(K-418) Rapidly assessing the cellular composition of neural stem and progenitor cell populations using qRT-PCR

Neural stem and progenitor cells (NSPCs) differentiate into neurons, astrocytes, and oligodendrocytes during embryonic development to form the central nervous system (CNS). Neurons use electrical and chemical signals to send information throughout the body, astrocytes are involved in regulating neuronal synapses, neuronal circuits, and behavior, and oligodendrocytes myelinate neuronal axons to aid the speed and efficiency of neuronal communication. Due to the poor regenerative properties of CNS tissue, NSPC transplantation has therapeutic potential for CNS diseases and injuries. However, previous transplantation studies found variable effects of NSPCs for alleviating CNS ailments. This is in part due to difficulties in predicting transplanted NSPCs’ fate, which leads to variability in transplants. To improve NSPC therapeutic potential, we are testing sorting strategies to enrich cells tied to specific fates (neuron, astrocyte or oligodendrocyte). We used a label-free, dielectrophoresis-based microfluidic device (HOAPES device) to sort NSPCs into neuron- and astrocyte-biased sub-populations. The goal of my project is to design a reliable qRT-PCR assay to rapidly identify fate-biased NSPCs by their gene expression signatures to assess cell fate potential in the sorted NSPC populations. I hypothesize that differences in mRNA expression for neuron-, astrocyte-, and oligodendrocyte-biased NSPCs can be detected using qRT-PCR and that specific combinations of expressed genes will reliably predict the cellular composition of NSPC populations.

The following workflow was used: (1) Design primers based on (a) highly expressed genes found in the literature or from our scRNAseq data that are associated with early progenitors in neuron, astrocyte, and oligodendrocyte lineages and (b) consistently expressed housekeeping genes to be used as controls. Short oligonucleotide DNA primers were customized through NCBI’s Primer-BLAST or used sequences from Origene. All primers were ordered from IDT. (2) Use PCR and gel electrophoresis of amplified products to find the appropriate annealing temperatures for each set of primers. (3) Generate fate-biased mNSPC samples utilizing the following methods: (a) Sort using the HOAPES device. (b) Use NSPCs isolated from different embryonic stages of cortical development (embryonic days E12 and E16) and anatomical structures (cortex and ganglionic eminence) known to be enriched in different fate biased NSPCs. (c) Culture cells, in some cases using modified culture medium known to enrich for a particular fate bias. (4) Extract RNA and make cDNA using the Aurum Total RNA Mini Kit from Bio-Rad Laboratories. (5) Verify the cellular composition of each batch of cells by differentiating mNSPCs and using immunocytochemistry (ICC) to detect mature neuron (MAP2 and DCX), oligodendrocyte (O4), and astrocyte (GFAP) markers. (6) Perform qRT-PCR to compare gene expressions among experimental groups using the PowerUp SYBR Green Master Mix and the Applied Biosystems QuantStudio 6 or 7 RT-PCR systems. (7) Normalize expression to housekeeping genes and use the qRT-PCR data to identify genes reliably expressed by neuron, astrocyte, and oligodendrocyte progenitors (by matching ICC results).

Multiple sources of NSPCs enriched for neuron, astrocyte, or oligodendrocyte progenitors were screened to determine markers of fate detected by qRT-PCR. The HOAPES device uses a label-free, dielectrophoresis-based approach to separate subpopulations of NSPCs based on fate potential and HOAPES-sorted NSPCs were used to assess neuron and astrocyte biased progenitors. Neurons, astrocytes, and oligodendrocytes are generated during development in particular brain regions at specific time points. NSPCs were isolated from E12 cortex for enriched neuron progenitors, E16 cortex for astrocyte progenitors, and E16 ganglionic eminence for oligodendrocyte progenitors.:

To determine markers for neuron-biased cells, I tested NeuroD1, Dclk2, Tbr2, Ngn1, and Ngn2. I also tested Mgat3, which encodes an N-linked glycosylation enzyme that blocks the enzyme Mgat5, which we found encourages astrocyte formation. From these, I found NeuroD1, Dclk2, and Mgat3 detected neuron-biased progenitors in both HOAPES sorted cells and E12 vs. E16 NSPCs (Fig. 1A). To determine markers for astrocyte-biased cells, I tested Egfr, Sparcl1, Id3, Asef, Glast, Fapb7, Mfge8, and Aqp4. From these, I found Egfr and Sparcl1 detected astrocyte-biased progenitors in HOAPES sorted cells and E16 vs. E12 NSPCs (Fig. 1B). To determine markers for oligodendrocyte-biased cells, I used NSPCs isolated from the E16 ganglionic eminence and also cultured the cells in media containing PDGF to support oligodendrocyte progenitors. I tested Pdgfra, Sox10, and Pcdh15 as potential markers. Of these, Pdgfra and Pcdh15 detected oligodendrocyte-biased progenitors cultured from the ganglionic eminence (Fig. 1C).:

I determined qRT-PCR markers that accurately assess the fate potential for each NSPC lineage and also identified Mgat3 as a novel marker for neuron-biased progenitors. This qRT-PCR assay can quickly and reliably assess the cellular composition of NSPCs, expediting the workflow of NSPC studies and transplantations. Future studies will focus on identifying additional primers based on scRNAseq data from our lab, repeating experiments to increase the number of biological repeats, and testing other cell types such as human iPSC-derived NSPCs.

A rapid qRT-PCR assay for cell fate will improve the consistency of cell transplantation for recovery from debilitating CNS ailments by providing a quick assessment of cellular composition that can help optimize NSPC transplants.