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

Summary

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable naïve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

Abstract

Naïve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a naïve-like pluripotency with improved functionality. This chemical naïve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3β, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently naïve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. naïve-like states are outlined.

Introduction

The 2i (MEK/ERK and GSK3β inhibitor) culture system was originally developed to refine heterogeneous serum-based mouse embryonic stem cells (mESC) cultures to a uniform ground state of pluripotency akin to the mouse preimplantation epiblast¹. However, 2i does not support the stable maintenance of human pluripotent stem cell (hPSC) lines². The various complex small molecule, growth factor-supplemented, and transgenic approaches have recently been reported to capture putatively similar human naïve-like pluripotent molecular states². However, many of the &#....

Protocol

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-....

Results

This protocol optimizes efficient naïve-like reversion with LIF-3i in both feeder-dependent and feeder-independent lineage-primed conventional hPSC cultures (Figure 1). The detailed protocol, described herein, outlines sequential adaptation to LIF-3i starting from either feeder-dependent or feeder-free conventional hPSC conditions (e.g. E8 medium).

Representative results for the LIF-3i reve.......

Discussion

The LIF-3i system applies a modified version of the classic murine 2i naïve reversion cocktail¹ to human pluripotent stem cells. The self-renewal of hPSC (which cannot expand in 2i alone) is stabilized in LIF-2i by supplementing this cocktail with the tankyrase inhibitor XAV939. LIF-3i culture allows bulk and efficient reversion of the conventional hPSC to a pluripotent state resembling the human preimplantation epiblast³. Although the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti- SSEA-1/CD15 antibody, APC conjugated	BD Biosciences	561716	use 5µL per assay (FACS)
anti- TFCP2L1 antibody	Sigma Aldrich	HPA029708	use at a 1:100 dilution (immunostainings)
anti-beta-Actin antibody	Abcam	ab6276	use at 1:5000 (Western blot)
anti-CD146 antibody,  PE conjugated	BD Biosciences	550315	use 5µL per assay (FACS)
anti-CD31 antibody, APC conjugated	eBioscience	17-0319-42	use 2µL per assay (FACS)
anti-CD31 microbead kit	Miltenyi Biotec	130-091-935
anti-NANOG antibody	Abcam	ab109250	use at a 1:100 dilution (immunostainings)
anti-NR5A2 antibody	Sigma Aldrich	HPA005455	use at a 1:100 dilution (immunostainings)
anti-p44/42 MAPK (Erk1/2) antibody	Cell Signaling	4695	use at 1:1000 (Western blot), for detection of total protein
anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204 antibody	Cell Signaling	4370	use at 1:1000 (Western blot)
anti-phospho-STAT3 (Tyr705) antibody	Cell Signaling	9145	use at 1:1000 (Western blot)
anti-rabbit immunoglobulin antibody, biotinylated	Agilent	E0432	use at a 1:500-1:1000 dilution (immunostainings)
anti-SSEA-4 antibody, APC conjugated	R&D System	FAB1435A	use 5µL per assay (FACS)
anti-SSEA-4 GloLIVE antibody, NL493 conjugated	R&D System	NLLC1435G	use at 1:50 dilution (live and fixed immunostainings)
anti-STAT3 antibody	Cell Signaling	9139	use at 1:1000 (Western blot), for detection of total protein
anti-STELLA/DPPA3 antibody	Millipore	MAB4388	use at a 1:50 dilution (immunostainings)
anti-TRA-1-60 GloLIVE antibody, NL557 conjugated	R&D System	NLLC4770R	use at  a 1:50 dilution (live and fixed immunostainings)
anti-TRA-1-60 StainAlive Antibody, DyLight 488 conjugated	Stemgent	09-0068	use at a 1:100 dilution (live and fixed immunostainings)
anti-TRA-1-81 StainAlive Antibody, DyLight 488 conjugated	Stemgent	09-0069	use at a 1:100 dilution (live and fixed immunostainings)
anti-TRA1-60 antibody, PE conjugated	BD Biosciences	560193	use 10µL per assay (FACS)
anti-TRA1-81 antibody, PE conjugated	BD Biosciences	560161	use 10µL per assay (FACS)
APEL2-Li	StemCell Technologies	5271
Bovine Serum Albumin	Sigma Aldrich	A3311
CellAdhere dilution buffer	StemCell Technologies	7183	dilutent for Vitronectin XF™ matrix
CF1 mouse	Charles river	023
CHIR99021	R&D System	L5283	reconstitute at 100mM in DMSO
confocal microscope system	Zeiss	LSM 510
cord blood CD34+ derived iPSC line	Thermo Fisher Scientific	A18945	also referred as 6.2 line
Corning Costar tissue culture-treated 6-well plates	Corning	3506
Countess  cell counting chamber slide	Thermo Fisher Scientific	C10228
Countess automated cell counter	Thermo Fisher Scientific	AMQAX1000
DMEM (Dulbecco's Modified Eagle Medium)	Thermo Fisher Scientific	11995-065
DMEM-F12	Thermo Fisher Scientific	11330-032
DMSO (dimethyl sulfoxide)	Sigma Aldrich	D2650
DR4 mouse	The Jackson Laboratory	3208
Essential 8 (E8) medium	StemCell Technologies	5940
Fetal bovin serum (FBS)	Thermo Fisher Scientific	SH30071.03
Forskolin	Stemgent	04-0025	reconstitute at 100mM in DMSO
Gelatin (porcine)	Sigma Aldrich	G1890-100G	resuspend in water and sterilize with an autoclave
KnockOut Serum Replacement	Thermo Fisher Scientific	10828-028
L-Glutamine (100X)	Thermo Fisher Scientific	25030-081
MEM Non-essential amino acid (MEM NEAA) (100X)	Thermo Fisher Scientific	11140-050
mTeSR1 medium	StemCell Technologies	85850
Nalgene cryogenic vials	Thermo Fisher Scientific	5000-0020
Nunc Lab-Tek II Chamber Slide System	Fisher Scientific	154534
Paraformaldehyde (PFA) solution , 4% in PBS	USB Corporation	19943
PD0325901	Sigma Aldrich	PZ0162	reconstitute at 100mM in DMSO
Penicillin/streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140-122
Phosphate buffered saline (PBS)	Biological Industries	02-023-1A
Purmorphamine	Stemgent	04-0009	reconstitute at 10mM in DMSO
recombinant human Activin A	Peprotech	AF-120-14E
recombinant human Bone morphogenetic protein (BMP)-4	Peprotech	120-05ET	resupend at 100ug/mL in 0.1% bovine serum albumin in PBS
recombinant human FGF-basic (bFGF)	Peprotech	100-18B	resupend at 100ug/mL in 0.1% bovine serum albumin in PBS
recombinant human LIF	Peprotech	300-05	resupend at 100ug/mL in 0.1% bovine serum albumin in PBS
SB431542	Stemgent	04-0010-05	reconstitute at 100mM in DMSO
Stemolecule Y27632 in Solution	Stemgent	04-0012-02	ROCK inhibitor in solution (10mM)
StemPro Accutase Cell Dissociation Reagent	Thermo Fisher Scientific	A11105-01
Streptavidin-Cy3 conjugate	Sigma Aldrich	S6402	use at 1:500-1:1000 dilution (immunostainings)
Thermo Scientific Mr. Frosty Freezing Container	Thermo Fisher Scientific	5100-0001
Vascular endothelial growth factor (VEGF)-165	Peprotech	100-21	resupend at 100ug/mL in 0.1% bovine serum albumin in PBS
Vitronectin XF matrix	StemCell Technologies	7180	dilute at 40µL/mL in CellAdhere™ dilution buffer
XAV939	Sigma Aldrich	X3004	reconstitute at 100mM in DMSO
β-mercaptoethanol	Thermo Fisher Scientific	21985-023	light sensitive