Name: chemical reversion of conventional human pluripotent stem cells to a na&#239;ve-like state with improved multilineage differentiation potency
Uploaded: 2018-06-10T15:00:00
Description: 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

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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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+Human+Naive+Pluripotency+in+the+Embryo+and+in+the+Dish.">Capturing Human Naive Pluripotency in the Embryo and in the Dish.</a> Stem Cells Dev. 26 (16), 1141-1161 (2017).</li><li>Zimmerlin, L., 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=Tankyrase+inhibition+promotes+a+stable+human+naive+pluripotent+state+with+improved+functionality.">Tankyrase inhibition promotes a stable human naive pluripotent state with improved functionality.</a> Development. 143 (23), 4368-4380 (2016).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells.">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> J Vis Exp. 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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=Comprehensive+Cell+Surface+Protein+Profiling+Identifies+Specific+Markers+of+Human+Naive+and+Primed+Pluripotent+States.">Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States.</a> Cell Stem Cell. 20 (6), 874-890 (2017).</li><li>Liu, X., 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=Comprehensive+characterization+of+distinct+states+of+human+naive+pluripotency+generated+by+reprogramming.">Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming.</a> Nat Methods. 14 (11), 1055-1062 (2017).</li><li>Choi, 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=Prolonged+Mek1/2+suppression+impairs+the+developmental+potential+of+embryonic+stem+cells.">Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells.</a> Nature. 548 (7666), 219-223 (2017).</li><li>Yang, Y., 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=Derivation+of+Pluripotent+Stem+Cells+with+In+Vivo+Embryonic+and+Extraembryonic+Potency.">Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.</a> Cell. 169 (2), 243-257 (2017).</li><li>Orlova, V. 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. C., 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=Dynamic+changes+in+the+copy+number+of+pluripotency+and+cell+proliferation+genes+in+human+ESCs+and+iPSCs+during+reprogramming+and+time+in+culture.">Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture.</a> Cell Stem Cell. 8 (1), 106-118 (2011).</li><li>Hussein, 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=Copy+number+variation+and+selection+during+reprogramming+to+pluripotency.">Copy number variation and selection during reprogramming to pluripotency.</a> Nature. 471 (7336), 58-62 (2011).</li></ol>

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.

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		<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.

chemical-reversion-conventional-human-pluripotent-stem-cells-to-naive

Research

JoVE Journal

Developmental Biology

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...