Name: direct induction of hemogenic endothelium and blood by overexpression of transcription factors in human pluripotent stem cells
Uploaded: 2015-12-03T10:30:00
Description: Watch this Scientific Journal Video about Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells at JoVE.com

We thank Matt Raymond for editorial assistance. This work was supported by funds from the National Institute of Health (U01HL099773, R01HL116221, and P51 RR000167) and The Charlotte Geyer Foundation.

1. Virus Preparations and Transcription Factor Combinations
<ol>
	<li>Prepare solutions with pSIN-EF1&#945; lentiviral expression plasmid containing protein-coding DNA for ETV2, GATA1, GATA2, TAL1 and LMO2 (Table 1).</li>
	<li>Measure the concentration and purity of plasmid preparations used for lentiviral production by recording UV absorption with a spectrophotometer at 230 nm, 260 nm, and 280 nm. Note: DNA preparations demonstrating A260/280 and A260/230 values greater than 1.8 are generally ...

<ol>
	<li>Zape, J. P., Zovein, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemogenic+endothelium:+origins,+regulation,+and+implications+for+vascular+biology.">Hemogenic endothelium: origins, regulation, and implications for vascular biology.</a> Semin Cell Dev Biol. 22, 1036-1047 (2011).</li><li>Swiers, G., Rode, C., Azzoni, E., de Bruijn, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+history+of+hemogenic+endothelium.">A short history of hemogenic endothelium.</a> Blood Cells, Mol &amp; Dis. 51, 206-212 (2013).</li><li>Kennedy, 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=T+lymphocyte+potential+marks+the+emergence+of+definitive+hematopoietic+progenitors+in+human+pluripotent+stem+cell+differentiation+cultures.">T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures.</a> Cell Rep. 2, 1722-1735 (2012).</li><li>Wang, 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=Endothelial+and+hematopoietic+cell+fate+of+human+embryonic+stem+cells+originates+from+primitive+endothelium+with+hemangioblastic+properties.">Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties.</a> Immunity. 21, 31-41 (2004).</li><li>Rafii, 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=Human+ESC-derived+hemogenic+endothelial+cells+undergo+distinct+waves+of+endothelial+to+hematopoietic+transition.">Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition.</a> Blood. 121 (5), 770-780 (2012).</li><li>Choi, K. D., 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=Identification+of+the+hemogenic+endothelial+progenitor+and+its+direct+precursor+in+human+pluripotent+stem+cell+differentiation+cultures.">Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures.</a> Cell Rep. 2, 553-567 (2012).</li><li>Vodyanik, M. A., Thomson, J. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukosialin+(CD43)+defines+hematopoietic+progenitors+in+human+embryonic+stem+cell+differentiation+cultures.">Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures.</a> Blood. 108, 2095-2105 (2006).</li><li>Wang, 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=TGFbeta+inhibition+enhances+the+generation+of+hematopoietic+progenitors+from+human+ES+cell-derived+hemogenic+endothelial+cells+using+a+stepwise+strategy.">TGFbeta inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy.</a> Cell Res. 22, 194-207 (2012).</li><li>Uenishi, G., 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=Tenascin+C+promotes+hematoendothelial+development+and+T+lymphoid+commitment+from+human+pluripotent+stem+cells+in+chemically+defined+conditions.">Tenascin C promotes hematoendothelial development and T lymphoid commitment from human pluripotent stem cells in chemically defined conditions.</a> Stem Cell Rep. 3, 1073-1084 (2014).</li><li>Elcheva, I., 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=Direct+induction+of+haematoendothelial+programs+in+human+pluripotent+stem+cells+by+transcriptional+regulators.">Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators.</a> Nat Commun. 5, 4372 (2014).</li><li>Tiscornia, G., Singer, O., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+lentiviral+vectors.">Production and purification of lentiviral vectors.</a> Nat Protoc. 1, 241-245 (2006).</li><li>Vodyanik, M. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoendothelial+differentiation+of+human+embryonic+stem+cells.">Hematoendothelial differentiation of human embryonic stem cells.</a> Curr Protoc Cell Biol. Chapter 23, Unit 23.6 (2007).</li><li>Cao, F., 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=Comparison+of+gene-transfer+efficiency+in+human+embryonic+stem+cells.">Comparison of gene-transfer efficiency in human embryonic stem cells.</a> Mol Imgn and Biol. 12, 15-24 (2010).</li></ol>

The above-described method for hematopoietic differentiation of hPSCs by overexpression of TFs, represents a rapid and efficient approach for the generation of HE and myeloid and erytho-magakaryocytic progenitors from hESCs and iPSCs, thereby allowing the production of up to 30 millions blood cells from one million pluripotent stem cells10. This method exhibited consistent differentiation in multiple hESC and iPSCs lines10. During differentiation by ETV2 and GATA2, GATA1 factors as well as GATA2 and...

The unique ability of human pluripotent stem cells (hPSCs) to self-renew and to differentiate into cells of the three germ layers, including blood, make them a valuable tool for the mechanistic studies of hematopoietic development, modeling of blood diseases, drug screening, toxicity studies, and the development of cellular therapies. Because blood formation in the embryo proceeds from hemogenic endothelium (HE) through an endothelial hematopoietic transition1,2, the generation of HE in cultures would be essential to study the molecular mechanisms regulating the endothelial to hematopoietic transition and hematopoietic specification. Current methods for studies of HE are based on the induction of hematoendothelial differentiation in aggregates (EBs) with the addition of hematopoietic cytokines3-5, and coculture of hPSCs with hematopoiesis-supportive stromal cells6,7 or in two-dimensional cultures with extracellular matrices and cytokines8,9. These classical differentiation methods are based on the introduction of external signals acting at the cell surface and initiating cascades of molecular pathways that eventually lead to the activation of transcriptional program guiding hematoendothelial development. Thus, the efficiency of hPSCs differentiations in these systems relies on an effective induction of those signals, signal transduction to the nucleus, and the resulting activation of specific transcriptional regulators. In addition, the study of HE in conventional differentiation cultures requires the additional step of isolating HE cells using cell sorting. Here, we describe a simple protocol for the direct induction of HE and blood by overexpression of hematopoietic transcription factors. This method allows for the efficient induction of HE in a dish and direct observation of the endothelial to hematopoietic transition without the need for isolation of HE using a cumbersome cell sorting procedure.
Formation of HE and blood from human pluripotent stem cells can be efficiently induced by overexpressing just a few transcription factors (TFs). The optimal combination of TFs capable of inducing robust pan-myeloid hematopoiesis from hPSCs includes ETV2 and GATA1 or GATA2. In contrast, combination of GATA2 and TAL1 induces erythromegakaryocytopoiesis10. Programming hPSCs through overexpression of these factors differentiates hPSCs directly to the VE-cad+CD43-CD73- HE cells that gradually acquire the hematopoietic phenotype defined by the expression of the early hematopoietic marker CD437. This lentiviral-based method for the direct programming of human pluripotent stem cells method is applicable for the generation of HE and blood cells for mechanistic studies, studies of endothelial to hematopoietic transition, and transcriptional regulation of hematopoietic development and specification. Although the current protocol describes blood production using constitutive expression of the transgenes, similar results could be obtained using modified mRNA10.

Table 1. Induction of hPSCs differentiation with trancription factors and analysis of hemogenic endothelium and blood cells.
<table>
	<tbody>
		<tr>
			<td>pSIN4-EF1a-ETV2-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61061</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>pSIN4-EF1a-GATA2-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61063</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>pSIN4-EF1a-GATA1-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61062</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>pSIN4-EF1a-TAL1-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61062</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>pSIN4-EF1a-LMO2-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61064</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide (Polybrene)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>107689-10G</td>
			<td>Cationic polymer used to increase the efficiency of infection</td>
		</tr>
		<tr>
			<td>Y-27632 (Dihydrochloride) ROCK inhibitor</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td>RHO/ROCK pathway inhibitor Inhibits ROCK</td>
		</tr>
		<tr>
			<td rowspan="2">StemPro Accutase Cell Dissociation Reagent</td>
			<td rowspan="2"><a href="http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html">Life Technologies</a></td>
			<td rowspan="2">A11105-01</td>
			<td rowspan="2">Cell Dissociation Reagent</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Incomplete (growth factor- free) culture medium&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; mTeSR1 Custom formulation&#160;</td>
			<td>WiCell Research Institute (Madison, WI)</td>
			<td>MCF</td>
			<td><a name="RANGE!D12">Serum-free mTeSR1 medium without bFGF and TGFb&#160;</a></td>
		</tr>
		<tr>
			<td>human SCF</td>
			<td>Peprotech</td>
			<td>300-07</td>
			<td>Premium grade</td>
		</tr>
		<tr>
			<td>human TPO</td>
			<td>Peprotech</td>
			<td>300-18</td>
			<td>Research grade</td>
		</tr>
		<tr>
			<td>human FGF-basic</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Premium grade</td>
		</tr>
		<tr>
			<td>CD144 (VE-cad) FITC</td>
			<td>BD Biosciences</td>
			<td>560411</td>
			<td>Endothelial marker (FACS)</td>
		</tr>
		<tr>
			<td>CD226 PE</td>
			<td>BD Biosciences</td>
			<td>338305</td>
			<td>Hematopoietic (FACS)</td>
		</tr>
		<tr>
			<td>CD43 PE</td>
			<td>BD Biosciences</td>
			<td>560199</td>
			<td>Hematopoietic (FACS)</td>
		</tr>
		<tr>
			<td>CD73 APC</td>
			<td>R&#38;D Systems</td>
			<td>FAB5795A</td>
			<td>Endothelial marker (FACS)</td>
		</tr>
		<tr>
			<td>CD45 APC</td>
			<td>BD Biosciences</td>
			<td>555485</td>
			<td>Hematopoietic (FACS)</td>
		</tr>
		<tr>
			<td>7AAD</td>
			<td>Life Technologies</td>
			<td>A1310</td>
			<td>Live/Dead assay (FACS)</td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P6148-500G</td>
			<td>Cell fixation&#160;</td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T9284-500ML&#160;</td>
			<td>Permeabilization&#160;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Fisher Scientific</td>
			<td>SH3007003</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD43</td>
			<td>BD Biosciences</td>
			<td>551457</td>
			<td>Pure, primary antibody for Immunofluorescence (IF) staining</td>
		</tr>
		<tr>
			<td>Rabbit anti-human VE-cadherin</td>
			<td>BenderMedSystem</td>
			<td>BMS158</td>
			<td>Primary (IF)</td>
		</tr>
		<tr>
			<td>Anti-rabbit Alexa Fluor 488-conjugated</td>
			<td>JacksonResearch</td>
			<td>715-486-152</td>
			<td>Secondary (IF)</td>
		</tr>
		<tr>
			<td>Anti-mouse Alexa Fluor 594-conjugated</td>
			<td>JacksonResearch</td>
			<td>715-516-150&#160;</td>
			<td>Secondary (IF)</td>
		</tr>
		<tr>
			<td>DAPI nucleic acid stain</td>
			<td><a href="http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html">Life Technologies</a></td>
			<td>D1306&#160;</td>
			<td>Live/Dead assay (IF)</td>
		</tr>
		<tr>
			<td>Clonogenic medium MethoCult H4435 Enriched</td>
			<td>STEMCELL Technologies</td>
			<td>4435</td>
			<td>CFC-assay</td>
		</tr>
		<tr>
			<td>Wright Stain solution</td>
			<td>Sigma -Aldrich</td>
			<td>32857</td>
			<td>Staining cytospins</td>
		</tr>
	</tbody>
</table>

Table 2. hPSCs culture.
<table>
	<tbody>
		<tr>
			<td>Human Pluripotent Stem Cells (hPSCs)&#160;</td>
			<td>WiCell Research Institute (Madison, WI)</td>
			<td>hESCs (WA01, WA09) human Embryonic Stem cells; iPSCs (DF-19-9-7T, DF-4-3-7T) transgene-free induced Pluripotent Stem Cells</td>
			<td>hPSCs are able to self-renew and to differentiate into cells of three germ layers</td>
		</tr>
		<tr>
			<td>Complete serum-free medium for culture of hPSCs&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; mTeSR1 media&#160;&#160;</td>
			<td>WiCell Research Institute (Madison, WI)</td>
			<td>M500&#160;</td>
			<td>Serum-free&#160; medium with growth factors for feeder free culture of ESC/iPSCs</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences/ Corning&#160;</td>
			<td>356234</td>
			<td>Matrix for maintenance of human ESC/iPSCs</td>
		</tr>
		<tr>
			<td>DMEM/F-12, powder&#160;</td>
			<td><a href="http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html">Life Technologies</a></td>
			<td>12500-062</td>
			<td>Basal Medium&#160;</td>
		</tr>
		<tr>
			<td>HyClone Dulbecco&#39;s PBS powder</td>
			<td>Fisher Scientific</td>
			<td>dSH30013.04</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td rowspan="2">Dispase II, powder</td>
			<td rowspan="2"><a href="http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html">Life Technologies</a></td>
			<td rowspan="2">17105-041</td>
			<td rowspan="2">Neutral protease, Cell dissociation</td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>
Table 3. Primers for detection of virus genomic integration.
<table>
	<tbody>
		<tr>
			<td>Primer&#39;s Name</td>
			<td>Forward (Fwd)&#160; 5&#8217; --&#62;3&#8217;</td>
			<td>Reverse (Rev) 5&#8217;--&#62;3&#8217;</td>
			<td>Discription&#160;</td>
		</tr>
		<tr>
			<td>pSIN EF1a Fwd</td>
			<td>TTC CAT TTC AGG TGT CGT GA</td>
			<td>--</td>
			<td>EF1a promoter sequence</td>
		</tr>
		<tr>
			<td>GATA1 Rev</td>
			<td>--</td>
			<td>TCC CTG TAG TAG GCC AGT GC</td>
			<td>&#160;Coding Region&#160;</td>
		</tr>
		<tr>
			<td>GATA2 Rev</td>
			<td>--</td>
			<td>GGT TGG CAT AGT AGG GGT TG</td>
			<td>&#160;Coding Region&#160;</td>
		</tr>
		<tr>
			<td>TAL1 Rev</td>
			<td>--</td>
			<td>AGG CGG AGG ATC TCA TTC TT</td>
			<td>&#160;Coding Region&#160;</td>
		</tr>
		<tr>
			<td>LMO2 Rev</td>
			<td>--</td>
			<td>GGC CCA GTT TGT AGT AGA GGC</td>
			<td>&#160;Coding Region&#160;</td>
		</tr>
		<tr>
			<td>ETV2 Rev</td>
			<td>--</td>
			<td>GAA CTT CTG GGT GCA GTA AC&#160;</td>
			<td>&#160;Coding Region&#160;</td>
		</tr>
	</tbody>
</table>

direct induction of hemogenic endothelium and blood by overexpression of transcription factors in human pluripotent stem cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

The schematic diagram of HE and blood induction from hPSCs by overexpression of transcription factors is shown in Figure 1. ETV2 with GATA1 or GATA2 combination induces pan-myeloid hematopoiesis, while a GATA2, TAL1 +/- LMO2 combination induces predominantly erythro-megakaryocytic hematopoiesis. Both TF combinations directly induced HE cells that subsequently transformed into blood progenitors with a distinct spectrum of hematopoietic differentiation. Differentiation of hPSCs from the pluripotent state t...

Watch this Scientific Journal Video about Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells at JoVE.com

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

direct-induction-hemogenic-endothelium-blood-overexpression

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Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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 ...

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 ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

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.