Name: human pluripotent stem cell culture on polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness under xeno-free conditions
Uploaded: 2018-02-03T15:00:00
Description: Watch this Scientific Journal Video about Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions at JoVE.com

This research was partially supported by the Ministry of Science and Technology, Taiwan under grant numbers 106-2119-M-008 -003, 105-2119-M-008-006, and 104-2221-E-008-107-MY3. This research was also supported by the Taiwan Landseed Hospital Project (NCU-LSH-105-A-001). A Grant-in-Aid for Scientific Research (number 15K06591) from the Ministry of Education, Culture, Sports, Science and Technology of Japan is also acknowledged. A. Higuchi would like to acknowledge for the International Scientific Partnership Program (ISPP-0062) Vice Rectorate for Graduate Studies and Research, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia.

The experiments in this study were approved by the ethics committees of the Taiwan Landseed Hospital (IRB-13-05) and the National Central University. All experiments were conducted in accordance with all relevant and applicable governmental and institutional guidelines and regulations during this study.
1. Solution and Media Preparation
<ol>
	<li>Polymer purification
	<ol>
		<li>Purify P-IA with carboxylic acid group with a degree of hydrolysis of &#62;96.5% by washing P-IA ...

<ol>
	<li>Higuchi, A., Ling, Q. D., Chang, Y., Hsu, S. T., Umezawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+cues+of+biomaterials+guide+stem+cell+differentiation+fate.">Physical cues of biomaterials guide stem cell differentiation fate.</a> Chem. Rev. 113, 3297-3328 (2013).</li><li>Higuchi, 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=Physical+cues+of+cell+culture+materials+lead+the+direction+of+differentiation+lineages+of+pluripotent+stem+cells.">Physical cues of cell culture materials lead the direction of differentiation lineages of pluripotent stem cells.</a> J. Mater. Chem. B. 3, 8032-8058 (2015).</li><li>Wen, J. 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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=The+combined+influence+of+substrate+elasticity+and+surface-grafted+molecules+on+the+ex+vivo+expansion+of+hematopoietic+stem+and+progenitor+cells.">The combined influence of substrate elasticity and surface-grafted molecules on the ex vivo expansion of hematopoietic stem and progenitor cells.</a> Biomaterials. 34, 7632-7644 (2013).</li><li>Higuchi, A., Iijima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DSC+investigation+of+the+states+of+water+in+poly(vinyl+alcohol)+membranes.">DSC investigation of the states of water in poly(vinyl alcohol) membranes.</a> Polymer. 26, 1207-1211 (1985).</li><li>Higuchi, A., Iijima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DSC+investigation+of+the+states+of+water+in+poly(vinyl+alcohol-co-itaconic+acid)+membranes.">DSC investigation of the states of water in poly(vinyl alcohol-co-itaconic acid) membranes.</a> Polymer. 26, 1833-1837 (1985).</li><li>Higuchi, 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=Long-term+xeno-free+culture+of+human+pluripotent+stem+cells+on+hydrogels+with+optimal+elasticity.">Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity.</a> Sci Rep. 5, 18136 (2015).</li><li>Wang, P. 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=Pluripotency+maintenance+of+amniotic+fluid-derived+stem+cells+cultured+on+biomaterials.">Pluripotency maintenance of amniotic fluid-derived stem cells cultured on biomaterials.</a> J Mater Chem B. 3, 3858-3869 (2015).</li><li>Muduli, 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=Proliferation+and+osteogenic+differentiation+of+amniotic+fluid-derived+stem+cells.">Proliferation and osteogenic differentiation of amniotic fluid-derived stem cells.</a> J Mater Chem B. 5, 5345-5354 (2017).</li><li>Muduli, 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=Stem+cell+culture+on+polyvinyl+alcohol+hydrogels+having+different+elasticity+and+immobilized+with+ECM-derived+oligopeptides.">Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides.</a> JPoly Eng. 37, 647 (2017).</li><li>Chen, Y. 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A., Chang, Y., Umezawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+for+the+feeder-free+culture+of+human+embryonic+stem+cells+and+induced+pluripotent+stem+cells.">Biomaterials for the feeder-free culture of human embryonic stem cells and induced pluripotent stem cells.</a> Chem Rev. 111 (5), 3021-3035 (2011).</li><li>Higuchi, 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=Design+of+polymeric+materials+for+culturing+human+pluripotent+stem+cells:+Progress+toward+feeder-free+and+xeno-free+culturing.">Design of polymeric materials for culturing human pluripotent stem cells: Progress toward feeder-free and xeno-free culturing.</a> Prog Polym Sci. 39, 1348-1374 (2014).</li><li>Higuchi, 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=Stem+cell+therapies+for+myocardial+infarction+in+clinical+trials:+bioengineering+and+biomaterial+aspects.">Stem cell therapies for myocardial infarction in clinical trials: bioengineering and biomaterial aspects.</a> Lab Invest. 97, 1167-1179 (2017).</li><li>Higuchi, 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=Stem+cell+therapies+for+reversing+vision+loss.">Stem cell therapies for reversing vision loss.</a> Trends Biotechnol. 35, 1102-1117 (2017).</li></ol>

P-IA-oligoECM and P-IA-ECM hydrogels with varying stiffness were developed for the long-term expansion of human ES and iPS cells maintaining their pluripotency for over ten passages in xeno-free conditions, as well as for the culture of human AFS cells, ADS cells, and hematopoietic stem cells25,28,32. P-IA hydrogels immobilized with oligoECM are an excellent candidate for cell cultivation materials to investigate the effect of c...

The fate of stem cell differentiation into a specific lineage of cells and the long-term proliferation of stem cells, especially human induced pluripotent (iPS) cells and human embryonic stem (ES) cells, is known to be regulated by inhibitors, growth factors, and/or small bioactive molecules in culture media. Recently, the physical cues of the biomaterials, particularly the stiffness of cell culture biomaterials, have been recognized to be an important factor guiding the fate of stem cell proliferation and differentiation1,2,3,4,5,6. Therefore, several researchers have started to investigate the fate of stem cells, which are cultured on hydrogels, on differentiation, mainly using polyacrylamide hydrogels with varying stiffness.
The stiffness of biomaterials can control focal adhesions, cell morphology, cell phenotype, and stem cell adhesion, especially in two-dimensional (2-D) cultivation1,2,3,5. Mechano-sensing of biomaterials by stem cells is generally controlled by focal adhesion signaling via integrin receptors. NMMIIA, nonmuscle myosin IIA-dependent contractility of the cytoskeleton of actin plays a critical role in the mechanosensing process of stem cells in 2-D cell cultivation systems3,4,5,7,8,9,10,11.
Engler and his colleagues developed an interesting notion that adult stem cells, such as bone marrow stem (BMS) cells cultivated on cell culture biomaterials with a similar stiffness to that of specific tissues, tend to differentiate into cells originated from specific tissues5. BMS cells incubated on 2-D soft polyacrylamide hydrogels coated with collagen type I (with a stiffness comparable to that of brain tissues) in expansion media were spontaneously induced to differentiate into early neuron lineages, whereas BMS cells cultured on hydrogels with a stiffness similar to that of muscle or collagenous bone tissues were found to induce differentiation into early lineages of myocytes and osteoblasts, respectively, on 2-D polyacrylamide hydrogels3,5. Many researchers have investigated the stem cell fate of differentiation cultured on polyacrylamide hydrogels immobilized with collagen type I12,13,14,15,16,17,18,19,20,21. However, it should be mentioned that some contradictory reports1,18,22,23,24 exist for the well-known idea suggested by Engler et al.5 This is because Engler&#39;s idea5 was developed solely on polyacrylamide hydrogels and their results have originated from specific characteristics of the biomaterial (polyacrylamide), and not solely from the physical cue (stiffness) of the biomaterial. Therefore, it is important to develop another type of hydrogel, of which the stiffness can be controlled by crosslinking of the hydrogels. For this purpose, bioinert hydrogels were developed, which were prepared from polyvinyl alcohol-co-itaconic acid (P-IA) with a different stiffness, which was controlled by the crosslinking degree with a changing crosslinking time25,26,27,28,29,30,31,32. The stem cells can be cultivated on nonmodified P-IA hydrogels, as well as P-IA hydrogels grafted with extracellular matrices (ECMs) and oligopeptides. In a previous study25, human hematopoietic stem cells (hHSCs) from umbilical cord blood were cultivated on P-IA hydrogels with different stiffness values ranging from a 3 kPa to 30 kPa storage modulus where fibronectin or an oligopeptide derived from fibronectin (CS1, EILDVPST) was grafted onto the P-IA hydrogels. High ex vivo fold expansion of hHSCs was observed in the P-IA hydrogels grafted with CS1 or fibronectin, which displayed an intermediate stiffness ranging from 12 kPa to 30 kPa25.
Human iPS and ES cells cannot be cultivated on conventional tissue culture polystyrene (TCP) dishes33,34 because human ES and iPS cells require specific binding to ECMs, such as vitronectin or laminin to maintain their pluripotency during long-term culture. Therefore, several structures of oligopeptide-grafted P-IA hydrogels with optimal stiffness characteristics were designed and prepared in formations of a single chain, a single chain with a joint segment, a dual chain with a joint segment, and a branched-type chain32. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of ECMs. The P-IA hydrogels grafted with vitronectin-derived oligopeptides with a dual chain or joint segment, which have a storage modulus at approximately 25 kPa, supported the long-term culture of human ES and iPS cells for over 12 passages under xeno-free and chemical defined conditions32. The joint segment and dual chain with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells32. Here, a protocol for preparing P-IA hydrogels (with a storage modulus from 10 kPa to 30 kPa, which was measured under wet conditions in the air) grafted with and without oligopeptides or ECMs is described. How to culture and passage several stem cells (including amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells) is shown.

<table>
	<tbody>
		<tr>
			<td>GTPGPQGIAGQRGVV</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: Cyclic RGD, cRGD</td>
		</tr>
		<tr>
			<td>GACRGDCLGA</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: FN1</td>
		</tr>
		<tr>
			<td>KGGAVTGRGDSPASS</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: CS1</td>
		</tr>
		<tr>
			<td>EILDVPST</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN1</td>
		</tr>
		<tr>
			<td>KGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: HBP1</td>
		</tr>
		<tr>
			<td>GKKQRFRHRNRKG</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: HBP2C</td>
		</tr>
		<tr>
			<td>CGGGKKQRFRHRNRKG</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN1G</td>
		</tr>
		<tr>
			<td>GGGGKGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN2C</td>
		</tr>
		<tr>
			<td>GCGGKGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Extracellular matrix 
			Abbreviation: rVN</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Thermo Fisher scientific</td>
			<td>A14700</td>
			<td>Specification: Extracellular matrix 
			Abbreviation: FN</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F2006</td>
			<td>Specification: Commercially available coating material 
			Abbreviation: Synthemax II</td>
		</tr>
		<tr>
			<td>Synthemax II</td>
			<td>Corning</td>
			<td>3535</td>
			<td>Specification: Polymer</td>
		</tr>
		<tr>
			<td>Polyvinylalcohol-co-itaconic acid</td>
			<td>Japan VAM &#38; Poval</td>
			<td>AF-17</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Na2SO4</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Sigma-Aldrich</td>
			<td>339741</td>
			<td>Specification: Chemical 
			Abbreviation: EDC</td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#39;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>E7750</td>
			<td>Specification: Chemical 
			Abbreviation: NHS</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>56480</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Specification: Cell culture consumable</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>3008</td>
			<td>Specification: Cell culture consumable</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma-Aldrich</td>
			<td>SI-D4693</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>Essential 6</td>
			<td>Thermo Fisher scientific</td>
			<td>A1516401</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>Essential 8</td>
			<td>Thermo Fisher scientific</td>
			<td>A1517001</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher scientific</td>
			<td>11330-032</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher scientific</td>
			<td>12800-017</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>MCDB 201</td>
			<td>Sigma-Aldrich</td>
			<td>M6770</td>
			<td>Specification: ES cell</td>
		</tr>
		<tr>
			<td>Human ES cell</td>
			<td>WiCell Research Institute, Inc..</td>
			<td>WA09</td>
			<td>Specification: iPS cell</td>
		</tr>
		<tr>
			<td>Human iPS cell</td>
			<td>Riken Cell Bank</td>
			<td>HS0077</td>
			<td>Specification: 35 mm 
			Abbreviation: TCP</td>
		</tr>
		<tr>
			<td>TCP dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Specification: 60 mm 
			Abbreviation: TCP</td>
		</tr>
		<tr>
			<td>TCP dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td>Specification: 24 well dish</td>
		</tr>
		<tr>
			<td>24 well dish</td>
			<td>Corning</td>
			<td>353047</td>
			<td>Specification: Blocking agent</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8806</td>
			<td>Specification: Detection reagent</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase live stain</td>
			<td>Thermo Fisher scientific</td>
			<td>A14353</td>
			<td>Specification: Detection reagent</td>
		</tr>
		<tr>
			<td>Hematoxylin &#38; eosin</td>
			<td>Sigma-Aldrich</td>
			<td>1.05175</td>
			<td>Specification: EB formation dish</td>
		</tr>
		<tr>
			<td>6-well ultralow attachment dish</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Specification: Coating material</td>
		</tr>
		<tr>
			<td>gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td>Specification: Coating material</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Specification: Mice</td>
		</tr>
		<tr>
			<td>NOD-SCID mice</td>
			<td>National Applied Research Laboratories</td>
			<td>None</td>
			<td>Specification: Serum</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biological Industries</td>
			<td>04-001-1A</td>
			<td>Specification: Antibiotic</td>
		</tr>
		<tr>
			<td>antimycotic antibiotic</td>
			<td>Thermo Fisher scientific</td>
			<td>15240-062</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Nanog</td>
			<td>Invitrogen</td>
			<td>MA1-017</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for SSEA4</td>
			<td>Abcam</td>
			<td>ab16287</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for OCT3/4</td>
			<td>Invitrogen</td>
			<td>PA5-27438</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Sox2</td>
			<td>Invitrogen</td>
			<td>48-1400</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Smooth Muscle Actin</td>
			<td>Invitrogen</td>
			<td>PA5-19465</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for AFP</td>
			<td>Invitrogen</td>
			<td>PA5-21004</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody GFAP</td>
			<td>Invitrogen</td>
			<td>MA5-15086</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 &#8211; conjugated Goat anti-Mouse antibody</td>
			<td>Invitrogen</td>
			<td>A-21422</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 &#8211; conjugated Goat anti-Rabbit antibody</td>
			<td>Invitrogen</td>
			<td>A-11008</td>
			<td>Specification: Antibody</td>
		</tr>
	</tbody>
</table>

human pluripotent stem cell culture on polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness under xeno-free conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

P-IA hydrogels grafted with ECM-derived oligopeptide (oligoECM) or ECM with different elasticities were prepared by following the reaction scheme, as seen in Figure 1A, using different types of oligoECM (Figure 1B). The elasticities of the hydrogels were regulated by the applied crosslinking intensity (time) (Figure 1C). P-IA hydrogels grafted with vitronectin-derived oligopeptides, which has a stora...

Watch this Scientific Journal Video about Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions at JoVE.com

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

The overall goal of this experiment is to prepare either unmodified or oligopeptide or extra-cellular matrix-grafted polyvinyl alcohol-co-itaconic acid hydrogels with the optimal elasticity for culturing and passaging stem cells. This method can help answer key questions in the stem cell research field about long term content of stem cells under xeno-free conditions. The main advantage of this technique is that it provides a platform to investigate a different hydrogel elasticity on stem cell differentiation into specific lineages.Demonstrating the procedure will be me, Jia-Sin Yang and Yeh-Chia Tseng, graduate students from Professor Higuchi's laboratory. To begin the procedure, place 20 grams of P-IA and 200 milliliters of ethanol in a 500 milliliter conical flask. Stir the mixture for 24 to 30 hours, replacing the ethanol every eight to ten hours.Collect the purified P-IA on Buchner funnel. Dry the P-IA under vacuum at room temperature for 24 hours. Next, over the course of 15 minutes, slowly dissolve 15 milligrams of purified P-IA in 100 milliliters of ultra-pure water.Heat the solution to above 60 degrees Celsius and stir for one hour. Confirm that the solution has become clear before proceeding. Then, turn off the heat.Allow the P-IA solution to cool to room temperature on the cooling hot plate while stirring. Continue stirring the solution at room temperature for 48 hours. Then, leave the P-IA solution undisturbed at room temperature for 20 to 24 hours to ensure that visible air bubbles are not present in the solution.Next, dispense one milliliter of the P-IA solution into each of eight 35 milliliter tissue culture-treated dishes. Dry the dishes in an oven at 45 degrees Celsius for two days to produce P-IA films in the dishes. Next, add to 10 milliliters of ultra-pure water 100 microliters of a high-purity aqueous 25%glutaraldehyde solution, two grams of sodium sulfate and 100 microliters of sulfuric acid.Stir the mixture well to obtain the crosslinking solution. Gently add one milliliter of the crosslinking solution to each dish, being careful not to damage the fragile P-IA films. Leave each film immersed in the crosslinking solution for half, one, two, four, six, twelve, twenty four or forty eight hours to obtain the hydrogels.Rinse each hydrogel with one milliliter of ultra-pure water after its crosslinking period. Sterilize each hydrogel by immersion in an aqueous 75%ethanol solution for one minute. Rinse each sterilized hydrogel with ultrapure water six times.Cover the hydrogels in fresh ultra-pure water, place the lids on the dishes and store the hydrogel dishes in a clean area until ready for activation and grafting. Next, activate each sterilized hydrogel by immersion in one milliliter of an aqueous solution containing 10 milligrams per milliliter each of EDC and NHS, either for one hour at 37 degrees Celsius or four hours at four degrees Celsius. Then, rinse each activated hydrogel with one milliliter of Ph 7.2 phosphate-buffered saline three times.Immerse each hydrogel in one milliliter of a solution of either oligopeptides or ECMs in PBS for 24 hours at four degrees Celsius. Finally, wash each hydrogel with one milliliter of ultra-pure water three times after grafting is complete. Use standard method to culture human ES or IPS cells on a membrane matrix in six centimeter culture dishes until near confluent.Then, incubate the cells in DMMF-12 supplemented with two milligrams per milliliter of dispase-2 at 37 degrees Celsius for eight to ten minutes. Rinse the cells twice with DMMF-12. Add two milliliters of DMMF-12 to each culture dish and attach weekly adherent colonies with the pipette or cell scraper.Collect the cells in 15 milliliter centrifuge tubes and centrifuge the cells at 160 times G for five minutes at 37 degrees Celsius. Discard the supernatant and re-suspend the cells in one milliliter of xeno-free eight component medium. Count the cell density in the suspension.Then, obtain culture dishes or multi-well plates containing sterilized P-IA hydrogels grafted with either oligopeptides or ECMs. Load the appropriate volumes of the cell suspension onto the hydrogels. Passage the cells on grafted P-IA hydrogels until 80%confluent the desired number of times.To begin the immuno-staining process for a 24 well plate of passaged human ES or IPS cells on grafted hydrogels, add 0.5 milliliters of four per cent paraformaldahyde by volume to each well. Incubate the cells at four degrees Celsius for 15 minutes to fix the cells. Aspirate the paraformeldehyde solution from the wells.Add one milliliter of PBS to each well and aspirate the PBS. Perform this PBS rinse three times in total. Then, add 0.5 milliliters of 0.3%octoxynol 9 by volume in PBS to each well.Incubate for 30 minutes at room temperature. Aspirate the octoxynol 9 solution. Add 300 microliters of two per cent BSA and PBS to each well and incubate for another 30 minutes at room temperature.Aspirate the two per cent BSA solution from the cells and add the primary antibodies. Incubate the cells for one day at four degrees Celsius. Then, aspirate the primary antibody solutions and wash the cells with 300 microliters of 05 per cent polysorbate 20 in PBS three times.Add 300 microliters of secondary antibodies specific to the primary IDG subtype in two per cent BSA to each well. Incubate the cells for one hour at room temperature in the absence of light. Finally, wash the cells with 300 microliters of 05 per cent polysorbate 20 and PBS three times in the dark.Image the stained cells with fluorescence microscopy. P-IA hydrogels crosslinked for 24 hours and grafted with vitronectin-derived oligopeptides, supported cultures of human ES and IPS cells for at least 10 to 20 passages. Human ESLs cultured on commercially available coated dishes were found to be more easily differentiated at the edges of the colonies.Differentiated cells were not observed on the hydrogels, indicating that human ESLs maintained their pluripotency. Pluripotency proteins were satisfactorily expressed on human ES cells cultured on hydrogels grafted with three different Vitronectin-derived oliogopeptide designs for ten passages. Satisfactory expression was also observed on recomibinent Vitronectin-coated dishes.Similar results were observed for human IPS cells. Both human ES and IPS cells differentiated into cells derived from all three germ layers after cultivation on P-IA hydrogels and on recombinant Vitronectin-coated dishes for at least ten passages. Human ES cells cultivated on hydrogels grafted with Vitronectin-derived oligopeptides were injected into NOD mice.The harvested teratomas exhibited cells originating from all three germ layers indicating that pluripotency is maintained in vivo. Following this procedure, methods like media screening can be performed to answer additional questions like the effect of calcium arterial and stem cell differentiation and proliferation. After this development, this technique paved the way for researchers in the field of stem cell co-division to explore tissue or organ regeneration in neurodegenerative medicine.Once mastered, this technique can be done in four days if it's performed properly. After watching this video, you should have a good understanding of how to prepare P-IA hydrogel dishes, both with and without ECM. While attempting this procedure, remember to set up a clean environment for preparing P-IA hydrogel dishes.Don't forget that working with ethanol, hot water and the sulfuric acid can be extremely hazardous. The appropriate lab coat, gloves and the protective glasses should always be worn while performing this procedure.

human-pluripotent-stem-cell-culture-on-polyvinyl-alcohol-co-itaconic

Research

JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 &#181;m diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with &gt;95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had &lt;50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...