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Chapter 3. Temporal Profile of Proneural and Region-Specific Markers during Generation of Induced Neural Precursor Cells using Non-Viral Plasmid Transfection

Introduction

We have previously shown that transient ectopic expression of neural-promoting transcription factors, SOX2 and PAX6 (Maucksch, Firmin et al. 2012, Connor, Firmin et al. 2015), in aHDFs is sufficient to reprogram them into neural stem/precursor cells, which can be further differentiated into region-specific neuronal populations. Characterisation of the iNPs shows they express a range of neural positional markers: SOX1, SOX2, SIX3, PAX6, NGN2, IRX3, HOXB9, NKX6.1, and ASCL1 at days 65, 75, and 85 post transfection (Maucksch, Firmin et al. 2012). Upon differentiation, the plasmid-derived iNPs give rise to GFAP+ astrocytes and functional mature neurons expressing TUJ1, MAP2, NSE, and the subtype-specific markers VGLUT1, TH, Calbindin, DARPP32, and GAD65/67 (Maucksch, Firmin et al. 2012, Connor, Firmin et al. 2015).
The relative mRNA levels vary depending on the time the iNPs were collected, indicating that a temporal change of proneural and region-specific marker expression may have occurred during reprogramming of iNPs (Maucksch, Firmin et al. 2012). Therefore, the aim of this chapter is to investigate the temporal profile of proneural and region-specific gene expression during reprogramming. This study focuses on the genes that are known to regulate human neural development processes, based on both the timing of human neural development and the region-specific identity of the developing human brain (Figure 3-1). These include specification of neural induction and the formation of the forebrain, midbrain, and hindbrain.
Human neural development originating from hiPSCs and hESCs shares a similar gene profile with variable potency (Hu, Weick et al. 2010). This chapter aims to determine whether or not the direct reprogramming of iNPs using plasmid transfection resembles the sequential gene profile observed during human neural development.

Methods

HDFs (line #2352) were proliferated and plated out (500,000 per well for 6-well plates) for plasmid transfection with both pLVX-SOX2-zsGreen and pLVX-PAX6-tdTomato (Chapter 2.4.1). During reprogramming, transfected cells were maintained in reprogramming media without replating for 31 days after transfection (Day 3 – 31 post transfection) followed by weekly replating of the cells for days 31 – 66 post transfection (passage 1 – 5). At each time point (except day 66 post transfection), biological triplicate samples were collected (Figure 3-2).
After cell collection, RNA was extracted (Section 2.8.1) and ran through a TapeStation (Agilent; 2200) to confirm the integrity of the RNA. The expression of genes (Table 2-8) controlling neural induction, forebrain, midbrain, and hindbrain specification was examined using Taqman assays (Section 2.8.3).

Quantitative PCR Analysis

The expression of mRNA for selected neural markers was measured by qPCR using the TaqMan system (Applied Biosystems; Section 2.8.3). The amplification efficiency of Taqman Gene Expression Assays are tested extensively by Applied Biosystems, and all Taqman Gene Expression Assays have a high (>90%) amplification efficiency (AppliedBiosystems 2004). In this study, the normalisation of gene expression was determined using a single housekeeping gene, 18S rRNA; it has been validated by our research assistant Ms. Erin Firmin that 18S is expressed abundantly and stably in cells throughout the reprogramming process.
Fold changes in gene expression were calculated relative to those of the initial aHDF samples using the Ct method (Livak and Schmittgen 2001). The neural markers in human fibroblasts are not expressed or expressed in a low levels (Janmaat, de Rooij et al. 2015, Huang, Miller et al. 2016, Li, Sun et al. 2016), therefore the normalization of proneural and region-specific gene expression relative to aHDFs may create variability. However, the purpose of this study is to demonstrate the generation of iNPs from aHDFs by characterising the change in neural-specific gene expression relative to the original cell source during reprogramming, rather than to compare the characteristics of aHDF-derived iNPs with other cell lines, e.g. hiPSC-derived iNPs or hESC-derived iNPs. Hence, aHDFs are appropriate as the baseline for determination of gene changes (Cheng, Hu et al. 2014, Zhu, Ambasudhan et al. 2014, Li, Sun et al. 2016, Shahbazi, Moradi et al. 2016).
Triplicate qPCR values from each time point were expressed as mean ± SEM. Differences between means were assessed by one-way analysis of variance (ANOVA) and Dunnett’s post hoc tests, and p 05 was considered statistically significant. The statistical significance does not reflect the fold change of gene expression relative to the control aHDFs, hence this study also employs a biologically meaningful assessment by using an accepted (2-fold) cut off (DeRisi, Penland et al. 1996, Schena, Shalon et al. 1996, Hui and Feng 2013) to analyse the qPCR data. However, it must considered that a minimal change in transcription factor levels may still have a huge effect on downstream targets. Analysis of gene expression based on either p-value or fold change cut off can alter the interpretation of gene expression significantly (Dalman, Deeter et al. 2012). The most stringent criteria would be a combination of both p-value and fold change cut-offs in order to give a statistically and biologically meaningful assessment (Dalman, Deeter et al. 20

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Results
Co-localization and Detection of Transgene Protein

The expression plasmids pLVX-SOX2-zsGreen and pLVX-PAX6-tdTomato have respectively the transgenes SOX2 and PAX6 located upstream of their fluorescent detection protein, zsGreen or tdTomato (See Appendix 7.2). To confirm that the expression of SOX2 or PAX6 transgenes co-localized with the corresponding fluorescent detection proteins, HDF cells were transfected with either pLVX-SOX2-zsGreen or pLVX-PAX6-tdTomato and fixed one day post transduction for immunocytochemistry with SOX2 or PAX6 antibodies (Chapter 2.7). The results showed co-localisation of the nuclear marker DAPI with both SOX2 and PAX6 which confirmed nuclear expression of both transgenes (Figure 3-3). In addition, the fluorescent detection proteins zsGreen and tdTomato were expressed in both the nucleus and cytoplasm of transduced cells (Figure 3-3). These results indicate that the fluorescent detection proteins zsGreen or tdTomato were co-expressed with the transgenes SOX2 and PAX6 as expected, and the expression of zsGreen or tdTomato represented SOX2 and PAX6 expression, respectively.

Morphological Changes for Transfection of Reprogrammed Cells

HDFs were plated out as per Section 2.4.1 one day prior to plasmid transfection to allow the fibroblasts to adhere to the bottom of the plates. Transfected HDFs were allowed to recover in HDF growth media for 3 days after transfection, before culturing in neural reprogramming medium (Section 2.5.1). Due to the cytotoxicity of Lipofectamine transfection, severe cell death was observed in culture shortly after transfection (days 1 – 3 post transfection) (Figure 3-4; D3), then, cell numbers recovered (days 3 – 31 post transfection) (Figure 3-4; D17 – D31).
When the cells were observed under a fluorescence microscope, the cultures exhibited a mixture of cells expressing either SOX2-zsGreen (green), PAX6-tdTomato (red) or neither, and a population of cells expressing both SOX2-zsGreen and PAX6-tdTomato (yellow). From days 3 – 10 post transfection, the fluorescence was downregulated (Error! Reference source not found.) indicating the transient nature of the plasmid transfection.
After the initial cell replate at day 31 post transfection, the cells exhibited morphological changes (Figure 3-4). Cells changed from having an elongated and thin fibroblast morphology to a round morphology that formed networks (days 33 – 38 post transfection). After the 2nd to 4th replates (days 38, 45, and 52 post transfection), the cells generated colonies resembling the neurospheres commonly observed for neural precursor cells (Marshall, Reynolds et al. 2007) 2 days after each replate (at days 40, 47, and 54 post transfection). However, 7 days after each replate (at days 45, 52 and 59 post transfection) a monolayer cell culture with networks was again observed.
These results demonstrate that transient ectopic expression of SOX2 and PAX6 by plasmid transfection can generate cells with a morphology characteristic of neural precursors. It is importantly to note that, colony formation was achieved in the absence of a feeder layer.

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Chapter 1. Introduction
1.1. Overview
1.2. Stem Cells
1.3. Human Embryonic Neurogenesis
1.4. Reprogramming
1.5. Summary
Chapter 2. Methods 
2.1. Cell Culture
2.2. Molecular Cloning
2.3. Lentiviral packaging
2.4. Gene Delivery Methods
2.5. Reprogramming
2.6. Differentiation of Induced Neural Precursor Cells
2.7. Immunocytochemistry
2.8. Gene Expression Analysis
2.9. Fluorescence-Activated Cell Sorting (FACS)
Temporal Profile of Proneural and Region-Specific Markers during Generation Chapter 3. of Induced
3.1. Introduction
3.2. Methods
3.3. Results
3.4. Discussion
3.5. Conclusions
Chapter 4. Temporal Profile of Proneural and Region-Specific Markers during Generation of Induced Neural Precursor Cells using Lentiviral Transduction 
4.1. Introduction
4.2. Methods
4.3. Results
4.4. Discussion
4.5. Conclusion
Investigating the Requirement of SOX2 and/or PAX6 for the Direct Chapter 5. Reprogramming of iNPs
5.1. Introduction
5.2. Methods
5.3. Results
5.5. Conclusion
Chapter 6. General Discussion 
6.1. Introduction
6.2. Summary of Main Findings
6.3. Future Perspectives
6.4. Conclusion
Chapter 7. Appendix
7.1. Plasmid Constructs
7.2. Vector Constructs
7.3. Insert Sequences of PAX6
Chapter 8. References 
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Direct Generation of Region-Specific Induced Neural Precursors from Adult Human Fibroblasts using Non-Viral and Viral Methods

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