READ ME File for the Thesis 'A 3D- Induced Pluripotent Stem Cell-Derived Human Neural Culture Model to Study Certain Molecular and 
Biochemical Aspects of Alzheimer’s Disease.'

Dataset DOI: https://doi.org/10.5258/SOTON/D2234 


ReadMe Author: Preeti Prasannan, University of Southampton 

Date of data collection: 2018-2020

Information about geographic location of data collection: UNIVERSITY OF SOUTHAMPTON, UK.


This dataset contains: 
1. 2D-NSC-RA EXPERIMENT
2. IMMUNOFLUORESCENCE DATA
3. WESTERNBLOT DATA

1. CHAPTER 2-2D-NSC Retinoic Acid (RA) Experiment

This dataset contains all the experimental data used to generate the figures included in Chapter 2- Human Neural 3D culture - Optimization and 
Validation of 3D Culture method, of the Thesis. 

Description:

Human foetal Neural Stem Cells treated with different concentrations of RA were immunostained for MAP2, Nestin and GFAP at different timepoints and imaged using a Confocal Laser Scanning Microscope-SP8. Positive cells were manually counted for each stain, concentration and timepoint using ImageJ. Graphpad Prism was used for statistical analysis of the data.

Number of variables: Four different concentrations of RA used to treat cells- 0.1µM, 0.5µM, 1µM and 2µM RA. 
Timepoints 1. MAP2 and NESTIN - day0, 2.5weeks and 8weeks and 
           2. GFAP and NESTIN - 8weeks.
Abbrevations used- RA - RETINOIC ACID
                 - T0-Day 0 (no treatments)
                 - T2.5WK-2.5 WEEKS
		     - T8WK- 8 WEEKS
		     - N= NESTIN
		     - M= MAP2
                 - G= GFAP 
                 - += POSITIVE
                 - -= NEGATIVE
Reference: Figures 2.5, 2.7, and 2.9.


2. CHAPTER 3-IMMUNOFLUORESCENCE DATA - 2D AND 3D CELL CULTURES

This dataset contains all the Immunofluorescence data used to generate the figures included in Chapter 3- 2D Cultures verses 3D Cultures, of the Thesis.  

Description:

Five iPSC derived NSC cell lines: AD-iPSCs with Presenilin 1 mutation HAD2, HAD3, HAD4 and controls HN9 and HN9 were differentiated into neurons and astrocytes both in 2D and 3D culture systems. The cultures were analysed at different timepoints- day 0, 6weeks and 12 weeks for different markers such as MAP2, Nestin, B3 Tubulin and GFAP using Immunofluorescence technique. A Confocal Laser Scanning Microscope-SP8 was used for imaging and ImageJ software was used to measure total positive area of stained cells. Graphpad Prism was used for statistical analysis of the data.

Reference: Figures 3.3 to 3.21. 

3. CHAPTER 3 and 4- WESTERN BLOT DATA - 2D AND 3D CELL CULTURES

This dataset contains all the Western Blot data used to generate the figures included in:
 Chapter 3- 2D Cultures verses 3D Cultures and
 Chapter 4 -Early Pathological Changes, of the Thesis. 

Description:

Five iPSC derived NSC cell lines: AD-iPSCs with Presenilin 1 mutation HAD2, HAD3, HAD4 and controls HN8 and HN9 were differentiated into neurons and astrocytes both in 2D and 3D culture systems. Total lysates from these cultures were analysed at different timepoints- 6weeks (2D and 3D) and 12 weeks (3D) for different markers such as Total Tau and PHF1 using Westernblot technique. Fractionated lysates from the 3D cultures at 6 weeks timepoints were analysed to study aggregation of Tau and its isoforms 3R Tau and 4R Tau. Licor Imaging System with Odessey software was used to detect signals. Blots were processed using ImageJ. Graphpad Prism was used for statistical analysis of the data.

Abbrevations used : A,B,C,D and E - different sets of Westernblot gels run

Reference: Figures 3.23, 4.4 to 4.12.