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

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 cells^2,3,4 and ultimately the development of induced pluripotency⁵ through ectopic expression of OCT4, SOX2, KLF4, and c-MYC. Since then, iPSC technology has become an indispensable platform for investigating human development, genetic disorders, and precision medicine. Early reprogramming methods used retroviral or lentiviral vectors, but their integration-related risks limited clinical application. Consequently, the development of integration-free reprogramming methods became a priority for clinical translation. These include the use of Sendai virus⁶, synthetic mRNA⁷, episomal vectors based on the oriP/EBNA1 (Epstein-Barr nuclear antigen-1) mechanism⁸ and chemical reprogramming^9,10,11,12 using only small-molecule compounds.

Aneuploidy is the leading cause of miscarriage and congenital birth defects in humans. Understanding the cellular and molecular mechanisms underlying aneuploidy-related developmental abnormalities requires appropriate cellular models that faithfully recapitulate the genetic background of these conditions. Amniotic fluid cells (AFCs) represent an attractive and efficient starting material for patient-specific iPSC generation^13,14,15, particularly in the context of prenatally diagnosed genetic disorders^16,17. Residual amniotic fluid obtained during routine amniocentesis provides access to fetal-derived cells without requiring additional invasive tissue biopsy after birth. Compared with adult somatic cells, AFCs generally exhibit higher proliferative capacity, longer telomeres, lower cumulative environmental mutational burden, and greater developmental plasticity, all of which facilitate cellular reprogramming^18,19. Importantly, AFCs faithfully preserve the chromosomal constitution of the developing fetus, making them an ideal source for establishing disease-specific iPSC models of chromosomal abnormalities, including trisomy 21²⁰, trisomy 18²¹, Turner syndrome, and Klinefelter syndrome²².

This protocol provides a comprehensive guide for generating integration-free iPSCs from aneuploid amniotic fluid cells using episomal vectors. The method includes detailed procedures for cell isolation, reprogramming, clone selection, and characterization. Compared with other integration-free reprogramming methods, the episomal plasmid system provides a practical balance between genomic safety, technical simplicity, and cost-effectiveness, making it particularly suitable for routine laboratory use and large-scale biobanking applications. The protocol is applicable to both cryopreserved AFCs from established biobanks and fresh residual amniotic fluid samples obtained during routine prenatal diagnosis. Although reported episomal reprogramming efficiencies for AFCs are relatively low, and spontaneous chromosome rescue may occur during reprogramming or prolonged culture, particularly in certain aneuploidies, the workflow is readily applicable to the establishment of disease-specific iPSC biobanks, studies of chromosome biology and early human development, and downstream differentiation into lineage-specific cell types. This approach is particularly well-suited for generating integration-free, clinically relevant iPSC lines from prenatal samples for disease modeling and mechanistic studies of chromosomal abnormalities.

Protocol

All procedures involving human amniotic fluid samples were conducted in accordance with the Declaration of Helsinki and were approved by the Institutional Review Board (IRB)/Human Research Ethics Committee of Peking University Third Hospital (Protocol No. M2023406). The aneuploid samples were sourced from the Biobank of the Center for Reproductive Medicine at Peking University Third Hospital, a state-certified clinical repository specializing in human developmental and reproductive resources. These specimens consist of residual amniotic fluid originally collected during routine amniocentesis for clinical prenatal diagnosis, which would have otherwise been discarded. Written informed consent was obtained from all donors prior to collection, explicitly authorizing the storage of residual samples in the Biobank and their subsequent use for research. All samples were strictly anonymized to protect patient privacy. Samples with confirmed aneuploid karyotypes identified through clinical G-banding analysis, including Trisomy 21 (Down syndrome, N = 2), Trisomy 18 (Edwards syndrome, N = 1), Monosomy X (Turner syndrome, N = 1), and Klinefelter syndrome (N = 2), were selected for the induction of iPSCs. Detailed information on the aneuploid cell lines used in reprogramming is summarized in Table 1.

1. Thawing and primary culture of biobanked amniotic fluid cells

NOTE: This section details the standardized procedure for retrieving and recovering aneuploid AFCs from the Biobank. The cultured cells are expanded to obtain sufficient populations for subsequent episomal reprogramming.

1. Standardized thawing procedure
   1. Media equilibrating: Warm complete AmnioType-1 medium to 37 °C before use.
   2. Sample retrieval: Retrieve a cryovial containing approximately 300,000 AFCs cryopreserved in cell freezing medium from the liquid nitrogen storage. Immediately transfer the vial to the laboratory on dry ice to prevent premature thawing.
   3. Rapid thawing: Submerge the lower half of the cryovial in a 37 °C water bath and agitate gently. Stop the thawing process when only a small, visible ice crystal remains.
      ​NOTE: Avoid complete thawing. This prevents the cells from being exposed to the toxic effects of warm DMSO.
   4. Dilution: In a laminar flow hood, transfer the cell suspension dropwise into a 15 mL conical tube containing 4 mL of pre-warmed AmnioType-1 medium.
   5. Centrifugation: Centrifuge the suspension at 300 × g for 5 min at room temperature.
   6. Seeding: Aspirate the supernatant and gently resuspend the cell pellet in 2 mL of fresh AmnioType-1 medium. Transfer the cells to the 6-well plate or T25 flask pre-coated with 0.1% Gelatin at a density of 30,000–50,000 cells/cm².
   7. Initial incubation: Place the cells in a humidified incubator at 37 °C and 5% CO₂. Examine the flasks under an inverted phase-contrast microscope on Day 3 post-seeding to confirm initial cell attachment.
2. Expansion and media transition of primary AFCs
   1. Fibroblast culture (FC) medium preparation: To prepare 100 mL of homemade fibroblast culture medium, combine 82 mL of high-glucose DMEM with 15 mL of Fetal Bovine Serum (FBS). Add 1 mL of 100× GlutaMAX, 1 mL of 100× Non-essential Amino Acids (NEAA), and 1 mL of Penicillin-Streptomycin (P/S). Filter the medium through a 0.22 µm vacuum filtration system in a sterile environment.
   2. During expansion, gradually change the media of AFC every 3 days from specialized clinical media (AmnioType-1) to a standardized fibroblast culture system (FC medium, homemade) to ensure metabolic stability before electroporation.
      1. Perform a gradual media transition:
         1. Day 3 (75% AmnioType-1: 25% FC): Aspirate the existing AmnioType-1 medium. Add a mixed medium consisting of 75% AmnioType-1 and 25% FC medium.
         2. Day 6 (50% AmnioType-1: 50% FC): Aspirate and replace with a 1:1 ratio of AmnioType-1 and FC medium.
         3. Day 9 (25% AmnioType-1: 75% FC): Aspirate and replace with a mixture of 25% AmnioType-1 and 75% FC medium.
         4. Day 12 (100% FC): Aspirate and switch to 100% FC medium.
            NOTE: This gradual adaptation reduces cellular shock and prevents the detachment of sensitive aneuploid clones.
   3. Observe colony morphology daily. Amniotic fluid contains a heterogeneous population of cells. Under phase-contrast microscopy, identify the two primary types: F-type (fibroblast-like, spindle-shaped) and E-type (epithelial-like, rounded). The fibroblast-like mesenchymal subpopulation is optimal for reprogramming.
      ​NOTE: Monitor the cells closely for signs of senescence.
3. Passaging of primary AFCs
   1. Once the cells reach 80%–90% confluence (typically Day 10–14), aspirate the medium from the flask and wash once gently with 2 mL of Dulbecco's phosphate-buffered saline (DPBS) without Ca²⁺/Mg²⁺.
   2. Add 1 mL of 0.25% Trypsin-EDTA solution for a single well of a 6-well plate to cover the cell monolayer.
   3. Incubate at 37 °C for 4–5 min until cells detach completely.
   4. Neutralize the dissociation reagent by adding 3 mL of FC medium. Transfer the suspension to a 15 mL conical tube.
   5. Centrifuge at 300 × g for 5 min at room temperature.
   6. Aspirate the supernatant and resuspend the cell pellet in 1 mL fresh FC medium. Count viable cells using a hemocytometer or automated cell counter with trypan blue exclusion.
   7. Seed cells into a new T25 flask at a density of 30,000–50,000 cells/cm² in complete medium (usually at a 1:2 or 1:3 ratio).
   8. Expand cells to passage 3–5 before using them for reprogramming. Do not use cells beyond passage 6.
      NOTE: Cell quality at the time of electroporation is the single most important determinant of reprogramming success. Use only healthy, log-phase growing F-type AFC with no signs of senescence or abnormal morphology.

2. Electroporation of episomal plasmids into AFCs using a cell electroporation system

1. Coat culture vessels with basement membrane matrix
   1. Thaw the basement membrane matrix (Matrigel) overnight at 4 °C on ice.
   2. Chill sterile DPBS or DMEM/F-12 on ice. Pre-cool pipette tips and tubes on ice for 30 min.
   3. Dilute the basement membrane matrix 1:200 in ice-cold DMEM/F-12 using cold tips and tubes (e.g., 30 µL matrix + 6 mL DMEM/F-12 for a 6-well plate).
   4. Mix thoroughly but gently by pipetting up and down 5–6 times. Avoid introducing air bubbles.
   5. Add the diluted coating solution to culture vessels at 1 mL per well for a 6-well plate.
   6. Incubate the coated vessels at 37 °C for at least 1 h, or at room temperature for 2 h.
   7. Aspirate the coating solution immediately before plating cells. Add 2 mL Antibiotic-free FC medium plus 10 μM Y-27632 (ROCK inhibitor) and return the plate to a 37 °C incubator. Do not allow the coated surface to dry.
      ​NOTE: Coated plates can be stored at 4 °C sealed with parafilm for up to 7 days. Equilibrate to room temperature before use.
2. Thaw and prepare episomal reprogramming plasmids
   1. Plasmid selection: Use the following episomal plasmids for reprogramming: pCXLE-hOCT3/4-shp53, pCXLE-hSK, pCXLE-hUL, and pCXLE-EBNA1.
      NOTE: (Optional) For optimization of electroporation conditions, pCE-GFP may be electroporated in parallel under identical settings. GFP expression during the first 48–72 h provides a convenient indicator of transfection efficiency, while the gradual loss of GFP signal during subsequent passaging can be used to monitor transient plasmid dilution.
   2. Quality control (QC): Verify the DNA concentration and purity using a spectrophotometer. Ensure the A260/280 ratio is between 1.8 and 1.9. Ensure that the concentration of each plasmid is greater than 1 µg/µL and sterile.
      ​NOTE: It is critical to ensure that all plasmids are prepared using endotoxin-free methods. Residual endotoxins can lead to high cytotoxicity and significantly reduced reprogramming efficiency. Store the plasmids in small aliquots at -20 °C to avoid repeated freeze-thaw cycles.
   3. For a 100 µL reaction, prepare 5 µg of the plasmid mixture (equal weight ratio of pCXLE-hOCT3/4-shp53, pCXLE-hSK, pCXLE-hUL, and pCXLE-EBNA1).
3. Set up the electroporation parameters.
   1. Initialize the electroporation device. Set the pulse parameters to the optimized configuration for primary adherent cells: Pulse voltage: 1,200 V; Pulse width: 20 ms; Number of pulses: 2.
   2. Fill the pipette station with 2mL Buffer E, ensuring the electrode is fully submerged.
      ​NOTE: If initial experiments yield cell viability below 50%, consider testing alternative parameter sets: 950 V / 20 ms / 2 pulse (gentler). These alternatives sacrifice some efficiency for improved survival.
4. Prepare AFCs for electroporation
   1. On Day 0, verify that cells display a healthy, spindle-shaped morphology under the microscope with no signs of overgrowth, senescence, or contamination. Ensure the cells have reached 90% confluence and are in the active log-phase of growth.
   2. Just before the electroporation procedure, aspirate the Matrigel coating and replace it with 2 mL of the pre-warmed antibiotic-free FC medium plus 10 μM Y-27632 (ROCK inhibitor). Pre-warm this antibiotic-free FC medium to 37 °C in an incubator for at least 20 min before harvesting the cells for the electroporation system.
      NOTE: Do NOT add Penicillin-Streptomycin (P/S) or any other antibiotics to this specific batch of medium. Post-electroporation, the cell membrane remains transiently permeable and vulnerable. The presence of antibiotics can lead to increased apoptosis.
   3. Aspirate the medium of AFCs for electroporation and wash the monolayer once with DPBS without Ca²⁺/Mg²⁺.
   4. Add 2 mL (for T25 flask) of TrypLE Select Enzyme and incubate at 37 °C for 4–5 min.
   5. Neutralize with 6 mL of FC complete medium and transfer to a 15 mL conical tube. Count the cells using an automated cell counter.
   6. Centrifuge at 300 × g for 5 min at room temperature.
   7. Completely remove the supernatant using a 200 μL pipette tip, taking care not to disturb the small cell pellet.
   8. Resuspend the cell pellet in Buffer R at a final density of 1.0 × 10⁷ to 1.4 × 10⁷ cells/mL.
      ​NOTE: Aspirate the supernatant as completely as possible to ensure the cell pellet is dry. Residual culture medium can dilute the resuspension buffer, which may reduce electroporation efficiency. Use Buffer R (for adherent cells) for AFCs. Verify buffer selection against the manufacturer's recommendations.
5. Perform electroporation.
   1. Aliquot 110 μL of the cell suspension (containing 1.0–1.4 × 10⁶ cells) into a sterile 1.5 mL microcentrifuge tube.
      NOTE: Add an extra 10 μL of Buffer R to the final volume to ensure there is sufficient cell suspension, which helps in avoiding the introduction of air bubbles during aspiration.
   2. Add 5 μg plasmid mix to the 110 μL cell aliquot. Mix by gentle pipetting 3 times.
   3. Attach a sterile 100 μL electroporation tip to the pipette. Ensure the tip clicks securely.
   4. Load 100 μL of the cell/DNA suspension, ensuring no visible air bubbles are present.
      NOTE: Air bubbles cause arcing during the electric pulse, which kills cells and damages the tip. If bubbles are visible, expel the mixture back into the tube and repeat aspiration slowly.
   5. Insert the loaded pipette vertically into the Buffer tube in the station until it clicks into place. Verify that all parameters are correct on the touchscreen, then press the Start button to deliver the pulse sequence.
   6. Wait for the screen to display "Complete" (typically within 1–2 s). Immediately remove the pipette from the station and dispense the electroporated cell suspension directly into 2–3 wells of a coated 6-well plate containing pre-warmed antibiotic-free FC medium + 10 μM Y-27632.
   7. Discard the used tip safely. Repeat steps for additional wells or replicates as needed. Place the 6-well plate in a 37 °C, 5% CO₂ humidified incubator overnight.

3. Post-electroporation reprogramming culture

NOTE: This section describes the sequential media changes and achieving full reprogramming of AFCs into iPSCs. The timeline spans approximately 25 days from electroporation to colony picking.

1. Approximately 16–18 h after electroporation, carefully aspirate the medium without dislodging any surviving adherent cells. Gently add 2 mL of pre-warmed FC medium (containing P/S) to each well to allow the cells to stabilize for the first 24 h. Return the plate to the incubator.
2. From Days 2 to 6, replace half of the medium every other day: carefully aspirate 1 mL of spent medium and add 1 mL of fresh TeSR-E8 (E8) medium.
   NOTE: Partial medium change preserves secreted autocrine factors that support early reprogramming events. Aspirate slowly from the edge of the well to minimize disturbance of loosely attached transitioning cells.
3. On Days 7, perform a full medium replacement with 2 mL of E8 every other day.
4. Observe morphological changes under the microscope daily. Expect partially reprogrammed cobblestone-like clusters to appear around Day 10–14.
   NOTE: (Optional) Supplement cultures with 25–100 μM sodium butyrate between Days 9–15 if colony formation is poor²³.
5. Replace the E8 medium daily from Day 15 onward.
6. Starting around Day 18, inspect the wells daily for the emergence of true iPSC colonies. A complete timeline of the reprogramming workflow from Day 0 electroporation to colony picking is provided in Table 2.

4. Mechanical picking of iPSC colonies and expansion of established iPSC lines

NOTE: Distinguish true iPSC colonies from reprogrammed or non-reprogrammed cells based on the morphological criteria below. Identify "ideal" colonies based on the following morphological hallmarks: (1) Border definition: Look for a sharp, smooth, and circular edge that clearly demarcates the colony from the surrounding amniotic fluid cells; (2) Cellular density: Ensure cells within the colony are small, tightly packed, and possess a high nucleocytoplasmic ratio with prominent nucleoli; (3) Refractivity: High-quality colonies appear bright and highly refractive under phase-contrast; (4) Flatness: The colony should grow as a flat, two-dimensional sheet rather than a three-dimensional "clump". Partially reprogrammed colonies often appear earlier than true iPSC colonies and tend to be larger, thicker, and more three-dimensional. They will spontaneously differentiate upon picking and passaging. Do not pick colonies that show "vacuoles," ragged edges, or signs of central differentiation.

1. Mechanically pick and transfer iPSC colonies
   1. Prepare a Matrigel coated 24-well plate with 0.5 mL per well of E8 medium supplemented with 10 μM Y-27632. Keep at 37 °C until use.
   2. Place the culture plate under a stereomicroscope inside a Class II biosafety cabinet. Sterilize the stereomicroscope objective and stage with 70% ethanol before bringing the plate near the field.
   3. Identify colonies that display correct morphology (flat, compact, clear-edged).
   4. Using a sterile 10 μL pipette tip held at a shallow angle (approximately 30° from horizontal), gently score around the perimeter of a selected colony to define its border. Cut the colony into around 3 fragments by making parallel incisions across its diameter. Use the same pipette tip to gently scrape and lift the colony fragments from the substrate, and transfer the fragments into one well of the prepared 24-well plate.
   5. Repeat steps for each colony, placing fragments into separate wells.
   6. Place the 24-well plate in the incubator. Leave undisturbed for 24 h to allow fragment attachment.
   7. Beginning on Day 2 post-picking, replace the medium daily with E8 medium without Y-27632.
   8. When picked colonies expand to 70–80% confluence (typically 3–6 days), passage using Accutase.
2. Passaging and expansion of established iPSC Lines
   1. Aspirate the medium from the iPSC-containing well. Add Accutase to cover the monolayer. Incubate at room temperature for 2–6 min. Observe the colony edges under the microscope; cells should begin to pull apart, and gaps appear between cells.
   2. Aspirate the Accutase solution completely. Add 1 mL of E8 medium + 10 μM Y-27632. Gently pipette the medium across the colony surface 5–8 times using a P1000 pipette to dissociate the colonies into small clumps of 2–8 cells.
      NOTE: Avoid over-dissociating iPSCs, which dramatically reduces survival. The goal is uniform small clumps, not a single-cell suspension.
   3. Transfer the clumps with 1 mL of E8 medium + 10 μM Y-27632 evenly into a freshly matrigel coated 12-well plate. Place the plate in the incubator.
   4. Change medium daily with E8 medium (without Y-27632 after Day 1).
   5. Continue passaging every 3–4 days at a split ratio of 1:2 to 1:5, depending on confluence.
   6. Culture iPSC lines for a minimum of 10 passages before downstream applications to ensure stable episomal clearance.

5. Characterization of aneuploid iPSC lines

NOTE: Perform the following validation assays on established iPSC lines (passage 10 or later) to confirm pluripotency, karyotype integrity, and episomal vector clearance.

1. Confirm pluripotency marker expression.
   1. Validate the expression of core pluripotency markers using immunofluorescence staining, flow cytometry, or both. Expected marker profiles are summarized in Table 3.
      NOTE: Although immunofluorescence staining provides initial confirmation of pluripotency, RT-qPCR and RNA-seq are recommended for broader evaluation of pluripotency-associated gene expression, including LIN28A, DPPA4, and KLF4.
2. Karyotype analysis by G-banding
   ​CRITICAL: Reprogramming can induce trisomy rescue in a subset of clones. Screen multiple clones (5–10 per parental line) to identify both aneuploid-maintained and euploid-rescued derivatives.
   1. Harvest iPSCs at passage 10–15 at 70%–80% confluence.
   2. Add 0.1 μg/mL colcemid to the culture medium. Incubate at 37 °C for 2–4 h to arrest cells in metaphase.
      NOTE: Colcemid is a mitotic poison. Handle with gloves in a biosafety cabinet. Dispose of colcemid-containing waste as hazardous material.
   3. Dissociate cells to a single-cell suspension using Accutase or equivalent. Centrifuge at 300 × g for 5 min.
   4. Resuspend the pellet in 5 mL of pre-warmed hypotonic solution (0.075 M potassium chloride). Incubate at 37 °C for 15–20 min.
   5. Add 1 mL of freshly prepared fixative (methanol: acetic acid, 3: 1 volume ratio) to the tube while gently mixing. Centrifuge at 300 × g for 10 min.
   6. Aspirate the supernatant. Add 5 mL of fresh fixative. Mix gently and incubate for 10 min. Centrifuge again.
   7. Repeat the fixation step (Step 6) once more for a total of 3 fixations.
   8. After the final centrifugation, resuspend the pellet in 0.5–1 mL of fresh fixative to achieve an appropriate cell density.
   9. Drop 2–3 drops of the cell suspension onto clean, wet glass slides from a height of approximately 30 cm. Air-dry.
   10. Bake slides at 80 °C for 2 h or 65 °C overnight to age chromosomes.
   11. Perform G-banding: treat slides with 0.025% trypsin solution for 30–60 s, rinse, and stain with 5% Giemsa solution for 1–5 min.
   12. Analyze at least 20 well-spread metaphases per clone under a bright-field oil-immersion microscope (100× objective).
   13. Document the karyotype using ISCN nomenclature (e.g., 47,XX,+21 for female trisomy 21; 45,X for Turner syndrome).
       ​NOTE: G-banded karyotyping detects major chromosomal abnormalities but has limited resolution. Complementary assays, such as Copy Number Variation (CNV) analysis, Whole Exome Sequencing (WES), or Whole Genome Sequencing (WGS), are recommended to identify submicroscopic genetic changes and confirm iPSC genomic integrity.
3. Verify episomal vector clearance by PCR.
   ​NOTE: This step confirms that all episomal plasmids have been lost from the iPSC line by PCR amplification of vector-specific sequences.
   1. Extract genomic DNA from iPSCs (passage ≥ 10) and from untransfected control AFCs using a standard column-based extraction kit.
   2. Set up a 25 μL PCR reaction. Perform PCR using Orip/GAPDH primer pairs under the cycling conditions (94 °C for 2 min; followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; final extension at 72 °C for 5 min). Include positive control (plasmid template) and negative control (genomic DNA from non-transfected cells) in every PCR run.
   3. Resolve PCR products on a 2% agarose gel with an appropriate DNA ladder.
   4. Interpret results: iPSC lines with cleared episomes show no amplification bands at 544 bp. The positive control must show bands of expected size. The negative control must show no bands.
4. Mycoplasma detection by PCR
   1. Collect 0.5–1 mL of culture supernatant after at least 24 h of incubation from iPSC cultures before medium replacement and centrifuge at 12,000 × g for 5 min to remove cellular debris.
   2. Use the clarified supernatant as the template for PCR with the PCR Mycoplasma Detection Kit, following the manufacturer's instructions.
   3. Separate PCR products by 2% agarose gel electrophoresis. Proceed with downstream experiments only if the iPSC cultures are confirmed to be mycoplasma-free.
      ​NOTE: If parental AFCs are not obtained from a certified biobank with documented sterility and mycoplasma testing, routine mycoplasma screening before reprogramming is strongly recommended as an initial quality-control step.
5. Trilineage differentiation potential assay (Optional)
   1. Confirm in vitro differentiation capacity using either directed trilineage differentiation or embryoid body (EB) formation, followed by germ-layer-specific marker analysis. Alternatively, perform in vivo teratoma formation in immunodeficient mice for gold-standard pluripotency validation.
      NOTE: For detailed teratoma assay or directed differentiation protocols, refer to published standard operating procedures²⁴. These assays are recommended but may be omitted if comprehensive in vitro marker analysis and functional differentiation data are provided.

6. Single-cell clonal derivation and verification

NOTE: To ensure that each iPSC line originates from a single cell, a two-stage clonal verification process is recommended. After mechanical picking and expansion, a secondary single-cell clone seeding after the karyotype confirmation is recommended. Verification that each iPSC line originates from a single cell is a critical requirement for iPSC generation. It ensures the removal of parental somatic mosaicism and confirms that the diagnostic aneuploid signature is uniform across the entire population.

1. Single-cell seeding
   1. Dissociate and resuspend karyotype-confirmed iPSC colonies into a single-cell suspension using Accutase for 2–6 min at 37 °C.
   2. Use E8 medium supplemented with 10 µM Y-27632 to maximize the survival of isolated single cells. Perform single-cell deposition using one of the following methods:
      1. Limiting dilution: Dilute the suspension to a concentration of 0.5–1 cell per 100 µL. Seed 100 µL into each well of a Matrigel-coated 96-well plate.
      2. Fluorescence-Activated Cell Sorting (FACS): Sort single viable cells directly into the Matrigel-coated 96-well plate wells.
2. Monoclonality verification and expansion
   1. Initial inspection: At 24–48 h post-seeding, use a high-resolution phase-contrast microscope to inspect every well. Mark only those wells that contain a single, clearly defined attachment event. Exclude wells with multiple clusters or no cells.
   2. Colony growth: Maintain the identified single cells in E8 medium without ROCK inhibitor after the first 24 h.
   3. Sequential expansion: Once the single-cell-derived colony reaches 70% confluence, sequentially expand the cells from the 96-well plate to 24-well, 12-well, and finally 6-well formats.
      NOTE: For cell therapy research, photographic evidence of the single-cell stage (Day 0 or Day 1) is often required as part of the Certificate of Analysis to prove the lineage-pure status of the iPSC lines.
      Potential causes of unsuccessful reprogramming, suboptimal colony formation, and challenges during iPSC clone establishment are summarized together with practical solutions in Table 4.

Results

Morphological progression during reprogramming

Primary amniotic fluid cells displayed heterogeneous morphologies, including E-type and F-type cells (Figure 1A). F-type cells should be expanded and used for reprogramming. Following electroporation of AFCs with Epi5 episomal plasmids, a characteristic sequence of morphological changes is observed. On Day 0, AFCs display typical spindle-shaped, fibroblast-like morphology. By Day 3–5, the first morphological evidence ...

Discussion

This protocol describes a non-integrating, feeder-free method for generating aneuploid human iPSCs from amniotic fluid cells using episomal plasmids delivered via electroporation. Several critical steps determine the success of reprogramming. First, the quality of the starting AFC population is paramount. In clinical cytogenetic laboratories, AFCs are typically expanded in media aiming to accelerate cell proliferation for prenatal karyotype analysis rather than long-term culturing. The impact on subsequent ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Thermo Fisher (Gibco)	25200056	Cell dissociation for AFC passaging
100bp Plus II DNA Ladder	TransGen Biotech	BM321-01	DNA Ladder
-80 C Freezer	Thermo Fisher	FDE60086fv	Long-term plasmid DNA and sample storage
Accutase Cell Detachment Solution	StemCell Technologies	07920	Gentle iPSC dissociation into small clumps
Acetic acid (glacial)	Sigma-Aldrich	338826	Fixative component for karyotyping (MeOH:AcOH 3:1)
Agarose	Sigma-Aldrich	A9539	Gel electrophoresis for PCR products (2%)
AmnioType-1 Medium	VivaCell BIOSCIENCES	C3620-0100	Primary AFC culture medium
Automated Cell Counter / Hemocytometer	Countstar	TC20	Viable cell counting with trypan blue exclusion
Bright-Field Microscope (100x oil)	Zeiss	Axiovert5	Metaphase spread analysis for karyotyping
Centrifuge (swing-bucket rotor)	Eppendorf	5810R	Cell pelleting at 300 x g
Class II Biosafety Cabinet	ESCO	AC2-4S8-CN	Sterile workspace for all cell culture operations
CO2 Incubator (humidified)	There scientific	3131	Maintain 37 C, 5% CO2 environment
Colcemid (10 ug/mL)	Capricorn scientific	COL-H	Metaphase arrest for karyotyping (0.1 ug/mL, 2-4 h)
CryoStor CS10 Cell Freezing Medium	STEMCELL Technologies	07959	Cell Freezing Medium
DAPI	Merck Millipor	10236276001	Nuclear stainning
DMEM (high glucose)	Thermo Fisher (Gibco)	11965092	Base for fibroblast culture (FC) medium
DMEM/F-12	Thermo Fisher (Gibco)	11330032	Matrigel dilution buffer (1:200)
DPBS without Ca2+/Mg2+	Thermo Fisher (Gibco)	14190250	Washing buffer
Episomal plasmid (pCE-GFP)	Addgene	#41858	Non-integrating reprogramming vector (GFP control)
Episomal plasmid (pCXLE-hOCT3/4-shp53-F)	Addgene	#27077	Non-integrating reprogramming vector (OCT4 + shp53)
Episomal plasmid (pCXLE-hSK)	Addgene	#27078	Non-integrating reprogramming vector (SOX2 + KLF4)
Episomal plasmid (pCXLE-hUL)	Addgene	#27080	Non-integrating reprogramming vector (L-MYC + LIN28)
Fetal Bovine Serum	HyClone	SH30071.03	Serum supplement for AFC expansion (FC medium, 15%)
Fluorescence Microscope	Leica	Sp8	TRA-1-60/81 live-staining and IF visualization
Gel Electrophoresis System	Bio-Rad	164-5056	Agarose gel analysis of PCR products
Gelatin (0.1%)	Sigma-Aldrich	G1393	Culture vessel coating for AFC attachment
Genomic DNA Extraction Kit	TIANGEN	4992199	PCR-quality DNA isolation from iPSCs
Giemsa Stain Solution	Sigma-Aldrich	GS500	Chromosome G-banding stain (5%)
GlutaMAX (100x)	Thermo Fisher (Gibco)	35050061	Supplement for FC medium
Inverted Phase-Contrast Microscope	Zeiss	Axiovert5	Daily monitoring of culture and colony morphology
Liquid Nitrogen Storage Tank	Thermo Scientific	CY50985	Cryopreservation of cells
Matrigel Basement Membrane Matrix	Corning	354230	Culture vessel coating for iPSC attachment
Methanol (anhydrous)	Sigma-Aldrich	34860	Fixative component for karyotyping (MeOH:AcOH 3:1)
NANOG Antibody (Rabbit)	Abcam	ab109250	Immunofluorescence; nuclear pluripotency marker
Neon NxT Electroporation Kit (100 uL)	Thermo Fisher (Invitrogen)	N10025	Electroporation; contains Buffers R, T, E and 100 uL tips
Neon NxT Electroporation System	Thermo Fisher (Invitrogen)	NEON18S	Delivery of episomal plasmids via electroporation
Non-Essential Amino Acids (NEAA, 100x)	Thermo Fisher (Gibco)	11140050	Supplement for FC medium
OCT4 Antibody (Rabbit)	Abclonal	A7920	Immunofluorescence; nuclear pluripotency marker
PCR Master Mix (high fidelity)	TIANGEN	4992920	Episomal clearance verification by PCR
pCXWB-EBNA1	Addgene	#37624	Non-replicating episomal expression of EBNA1
PE anti-human TRA-1-81 Antibody	BioLegend	330708	Pluripotency staining
Penicillin-Streptomycin (100x)	Thermo Fisher (Gibco)	15140122	Antibiotic supplement (NOT used post-electroporation)
Phalloidin-iFluor 647 Reagent	Abcam	ab176759	F-actin (actin filaments) labeling
Potassium chloride (KCl)	Sigma-Aldrich	P5405	Hypotonic solution for karyotyping (0.075 M)
Real-time PCR System	Thermo Fisher Scientific	QuantStudio3	qPCR for marker gene verification
Sodium Butyrate (NaB)	Sigma-Aldrich	B5887	HDAC inhibitor; 25-100 uM for enhancing reprogramming
SOX2 Antibody (Mouse)	R&D Systems	MAB2018	Immunofluorescence; nuclear pluripotency marker
SSEA-4 Antibody	BioLegend	330408	Immunofluorescence; cell surface pluripotency marker
Stereomicroscope	Nikon	SMZ745	Mechanical colony picking under magnification
TeSR-E8	STEMCELL Technologies	05990	Feeder-free iPSC maintenance medium
TRA-1-60 Antibody	Merck Millipor	MAB4360	Pluripotency staining
Trans DNA Marker II	TransGen Biotech	BM411-01	DNA Ladder
TransDetect PCR Mycoplasma Detection Kit	TransGen Biotech	FM311-01	Mycoplasma test
TrypLE Select Enzyme (1x)	Thermo Fisher (Gibco)	12563011	Gentle cell dissociation for adherent AFCs
Trypsin (0.025%)	Sigma-Aldrich	T4799	G-banding pretreatment of slides (30-60 s)
VeritiPro 96-well Thermal Cycler	Thermo Fisher Scientific	A48141	PCR for episomal clearance verification
Water Bath (37 °C)	Shanghai Zhixin Experimental Instrument Technology Co., Ltd.	ZX-S22	Thawing media and reagents
Y-27632 Dihydrochloride (ROCK inhibitor)	Tocris	1254	Enhances iPSC survival post-dissociation (10 uM)