Single-cell controlled human induced pluripotent stem cell differentiation to pericytes by CMOS-MEA-assisted electroporation.

Vascularization is important for tissue morphogenesis, but blood vessel cells in different tissues show distinct gene expression patterns that influence their tissue-specific specialization. For instance, the blood-brain barrier (BBB), which consists of endothelial cells (EC), pericytes (PC), and astrocytes, regulates the exchange of substances between the brain and other tissues. In this barrier, PCs are specialized cells located on the periphery of endothelial cells where they play essential roles in vascular stability, BBB integrity, blood pressure regulation, and angiogenesis1. However, the differentiation of PCs from human induced pluripotent stem cells (hiPSCs) with high brain-specific fidelity remains a challenge in vascular tissue engineering, despite several studies reporting the generation of brain PCs.  Identification of these cells relies on morphological assessment, proximity to ECs, and expression of markers such as PDGFRβ, CD13, or CD1461,2. Recent studies have shown that overexpression of NKX3.1 in iPSCs directs their differentiation toward mural progenitor cells3. NKX3.1 is a transcription factor essential for the proper development and function of iPSC-derived PCs3,4. Another recent study indicates that Cxcl12, along with Nkx3.1, is required for early PC differentiation, as it recruits endothelial progenitor cells, thereby initiating blood vessel formation5. This evidence suggests a strong role for CXCL12 in PC differentiation. Furthermore, overexpression of the Notch3 intracellular domain (N3ICD) has been shown to be sufficient to direct the differentiation of hiPSC-derived neural crest cells into PC-like cells with enhanced functionality6. At imec, we developed a Microelectrode Array (MEA) chip using complementary metal-oxide-semiconductor (CMOS) technology (CMOS-MEA) that enables precise electroporation to deliver different molecules at much lower voltages (~1 V) than the traditional suspension method, resulting in high efficiency and low cell death rates7,8. We aim to leverage the platform's ability to obtain PCs via iPSC differentiation, driven by controlled overexpression of these factors, yielding PCs that exhibit brain PC characteristics. Furthermore, the subcellular size of the electrodes on the CMOS-MEA platform enables single-cell-resolution electroporation, allowing micropatterning of differentiated cells. Once the electroporation protocol is established for PC differentiation, we will use it together with the EC differentiation protocol to perform co-transfections and create 2D micropatterned vasculature-like EC and PC co-cultures. 

1. Winkler, E. A., Bell, R. D. & Zlokovic, B. V. Central nervous system pericytes in health and disease. 

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3. Lee, U. et al. Robust differentiation of human pluripotent stem cells into mural progenitor cells via transient activation of NKX3.1. Nat Commun 15, 8392 (2024). 

4. Bose, A. et al. Tumor-Derived Vascular Pericytes Anergize Th Cells. The Journal of Immunology 191, 971–981 (2013). 

5. Ahuja, S. et al. The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors. PLoS Biol 22, e3002590 (2024). 

6. Gastfriend, B. D. et al. Notch3 directs differentiation of brain mural cells from human pluripotent stem cell-derived neural crest. Sci Adv 10, eadi1737 (2024). 

7. Duckert, B. et al. High-definition electroporation: Precise and efficient transfection on a microelectrode array. J Control Release 352, 61–73 (2022). 

8. Duckert, B., Lambrechts, D., Braeken, D., Lagae, L. & Fauvart, M. Optimizing mRNA transfection on a high-definition electroporation microelectrode array results in 98% efficiency and multiplexed gene delivery. Biosens Bioelectron 241, 115634 (2023).