Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Naïve-like State with Improved Multilineage Differentiation Potency

All animal procedures were performed in accordance with animal care guidelines and protocols approved by the Johns Hopkins School of Medicine Institute of Animal Care and Use Committee (IACUC).

1. Preparation of Mouse Embryonic Fibroblasts (MEF) for Feeder-dependent Conventional (hESC medium/MEF) or Naïve-reverted (LIF-3i medium/MEF) hPSC Culture

1. Purchase or prepare in-house low-passage supplies of MEF feeders from CF1 or CF1 x DR4 hybrid E13.5 mouse embryos following published protocols⁴.
   1. Cryopreserve low passage (p1-p2) MEF cultures pre- (for long term storage) or post- (for up to 6 months) irradiation and store in liquid nitrogen as previously described⁴.
   2. Irradiate (5,000 rad) bulk expanded MEF under p5 using a γ- or X-ray- irradiator and prepare aliquots at 1.5 x 10⁶ cells per vial for short-term storage (less than 6 months) at -80 °C in an ultra-low temperature freezer.
   3. Plate between 1.2 x 10⁶ (freshly irradiated non-cryopreserved) and 1.5 x 10⁶ (irradiated cryopreserved) MEF per one 6-well gelatinized plate for preparing feeder cultures, as described below.
2. Prepare gelatin-coated 6-well sterile tissue culture-treated plates by adding 1.5 mL of sterile 0.1% gelatin solution to each well in a biological safety cabinet.
3. Incubate gelatin-coated plates at 37 °C for at least 1 h or overnight in a laboratory CO₂ incubator.
4. On the next day, thaw low passage (e.g., P2 to P4) DMSO-cryopreserved and irradiated (5,000 rad) MEF according to the steps indicated below.
   NOTE: Non-irradiated MEF should also be thawed, expanded, and irradiated immediately prior to usage.
5. Place a cryopreserved MEF aliquot in a 37 °C water bath. Upon thawing, sterilize the tube with ethanol and immediately dilute the DMSO cryoprotectant at least 10-fold with MEF medium (Table 1) within a biosafety cabinet (e.g. transfer 1 mL DMSO-MEF aliquot into 9 mL MEF medium in a sterile 15 mL conical).
6. Centrifuge the diluted MEF cells at 200 x g for 5 min in sterile 15 mL conical tubes.
7. In a biosafety cabinet, aspirate and discard the cell-free supernatant, and resuspend the cell pellet in 1–2 mL fresh MEF medium.
8. Gently discard the gelatin solution and add 2 mL of MEF re-suspended in the MEF medium to each well of a gelatinized 6-well plate, as indicated above. Count MEF cells and plate 1.2 x 10⁶ (freshly irradiated non-cryopreserved) or 1.5 x 10⁶ (irradiated cryopreserved) MEF per one 6-well gelatinized plate.
   NOTE: All cell culture plates that are transferred from the CO₂ incubator to the biosafety cabinet can be gently wiped with ethanol-sprayed paper but should not be directly sprayed on with 70% ethanol solution to avoid ethanol dissemination in the wells.
9. Incubate MEF plates at 37 °C overnight in a laboratory CO₂ incubator (5% CO₂, humid atmosphere) to allow attachment, prior to use.

2. Bulk Stabilization of Conventional hPSC Cultures for Subsequent Naïve Reversion with a Brief LIF-5i Adaptation Step

1. Validate all conventional hPSC lines for possessing a normal karyotype by G-banding, prior to beginning LIF-5i/LIF-3i reversion.
   NOTE: LIF-3i reversion of high-passage conventional hPSC lines (e.g., P>40–50) should be avoided, since these cultures may already harbor genomic aberrations that may negatively impact stable, efficient, and bulk LIF-3i reversion of primed, convetional hPSC lines.
2. Maintain and expand conventional hPSC cultures with validated normal karyotypes in either a MEF-based culture system (as outlined in Section 1), or a feeder-free culture system (according to the investigator's preference).
   NOTE: Both feeder-dependent hPSC/MEF cocultures (e.g., hESC medium (Table 1) supplemented with 4–10 ng/mL bFGF) or feeder-free (e.g., E8 ⁵ or mTSER ⁶ media (according to manufacturer's instructions on vitronectin-coated plates) methods are compatible with bulk naïve reversion using the LIF-5i/LIF-3i/MEF system (Figure 1). Non-enzymatic methods are preferred for the passaging of conventional hPSC prior to preparing them for reversion. LIF-5i and LIF-3i media formulations do not contain antibiotics or antifungal agents. Standard operation rules for the biosafety cabinet sterility and maintenance are expected to be rigorously observed to avoid any bacterial or fungal contamination.
3. For the MEF- based culture system, prepare MEF feeders in the gelatinized 6-well plate as described in Section 1 at least one day before passaging of LIF-5i-adapted conventional hPSC cultures.
4. After conventional hPSC cultures have reached ~50% confluency (i.e., 3–5 days after initial plating), replace the standard hESC culture medium with LIF-5i medium (2 mL per well; Table 1).
   NOTE: Perform these steps in a biosafety cabinet.
5. Culture and maintain conventional hPSC/MEF for up to 2 days in LIF-5i in the CO₂ incubator (5% CO₂, humid atmosphere). Change the LIF-5i medium daily to adapt them for their subsequent passage and stable reversion in LIF-3i.
6. Alternatively, for the feeder-free conventional cultures (e.g., in E8), culture hPSC in LIF-5i only overnight before passaging the next morning.
   NOTE: LIF-5i and LIF-3i cultures can both be maintained with either atmospheric (21% O₂) or physiologic (5% O₂) oxygen levels in the CO₂ incubator (5% CO₂, humid atmosphere).
7. Prior to passaging, place the culture plates in a biosafety cabinet, wash LIF-5i-adapted conventional hPSC once with PBS, and add 1 mL of cell dissociation reagent to each well. Incubate for 5 min at 37 °C in a CO₂ incubator, and gently triturate with a pipette into a single cell suspension back in the biosafety cabinet.
   NOTE: Non-enzymatic dissociation buffers may alternatively be used for preparing single cells for passaging.
8. Collect the cell suspension in hESC medium (at least 2-fold dilution) in a sterile 15 mL conical tubes, and gently triturate cells by pipetting to obtain a single cell suspension.
9. Centrifuge at 300 x g for 5 min, aspirate/discard the supernatant, and resuspend the cell pellet in 1–2 mL of the LIF-5i medium in the biosafety cabinet. Count the cells using a hemocytometer or an automatic cell counter.
10. Wash the pre-plated MEF plate twice with PBS (2 mL per well in 6-well plates) and distribute 1–2 x 10⁶ cells (in 2 mL LIF-5i medium) onto 1 well of the PBS-washed MEF plate.
11. Adjust and optimize initial plating densities for each individual hPSC line to be naïve-reverted, as replating efficiencies can be highly variable. Place the plate in a CO₂ incubator (5% CO₂, humid atmosphere).
    NOTE: The routine use of anti-apoptotic reagents is not recommended for most hPSC lines with this method. The LIF-5i system already significantly enhances initial bulk clonal re-plating efficiencies of conventional hPSC lines. However, a one-time use of ROCK inhibitor (5–10 μM Y-27632) may further improve the initial LIF-5i clonal re-plating efficiency of conventional hPSC cultures in the first passage for some unstable lineage-primed hPSC lines with the propensity for spontaneous differentiation.
12. If starting from feeder-free cultures (e.g., E8), directly transfer one well of the dissociated conventional hPSC adapted in LIF-5i into one irradiated MEF-plated well (i.e., 1:1 passage).
13. The next day, gently swirl the plate to lift all non-attached cells, aspirate the medium (PBS wash is optional) and replace with 2 mL of LIF-5i medium. Perform this step in a biosafety cabinet daily for 3–5 days or until cells are 60–70% confluent (Figure 1). Place the plate in a CO₂ incubator (5% CO₂, humid atmosphere).

3. Long-term Maintenance and Expansion of N-hPSC in LIF-3i Medium

1. Following initial LIF-5i adaptation, passage subsequent stable LIF-3i cultures every 3–4 days in a biosafety cabinet, or when cultures become 60–70% confluent (Figure 1).
   NOTE: LIF-3i cultures require rigorous maintenance and allowing N-hPSC cultures to reach high confluency/cell density from prolonged culture (e.g., >4 days) decreases subsequent clonal re-plating efficiency, and promotes spontaneous differentiation.
2. In a biosafety cabinet, discard the culture medium and wash each well of LIF-5i/LIF-3i cultures by gently adding 2 mL of PBS. Discard PBS and add 1 mL of cell detachment solution. Incubate for 5 min at 37 °C in a CO₂ incubator (5% CO₂, humid atmosphere).
3. Collect the cell suspension, add hESC medium (without inhibitors or growth factors (Table 1); at least 2-fold dilution) to recover all hPSC and gently triturate cells by pipetting to obtain a single cell suspension. Transfer the suspension to a sterile 15 mL conical tube.
4. Centrifuge at 300 g for 5 min and aspirate/discard the supernatant. Re-suspend the cell pellet in LIF-3i medium. Count the cells using a hemocytometer or an automatic cell counter.
5. Plate ~2 x 10⁵ cells per well onto the irradiated MEF in the gelatinized 6-well plate for routine passaging of LIF-3i cultures. For the initial LIF-5i-adapted cultures, plate an initially higher density (4 x 10⁵ cells/well) prior to the first passage into LIF-3i/MEF.
6. Re-plate and distribute LIF-5i-adapted hPSC onto fresh PBS-washed irradiated MEF feeder plates (prepared the previous day as above) in the LIF-3i medium. Replace the LIF-3i medium daily.
7. Passage N-hPSC for least 4–7 continuous bulk passages in the LIF-3i medium prior to use of N-hPSC in functional studies or cryopreservation. Record the number of passages of N-hPSC in either conventional or LIF-3i media.
   NOTE: LIF-3i reversion of high-passage (e.g. >p40) lineage-primed, conventional hPSC lines is not recommended. An effort should be made to revert conventional hPSC lines at the lowest possible passage that they are available. Additionally, the use of LIF-3i-reverted hPSC that have undergone greater than 10 LIF-3i passages is not recommended for functional studies, since such N-hPSC cultures may harbor karyotypically-abnormal clones due to prolonged clonal cell culture selection. Fresh LIF-5i/LIF-3i reversions of low-passage conventional hPSC lines should be conducted for functional studies, if stocks of N-hPSC with <10 passages in LIF-3i are not available.

4. Cryopreservation and Thawing of LIF-3i-reverted N-hPSC

1. Expand reverted N-hPSC for at least 5-7 passages in LIF-3i, as indicated above, prior to use in functional studies or long-term cryopreservation. Record the number of passages in conventional conditions and in LIF-3i conditions on each cryopreserved vial.
   NOTE: Excess LIF-3i-reverted N-hPSC not used in functional assays can be cryopreserved at each passage, but freezing of lower post-reversion passages (e.g., <p3) may result in poor, or highly variable post-thaw recovery efficiencies.
2. In a biosafety cabinet, aspirate the culture medium, wash cells in PBS (2 mL per well), aspirate PBS and dissociate hPSC colonies into single cells using cell detachment solution (1 mL per well). Place the plate for 5 min at 37 °C in a CO₂ incubator (5% CO₂, humid atmosphere). Dilute the cell detachment solution with the LIF-3i medium (2-fold), collect hPSC in a sterile 15 mL conical, centrifuge cells at 200 x g for 5 min and resuspend the cell pellet in LIF-3i medium (1–2 mL per well-equivalent). Count the number of cells using a hemocytometer or an automatic cell counter.
3. Centrifuge N-hPSC in the LIF-3i medium (200 x g for 5 min) and resuspend cells in a biosafety cabinet in the freezing solution (Table 2), at a density of at least 1 x 10⁶ cells/mL.
4. Transfer cells into the long-term storage cryogenic tubes and place into a slow-freezing container. Allow the samples to freeze overnight in a -80 °C freezer.
5. The next day, transfer the cryovials into a liquid nitrogen freezer for long-term storage.
6. For thawing, place the frozen vial into a 37 °C water bath for ~2 min. Sterilize the vial (i.e., ethanol spray), transfer hPSC in a sterile 15 mL conical and slowly dilute the cells 10-fold in hESC medium (Table 1) supplemented with 5 μM of Rho-associated protein kinase (ROCK) inhibitor Y-27632 within a sterile biological safety hood cabinet.
7. Centrifuge at 200 x g for 5 min. In a biosafety cabinet, discard cell-free supernatant and resuspend the cell pellet in LIF-3i medium (1–2 mL) supplemented with 5 μM ROCK inhibitor Y-27632.
   NOTE: Exclusion of Y-27632 will result in poor post-thawing recovery efficiencies (Figure 2).
8. Transfer the thawed cells resuspended in LIF-3i/ROCK Inhibitor onto the PBS-washed MEF-plated wells. Cryopreserve LIF-3i cultures at a density of 1 x 10⁶ cells per vial. Thaw each of these vials onto feeders in one well of a gelatinized 6-well plate.
9. The next day, start regular LIF-3i medium expansion without Rho kinase inhibitor.

5. Feeder Removal for the Collection of N-hPSC Samples

1. To prepare samples from LIF-3i/MEF (or hESC/MEF conventional) cultures (i.e., for gene expression (e.g., quantitative RT-PCR, microarrays) or protein (e.g., Western blot) analyses, deplete LIF-3i cultures from MEF feeders using Magnetic Activated Cell Sorting (MACS) with anti-TRA-1-81 antibody coated microbeads according to the manufacturer's protocol.
2. Alternatively, utilize the following simple pre-plating method to deplete cultures of feeders, as described below.
3. In a sterile biological safety hood cabinet, discard the cell-free supernatant, wash the pellet with 2 mL sterile PBS per well. Detach adherent LIF-3i/MEF cultures using cell detachment solution (1 mL/well). Incubate for 5 min in a CO₂ incubator (5% CO₂, humid atmosphere). Collect hPSC in a sterile 15 mL conical. Wash 2-fold in hESC medium, transfer cells into the sterile 15 mL conical and centrifuge at 200 x g for 5 min.
4. In a biosafety cabinet, re-suspend each well of LIF-3i/MEF dissociated cells into 2 mL of the LIF-3i medium, and directly transfer onto a new well of a 6-well plate that has been freshly coated with 0.1% gelatin.
5. Incubate LIF-3i/MEF hPSC cells for 1 h at 37 °C in a CO₂ incubator (5% CO₂, humid atmosphere). Collect non-adherent cells with a pipette in a new conical tube. Gently add 2 mL of hESC medium to each well and swirl to collect the remaining non-adherent cells.
   NOTE: The majority of the irradiated MEF will attach to the gelatinized plate in 1 h, leaving the majority of the hPSC in suspension.
6. Combine and centrifuge cells at 300 x g for 5 min. Wash in PBS. Snap-freeze the cell pellet in liquid nitrogen after centrifugation, or alternatively re-suspend pellets in a lysing buffer that is compatible with the downstream protein or nucleic acid analysis (Figure 2B).
7. Perform characterizations of LIF-3i hPSC lines with a matching conventional primed isogenic hPSC control.
   NOTE: Because of intrinsic variability between and within primed cultures, relevant controls are prepared at a matching timepoint in culture or prior to naïve reversion. Detailed protocols and materials for downstream immunofluorescent bioimaging, flow cytometry, Western blotting, gene expression (RT-PCR assays and microarrays), methylation studies (dot blot, CpG methylation microarray), OCT4 proximal and distal enhancer predominance reporter assays and signaling inhibitor assays are provided elsewhere³.

6. Post Naïve reversion: Validation of Genomic Integrity and Retention of Parental Imprints of LIF-3i-reverted N-hPSC Prior to Use in Functional Assays

1. Screen the starting primed, conventional hPSC cultures for possession of a normal karyotype (e.g., with Giemsa-band staining analysis using published methods ⁷) before initiating LIF-5i/LIF-3i reversion.
   NOTE: This is to eliminate conventional hPSC populations that may harbor abnormal genomic alterations which may drive artefactual selective survival advantage in clonal LIF-3i conditions.
2. For optimal results, freshly revert conventional hPSC cultures to a naïve-like state with LIF-3i several weeks prior to their use in functional studies or directed differentiation.
   NOTE: Routine prolonged 'maintenance' culture in LIF-3i conditions for more than 10 passages following naïve reversion is not recommended. Routine expansion and maintenance of hESC and hiPSC lines should be performed using conventional culture systems (e.g., in E8, or MEF/hESC medium with bFGF).
3. Assess post-reverted N-hPSC lines for the retention of normal karyotypes 5–7 passages after LIF-3i reversion (e.g., with Giemsa-band staining analysis⁷, or any other method of choice).
4. Assess all reverted N-hPSC lines for retention of normal parental genomic imprints by a DNA methylation analysis of choice (e.g., protocols for CpG DNA microarray analysis of parental imprints in LIF-3i-reverted N-hPSC are provided elsewhere³) after 5–10 passages of LIF-3i reversion.

7. Post Naïve Reversion: Experimental Design Guidelines for Quantitative Directed Differentiation Assays Using LIF-3i-reverted N-hPSC

1. Directly utilize LIF-3i N-hPSC into established directed differentiation protocols without additional cell culture manipulations.
   NOTE: Re-priming (i.e., converting N-hPSC back to conventional primed conditions prior to their use in directed differentiation assays) is not necessary with the LIF-3i method and is not recommended.
2. To control for the impacts of the assay and interline variability in the functional testing of individual hPSC lines, cross-validate the lineage-specific differentiation potencies by employing independent differentiation protocols with at least three hPSC lines derived from independent genetic backgrounds (i.e., multiple donor-derived hiPSC and hESC).
3. For functional comparisons, set up sibling cultures, at equivalent passage number, and from the same (isogenic) hPSC line in parallel to the conventional lineage-primed and LIF-3i-reverted hPSC cultures. Maintain primed/naïve sibling isogenic hPSC cultures in their respective media (e.g., E8 vs. LIF-3i), and simultaneously differentiate using identical differentiation protocols and materials, to eliminate any experimental bias (Figure 4).
4. For isogenic primed vs. naïve hPSC comparisons, adjust and optimize initial plating densities for each individual differentiation assay.
   NOTE: Detailed protocols for neural progenitor, definitive endoderm and hemato-endothelial directed differentiation of LIF-3i-reverted N-hPSC are provided elsewhere ³. LIF-3i-reverted N-hPSC have more robust proliferative and differentiation capacity in the directed differentiation assays than conventional hPSC. N-hPSC typically requires a lower initial plating concentration than the conventional hPSC, and unlike their conventional primed hPSC counterparts, do not require the use of anti-apoptotic reagents to enhance their clonal survival following enzymatic digestion in differentiation assays.