Microfluidic cell squeezing enables the in vitro generation of induced neurons from human pluripotent stem cells through non-viral transcription factor delivery

# Microfluidic cell squeezing enables the in vitro generation of induced neurons from human pluripotent stem cells through non-viral transcription factor delivery This page is a text version of a Portal Biotechnologies publication. The bibliographic record is labeled in the text so it can be read without the surrounding website. ## Bibliographic record - Title: Microfluidic cell squeezing enables the in vitro generation of induced neurons from human pluripotent stem cells through non-viral transcription factor delivery - Document type: Poster - Year: 2021 - Outlet: ISSCR - Cells: iPSCs - Cargos: mRNA, CRISPR RNPs - Page URL: https://portal.bio/publications/2021-microfluidic-cell-squeezing-enables-the-in-vitro-generation-of-induced-neurons-from-h--ajkHbxEAAC4Ac-Gi - Markdown URL: https://portal.bio/publications/2021-microfluidic-cell-squeezing-enables-the-in-vitro-generation-of-induced-neurons-from-h--ajkHbxEAAC4Ac-Gi.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65cd22fd9be9a5b998b5d44d_ISSCR-2021-Regenerative-Medicine-Poster-Wu_FINAL.pdf ## Summary Chun-I Wu, Jonathan Gilbert, Marija Tadin-Strapps, Armon Sharei, Devin T Bridgen ## Publication text Microfluidic cell squeezing enables the in vitro generation of induced neurons from human pluripotent stem cells through non-viral transcription factor delivery Chun-I Wu, Jonathan Gilbert, Marija Tadin-Strapps, Armon Sharei, Devin T Bridgen SQZ Biotechnologies, Watertown MA ### Abstract Cell replacement therapies are a promising treatment for a variety of intractable diseases including neurodegenerative conditions. Traditional methods to generate cells for these therapies utilize sequential treatment of signaling molecules to differentiate human pluripotent stem cells into specific cell types. These methods are often inefficient and result in variable purity. However, one promising recent approach is cell fate reprogramming by expression of single or multiple transcription factors to drive cell differentiation more efficiently. Here we demonstrate our proprietary Cell Squeeze® technology’s ability to deliver transcription factors into induced pluripotent stem cells (iPSCs) to efficiently generate neurons preclinically. The Cell Squeeze® technology is uniquely positioned to engineer cell function through transcription factor delivery since it enables non-viral, cytosolic delivery of a variety of materials while preserving cell health and avoiding changes to gene expression. Furthermore, the use of the Cell Squeeze® technology allows for the precise timing and magnitude of delivery of one or multiple transcription factors for the delicate task of cell fate reprogramming. This vector-free platform has demonstrated the ability to process billions of cells per minute and is already in use clinically for an oncology target. The efficient non-viral generation of neurons from pluripotent stem cells preclinically illustrates the potential of using Cell Squeeze® technology to generate specific cell types through transcription factor delivery for cell replacement therapies. Applying SQZ™ Technology to Regenerative Medicine Cell Squeeze® Technology Preserves iPSC Pluripotent Gene and Protein Expression In Vitro Optimized Neurog2 mRNA Constructs Resulted in Higher Neurog2 Protein and Downstream Target Gene Expression Optimization of Cell Squeeze® Technology Pressure Conditions in iPSCs using GFP mRNA ### Summary We have demonstrated the potential to rapidly generate neuronal cells from iPSCs using microfluidic cell squeezing to deliver transient transcription factors in vitro. More specifically: 1. The CellSqueeze® process minimally alters iPSC gene and protein expression as compared to untreated cells. 1. Squeezing mRNA into iPSCs leads to robust protein expression as shown by GFP and Neurog2 production. 1. A single delivery of Neurog2 mRNA leads to rapid expression of the neuronal marker Tuj1. Subsequent cell maturation shows the cells can express mature neuronal markers MAP2 and NeuN after 14 days. 1. Codelivery of Neurog2 and PAC mRNA allowed for puromycin selection to eliminate undelivered iPSCs. 1. Sharei A, et al. "A vector-free microfluidic platform for intracellular delivery." PNAS, 2013 Feb 5;110(6):2082-7 1. Xue Y, et al. “Synthetic mRNAs drive highly efficient iPS cell differentiation to dopaminergic neurons." Stem Cells Translational Medicine, 2019;8:112-123 References18S ### Mt-atp6 ### Rpl37a ### Actb ### Gapdh ### Ppia ### Rplp0 ### Rps17 ### Rpl30 ### Ubc ### Pgk1 ### Casc3 ### Pum1 ### Abl1 ### Polr2a ### Tfrc ### Psmc4 ### Hprt1 ### B2m ### Ipo8 ### Pes1 ### Elf1 ### Mrpl19 ### Eif2b1 ### Gusb ### Ywhaz ### Pop4 ### Cdkn1b ### Hmbs ### Tbp ### Cdkn1a ### Gadd45a Ct Untreated Squeeze No obvious difference in Ct value of 32 housekeeping gene expressions between untreated and squeeze iPSCs after 24 hours. Total RNA was isolated for RT-qPCR at 24 hours post squeeze delivery.Oct4 Nanog Sox2 Myc ### Tert 0.0 0.5 1.0 1.5 Fold change compared to untreated Untreated squeeze Sox2Oct4 ### Ssea4 There is no obvious difference between iPSC marker protein expression between untreated and squeeze iPSCs after 24 hours. The cells were collected to analyze iPSC pluripotent marker expression (Oct4, Sox2, SSEA4) by flow cytometry. No significant difference in iPSC marker gene expression between untreated and squeeze iPSCs after 24 hours. Total RNA was isolated for RT-qPCR at 24 hours post squeeze delivery. Relative expression of each gene was normalized to MT-ATP6. Fold change was determined between squeeze and untreated iPSCs. Single Squeeze Delivery of Neurog2 mRNA Transcription Factor Leads to Rapid Tuj1 Expression within 24 Hours Merge Neurog2-Derived Neurons Express Mature Neuronal Markers MAP2 and NeuN at Day 14 Tuj1 (early neuron marker) ### Dapi (Nucleus staining) Merge Nanog (iPSC marker) Co-Delivery of Puromycin Resistance mRNA with Neurog2 mRNA Allows for Puromycin Selection Cells were fixed at 24 hours post squeeze delivery for immunofluorescence staining. Green arrows marked iPSCs express Nanog protein and red arrows marked cells express Tuj1 protein but lost Nanog expression. Cell nuclei were counterstained with DAPI. New Technologies (NT), Poster 318No SQZ % Live Cells %No SQZ ### % Gfp+ %No SQZ 10000 20000 30000 40000 ### Gfp Mfi Squeezed iPSC had high viability, with >80% delivery and high fluorescent MFI 24 hours after delivery with green fluorescent protein (GFP) mRNA using different pressures. GFP signal was assessed by flow cytometry. Human iPSCs GFP mRNA Squeezing Flow cytometry ### Gapdh Neurog2 No mRNA Neurog2_v1 Neurog2_v2 Neurog2_v3 Neurog2_v4 Neurog2_v5 Single squeeze mRNA delivery drives high levels of Neurog2 protein expression at 8 hours. Western blot analysis showed Neurog2_v5 mRNA results in the most robust protein expression. Single squeeze mRNA delivery drives high expression levels of Neurog2 downstream target genes. Total RNA was isolated for RT-qPCR at 12, 24, 36, and 48 hours post squeeze delivery. Neurog2_v5 mRNA more potently induce the downstream targets than other Neurog2 mRNA.12 hrs 24 hrs 36 hrs 48 hrs 10000 15000 NeuroD1 Normalized to squeeze delivery only fold increase12 hrs 24 hrs 36 hrs 48 hrs NeuroD4 Human iPSCs Neurog2 mRNA Squeezing Western blot & RT-qPCR Human iPSCs Neurog2 mRNA Squeezing ### Dapi (Nucleus staining) Merge ### Map2 (mature neuronal marker) NeuN (mature neuronal marker) Neurog2 mRNA-induced neurons from iPSCs were immunostained for mature neuronal markers MAP2 (green arrows) and NeuN (red arrows) at differentiation day 14. Cells nuclei were counterstained with DAPI. Non-neuronal cells were removed with 16 hours puromycin selection followed by 4 days of AraC treatment. Human iPSCs Neurog2 and PAC mRNAs Cell Squeeze® technology at day 14 iPSC were squeeze delivered with Neurog2 or Neurog2 with PAC (puromycin N-acetyltransferase) and treated with 16 hours of puromycin selection. Phase contrast images were taken after puromycin selection and showed that cells with PAC mRNA survived puromycin selection. Human iPSCs Neurog2 and PAC mRNAs Cell Squeeze® technology No material + No Puromycin Neurog2 + PAC mRNA + Puromycin Neurog2 mRNA + No Puromycin These data highlight the potential of the platform to rapidly reprogram cells through nonviral methods and generate clinically-relevant cell therapies. Cytosolic Transcription Factor Delivery (Cell Squeeze® Technology) Squeeze Process: Cells flow through a constriction smaller than the cell diameter. This process results in temporary disruption of the cell membrane to facilitate target material delivery. The cell membrane reseal rapidly and cause a minimal perturbation to the cells. Flexibility of cargo delivery: Ability to control the delivery magnitude and timing of single or multiple transcription factors. Non-integrative approach: Transient delivery avoids potential genomic integration. Minimal perturbation of baseline cell function Scalable for High Throughput: Treat more than 1010 cells/minute. Speed and efficiency potentially enabling allogenic or autologous approaches Unique SQZ™ Opportunity in Regenerative Medicine12 hrs 24 hrs 36 hrs 48 hrs NeuroD4 SA-co Neurog2 SA-co-m6AG Neurog2 SA-co-Kozak Neurog2 Neurog2_v5 Neurog2_v3 Neurog2_v2 ## Provenance Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.