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Elena Unlocks Immune Cell Potential with Simple Western and CRISPR/Cas9

"I used Simple Western because the protein samples I had were small. We only had a small number of [iPSC-derived macrophages] because the cell line does not proliferate."

- Elena Navarro-Guerrero, Ph.D., Senior Postdoctoral Researcher, Nuffield Department of Medicine, Target Discovery Institute

Elena Navarro-Guerrero, Ph.D.

MAKING THE MOST OF MACROPHAGES

Macrophages derived from human induced Pluripotent Stem Cells (iPSC) have enormous potential to model pathology and treat disease, but their stubbornness to genetic engineering has been a major roadblock until now. Dr. Elena Navarro-Guerrero has helped to unlock the potential of iPSC-derived macrophages with her work in the laboratory of Dr. Daniel Ebner, located in the Nuffield Department of Medicine, Target Discovery Institute at the University of Oxford. She applies CRISPR/Cas9 and lentiviral transduction to genetically engineer iPSC-derived macrophages with high efficiency to better understand their involvement in immune responses and increase their potential in cell & gene therapy applications. Simple Western™ from ProteinSimple has joined her in this journey.

BREAKING NEW GROUND WITH CRISPR/CAS9

Biology entered a revolution with the discovery and application of CRISPR/Cas9, a molecular toolkit with proven versatility to precisely make genetic modifications in many different cell types, even human cells. Once CRISPR/Cas9 introduces a genetic modification, phenotypic validation at the protein level is often required, and this has traditionally been accomplished by the Western blot. But when it comes to analyzing iPSC-derived macrophages, the Western blot falls critically short. As Elena describes, “we only had a small number of cells because the cell line does not proliferate.” In other words, realizing the exciting potential of CRISPR/Cas9 and lentiviral transduction was burdened with outdated and inefficient protein detection methods.

THE SIMPLE WESTERN SOLUTION TO SMALL SAMPLE SIZES

Seeking a solution for her small sample sizes, Elena turned to Simple Western, a relatively new immunoassay technology that is gaining momentum to replace the traditional Western blot for the specific detection of proteins. As an automated capillary-based immunoassay, Simple Western is poised to replace traditional Western blot partly because it can analyze very small sample sizes, as little as 3 μL, with picogram-level sensitivity. This makes analyzing small sample sizes like iPSC-derived macrophages possible. As she describes, “I was able to do several [Simple Western] analyses with a small sample, which I couldn’t have done by traditional Western blot.”

Using Wes, a member of the Simple Western instrument family, Elena could be confident that the genetic knockouts she made with CRISPR/Cas9 and lentiviral transduction were real. In a study published in 2021 in Scientific Reports, Wes analysis showed the absence of the 3 non-essential genes, HPRT1, PPIB, and CDK4, that she intended to knock out (FIGURE 1). One of the significant outcomes of her work is that this technique opens the door to the systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases, and cancer progression, which has never been done before.

Wes confirms knockouts of 3 essential genes (HPRT1, PPIB, and CDK4) in iPSC-derived macrophages performed by CRISPR/Cas9 and lentiviral transduction

Figure 1.  Wes confirms knockouts of 3 non-essential genes (HPRT1, PPIB, and CDK4) in iPSC-derived macrophages performed by CRISPR/Cas9 and lentiviral transduction. Data generated by Wes automatically appear in the Compass for Simple Western software, which controls Simple Western systems. The lane view shown here was adapted with permission from Navarro-Guerrero et al. (2021) Scientific Reports. (CC BY 4.0)

FAST, RELIABLE, AND MORE TARGETS PER SAMPLE THAN TRADITIONAL WESTERN BLOT

Elena noted that “Wes is faster than traditional Western blot.” Results are generated in as little as 3 hours, and it is fully automated. Compare this to traditional Western blot which can take a full day or more of manually intensive steps, and the time savings becomes clear. Unlike the cumbersome steps of traditional Western blot which are prone to error and introduce variability, Simple Western is fully automated and therefore more reliable and reproducible. Elena also noted that Simple Western “has enabled us to analyze several proteins in one sample.” Towards this end, the latest Simple Western systems like Jess and Abby push multiplex detection further with RePlex™, which performs two sequential immunoassays on the sample in the sample capillary. Simple Western and RePlex can even perform Total Protein Detection to normalize protein expression data. While chemiluminescence detection comes standard with all Simple Western systems, Jess also includes two channels of fluorescence detection, thereby pushing the multiplex functionality even further.

For more information on how Elena and her colleagues used Simple Western and CRISPR/Cas9 to engineer iPSC-derived macrophages, check out their 2021 publication in Scientific Reports:

Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages, E. Navarro-Guerrero, C. Tay, J. Whalley, S. Cowley, B. Davies, J. Knight, D. Ebner, Scientific Reports, 2021; 11:4245.

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