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Characterization of iPSCs and Derived Neuronal Stem Cells by Flow Cytometery Tavneet Gill, Jing Lui, Kasia Wilczek, Jan Nolta Institute for Regenerative Cures, CIRM Stem Scholars Internship, UC Davis, Sacramento, CA, 95817 Summer 2012 Flow cytometry facilitates the evaluation of distinct characteristics of cells, and it is utilized to confirm observations. Flow cytometry specializes in the compensations of different transactions including cell counting, cell sorting, protein engineering, and biomarker detection. Here we used flow cytometry to characterize iPSCs (induced Pluripotent Stem Cells). iPSCs are generated from adult cells that are “forced” into the expression of specific genes via induction. iPSCs have analogous nature to other pluripotent stem cells, such as human embryonic stem cells. With the input of selective cell markers, the analysis of their expressions with a flow cytometer can aid in iPSC characterization. One of the differentiation potential of iPSC is to produce neural cells. NSC, neural stem cells, are multipotent cells and have potential for cell Therefore, we also used flow cytometry for their characterization. Introduction Flow Cytometry Basics & FlowJo Flow cytometry uses components of fluidics, optics, and electronics to obtain essential information of specific cells. Fluidics focuses on the filing of cells in a systematic fashion through which a laser beam points. There are various detectors that are directed through this laser beam, consisting of the one single beam (forward scatter; x-axis), and many perpendicular beams (side scatter; y-axis). Subsequently, optics, another component of flow, utilizes lenses and filters for collection, separation, absorbance, and interference. Lenses manufacture light collection, which captures emission of light in the lens. Filters direct attention on absorbance as unwanted wavelengths are uncovered by the glass of the optical filters. Electronics aids in light detection, through the assistance of various detectors called PMTs (photomultiplier tubes). Once photons are collected to a proportional number of electrons by the PMTs, with addition of voltage, these electrons are amped. Once amplified it can be converted into a voltage pulse captured by the computer. The FlowJo Analysis software is utilized for the examination of data generated by modern flow cytometers that are written in the Flow Cytometer Standard (.fcs) format file extention. The display on the right shows the sequential steps during the analysis by this software. Figure 1: The figure displays the basic pathway of the cell from the application of fluidics to the utilization of electronics of the flow cytometer Step A Step B Step D Step C Flow Jo Analysis Figure 2 Step A: Display of all tubes analysed in the flow cytometer. Step B: Cluster identification and backgating display Step C: Histogram in selected channel and gate setting Step D: layout for presenting iPSC Characterization iPSCs are generated by forced expression of 4-6 transcription factors in somatic cells , including OCT3/4, SOX-2, Klf4, cMyc, Lin28, and Nanog. There are many approaches, including viruses, nonintegrating episomal vectors, synthetic mRNA and reprogramming proteins to deliver these genes. Here in this lab, we used retroviruses carrying OCT3/4, SOX-2, Klf4, cMyc to produce patient-specific iPSCs. The upper right figure shows flow data obtained from iPSC stained with Tra-1- 81, SOX-2 and OCT4. A dominant positive population was detected for each staining, showing most of the iPSC cells express those pluripotent markers. This is also confirmed by the immunostaining results, indicating the generated iPSC are self-renewal. Other methods are also applied to characterize iPSC, including teratoma formation assay, karyotyping, morphology examination, and In-vitro embryoid formation. EX- Xa Nanog OCT4 SOX2 SSEA4 Tra-1-60 NL-Xa Figure 3: Resuts from immunostaining indicate that the generated iPSC are self renewal. They are each markes with pliuripotent markers such as SSEA4, Tra-1-60, SOX2, OCT4, and Nanog. Stained with PE Tra- 1-81 and gated according to PE IgG isotype control Stained with PerCP- Cy5.5 SOX2 and gated by PerCP-Cy5.5 IgG isotype control Stained with unconjugated OCT4 and goat anti-Ms 488 second antibody. iPSC Characterization Figure 4: A dominant positive population was detected for each staining, showing most of the iPSC cells express those pluripotent markers. iPSC Culturing The iPSCs are cultured and maintained according to proper passaging procedures. First, the mouse embryonic fibroblasts (MEF) are seeded at a certain density on the culture plate. Good iPSC colonies are defined as compact and even structured with distinct edges. Once these colonies are selected and marked, a P200 pipette is used to scratch these colonies and break them apart. Those pieces are then harvested and seeded onto the MEF in iPSC medium . Overtime colonies begin to grow, and around 5-7 days later they are ready for passaging Figure 5: This illustrates the basic steps taken during iPSC culturing. This integrated process starts from the thawed MEF and leads all the way up to passaging Acknowledgements The CIRM Stem Scholars internship was by far one of the greatest learning experiences of my life. I would like to thank Jing Lui, Kasia Wilczek, Gerhard Bauer, Jan Nolta, and everyone else who had made this amazing opportunity possible. I have learned a legion of knowledge that will help me in the future and present. Thank you. NSC Characterization Neurogenesis in the adult brain originates for NSCs and these cells are the building blocks of many functionalities the brain impacts. These self-renewing multipotent cells are the generators of phenotypes of the nervous system. The purity of NSCs will determine their differentiation efficiency into either neurons or astrocytes. We again used a panel of markers to identify NSCs. As shown on the right, remarkably high fractions of NSCs expressed the neuroepithelial markers, nestin and SOX-1, but not the pluripotent marker, Tra-1-81. Stained with unconjugated nestin and goat anti-Ms 488 2 nd antibody. Stained with unconjugated SOX-1 and goat anti-Rb 488 second antibody. 2 nd antibody alone gating control. Stained with PE Tra-1-81 and gated according to PE IgG isotype control.
