Generation of Aneuploid Human Induced Pluripotent Stem Cells from Primary Amniotic Fluid Cells via Episomal Plasmid Electroporation

Zifan Song; Jing Wang; Peng Yuan; Xiaohui Zhu; Liying Yan; Jianying Guo

doi:10.3791/71975

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Method Article

Generation of Aneuploid Human Induced Pluripotent Stem Cells from Primary Amniotic Fluid Cells via Episomal Plasmid Electroporation

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DOI:

10.3791/71975

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September 3rd, 2026

Zifan Song¹^,²^,³^,⁴ , Jing Wang¹^,²^,³^,⁴ , Peng Yuan¹^,²^,³^,⁴ , Xiaohui Zhu¹^,²^,³^,⁴ , Liying Yan¹^,²^,³^,⁴ , Jianying Guo¹^,²^,³^,⁴

¹State Key Laboratory of Female Fertility Promotion, Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, ²National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, ³Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, ⁴Beijing Key Laboratory of Collaborative Innovation in Frontier Technologies for Population Quality

Summary

This protocol provides a reproducible method for generating integration-free aneuploid human iPSCs from primary amniotic fluid cells using episomal plasmid electroporation. It optimizes delivery and culture conditions to stabilize chromosomal abnormalities, offering a practical platform for creating disease-specific models for downstream research and clinical applications.

Abstract

The generation of patient-specific induced pluripotent stem cells (iPSCs) from amniotic fluid cells (AFCs) carrying defined chromosomal aneuploidies provides a powerful platform for modeling genetic disorders. However, establishing a reliable and reproducible reprogramming pipeline for aneuploid AFCs remains technically challenging due to the intrinsic genomic instability and variable proliferative capacity of these cells. Here, we present a comprehensive, non-integrating method for generating aneuploid human iPSCs from primary AFCs using episomal plasmid electroporation. This protocol details the complete workflow, encompassing cell thawing and expansion with a gradual media adaptation strategy, optimized plasmid delivery via electroporation system, sequential post-electroporation culture with mesenchymal-to-epithelial transition (MET)-directed media changes, and mechanical colony picking based on defined morphological criteria. We further describe validation procedures, including immunofluorescence staining for core pluripotency markers, G-banding karyotype analysis to confirm aneuploid karyotype maintenance, and PCR-based episomal vector clearance verification. This feeder-free, integration-free protocol yields aneuploid iPSC lines suitable for disease modeling, drug screening, and studies of chromosome biology.

Introduction

The advent of induced pluripotent stem cell technology has transformed regenerative medicine, disease modeling, and drug discovery by enabling the generation of patient-specific pluripotent cells without the ethical concerns associated with human embryonic stem cells (hESCs). The conceptual foundation of cellular reprogramming originated from Gurdon's demonstration of somatic cell nuclear transfer¹, followed by the establishment of embryonic stem cells²^,³^,⁴ and ultimately the development of induced pluripotency⁵through....