<meta charset="utf8"></head><body>العضيات الوعائية المشتقة من hiPSC لفحص الأدوية ونمذجة الأمراض

<ol>
	<li>Park, S. E., Georgescu, A., Huh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids-on-a-chip.">Organoids-on-a-chip.</a> Science. 364 (6444), 960-965 (2019).</li><li>Takebe, T., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+by+design.">Organoids by design.</a> Science. 364 (6444), 956-959 (2019).</li><li>Griffith, L. G., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+complex+3d+tissue+physiology+in+vitro.">Capturing complex 3d tissue physiology in vitro.</a> Nat Rev Mol Cell Bio. 7 (3), 211-224 (2006).</li><li>Lancaster, M. A., 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Pasca, A. 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=Functional+cortical+neurons+and+astrocytes+from+human+pluripotent+stem+cells+in+3d+culture.">Functional cortical neurons and astrocytes from human pluripotent stem cells in 3d culture.</a> Nat Methods. 12 (7), 671-678 (2015).</li><li>Takasato, 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=Kidney+organoids+from+human+iPS+cells+contain+multiple+lineages+and+model+human+nephrogenesis.">Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.</a> Nature. 526 (7574), 564-568 (2015).</li><li>Koike, H., 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=Modelling+human+hepato-biliary-pancreatic+organogenesis+from+the+foregut-midgut+boundary.">Modelling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary.</a> Nature. 574 (7776), 112-116 (2019).</li><li>Takebe, T., 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=Vascularized+and+functional+human+liver+from+an+iPSC-derived+organ+bud+transplant.">Vascularized and functional human liver from an iPSC-derived organ bud transplant.</a> Nature. 499 (7459), 481-484 (2013).</li><li>Osakada, 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=Toward+the+generation+of+rod+and+cone+photoreceptors+from+mouse,+monkey+and+human+embryonic+stem+cells.">Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.</a> Nat Biotechnol. 26 (2), 215-224 (2008).</li><li>Potente, M., Gerhardt, H., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+and+therapeutic+aspects+of+angiogenesis.">Basic and therapeutic aspects of angiogenesis.</a> Cell. 146 (6), 873-887 (2011).</li><li>Wimmer, R. A., Leopoldi, A., Aichinger, M., Kerjaschki, D., Penninger, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+blood+vessel+organoids+from+human+pluripotent+stem+cells.">Generation of blood vessel organoids from human pluripotent stem cells.</a> Nat Protoc. 14 (11), 3082-3100 (2019).</li><li>Wimmer, R. A., 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+blood+vessel+organoids+as+a+model+of+diabetic+vasculopathy.">Human blood vessel organoids as a model of diabetic vasculopathy.</a> Nature. 565 (7740), 505-510 (2019).</li><li>He, 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=Mapping+morphological+malformation+to+genetic+dysfunction+in+blood+vessel+organoids+with+22q11.2+deletion+syndrome.">Mapping morphological malformation to genetic dysfunction in blood vessel organoids with 22q11.2 deletion syndrome.</a> Biorxiv. , (2021).</li><li>He, 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+vascular+organoids+with+a+mosaic+akt1+mutation+recapitulate+proteus+syndrome.">Human vascular organoids with a mosaic akt1 mutation recapitulate proteus syndrome.</a> Biorxiv. , (2024).</li><li>Chen, 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=A+versatile+polypharmacology+platform+promotes+cytoprotection+and+viability+of+human+pluripotent+and+differentiated+cells.">A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.</a> Nat Methods. 18 (5), 528-541 (2021).</li><li>Ungrin, M. D., Joshi, C., Nica, A., Bauwens, C., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible,+ultra+high-throughput+formation+of+multicellular+organization+from+single+cell+suspension-derived+human+embryonic+stem+cell+aggregates.">Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.</a> PLoS ONE. 3 (2), e1565 (2008).</li><li>Hwang, Y. -. 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=Microwell-mediated+control+of+embryoid+body+size+regulates+embryonic+stem+cell+fate+via+differential+expression+of+wnt5a+and+wnt11.">Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of wnt5a and wnt11.</a> Proc Natl Acad Sci. 106 (40), 16978-16983 (2009).</li><li>Antonchuk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+embryoid+bodies+from+human+pluripotent+stem+cells+using+aggrewell+plates.">Formation of embryoid bodies from human pluripotent stem cells using aggrewell plates.</a> Methods Mol Biol. 946, 523-533 (2013).</li><li>Lippmann, E. 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=Derivation+of+blood-brain+barrier+endothelial+cells+from+human+pluripotent+stem+cells.">Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells.</a> Nat Biotechnol. 30 (8), 783-791 (2012).</li><li>Nishihara, H., 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=Differentiation+of+human+pluripotent+stem+cells+to+brain+microvascular+endothelial+cell-like+cells+suitable+to+study+immune+cell+interactions.">Differentiation of human pluripotent stem cells to brain microvascular endothelial cell-like cells suitable to study immune cell interactions.</a> Star Protoc. 2 (2), 100563 (2021).</li><li>Qian, T., 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=Directed+differentiation+of+human+pluripotent+stem+cells+to+blood-brain+barrier+endothelial+cells.">Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells.</a> Sci Adv. 3 (11), e1701679 (2017).</li><li>Stebbins, M. 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=Human+pluripotent+stem+cell-derived+brain+pericyte-like+cells+induce+blood-brain+barrier+properties.">Human pluripotent stem cell-derived brain pericyte-like cells induce blood-brain barrier properties.</a> Sci Adv. 5 (3), eaau7375 (2019).</li><li>Campisi, 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=3D+self-organized+microvascular+model+of+the+human+blood-brain+barrier+with+endothelial+cells,+pericytes+and+astrocytes.">3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes.</a> Biomaterials. 180, 117-129 (2018).</li><li>Osaki, T., Sivathanu, V., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+3D+vascular+and+neuronal+networks+in+a+microfluidic+platform.">Engineered 3D vascular and neuronal networks in a microfluidic platform.</a> Sci Rep. 8 (1), 5168 (2018).</li><li>Osaki, T., Uzel, S. G. M., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microphysiological+3D+model+of+amyotrophic+lateral+sclerosis+(ALS)+from+human+iPS-derived+muscle+cells+and+optogenetic+motor+neurons.">Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons.</a> Sci Adv. 4 (10), eaat5847 (2018).</li><li>Sakurai, 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=A+microengineered+vascularized+bleeding+model+that+integrates+the+principal+components+of+hemostasis.">A microengineered vascularized bleeding model that integrates the principal components of hemostasis.</a> Nat Commun. 9 (1), 509 (2018).</li><li>Kim, 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=Engineering+of+a+biomimetic+pericyte-covered+3d+microvascular+network.">Engineering of a biomimetic pericyte-covered 3d microvascular network.</a> PLoS ONE. 10 (7), e0133880 (2015).</li><li>Kosyakova, N., 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=Differential+functional+roles+of+fibroblasts+and+pericytes+in+the+formation+of+tissue-engineered+microvascular+networks+in+vitro.">Differential functional roles of fibroblasts and pericytes in the formation of tissue-engineered microvascular networks in vitro.</a> Biorxiv. , (2019).</li><li>Whisler, J. A., Chen, M. B., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+perfusable+microvascular+network+morphology+using+a+multiculture+microfluidic+system.">Control of perfusable microvascular network morphology using a multiculture microfluidic system.</a> Tissue Eng Part C Methods. 20 (7), 543-552 (2013).</li><li>Haase, K., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+on-chip+vascularization.">Advances in on-chip vascularization.</a> Regen Med. 12 (3), 285-302 (2017).</li><li>Wong, K. H. K., Chan, J. M., Kamm, R. D., Tien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+models+of+vascular+functions.">Microfluidic models of vascular functions.</a> Annu Rev Biomed Eng. 14 (1), 205-230 (2012).</li><li>Boerckel, J. D., Uhrig, B. A., Willett, N. J., Huebsch, N., Guldberg, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+regulation+of+vascular+growth+and+tissue+regeneration+in+vivo.">Mechanical regulation of vascular growth and tissue regeneration in vivo.</a> Proc Natl Acad Sci. 108 (37), E674-E680 (2011).</li><li>Homan, K. A., 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=Flow-enhanced+vascularization+and+maturation+of+kidney+organoids+in+vitro.">Flow-enhanced vascularization and maturation of kidney organoids in vitro.</a> Nat Methods. 16 (3), 255 (2019).</li><li>Mansour, A. A., 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=An+in+vivo+model+of+functional+and+vascularized+human+brain+organoids.">An in vivo model of functional and vascularized human brain organoids.</a> Nat Biotechnol. 36 (5), 432-441 (2018).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol (1000x)</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma-Aldrich</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure F(ab&#39;)2 Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immuno Research Labs</td>
			<td>711-546-152</td>
			<td>reconstitute with 0.25 mL 50% glycerol,&#160; store at -20 &#176;C for up to 1 year, dilution ratio for use: 1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure F(ab&#39;)2 Fragment Donkey Anti-Goat IgG (H+L)&#160;</td>
			<td>Jackson Immuno Research Labs</td>
			<td>705-606-147</td>
			<td>reconstitute with 0.25 mL 50% glycerol,&#160; store at -20&#176;C for up to 1 year, dilution ratio for use: 1:1000</td>
		</tr>
		<tr>
			<td>B27 supplement minus vitamin A (50x)</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20 &#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>ProSpec</td>
			<td>CYT-081</td>
			<td>reconstitute with sterile ddH2O at a concentration of 100 ng/&#181;L, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>CD31 antibody&#160;</td>
			<td>abcam</td>
			<td>ab28364</td>
			<td>dilution ratio: 1:400</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>022626001</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris Bioscience</td>
			<td>4423/10</td>
			<td>make the stock solution at 12 mM with DMSO, and store at -20 &#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>Chroman 1</td>
			<td>Tocris</td>
			<td>7163/10</td>
			<td>make the stock solution at 100 &#181;M with DMSO, and store at -20 &#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>Collagen I&#160;</td>
			<td>Corning</td>
			<td>40236</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>AXRMP/Ti2</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Elplasia Plates</td>
			<td>Corning</td>
			<td>4441</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL automated cell counter</td>
			<td>Thermo Fisher</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, powder</td>
			<td>Gibco</td>
			<td>12500062</td>
			<td>make 10x stock solution with biograde ddH2O and sterilize it with a 0.2 &#181;m filter. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Edi042A</td>
			<td>Cedars Sinai</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emricasan</td>
			<td>Medchemexpress LLC</td>
			<td>HY1039610MG</td>
			<td>make the stock solution at 1 mM with DMSO, and store at -20 &#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>ERG antibody</td>
			<td>Cell Singali Technology</td>
			<td>97249</td>
			<td>dilution ratio: 1:400</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Premium</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td>thaw at 4 &#176;C&#160; and aliquot, store at&#160; -20 &#176;C for up to five years, or store at 4 &#176;C for up to a month</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>ProSpec</td>
			<td>CYT557</td>
			<td>reconstitute with sterile ddH2O at a concentration of 100 ng/&#181;L, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Fluorodish Cell Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Tocris Bioscience</td>
			<td>1099</td>
			<td>reconstitute with DMSO at a concentration of 12 mM, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>Gibco</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement (100x)</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin, 100 kU</td>
			<td>MilliporeSigma</td>
			<td>375095100KU</td>
			<td>reconstitute with sterile ddH2O at a concentration of 4 kU/mL</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>13998123</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>Matrigel, GFR Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356230</td>
			<td>thaw at 4 &#176;C, aliquot and store at -80&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (NEAA, 100x)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water</td>
			<td>Corning</td>
			<td>46000CV</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus kit</td>
			<td>STEMCELL Technologies</td>
			<td>1001130</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>N2 supplement (100x)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>NaOH solution (1 M)</td>
			<td>Cytiva&#160;HyClone</td>
			<td>SH31088.01</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuroBasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements, ver 5.42</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson Immuno Research Labs</td>
			<td>17000121</td>
			<td>reconstitute with 10 mL sterile ddH2O, store at 4 &#176;C for up to two weeks</td>
		</tr>
		<tr>
			<td>paraformaldehyde, 20%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713S</td>
			<td>dilute with PBS</td>
		</tr>
		<tr>
			<td>PBST, 20x</td>
			<td>Thermofisher</td>
			<td>28352</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGFR&#946; antibody</td>
			<td>R&#38;D</td>
			<td>AF385</td>
			<td>dilution ratio: 1:400</td>
		</tr>
		<tr>
			<td>polyamine supplement (1000x)</td>
			<td>Sigma-Aldrich</td>
			<td>P8483</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapiclear clearing reagent, 1.49</td>
			<td>SUNJIN LAB</td>
			<td>RC149001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (7.5%)</td>
			<td>Gibco</td>
			<td>25080094</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate monohydrate</td>
			<td>Thermofisher</td>
			<td>J6228822</td>
			<td>dissolve with ddH2O for 1% (wt/v) stock solution</td>
		</tr>
		<tr>
			<td>TBST, 20x</td>
			<td>Thermofisher</td>
			<td>28360</td>
			<td></td>
		</tr>
		<tr>
			<td>trans-ISRIB</td>
			<td>Tocris Bioscience</td>
			<td>5284/10</td>
			<td>make the stock solution at 1 mM with DMSO, and store at -20&#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>Tritin X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Thermo Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF-A</td>
			<td>ProSpec</td>
			<td>CYT-116</td>
			<td>reconstitute with sterile ddH2O at a concentration of 100 ng/&#181;L, aliquot and store at -20&#176;C</td>
		</tr>
	</tbody>
</table>

vascular organoid generation from human-induced pluripotent stem cells

In vitro microphysiological models, including organoid and tissue-on-a-chip systems, have emerged over the past decade1,2,3. These three-dimensional (3D), human cell-derived systems address the limitations in conventional two-dimensional (2D) cell culture and animal models, such as the lack of many components in the physiological microenvironment and genetic differences across species, respectively. They provide more physiological insights and are more suitable for disease modeling and drug screening than conventional models. Among the microphysiological models, organoids derived from human induced pluripotent stem cells (hiPSCs) have been proven to recapitulate the architectural features of native human tissues effectively and have been developed to mimic different human organs, including the brain4,5, kidney6, liver7,8&#160;and retina9. Here, we particularly focus on the generation of vascular organoids from hiPSCs and outline a streamlined protocol that aims to minimize the variations between different organoid samples and batches, thereby improving the overall reproducibility.
Vasculature plays a crucial role in maintaining physiological homeostasis across all human organs. The formation of vasculature involves the processes of vasculogenesis and angiogenesis, orchestrated through the interactions between endothelial and mural (e.g., pericytes and vascular smooth muscle cells) cells and influenced by various stimuli10. Penninger, Wimmer&#160;and colleagues developed the first vascular organoid generation method11,12 that mimics these two processes. With the organoids generated from this method, their group12 and us13,14 have demonstrated the potential of vascular organoids for modeling vascular malfunctions in diseases. For example, in our recent works13,14, distinct phenotypes were observed, such as vessel sprouting and mural cell composition variations, between disease-mimicking vascular organoids and their wild-type counterparts. These examples highlight that the vascular&#160;organoid model could serve as a platform for drug screening by mimicking disease-related vascular malfunctions.
Built upon the initial vascular organoid generation works11,12, a revised protocol is presented that includes the use of microwell to facilitate homogenous embryoid body (EB) formation, a critical factor in minimizing the variations often seen within the same organoid generation batch. Additionally, the CEPT cocktail, a combination of chroman 1, emricasan, polyamines&#160;and the integrated stress response inhibitor (trans-ISRIB)15, is employed&#160;to improve the viability of hiPSCs and differentiated cells during the EB formation process. This modification is mainly to reduce the variations between different organoid samples and batches. Uniform vascular organoids can be produced with this roughly two-week-long protocol, where vasculogenesis is induced to help endothelium organize into primitive tubular networks on Day 1, followed by angiogenesis induction on Day 5 to develop complex vascular networks in organoids. On Day 12-15, the generated organoids should show mature vascular networks composed of both endothelium and mural cells.

<meta charset="utf8"></head><body>أضواء الكاتب: تحسين قابلية التكاثر في العضيات الوعائية باستخدام مثبطات ROCK وحبس Microwell

توليد العضوية الوعائية من الخلايا الجذعية متعددة القدرات التي يسببها الإنسان

This study introduces a modified method for scalable production of homogeneous vascular organoids from human IPSCs. By using novel ROCK inhibitors and the microwell confinement, the approach enhances reproducibility and minimize batch-to-batch variations, making it suitable for downstream morphological analysis and disease modeling applications. Single-cell sequencing technologies have been extensively utilized in organoid research to unravel the genomic, epigenomic, and the transcriptomic heterogeneity and dynamics within cellular populations.3D imaging technologies such as tissue clearing and the light-sheet microscopy facilitate rapid, high-resolution imaging of cellular morphology and the tissue architecture in a three-dimensional context. The throughput and the standardization are key challenges in organoid research. Manual handling is labor-intensive and limits the throughput, making robotic automation a promising solution.Standardization of organoid culture, including IPSC quality control and the differentiation protocol optimization can significantly enhance reproducibility and minimize variations between batches and cell lines.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

vascular-organoid-generation-from-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Bioengineering

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

860 Views.  Columbia University. تقدم هذه الدراسة طريقة معدلة للإنتاج القابل للتطوير للعضيات الوعائية المتجانسة من IPSCs البشرية. باستخدام مثبطات ROCK الجديدة وحبس الآبار الصغيرة ، يعزز هذا النهج قابلية التكاثر ويقلل من الاختلافات من دفعة إلى أخرى ، مما يجعله مناسبا للتحليل المورفولوجي ال?...

Video: أضواء الكاتب: تحسين قابلية التكاثر في العضيات الوعائية باستخدام مثبطات ROCK وحبس Microwell

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Consistent Generation of Human Vascular Organoids Using Microwell Technology

To begin, place a 50 milliliter tube on ice and add collagen one solution along with other components sequentially. Shake the tube to mix thoroughly. Using a P10 pipette, add one molar sodium hydroxide solution dropwise while shaking the mix solution to adjust the pH to 7.3.Add 1.25 milliliters of basement membrane matrix to the five milliliter collagen one mixture and mix the solution gently. Next place a 12-well plate on ice for two minutes. Using wide bore tips, add the collagen one basement membrane matrix mixture slowly to the 12-well plate.Place the 12-well plate in the 37 degree Celsius incubator for two hours to solidify the first layer of hydrogel. Keep the remaining collagen one basement membrane matrix mixture on ice for later use. Then transfer approximately 60 cell aggregates into a 1.5 milliliter microcentrifuge tube.And cool the tube on ice for five minutes. Using a P200 pipette tip, carefully remove the medium from the tube. Add 1.5 milliliters of the collagen one basement membrane mixture, dropwise to the cell aggregates, and gently mix them using a wide bore P200 pipette tip to resuspend.Transfer the plate with the first layer of hydrogel from the incubator to the biosafety cabinet. Add 1.5 milliliters of the aggregate suspension onto the cured hydrogel layer and gently shake the plate to distribute the aggregates evenly. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for two hours.Prewarm vascular differentiation medium two in a 37 degrees Celsius water bath for 20 minutes. Add two milliliters of medium to each well and culture the cells at 37 degrees Celsius with 5%carbon dioxide. Change the medium every three days with fresh pre-warmed vascular differentiation medium two.Organoids expressed CD31, ERG1, and PDGFR beta, indicating the presence of vascular endothelial cells and parasites. Vascular networks were visualized via whole mount immunofluorescence microscopy after tissue clearing. Mature organoids stained on day 17 showed vascular networks encapsulating the spheroids within the gel block.Organoids retrieved on day 28 had vascular networks wrapping around the spheroids instead of radially expanding.