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Engineering Cell Fate Through Multiplexed Transcription Factor Delivery

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Who this is for:

  • Stem cell and regenerative medicine scientists: Researchers engineering hematopoietic, mesenchymal, or pluripotent stem cells (HSCs, MSCs, iPSCs)
  • Cell therapy developers: R&D teams in biotech/pharma working on cell-based therapies who need scalable, gentle transfection of stem cells

Opportunities

  • Minimal disruption of cell state and function: Transcriptomic profiling confirms minimal gene dysregulated at 24 h post-delivery in unstimulated primary cells, in stark contrast to the thousands of genes affected by electroporation. This allows precise modulation of cell behavior without unintended consequences.
  • Preserve differentiation capacity post-delivery: Stem cells retain their full differentiation potential following mechanoporation. For instance, delivery of transcription factors (e.g., NGN2) into iPSCs drives targeted lineage commitment with rapid marker expression, all while maintaining a stable gene expression profile.
  • Maintain engraftment potential: Stem cells engineered using mechanoporation retain high viability and functional attributes essential for hematopoietic reconstitution. Unlike harsher transfection methods, mechanoporation avoids activation or exhaustion signatures.
  • Broad cargo compatibility with sensitive stem cells: Achieve efficient intracellular delivery of mRNA, siRNA, CRISPR RNPs, proteins, and other biologics into fragile populations such as iPSCs, CD34⁺ HSCs, etc. Mechanoporation enables this without compromising cell viability, phenotype, or function.

Results Obtained

  • Initiated the conversion of human induced pluripotent stem cells (iPSCs) into neurons using Ngn2 mRNA
  • iPSCs maintain expression of housekeeping and pluripotency genes post-boost
  • Efficient delivery in iPSCs and MSCs of Ngn2 mRNA, cRNA, DELs, and siRNA

How We Did It

Portal uses mechanoporation to deliver virtually any cargo to diverse cell types while maintaining cell health. This enables intracellular delivery of impermeable molecules without the complications of electroporation or other delivery methods. Learn more about mechanoporation here! 

iPSCs Express Early Neuronal Markers 12hrs After Boosting With Ngn2 mRNA

We demonstrated a method for initiating the conversion of human induced pluripotent stem cells (iPSCs) into neurons in just 12 hours using mechanoporation. We delivered neurogenin 2 (Ngn2) mRNA directly into human iPSCs using the Gateway system. To track delivery efficiency, we co-delivered dextran molecules and confirmed successful cellular uptake through flow cytometry analysis. Following the boosting, cells were harvested 12 hours later for RNA extraction and RT-qPCR analysis. 

The data shows that the Ngn2 mRNA boost significantly increased expression of Ngn2 itself (approximately 10,000-fold relative to control, Fig. 2), as well as downstream neuronal transcription factors NeuroD1 (approximately 40-fold increase, Fig. 3) and NeuroD4 (approximately 10-fold increase, Fig. 4). These results indicate that the mechanoporation-delivered Ngn2 mRNA successfully initiated a neuronal differentiation cascade, with the boosted cells expressing early neuronal markers within just 12 hours of treatment, demonstrating this is an efficient method for directing iPSCs toward a neuronal fate.

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Fig 2
Fig 2: Ngn2 mRNA Expression: RT-qPCR analysis reveals approximately 10,000-fold upregulation of Ngn2 mRNA in mechanoporation-boosted iPSCs compared to untreated controls 12 hours post-treatment.
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Fig 3: NeuroD1 Expression: Downstream neuronal transcription factor NeuroD1 shows approximately 40-fold increased expression in Ngn2-boosted cells versus controls at 12 hours, demonstrating activation of the neuronal differentiation cascade.
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Fig 4: NeuroD4 Expression: Expression analysis of neuronal marker NeuroD4 shows approximately 10-fold upregulation in boosted cells compared to controls at 12 hours post-mechanoporation, demonstrating activation of the neuronal differentiation cascade.

iPSCs maintain expression of housekeeping and pluripotency genes post-boost

We investigated whether induced pluripotent stem cells (iPSCs) maintain their characteristic gene expression profiles after undergoing mechanoporation. We cultured iPSCs for 48 hours, then extracted total RNA from both untreated control cells and boosted cells. Using RT-qPCR, we analyzed the expression of numerous housekeeping genes (Fig. 5) and key pluripotency markers (Fig. 6). The data shows that boosted cells maintained expression levels comparable to untreated controls across all tested genes. Housekeeping genes showed consistent expression with Ct values mostly between 15-30, while pluripotency markers Oct4, SOX2, Nanog, Klf4, Myc, and TERT displayed relative expression levels normalized to ACTB that were similar after boosting. The minimal variation between treated and untreated samples, as indicated by the error bars, suggests that the boost treatment does not significantly alter the fundamental cellular identity or pluripotent state of iPSCs. 

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Fig 5: Housekeeping gene expression remains stable in iPSCs following boosting. RT-qPCR analysis of housekeeping gene expression in untreated (light purple) and boosted (dark purple) iPSCs 48 hours post boosting. Ct values are shown for many housekeeping genes.

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Fig 6: Relative mRNA expression of core pluripotency transcription factors in untreated (light purple) and mechanoporated (dark purple) iPSCs, normalized to ACTB expression. Cells were mechanoporated and cultured for 48 hours prior to RNA extraction and RT-qPCR analysis. No significant differences were observed between treatment groups, indicating maintenance of the pluripotent state. 

iPSC: mRNA, siRNA, cRNA, and DELs 

Several proof of concept studies were performed in iPSCs to demonstrate successful delivery of mRNA, cRNA, and DELs. iPSCs have been successfully boosted with GFP mRNA & B2M siRNA demonstrating effective multiplexed delivery (Fig xx). Additionally, GFP cRNA expression has been demonstrated in iPSCs (learn more about the usefulness of cRNA here!) along with successful delivery of DNA encoded libraries (DELs).

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Fig 7: Percentage of iPSCs expressing GFP and exhibiting B2M knockdown following multiplexed delivery. iPSCs were either left untreated or boosted with GFP mRNA and B2M siRNA via mechanoporation. Flow cytometry analysis at 40h post-treatment shows 73% efficiency in the boosted condition compared to 0% in untreated controls. Histograms compare untreated (top) versus boosted (bottom) iPSCs at 40h post-mechanoporation. Left panels show GFP expression levels, right panels show B2M expression levels. Boosted cells demonstrate increased GFP expression and decreased B2M expression compared to untreated controls demonstrating the activity of the GFP mRNA and B2M siRNA.
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Fig 8: Representative fluorescence microscopy images of iPSCs post-boost. Top panels show untreated iPSCs with minimal GFP signal (left: GFP channel, right: transmitted light). Bottom panels show boosted iPSCs with robust GFP expression throughout the cell population (left: GFP channel showing widespread green fluorescence, right: transmitted light).
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Fig 9:  iPSCs were mechanoporated with circular RNA (cRNA) encoding green fluorescent protein (GFP). Flow cytometry analysis performed 48 hours post-boost shows robust GFP expression. The iPSCs demonstrated GFP positivity in approximately 80% of live cells post-treatment, compared to negligible expression in untreated controls.
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Fig 10: iPSCs were mechanoporated with a fluorescently-tagged DNA encoded library (DEL) probe. Delivery efficiency was assessed immediately after treatment using flow cytometry to detect the fluorescent tag. Results demonstrate that mechanoporation efficiently delivered DEL probes into iPSCs, with approximately 80% of live cells exhibiting fluorescence compared to untreated controls. Histogram analysis further confirms a clear shift in fluorescence intensity post-mechanoporation.

HSC & MSC: mRNA, siRNA

Several proof of concept studies were performed in HSCs & MSCs to demonstrate successful delivery of mRNA & siRNA. MSCs have been successfully boosted with GFP mRNA & HSCs with B2M siRNA demonstrating the usefulness of mechanoporation when working with HSCs.

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Fig 11: MSCs were mechanoporated with mRNA encoding gGFP. GFP expression was analyzed by flow cytometry 20 hours post-treatment, comparing two mechanoporation conditions using pore sizes of 10 µm and 11 µm. High transfection efficiency was achieved, with approximately 80-90% of MSCs expressing GFP under both pore size conditions. Fluorescence histograms confirmed robust GFP 
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Fig 12: Cell morphology assessed by microscopy indicated that mechanoporation preserved overall MSC health and structure, supporting the method's compatibility with delicate stem cell populations.
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Fig 13:  Human CD34+ hematopoietic stem cells HSCs were mechanoporated with siRNA targeting B2M. After 40 hours, flow cytometry analysis was performed to evaluate both cell viability and the knockdown efficiency of B2M expression. Results showed high cell viability post-treatment, indicating minimal cytotoxicity. Additionally, significant knockdown of B2M was achieved, with approximately 50% reduction compared to untreated controls

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