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

The LIF-3i method reverts conventional human pluripotent stem cells into a more primitive pluripotent state with biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This method improves the functionality of conventional human pluripotent stem cells by decreasing the lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Using this method, chemically-reverted human pluripotent stem cells maintain karyotypic and epigenomic stability following extended passage, including at imprinted loci and can be differentiated directly with improved efficiencies without requiring a re-priming step.

The LIF-3i method will open new avenues of investigation into early human developmental biology and may advance regenerative medicine by improving human pluripotent stem cell differentiation, gene targeting, and the generation of interspecies chimeras. To begin the protocol, maintain and expand conventional human pluripotent stem cells or hPSCs with validated normal karyotypes in co-cultures with a mouse embryonic fibroblast or MEF-based feeder culture system or in a feeder-free culture system. Prepare MEF feeders in a previously-prepared gelatinized six-well plate at least one day before passaging the LIF-5i-adapted conventional hPSC cultures.

After the conventional hPSC cultures have reached about 50%confluency, replace the standard hESC culture medium with two milliliters per well of Leukemia Inhibitory Factor 5i or LIF-5i medium. Culture and maintain the conventional hPSCs and MEFs for up to two days in LIF-5i in a CO2 incubator. Change the LIF-5i medium daily to adapt them for their subsequent passage and stable reversion in LIF-3i.

For the feeder-free conventional cultures, culture hPSC in LIF-5i only overnight before passaging the next morning. Prior to passaging, wash the LIF-5i-adapted conventional hPSCs once with PBS and add one milliliter of cell-dissociation reagent to each well. Next, incubate the plate for five minutes at 37 degrees Celsius in a CO2 incubator.

After incubation, gently triturate the cells with a pipette into a single cell suspension back. Collect the cell suspension in the hESC medium in at least a two-fold dilution in a sterile, 15-milliliter conical tube and gently triturate the cells by pipetting to obtain a single cell suspension. Centrifuge the cells at 300 g for five minutes.

After the spin, aspirate and discard the supernatant and re-suspend the cell pellet in one to two milliliters of LIF-5i medium. Then, count the cells. Wash the pre-plated six-well MEF plate twice with two milliliters of PBS per well.

Distribute one to two times 10 to the sixth cells in two milliliters LIF-5i medium into one well of the PBS-washed MEF plate. Place the plate in a CO2 incubator. The next day, gently swirl the plate to lift all non-attached cells and aspirate the medium and replace the medium with two milliliters of LIF-5i medium.

Repeat this step daily for three to five days or until the cells are 60 to 70%confluent. This protocol supports rapid and bulk naive-like reversion of a large range of human embryonic and induced pluripotent stem cell lines as long as human pluripotent stem cells preparations with marked differentiation or abnormal karyotypes are meticulously excluded. Following the initial LIF-5i adaptation, passage the subsequent stable LIF-3i cultures every three to four days in a bio-safety cabinet or when the cultures become 60 to 70%confluent.

Discard the culture medium and wash each well of the LIF-5i-LIF-3i cultures by gently adding two milliliters of PBS. Discard the PBS and add one milliliter of cell detachment solution. Incubate the cells for five minutes at 37 degrees Celsius in a CO2 incubator.

After incubation, collect the cell suspension, add untreated hESC medium in a two-fold dilution, and gently triturate the cells by pipetting to obtain a single cell suspension. Transfer the suspension to a sterile 15-milliliter conical tube. Centrifuge the cells at 300 g for five minutes and aspirate and discard the supernatant.

Re-suspend the pellet in LIF-3i medium, then count the cells. Next plate two times 10 to the five cells per well onto the irradiated MEF in the gelatinized six-well plate for routine passaging of LIF-3i cultures. For the initial LIF-5i-adapted cultures, place an initially higher density prior to the first passage into LIF-3i MEF.

Individual human pluripotent stem cell lines should be optimized and adjusted to high initial plating densities if required to maintain continuous bulk passaging during the early passages of LIF-3i reversion culture. Re-plate and distribute the LIF-5i-adapted hPSCs onto fresh, PBS-washed, irradiated MEF-feeder plates in the LIF-3i medium. Replace the LIF-3i medium daily.

Passage N-hPSC for at least four to seven 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. Immunofluorescent stains and western blot detected the expression of the activated phosphorylated and total isoforms of STAT3 and Erk1/2, key markers of molecular pluripotency.

Hematoxylin and eosin stains of teratoma microsections revealed robust differentiation into all three germ layers with well-structured ectoderm, mesoderm, and endoderm lineages. After watching this video, you should have a good understanding of how to efficiently revert conventional human pluripotent stem cells in bulk to a naive-like state using the sequential universal LIF-5i to LIF-3i chemical method. Before attempting this protocol, it is important to begin with a low passage of conventional human pluripotent stem cell lines which conform to normal karyotypes.

Conventional human pluripotent stem cell cultures should be mostly undifferentiated and contain few spontaneously-differentiated cells before beginning. The initial, brief LIF-5i adaptation step significantly enhances the clonal expansion of conventional human pluripotent stem cells and permits them to be subsequently naive reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking or sub-cloning rare naive human pluripotent stem cell colonies later. The further optimization of this tankyrase inhibitor-utilizing chemical method in defined feeder-free GMP-compliant culture conditions will facilitate efficient, clinically-useful generation of a broad array of functional and engraftable cell types for therapeutic use.