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 of reprogramming appears: small clusters of epithelial-like cells emerge within the mesenchymal cell population, marking the onset of MET. These transitional cells are smaller and more compact than the surrounding mesenchymal cells. Between Day 7–10, the cell clusters exhibit increased compaction, reflecting a transition to a characteristic epithelial morphology. Fully reprogrammed iPSC colonies begin to emerge between Day 14–16. By Day 21–25, mature iPSC colonies are ready for picking (Figure 1B). The pCE-GFP plasmid was electroporated in parallel with reprogramming plasmids to assess DNA delivery efficiency (Figure 1C). GFP expression can be used as a visual indicator of successful electroporation and plasmid persistence during early culture.

Distinguishing fully reprogrammed iPSC colonies from partial reprogramming

A critical skill in this protocol is the ability to distinguish fully reprogrammed iPSC colonies from partially reprogrammed colonies, as picking the latter results in the failed establishment of stable iPSC lines. Table 5 summarizes the key morphological and molecular differences between these two colony types. 

Figure 2 illustrates a side-by-side comparison. Partially reprogrammed cells often form loose aggregates that lack clear boundaries. These clusters may show some degree of compaction but typically retain mesenchymal features or exhibit irregular, "fuzzy" margins, indicating incomplete epigenetic resetting (Figure 2, left). During primary reprogramming, colonies with typical iPSC-like morphology may contain areas of spontaneous differentiation (Figure 2, middle). Morphologically defined undifferentiated regions can be manually selected and picked for further expansion. Successfully established iPSC lines should exhibit classic compact pluripotent morphology, characterized by a high nucleus-to-cytoplasm ratio, prominent nucleoli, and extremely tight cell-cell junctions (Figure 2, right).

Typically, dozens to hundreds of colonies were observed after reprogramming by electroporation with 1–2 × 10⁶ AFCs. From each AFC line, 24 single colonies were manually picked, resulting in 1-9 expandable iPSC clones (Table 1).

Pluripotency marker validation

Established iPSC lines should express the full complement of pluripotency markers. Figure 3 shows representative immunofluorescence and FACS results for an aneuploid iPSC line with Klinefelter syndrome (47,XXY) at passage 15. Nuclear markers OCT4, NANOG, and SOX2 show uniform expression across the colony, while cell surface marker TRA-1-60 displays characteristic ring-like membrane staining (Figure 3A). FACS analysis further quantified high expression levels of SSEA-4 and TRA-1-81 (Figure 3B). These expression patterns are consistent with established criteria for bona fide pluripotent stem cells, confirming that aneuploidy does not impair the acquisition of pluripotency at the marker level.

Karyotype confirmation of aneuploid iPSCs

G-banding karyotype analysis is essential for every iPSC clone derived from aneuploid amniotic fluid cells. Figure 4 shows representative karyotype results.

A positive karyotype result is the identification of iPSC clones with the expected aneuploid karyotype confirmed in at least 20 metaphase spreads. A negative result is the finding of only euploid clones (46,XX or 46,XY) from an aneuploid source, which, while confirming protocol success in generating iPSCs, indicates complete trisomy rescue and necessitates repeating the single-cell clonal derivation or reprogramming to obtain aneuploid-maintained lines.

Episomal vector clearance verification

Episomal vectors are progressively diluted during passaging and become undetectable after prolonged culture in most established iPSC lines^8,25,26,27. Exogenous vector clearance is a hallmark of the non-integrating reprogramming approach. A positive result (episomal clearance confirmed) is the absence of oriP (544 bp) amplification bands at passage 10 or later. A negative result (incomplete clearance) shows persistent bands at passage 10, which typically resolves by passage 15–20 with continued passaging. GAPDH amplification was used as an internal control to confirm genomic DNA integrity and successful PCR amplification (Figure 5A). Rare cases of persistent episomal vectors beyond passage 20 may indicate genomic integration events, which indicate the clone should be discarded. To ensure the biological safety and experimental integrity of the generated aneuploid iPSC lines, a PCR-based screening for mycoplasma contamination is strongly recommended. Cell culture supernatants from all maintained monoclonal lines must be collected and analyzed via PCR using mycoplasma-specific primers. Any monoclonal strains testing positive (e.g., Clone #7 and #9) must be discarded immediately (Figure 5B).

In vitro trilineage differentiation potential

Confirmation of trilineage differentiation potential is a required criterion for validating iPSC lines. Figure 6 shows representative results from in vitro embryoid body differentiation of a trisomy 18 iPSC line. Expression of PAX6 (ectoderm), T-Brachyury and TBX6 (mesoderm), GATA6 and SOX17 (endoderm) confirms the capacity of aneuploid iPSCs to differentiate into all three germ layers (Figure 6).

Single-cell clonal derivation and verification

To establish high-fidelity aneuploid iPSC models, a two-round single-cell derivation and verification workflow was implemented to minimize cellular heterogeneity and exclude potential chimerism. Following initial expansion, karyotype-normal clones were dissociated and re-seeded at single-cell density. Microscopic inspection at 24–48 h confirmed the presence of single-cell-derived attachment events, which were subsequently expanded to establish validated monoclonal lines (Figure 7A).

A positive result is the successful expansion of a single colony from a single cell in a 96-well plate, confirmed by daily microscopic monitoring showing only one colony per well (Figure 7B). After expansion, reconfirmation of karyotype and pluripotency markers should match the original clone. A negative result is the observation of multiple colonies per well or failure of single cells to survive and expand.

Figure 1: Morphological progression of amniotic fluid cell reprogramming using Epi5 episomal plasmids. Representative images showing the morphology of different types of primary AFC and reprogramming procedure at key time points. (A) Phase-contrast images showing the three primary morphologies of starting material: epithelial-like (E-type), fibroblast-like (F-type), and senescent AFCs. (B) Representative time-course images of the reprogramming process from Day 0 to Day 21, showing the transition from primary AFCs to the emergence of compact iPSC-like colonies. Day 0: AF-MSCs prior to electroporation, displaying typical spindle-shaped mesenchymal morphology. Day 3–5: Early MET stage; epithelial-like cells appear in clusters amid residual mesenchymal cells. Day 7–10: Primary reprogramming cells emerge. Day 14–16: Primary reprogramming colonies (MET colonies) with cobblestone-like morphology but ill-defined boundaries. Day 21–25: Fully reprogrammed iPSC colonies emerging with flat, tightly packed cells, distinct borders, a high nuclear-to-cytoplasm ratio, and prominent nucleoli. (C) Representative fluorescence images showing GFP expression after AFC electroporation with pCE-GFP. Scale bar represents 100 µm. Please click here to view a larger version of this figure.

Figure 2: Comparison of fully reprogrammed and partially reprogrammed colonies. Left: Partially reprogrammed colonies displaying loose aggregates and irregular borders. Middle: Primary reprogramming colonies with typical iPSC-like regions accompanied by areas of spontaneous differentiation. Right: Expanded colonies with typical iPSC morphology. Scale bars, 100 µm. Please click here to view a larger version of this figure.

Figure 3: Immunofluorescence analysis of pluripotency markers in aneuploid iPSCs. (A) Immunofluorescence staining of XXY-iPSCs for core pluripotency markers, including OCT4, SOX2, NANOG, and TRA-1-60 (green). Cytoskeletal structure is visualized with Phalloidin (red) and nuclei with DAPI (blue). Scale bar represents 50 µm. (B) Flow cytometry analysis (FACS) showing the gating strategy and quantitative expression of SSEA-4 and TRA-1-81 in the XXY-iPSC line. Please click here to view a larger version of this figure.

Figure 4: G-banding karyotype analysis of aneuploid iPSC lines. G-banded karyotype analysis and corresponding metaphase spreads confirming the stable maintenance of chromosomal abnormalities in generated lines, including 46,XX (Normal female), 46,XY (Normal male), 47,XXY (Klinefelter syndrome), 45,X (Turner syndrome), 47,XX,+21 (Down syndrome), and 47,XX,+18 (Edwards syndrome). Please click here to view a larger version of this figure.

Figure 5: PCR-based detection of episomal vector clearance and mycoplasma test in aneuploid iPSC lines. Agarose gel electrophoresis (2%) of PCR products amplified with oriP/GAPDH or mycoplasma primer set. (A)Total vector clearance in iPSC clone at different passages. (B) PCR-based detection of mycoplasma contamination across 9 independent clones. Clone #7 (faint) and #9 (strong) show mycoplasma contamination and must be discarded. PC, positive control; NC, negative control. Please click here to view a larger version of this figure.

Figure 6: In vitro trilineage differentiation of aneuploid iPSCs. (A) Phase-contrast micrograph illustrating the morphology of embryoid bodies (EB) at Day 10 during the spontaneous differentiation process following attachment to a gelatin-coated surface. Scale bar represents 100 µm. (B) RT-qPCR analysis quantifying the pluripotency exit and subsequent lineage commitment. Results show a significant increase in the mRNA relative expression of hallmark markers for three embryonic germ layers at Day 10 compared to undifferentiated iPSCs: Endoderm (GATA6, SOX17), Mesoderm (T, TBX6), and Ectoderm (PAX6). Data are presented as mean ± SD. Please click here to view a larger version of this figure.

Figure 7: Single-cell clonal derivation workflow for aneuploid iPSC lines. (A) Schematic workflow illustrating Round 1 (initial derivation and karyotype confirmation) and Round 2 (secondary single-cell seeding via limiting dilution or FACS to ensure monoclonality) clonal verification process. Stage 1: Mechanical picking of individual iPSC colonies, expansion to 6-well plates, and karyotype verification. Stage 2: For karyotype-confirmed aneuploid clones, single-cell seeding by limiting dilution or FACS into 96-well plates to ensure clonal origin from a single cell. Only wells with single colony growth are expanded. (B) Representative image of 96-well plates during the clonal expansion phase, with successfully identified single-cell-derived colonies circled for sequential expansion. Please click here to view a larger version of this figure.

Genetic background	Karyotype	Number of AFC lines tested	Number of single colonies picked after reprogramming	Number of successfully established iPSC clones
Trisomy 21	47,XX,+21	2	48	9
Trisomy 18	47,XX,+18	1	24	8
Klinefelter syndrome	47,XXY	2	48	6
Monosomy X	45,X	1	24	1
Normal female	46,XX	1	24	2
Normal male	46,XY	2	48	6
Single colonies were manually picked based on colony morphology after reprogramming and expanded for further characterization. The number of successfully established iPSC clones indicates colonies that could be expanded and maintained after picking.

Table 1: Details of AFC lines used in somatic cell reprogramming.

Day	Medium Composition	Key Observation
0	FC medium without antibiotic + Y27632	Electroporation
1	FC medium	Surviving cells begin recovery
2 – 6	FC to E8 (half-change)	MET begins; cells become compact; smaller, rounder cells appearing in clusters
7 – 8	E8 (full change every other day)	Early colony formation
9 – 14 (optional)	E8 (+ 25 μM NaB)	Cobblestone-like clusters with indistinct borders; variable sizes
15-18	E8 (daily)	True iPSC colonies emerge (Day 18–25) Flat, tightly packed colonies with sharp borders, high nuclear-to-cytoplasmic ratio
19 – 25	E8 (daily)	Colonies exceed 400 μm in diameter; uniform flat morphology; ready for picking

Table 2: Summary of the complete reprogramming timeline from Day 0 to colony picking.

Marker	Cellular Localization	Detection Method
OCT4	Nuclear	Immunofluorescence / Flow cytometry
NANOG	Nuclear	Immunofluorescence / Flow cytometry
SOX2	Nuclear	Immunofluorescence / Flow cytometry
SSEA-4	Cell surface	Immunofluorescence / Flow cytometry
TRA-1-60	Cell surface	Flow cytometry / Live stain
TRA-1-81	Cell surface	Flow cytometry / Live stain

Table 3: Expected pluripotency marker expression profiles of established iPSC clones.

Problem	Possible Cause	Solution
Low cell viability (< 40%) after electroporation	1. Pulse parameters too harsh for this cell line	1. Test gentler parameters
	2. Cells were overconfluent (> 90%)	2. Ensure 75–90% confluence on Day 0
	3. Air bubbles caused arcing	3. Aspirate slowly; inspect tip for bubbles before pulsing
	4. Degraded or incorrect resuspension buffer	4. Use fresh Buffer R from kit
	5. Cell quality poor prior to electroporation	5. Use only P3–P5 cells with healthy morphology
No iPSC colonies observed by Day 25	1. Medium was inactive	1. Use fresh prepared E8; add NaB at a concentration of 25–100 μM from Day 9 to Day14
	2. Plasmid DNA degraded or incorrectly thawed	2. Verify plasmid integrity on agarose gel
	3. Contaminating differentiated cells overgrowing	3. Manually remove differentiated areas before they spread；passage the cells to reduce density
	4. Matrix coating failed or deteriorated	4. Prepare fresh coating; keep on ice
iPSC colonies differentiate spontaneously after picking	1. Colonies were picked too early (before Day 18)	1. Wait for colonies > 400 μm with mature flat morphology
	2. Matrix coating insufficient or degraded	2. Ensure fresh coating; do not let dry before use
	3. Passaged as single cells without ROCK inhibitor	3. Always use Y-27632 for first 24 hours after passaging
Karyotype shows loss of expected aneuploidy (trisomy rescue)	1. Spontaneous chromosome elimination intrinsic to reprogramming process	1. Screen multiple clones (≥ 5–10) per line; rescued clones serve as isogenic euploid controls
	2. Late-passage starting cells more prone to rescue	2. Use only P3–P5 cells with healthy morphology
PCR detects episomal sequences at late passage (P10+)	1. Insufficient passaging since reprogramming	1. Continue passaging to P15–P20; re-test by PCR
	2. EBNA-1 expression persistently maintaining plasmids	2. Extended culture usually resolves; monitor every 5 passages
	3. False-positive from residual plasmid contamination in prep	3. Include proper negative controls; use column-based DNA cleanup
Arcing/spark during electroporation pulse	1. Air bubbles trapped in the tip	1. Aspirate mixture slowly; tap tube to release bubbles before loading
	2. High salt concentration in DNA sample	2. Use endotoxin-free plasmid preparation
	3. Cellular debris clogging the tip orifice	3. Filter cell suspension through 40 μm cell strainer
	4. Tip damaged or reused beyond limit	4. Use each tip maximum 2 times; inspect for damage

Table 4: Troubleshooting guide for common issues during AFC reprogramming and iPSC clone establishment.

Feature	Partially Reprogrammed Colonies	Fully Reprogrammed iPSC Colonies
Time of emergence	Appears early (Day 7–10)	Variable, typically appears later (Day 14–18)
Colony border	Irregular edges	Sharp, well-defined edges
Cell morphology	Larger cells, heterogeneous	Small, uniform, high  nuclear-to-cytoplasm ratio
Cell packing	Loosely packed	Tightly packed, cobblestone
Nucleoli	Inconspicuous	Prominent nucleoli
Pluripotency markers	Negative or weak/focal	Strong, uniform
Colony stability	Degenerate or differentiate upon passage	Stable, expandable

Table 5: Distinguishing features of fully reprogrammed vs. partially reprogrammed colonies.

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 iPSC generation efficiency remains incompletely understood. Therefore, the culture medium composition should be considered during sample preparation, and low-passage, highly proliferative AFC populations should be enriched after thawing to ensure optimal cell quality before electroporation. Second, electroporation conditions should be optimized to maximize DNA delivery while preserving cell viability. In the present protocol, electroporation at 1200 V, 20 ms, and 2 pulses consistently produced satisfactory transfection efficiency and reproducible colony formation. Minor optimization may occasionally be required for individual donor samples. Excessive cell death during the first 24–48 h after electroporation is frequently associated with poor starting cell quality, incomplete removal of residual culture medium before resuspension in electroporation buffer, or prolonged handling during electroporation. Third, the timing of colony picking is critical: colonies appearing before Day 14 are typically partially reprogrammed, whereas true iPSC colonies emerge between Days 15 and 25 and display the characteristic flat, compact morphology with clearly defined edges. Spontaneous differentiation following colony emergence is commonly caused by premature colony picking, delayed medium replacement, or excessive colony density, and timely colony picking helps maintain colony quality. In addition, sodium butyrate may be included as an optional optimization between Days 9 and 15 for AFC lines exhibiting relatively poor reprogramming performance²³. Finally, single-cell cloning via limiting dilution or FACS into 96-well plates is necessary after initial karyotype verification to ensure clonal origin.

The role of p53 in reprogramming is methodology-dependent. In OSKM-based episomal reprogramming, transient p53 suppression enhances cell survival during the initial stress response and facilitates MET²⁷, whereas p53 serves as a genomic safeguard in chemical reprogramming²⁸. Given the efficiency of electroporation-based OSKM reprogramming for rare clinical samples, this approach remains valuable. To minimize potential genomic risks, established iPSC lines should undergo routine karyotyping and, when appropriate, CNV analysis and WES/WGS before downstream applications.

A particularly important limitation for aneuploid iPSC generation is the phenomenon of trisomy rescue during reprogramming. Akutsu et al.²⁹ demonstrated that the rescue frequency varies dramatically by chromosome type. This variability means that for certain aneuploidies, a large number of clones must be screened by karyotyping to identify those that have maintained the original chromosomal abnormality. Conversely, the same phenomenon can be exploited to generate isogenic euploid control lines from the same reprogramming experiment, providing a powerful internal control for studying aneuploidy-specific phenotypes.

The protocol described here provides a robust and reproducible platform for establishing patient-specific aneuploid iPSC lines from residual prenatal diagnostic samples. These cell lines can be applied to studies of chromosome biology, developmental mechanisms, disease modeling, functional genomics, genome editing, and high-throughput drug screening following directed differentiation into disease-relevant cell types. In regenerative medicine, the transgene-free iPSCs meet the safety requirements for potential future clinical translation. This protocol, by providing a standardized and reproducible workflow, facilitates broader adoption of aneuploid iPSC technology across these diverse applications.

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)