This work was supported by grants from NIH/NEI (R01EY023962), NIH/NICHD (R01HD082098), RPB Stein Innovation Award, The Maryland Stem Cell Research Fund (2018-MSCRFV-4048, 2014-MSCRFE-0742), Novo Nordisk Science Forum Award, and The Moseley Foundation.

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 Na&#239;ve-reverted (LIF-3i medium/MEF) hPSC Culture
<ol>
	<li>Purchase or prepare in-house low-passage supplies of MEF feeders from CF1 or CF1 x DR4 hybrid E13.5 mouse embryos following ...

<ol>
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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation,+expansion+and+functional+analysis+of+endothelial+cells+and+pericytes+derived+from+human+pluripotent+stem+cells.">Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells.</a> Nat Protoc. 9 (6), 1514-1531 (2014).</li><li>Park, T. S., Zimmerlin, L., Zambidis, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+simultaneous+generation+of+hematopoietic+and+vascular+progenitors+from+human+induced+pluripotent+stem+cells.">Efficient and simultaneous generation of hematopoietic and vascular progenitors from human induced pluripotent stem cells.</a> Cytometry A. 83 (1), 114-126 (2013).</li><li>Park, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+progenitors+from+cord+blood-derived+induced+pluripotent+stem+cells+possess+augmented+capacity+for+regenerating+ischemic+retinal+vasculature.">Vascular progenitors from cord blood-derived induced pluripotent stem cells possess augmented capacity for regenerating ischemic retinal vasculature.</a> Circulation. 129 (3), 359-372 (2014).</li><li>Taapken, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karotypic+abnormalities+in+human+induced+pluripotent+stem+cells+and+embryonic+stem+cells.">Karotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells.</a> Nat Biotechnol. 29 (4), 313-314 (2011).</li><li>Mitalipova, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+genetic+integrity+of+human+embryonic+stem+cells.">Preserving the genetic integrity of human embryonic stem cells.</a> Nat Biotechnol. 23 (1), 19-20 (2005).</li><li>Beers, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+and+colony+expansion+of+human+pluripotent+stem+cells+by+enzyme-free+dissociation+in+chemically+defined+culture+conditions.">Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.</a> Nat Protoc. 7 (11), 2029-2040 (2012).</li><li>Laurent, L. 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Under a licensing agreement between Life Technologies and the Johns Hopkins University (JHU), Dr. Zambidis is entitled to a share of royalty received by the University for licensing of stem cells. The terms of this arrangement are managed by JHU in accordance with its Conflict of Interest policies. This does not alter authors' adherence to journal policies on sharing data and materials.

The LIF-3i system applies a modified version of the classic murine 2i na&#239;ve 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...

The 2i (MEK/ERK and GSK3&#946; 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 na&#239;ve-like pluripotent molecular states2. However, many of the &#34;na&#239;ve-like&#34; states created with these methods also exhibited karyotypic instability, epigenomic defects (e.g., global loss of parental genomic imprinting), or impaired differentiation potential.
In contrast, the cocktail of triple chemical inhibition of GSK3&#946;, ERK and tankyrase signaling and leukemia inhibitory factor (LIF-3i) was sufficient for the stable na&#239;ve-like reversion of a broad repertoire of conventional hPSC lines3. LIF-3i-reverted na&#239;ve hPSC (N-hPSC) maintained normal karyotypes and increased their expressions of na&#239;ve-specific human preimplantation epiblast genes (e.g.,NANOG, KLF2, NR5A2, DNMT3L, HERVH, Stella (DPPA3), KLF17, TFCP2L1). LIF-3i reversion also conferred hPSC with an array of molecular and biochemical characteristics unique to mESC-like na&#239;ve pluripotency that included increased phosphorylated STAT3 signaling, decreased ERK phosphorylation, global 5-methylcytosine CpG hypomethylation, genome-wide CpG demethylation at embryonic stem cell (ESC)-specific gene promoters, and dominant distal OCT4 enhancer usage. Moreover, in comparison to other na&#239;ve reversion methods that resulted in aberrantly hypomethylated imprinted genomic loci, LIF-3i-reverted N-hPSC were devoid of systematic loss of imprinted CpG patterns or loss of DNA methyltransferase expression (e.g., DNMT1, DNMT3A, DNMT3B)3.
A direct LIF-3i culture of a broad array of conventional human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) grown on either feeders or E8 feeder-free conditions achieved rapid and bulk reversion to a na&#239;ve epiblast-like state. However, direct LIF-3i na&#239;ve reversion may be inefficient in some unstable conventional hPSC lines due to the inherent genomic and lineage-primed variabilities arising from the genetically diverse donor backgrounds.
Thus, to broaden the utility of the LIF-3i method, a stepwise optimization was developed and is presented herein, that allows universal na&#239;ve reversion with almost any conventional hESC or transgene-free hiPSC line cultured on feeders. This universalized na&#239;ve reversion method employs a transient initial culture step in conventional hPSC that supplements the LIF-3i cocktail with two additional small molecules (LIF-5i) that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling. One initial passage of conventional hPSC in LIF-5i adapts them to subsequent stable LIF-3i reversion in bulk quantities. Initial LIF-5i adaptation significantly augments the initial single cell clonal proliferation of conventional hPSC grown on E8 or feeders (prior to their subsequent stable, continuous passage in LIF-3i alone). Conventional hPSC lines adapted first to one passage in LIF-5i tolerate subsequent bulk clonal passaging of na&#239;ve-reverted cells in LIF-3i conditions, which obviates the need for picking and subcloning of the rare stable colonies, or the routine use of anti-apoptotic molecules or Rho-associated protein kinase (ROCK) inhibitors.
The LIF-3i method has been successfully employed to stably expand and maintain a broad repertoire of &#62;30 independent, genetically-diverse conventional hPSC lines for &#62;10&#8211;30 passages using either non-enzymatic or enzymatic dissociation methods, and without evidence of induction of chromosomal or epigenomic abnormalities, including abnormalities at imprinted gene loci. Additionally, sequential LIF-5i/LIF-3i culture is the only na&#239;ve reversion method that has thus far been reported that improves the functional pluripotency of a broad repertoire of conventional hPSC lines by decreasing their lineage-primed gene expression and dramatically improving their multipotent differentiation potency. The LIF-3i na&#239;ve reversion method erases the inherent interline variability of differentiation of lineage-primed, conventional hPSC lines, and will have a great utility of application in regenerative medicine and cellular therapies.

<table border="0" cellpadding="0" cellspacing="0" style="width:1024px;" width="1023">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti- SSEA-1/CD15 antibody, APC conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">561716</td>
			<td style="width:371px;">use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti- TFCP2L1 antibody</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">HPA029708</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-beta-Actin antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab6276</td>
			<td>use at 1:5000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD146 antibody,&#160; PE conjugated&#160;</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">550315</td>
			<td>use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD31 antibody, APC conjugated</td>
			<td style="width:161px;">eBioscience</td>
			<td style="width:119px;">17-0319-42</td>
			<td>use 2&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD31 microbead kit</td>
			<td style="width:161px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-091-935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-NANOG antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab109250</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-NR5A2 antibody</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">HPA005455</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-p44/42 MAPK (Erk1/2) antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">4695</td>
			<td>use at 1:1000 (Western blot), for detection of total protein</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204 antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">4370</td>
			<td>use at 1:1000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-phospho-STAT3 (Tyr705) antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">9145</td>
			<td>use at 1:1000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-rabbit immunoglobulin antibody, biotinylated</td>
			<td style="width:161px;">Agilent</td>
			<td style="width:119px;">E0432</td>
			<td>use at a 1:500-1:1000 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-SSEA-4 antibody, APC conjugated&#160;</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">FAB1435A</td>
			<td>use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-SSEA-4 GloLIVE antibody, NL493 conjugated</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">NLLC1435G</td>
			<td>use at 1:50 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-STAT3 antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">9139</td>
			<td>use at 1:1000 (Western blot), for detection of total protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-STELLA/DPPA3 antibody</td>
			<td style="width:161px;">Millipore</td>
			<td style="width:119px;">MAB4388</td>
			<td>use at a 1:50 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA-1-60 GloLIVE antibody, NL557 conjugated&#160;</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">NLLC4770R</td>
			<td>use at&#160; a 1:50 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-TRA-1-60 StainAlive Antibody, DyLight 488 conjugated</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">09-0068</td>
			<td>use at a 1:100 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-TRA-1-81 StainAlive Antibody, DyLight 488 conjugated</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">09-0069</td>
			<td>use at a 1:100 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA1-60 antibody, PE conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">560193</td>
			<td>use 10&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA1-81 antibody, PE conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">560161</td>
			<td>use 10&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">APEL2-Li</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">5271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Bovine Serum Albumin</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">A3311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CellAdhere dilution buffer</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">7183</td>
			<td>dilutent for Vitronectin XF&#8482; matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CF1 mouse</td>
			<td style="width:161px;">Charles river</td>
			<td style="width:119px;">023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CHIR99021</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">L5283</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">confocal microscope system</td>
			<td style="width:161px;">Zeiss</td>
			<td style="width:119px;">LSM 510</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">cord blood CD34+ derived iPSC line</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A18945</td>
			<td>also referred as 6.2 line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Corning Costar tissue culture-treated 6-well plates</td>
			<td style="width:161px;">Corning</td>
			<td style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Countess&#160; cell counting chamber slide</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C10228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Countess automated cell counter</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)&#160;</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11995-065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">DMEM-F12</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">DMSO (dimethyl sulfoxide)</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">DR4 mouse</td>
			<td style="width:161px;">The Jackson Laboratory</td>
			<td align="right" style="width:119px;">3208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Essential 8 (E8) medium</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">5940</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Fetal bovin serum (FBS)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Forskolin</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0025</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Gelatin (porcine)</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td>resuspend in water and sterilize with an autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">KnockOut Serum Replacement</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">L-Glutamine (100X)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">MEM Non-essential amino acid (MEM NEAA) (100X)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">mTeSR1 medium</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">85850</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Nalgene cryogenic vials</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">5000-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Nunc Lab-Tek II Chamber Slide System</td>
			<td style="width:161px;">Fisher Scientific</td>
			<td style="width:119px;">154534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Paraformaldehyde (PFA) solution , 4% in PBS</td>
			<td style="width:161px;">USB Corporation&#160;</td>
			<td style="width:119px;">19943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">PD0325901</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">PZ0162</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Penicillin/streptomycin (10,000 U/mL)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Phosphate buffered saline (PBS)</td>
			<td style="width:161px;">Biological Industries</td>
			<td style="width:119px;">02-023-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Purmorphamine</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0009</td>
			<td>reconstitute at 10mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human Activin A</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">AF-120-14E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">recombinant human Bone morphogenetic protein (BMP)-4</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">120-05ET</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human FGF-basic (bFGF)</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human LIF</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">300-05</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">SB431542&#160;</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0010-05</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Stemolecule Y27632 in Solution</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0012-02</td>
			<td>ROCK inhibitor in solution (10mM)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A11105-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Streptavidin-Cy3 conjugate</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">S6402</td>
			<td>use at 1:500-1:1000 dilution (immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Thermo Scientific Mr. Frosty Freezing Container</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vascular endothelial growth factor (VEGF)-165</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">100-21</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vitronectin XF matrix</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">7180</td>
			<td>dilute at 40&#181;L/mL in CellAdhere&#8482; dilution buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">XAV939</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">X3004</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">&#946;-mercaptoethanol</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">21985-023</td>
			<td>light sensitive</td>
		</tr>
	</tbody>
</table>

chemical reversion of conventional human pluripotent stem cells to a na&#239;ve-like state with improved multilineage differentiation potency

Na&#239;ve 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 na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, 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 na&#239;ve-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. na&#239;ve-like states are outlined.

This protocol optimizes efficient na&#239;ve-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...

Watch this Scientific Journal Video about Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃ¯ve-like State with Improved Multilineage Differentiation Potency at JoVE.com

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&amp;#239;ve-like State with Improved Multilineage Differentiation Potency

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve 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.

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

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.

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JoVE Journal

Developmental Biology

9.8K Görüntüleme.  Johns Hopkins School of Medicine. 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 hu...