of nucleotides.

We replaced nucleoside bases with 5-methylcytidine for cytidine and pseudouridine for uridine. Our showed that the modified mRNA transcript led to an approximately five-fold increase in expression in comparison to the unmodified transcript Figure 1 c. Interestingly, the expression levels of interferon response genes at two weeks post-transfection were increased as much as those in embryonic stem cell ESC derived NSCs Supplementary Figure S1 b.

Second, we measured the of GFP-expressing cells over time. The highest of GFP-expressing cells was observed at 60 h, but the overlay data indicated the greatest effective fluorescence intensity at 48 h Figure 1 e. It seemed that the translated GFP was divided into daughter cells 48 h post-transfection.

Both transfection systems are known to facilitate high transfection efficiency with low cellular toxicity. Then, we also tested two types of PEI-conjugated carriers, which consist of cationic polymers and form complexes with mRNA. Taken together, we optimized the mRNA transfection protocol with a prominent intracellular delivery system that reduced cytotoxicity and activation of interferon response genes.

Based on the data above and our studies, we developed a direct conversion protocol for NSCs, as illustrated in Figure 2 a. After 14 days, we observed NSC-like colonies with 0. On both types of culture dishes, the cells could be maintained as a monolayer or as neurosphere, and their morphologies were similar to those of human ESC-derived NSCs Figure 2 c and Supplementary Figure S2 a. Moreover, UM-iNSC lines were expandable for more than 50 passages without changes in morphology or growth rate data not shown. To measure these, cells formed primary neurospheres through transfer from coated to non-coated culture dishes, and they were again dissociated and re-plated on non-coated culture dishes to form secondary neurospheres Figure 2 e.

Second, cells were stained for the proliferation marker Ki to further characterize the self-renewal property of UM-iNSCs. To further identify changes in gene expression patterns after the neural reprogramming, we performed global gene expression profiling between UM-iNSCs and ESC-derived NSCs using a microarray analysis with 34, probes in total. Here, 8. These differentially expressed genes were categorized using gene ontology GO function enrichment analysis Figure 4 d. The relative overexpressed genes in UM-iNSCs were related to mRNA surveillance pathway, MAPK aling pathway, and FoxO aling pathway in order of enrichment, whereas the relative downregulated genes were related to phagosome, graft- versus -host disease, and antigen processing and presentation.

Two-fold change difference boundaries are displayed as black lines. To verify the multipotency of UM-iNSCs, we differentiated the cells into neurons, astrocytes, and oligodendrocytes with specific proper conditions. After 7 days of neuronal differentiation, a-internexin and neuro-filament NFwhich are known as neuronal intermediate filament proteins, and doublecortin DCXwhich is known as a microtubule-associated neuronal migrating protein marker, were positively stained Figure 5 a,b.

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To further investigate whether it is possible to differentiate UM-iNSCs into mature neuronal subtypes, we stained the cells with neuronal maturation markers — neuronal nuclei NeuNmicrotubule-associated protein 2 MAP2 and choline acetyltransferase ChAT — after 14 days of neuronal differentiation.

The differentiated cells were positively stained at cell bodies and the nucleus Figure 5 c and Supplementary Figure S3 a,b. Using well-established differentiation protocols, we successfully differentiated cells into glial fibrillary acidic protein GFAP -positive astrocytes and oligodendrocyte transcription factor OLIG2 - and myelin basic protein MBP -positive oligodendrocytes Figure 5 d,e and Supplementary Figure S3 d.

By counting, GFAP-positive cells were Altogether, these data showed that UM-iNSCs can be differentiated into neurons, astrocytes, and oligodendrocytes, demonstrating their multipotency. Error bars represent standard deviation of triplicate reactions. studies suggested that SOX2 is the master regulatory gene for the preservation of properties of NSCs, including proliferation, self-renewal and neurogenesis, and therefore, SOX2 could play a crucial role in direct reprogramming of somatic cells into neural lineages 111636 — Furthermore, several studies have shown that neural reprogramming using single-factor SOX2 is possible using the viral method 8 In the meantime, cellular reprogramming techniques are advancing, and mRNA-based technologies for reprogramming are also actively being studied because mRNA-based gene regulation is not concerned with chromosomal-integration.

This study is notable for using IVT mRNA, which is an extremely valuable tool for therapeutic applications and for various clinical approaches. In this study, we suggested the best conditions for the highest transfection and translation efficiency by screening of the optimal concentration and interval of transfected IVT mRNA.

We optimized the concentration that led to maximum GFP-expressing cells without cell death and the most effective interval which reduced the immune response and sustained SOX2 protein expression. However, the low transfection and translation efficiencies of mRNA still remain a challenge.

Currently, IVT mRNAs are complexed with structural elements, such as nanoparticles, polymers, or cationic lipids, to further improve intracellular stability and translational efficiency 2343 Not only limitations of transfection and translation efficiency but also stochastic efficiency might have led to the failure of reprogramming Recently, various methods have been attempted to increase stochastic efficiency during the reprogramming process. More recently, chemical-mediated methods have been reported that make the cells lose original cellular features and gain multipotent stem cell-specific features by acting as epigenetic modifiers, cell death-related pathway regulators, and metabolic modifiers.

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Chemicals that play various roles in a cellular pathway have been suggested as options to increase the stochastic efficiency. Such chemicals that might enhance the cellular reprogramming efficiency have been investigated in other studies. In general, MSCs can be derived from umbilical cord blood UCBbone marrow BMand adipose tissue ADand reveal multipotent differentiations into chondrocytes, adipocytes, and osteocytes Moreover, whenever the patients need them, we can clinically use the cells because they can be cryopreserved in a cell bank.

Many researchers suggest UCB-MSCs are valuable and need to be studied for their clinical utility and regenerative medicine An introduction of appropriate genes is compulsory to kick-start the reprogramming process, and they have to force the cells to overcome the reprogramming barrier To clarify these limitations of reprogramming, such as cell type variations, whole-genome expression analyses of initial and intermediate states in the reprogramming processes are needed for understanding reprogramming efficiency.

Most neurodegenerative diseases caused by neuronal dysfunction involve loss of the neuronal population. Due to a lack of molecular studies and therapeutic treatments for diseases, cell therapy and disease modeling by direct reprogramming of disease-specific cells are actively being studied to develop an efficacious alternative. Although differentiated neurons from iNSCs have not been functionally proved in the present study, iNSCs were differentiated into mature neurons. As long as it is verified that differentiated neurons are functional, it has remarkable potential for use as a clinical approach, especially for patients with neurodegenerative diseases.

Thus, it will be valuable to study mRNA-based methods and application of synthetic mRNA for cellular reprogramming, and continuous study should be done to improve efficiency. Statement of Human and Animal Rights: This article does not contain any studies with human or animal subjects.

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