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 with ethanol. Place 20 g of P-IA into 200 mL of ethanol in a 500-mL conical beaker and agitate on a magnetic stirrer for 24-30 h. Exchange the ethanol with fresh ethanol every 8-10 h.</li>
		<li>Remove P-IA from the ethanol by filtration using a B&#252;chner funnel.</li>
		<li>Dry P-IA by vacuum drying at room temperature for 24 h. 
		NOTE: It is recommended to clean the trap in the vacuum drying system (by the removal of ethanol) frequently, especially during the initial few hours, because the trap tends to become clogged after the removal of a large amount of ethanol from P-IA.</li>
	</ol>
	</li>
	<li>Preparation of the P-IA solution 
	NOTE: Add the polymer very slowly into the solvent (water). It is recommended to take at least 15 min to add the P-IA into the solvent. If solvent is added into the polymer, the polymer would not be dissolved completely. Be careful not to generate explosive boiling of the P-IA solution. Use protective glasses during preparation of the P-IA solution. The heating process of the P-IA solution is essential to dissolve crystalline P-IA. It is suggested (and preferable) to prepare the P-IA solution in a relatively clean experimental room, if possible.
	<ol>
		<li>Dissolve the P-IA in pure water to a 0.050 weight% concentration for the cell cultivation experiment or a 0.50 weight% concentration for the rheometer measurement: for example, dissolve 50 mg of P-IA in 100 mL of pure water for cell culture and 500 mg of P-IA in 100 mL of deionized (DI) water for the rheometer measurements.</li>
		<li>Agitate the P-IA solution for 1 h on the hot plate. 
		NOTE: To avoid explosive boiling, do not heat the solution over 95 &#176;C. Explosive boiling of the polymer solution at a high temperature may generate skin burns. Therefore, perform the heating of P-IA very carefully, and monitor the temperature of the P-IA solution during the heating.</li>
		<li>After the P-IA solution was cooled at ambient temperature, agitate the P-IA solution at room temperature for 48 h. Leave hot P-IA solution on the hot plate without heating, and then leave it without agitation at room temperature for 20-24 h to ensure that air bubbles are not present in the P-IA solution.</li>
	</ol>
	</li>
	<li>Preparation of the crosslinking solution
	<ol>
		<li>Make the composition of the crosslinking solution 1.0 weight% glutaraldehyde from 25% aqueous glutaraldehyde solution, 20.0 weight% Na2SO4 using 99% &#62; purity, and 1.0 weight% H2SO4. For example, for one 6 or 12 well cell culture plate, add 100 &#181;L of glutaraldehyde solution, 2 g of sodium sulfate, and 100 &#181;L of sulfuric acid into 10 mL of pure water.</li>
	</ol>
	</li>
	<li>Human ES/PS cell culture media 
	NOTE: Use DMEM/F12 media, essential 6 media, and essential 8 media for the cell culture.
	<ol>
		<li>Return frozen 50X Essential 8 supplement slowly to the solution overnight by thawing in a 4 &#176;C refrigerator.</li>
		<li>Add one bottle of 50X Essential 8 supplement into one bottle of Essential 8 basal media. Separate the media into small aliquots (50 mL each) in 100 mL centrifugation tubes, then store the media at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. P-IA Hydrogel Dish Preparation
<ol>
	<li>P-IA film preparation
	<ol>
		<li>Inject a 1 mL aliquot of the P-IA solution into a 35 mm TCP dish, and dry the dish in a 45 &#176;C oven for 2 days to produce a P-IA film in a clean bench.</li>
	</ol>
	</li>
	<li>Crosslinking of the P-IA hydrogel dishes
	<ol>
		<li>Immerse the P-IA films into an aqueous crosslinking solution for 0.5, 1, 2, 4, 6, 12, 24, and 48 h. 
		NOTE: &#39;P-IA-X&#39; (e.g., P-IA-12 h) refers to a P-IA hydrogel crosslinked for X h (e.g., 12 h).</li>
		<li>After crosslinking, rinse the P-IA hydrogels with pure water at ambient temperature, and then keep hydrogels in pure water at ambient temperature in a clean bench.</li>
		<li>Sterilize the P-IA hydrogels by immersion in a 75.0 volume/volume% ethanol solution for 1 min, rinse the P-IA hydrogels in pure water six times, and then keep the P-IA hydrogels in pure water until used for cell cultivation.</li>
	</ol>
	</li>
	<li>Preparation of P-IA hydrogel dishes grafted with oligopeptide or ECM
	<ol>
		<li>Activate the P-IA hydrogels via immersion in 1 mL of an aqueous solution containing 10 mg/mL N-(3-Dimethylaminopropyl)-N&#39;-ethylcarbodiimide hydrochloride (EDC) and 10 mg/mL N-hydroxysuccinimide (NHS) for 1 h at 37 &#176;C or 4 h at 4 &#176;C.</li>
		<li>Rinse the P-IA hydrogels with 1 mL of phosphate buffered saline (PBS, pH 7.2) 3 times and immerse the P-IA hydrogels in a PBS solution containing 1 mL of oligopeptide (100-1,500 &#181;g/mL) or ECM (10-100 &#181;g/mL) for 24 h at 4 &#176;C. 
		NOTE: The oligopeptide sequences and ECMs used for stem cell culture are summarized in Table 1.</li>
		<li>After grafting the oligopeptide or ECM, wash the P-IA hydrogels with pure water 3 times. 
		NOTE: The P-IA hydrogels grafted with Y &#181;g/mL of oligopeptide or ECM (Z) are hereafter referred to as P-IA-Xh-Z orP-IA-Xh-Z-Y, where X refers to the crosslinking time (h), Y indicates the concentration of the oligopeptide or ECM, and Z indicates a different oligopeptide or ECM.</li>
	</ol>
	</li>
</ol>
3. Human ES/iPS Cell Culture
<ol>
	<li>Method of passage and maintenance of the undifferentiated human ES and iPS cells
	<ol>
		<li>Maintain human ES (e.g., WA09 or H9) cells or human iPS (e.g., HS0077) cells on Matrigel in Essential 8 media in 6 cm dishes using standard human ES/iPS cell culture protocols28,32.</li>
		<li>Incubate near-confluent human ES/iPS cells with 2.0 mg/mL dispase II in DMEM/F-12 media at 37 &#176;C for 8-10 min and then rinse human ES/iPS cells twice with DMEM/F12 media.</li>
		<li>After the addition of 2 mL of DMEM/F-12 media to human ES/iPS cell culture dishes, detach weakly adherent colonies using a cell scraper or by pipetting.</li>
		<li>Collect the human ES/iPS cells into 15 mL centrifugation tubes and centrifuge human ES/iPS cells at 160 &#215; g for 5 min at 37 &#176;C.</li>
		<li>After centrifugation, discard the DMEM/F12 and suspend the human ES/iPS cells in 1 mL of E8 media, and then count the cell density by using a cell counter.</li>
		<li>Inoculate the ES/iPS cells after the appropriate density adjustment (1-5 x 104 cells per cm2 for passaging or as indicated) into new culture dishes (P-IA hydrogels grafted with the oligopeptide or ECM).</li>
	</ol>
	</li>
</ol>
4. Characterization of Human ES/iPS Cell Characterization
<ol>
	<li>Evaluation of alkaline phosphatase (AP) activity
	<ol>
		<li>Measure the alkaline phosphatase (AP) activity of human ES/iPS cells using a standard alkaline phosphatase live staining.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Perform immunostaining of Tra-1-81, SSEA-4, Sox2, and Oct3/4 on hES and hiPS cells to investigate pluripotency following the conventional protocol28,32.</li>
		<li>Add a 0.5 mL volume of 4% (volume/volume) paraformaldehyde into each 24 well dish in which the human ES/iPS cells were cultured, and subsequently, incubate the dishes for 15 min at 4 &#176;C to fix the cells.</li>
		<li>Aspirate the paraformaldehyde solution from each well. Then, add 1 mL of PBS per well and aspirate PBS to rinse the cells. Perform the rinsing process 3 times.</li>
		<li>Permeabilize cell membranes by adding 0.5 mL of 0.3% (volume/volume) Triton-X 100 solution in PBS for 30 min at room temperature.</li>
		<li>Aspirate the Triton-X 100 solution and add 300 &#181;L of 2% (weight/volume) bovine serum albumin (BSA) in PBS (blocking buffer) into each well, and incubate for 30 min at room temperature.</li>
		<li>Remove the blocking buffer by pipetting, and subsequently, add the desired primary antibodies (Oct3/4 (1:200), Sox2 (1:200), SSEA-4 (1:200), and Tra-1-81(1:200) by pipette, see Table 2) into each well, where 1:200 indicates the antibodies were diluted with PBS 200-fold and incubated for one day at 4 &#176;C.</li>
		<li>Aspirate the primary antibody solution, and wash the cells with 300 &#181;L of 0.05% Tween 20 in PBS solution 3 times at room temperature.</li>
		<li>Add 300 &#181;L of secondary antibodies (1:200, see Table 2), which are specific to the primary IgG subtype, in 2% (weight/volume) BSA solution into each well and incubate for 1 h at room temperature in the dark.</li>
		<li>Wash the cells with 300 &#181;L of 0.05% Tween 20 in PBS solution 3 times at room temperature in the dark.</li>
		<li>Analyze the stained cells by fluorescence microscopy or confocal microscopy.</li>
	</ol>
	</li>
	<li>Embryoid body formation
	<ol>
		<li>Investigate pluripotency of human ES/iPS cells by embryoid body (EB) formation at passages 10 and 20. 
		NOTE: Near 80% confluence in 6-well plates is sufficient for the preparation of EB formation.</li>
		<li>Rinse human ES or iPS cells with 2 mL of DMEM/F-12 media twice, and then immerse the cells in 1.5 mL of Essential 6 media.</li>
		<li>Cut near 80% confluent human ES or iPS cells into approximately 32 pieces using 200 &#181;L tips. Next, detach the cells from the dishes by using a cell scraper.</li>
		<li>Collect human ES/iPS cells and transfer into a 6-well ultralow attachment dish.</li>
		<li>Exchange Essential 6 media by pipetting old media and adding fresh media every 2 days. 
		NOTE: Culture the cells in suspension for 2 weeks at 37 &#176;C with 5% CO2.</li>
		<li>After the EBs were homogenously suspended in Essential 6 media, transfer EBs to culture on 24-well TCP dishes coated with 0.1 weight% gelatin, and culture the cells in Essential 6 media for an additional week.</li>
		<li>Stain the cells with antibodies against markers of the cells derived from three embryonic germline layers [AFP (endoderm), GFA (ectoderm), &#946; III-Tubulin (ectoderm), and SMA (mesoderm)] and evaluate the cells by the immunostaining method described above.</li>
	</ol>
	</li>
	<li>Teratoma formation
	<ol>
		<li>Investigate pluripotency of human ES/iPS cells by teratoma formation at passages 10 and 20. 
		NOTE: 5 dishes of near 80% confluence in 6-well plates are sufficient for teratoma formation (at least 3 x 106 cells are necessary for the teratoma formation experiments).</li>
		<li>Rinse the cells with 2 mL DMEM/F-12 media twice, and then add 1.5 mL DMEM/F-12 media to the cell culture dishes.</li>
		<li>Cut nearly 80% confluent human ES or iPS cells into approximately 32 pieces by using 200 &#181;L tips. Next, detach the cells from the dishes by using a cell scraper.</li>
		<li>Collect human ES/iPS cells into a 15-mL centrifugation tube and centrifuge the cells at 160 &#215; g for 5 min at 37 &#176;C.</li>
		<li>After centrifugation, suspend the cell pellets in 100 &#181;L DMEM/F12, and then mix the cell pellets with 100 &#181;L Matrigel (1:1 volume ratio).</li>
		<li>Transfer the cell suspension into a -20 &#176;C pre-cooled syringe. Inject, in total, at least 3 &#215; 106 cells subcutaneously into male NOD-SCID (NOD.CB17-Prkdascid/JNarl) mice (5-8 weeks).</li>
		<li>After 5-8 weeks, dissect teratomas, fix with 4.0% (weight/volume) paraformaldehyde solution, and then store at 4 &#176;C.</li>
		<li>Fix the teratoma with paraffin, slice the paraffin-embedded teratomas, and stain with hematoxylin and eosin (H&#38;E) using a standard protocol28,32. 
		NOTE: Researchers may send fixed teratoma tissue to the company to fix the teratomas with paraffin, slice the paraffin-embedded teratomas, and stain the sample with H&#38;E, which is typically used in Department of Pathology in hospitals.</li>
	</ol>
	</li>
	<li>Human adipose-derived stem (ADS) cell culture
	<ol>
		<li>Warm culture media (DMEM containing 1% antimycotic antibiotic and 10% fetal bovine serum (FBS)), 0.25% of trypsin-EDTA, and PBS to 37 &#176;C in a water bath prior to use.</li>
		<li>Wash human ADS cells by pipetting 10 mL of PBS into each 6-cm culture dish where the cells are cultured.</li>
		<li>Add 1 mL of trypsin-EDTA solution (0.25%) into the 6-cm culture dishes and incubate the solution at 37 &#176;C for 5 min.</li>
		<li>Observe the cells in the 6 cm culture dishes under microscopy to confirm that the cells are detached.</li>
		<li>Collect the cells into a 15-mL centrifuge tube, and add an equal volume of culture media (DMEM containing 1% antimycotic antibiotic and 10% FBS) into the centrifuge tube to neutralize the trypsin-EDTA.</li>
		<li>Centrifuge the cells at 250 &#215; g for 5 min at 37 &#176;C.</li>
		<li>After centrifugation, discard the supernatant carefully by pipetting, without disturbing the cell pellet.</li>
		<li>Resuspend the cells in the culture media, and then seed the cells at the appropriate density (5-10 x 103 cells per cm2 for passaging or as indicated) into new culture dishes (P-IA hydrogels grafted with and without oligopeptides and ECM).</li>
	</ol>
	</li>
	<li>Human amniotic fluid stem (AFS) cell culture
	<ol>
		<li>Warm cultivation media (DMEM/MCDB 201 (2:3) containing 20% fetal bovine serum (FBS), 5 ng/mL bFGF and 1% antimycotic antibiotic), 0.25% of trypsin-EDTA, and PBS to 37 &#176;C in a water bath prior to use.</li>
		<li>Wash human AFS cells by pipetting 10 mL of PBS in each 6-cm culture dish where the cells are cultured.</li>
		<li>Add 1 mL of trypsin-EDTA solution (0.25%) into 6-cm culture dishes, and incubate the cells at 37 &#176;C for 5 min.</li>
		<li>Observe the cells on 6-cm culture dishes under microscopy to confirm the cells are detached.</li>
		<li>Collect the cells into a 15-mL centrifuge tube, and add an equal volume of culture media (DMEM/MCDB 201 (2:3) containing 20% fetal bovine serum (FBS), 5 ng/mL bFGF and 1% antimycotic antibiotic) into the centrifuge tube to neutralize the trypsin-EDTA.</li>
		<li>Centrifuge the cells at 250 &#215; g for 5 min at 37 &#176;C.</li>
		<li>After centrifugation, discard the supernatant carefully without disturbing the cell pellet by pipetting.</li>
		<li>Resuspend the cells in the cultivation media and then seed the cells at the appropriate density (5-10x103 cells per cm2 for passaging or as indicated) into new culture dishes (P-IA hydrogels grafted with and without oligopeptide and ECM).</li>
	</ol>
	</li>
</ol>

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A. 104 (9), 2356-2368 (2016).</li><li>Olivares-Navarrete, R., 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=Substrate+Stiffness+Controls+Osteoblastic+and+Chondrocytic+Differentiation+of+Mesenchymal+Stem+Cells+without+Exogenous+Stimuli.">Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli.</a> PLoS One. 12, e0170312 (2017).</li><li>Kumar, S. 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. 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=Xeno-free+culture+of+human+pluripotent+stem+cells+on+oligopeptide-grafted+hydrogels+with+various+molecular+designs.">Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs.</a> Sci Rep. 7, 45146 (2017).</li><li>Higuchi, A., Ling, Q. D., Ko, Y. 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>

The authors have nothing to disclose.

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&lt;br/&gt; Abbreviation: Cyclic RGD, cRGD</td></tr><tr><td>GACRGDCLGA</td><td>PH Japan</td><td>none</td><td>Specification: Oligopeptide&lt;br/&gt; Abbreviation: FN1</td></tr><tr><td>KGGAVTGRGDSPASS</td><td>PH Japan</td><td>none</td><td>Specification: Oligopeptide&lt;br/&gt; Abbreviation: CS1</td></tr><tr><td>EILDVPST</td><td>PH Japan</td><td>none</td><td>Specification: Oligopeptide&lt;br/&gt; Abbreviation: VN1</td></tr><tr><td>KGGPQVTRGDVFTMP</td><td>PH Japan</td><td>none</td><td>Specification: Oligopeptide&lt;br/&gt; Abbreviation: HBP1</td></tr><tr><td>GKKQRFRHRNRKG</td><td>PH Japan</td><td>none</td><td>Specification: Oligopeptide&lt;br/&gt; Abbreviation: HBP2C</td></tr><tr><td>CGGGKKQRFRHRNRKG</td><td>PH Japan</td><td>none</td><td>Specification: Oligopeptide&lt;br/&gt; Abbreviation: VN1G</td></tr><tr><td>GGGGKGGPQVTRGDVFTMP</td><td>PH Japan</td><td>none</td><td>Specification: Oligopeptide&lt;br/&gt; Abbreviation: VN2C</td></tr><tr><td>GCGGKGGPQVTRGDVFTMP</td><td>PH Japan</td><td>none</td><td>Specification: Extracellular matrix&lt;br/&gt; Abbreviation: rVN</td></tr><tr><td>Vitronectin</td><td>Thermo Fisher scientific</td><td>A14700</td><td>Specification: Extracellular matrix&lt;br/&gt; Abbreviation: FN</td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F2006</td><td>Specification: Commercially available coating material&lt;br/&gt; 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 &amp;amp; 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>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>239313</td><td>Specification: Chemical</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>339741</td><td>Specification: Chemical&lt;br/&gt; Abbreviation: EDC</td></tr><tr><td>N-(3-Dimethylaminopropyl)-N&#039;-ethylcarbodiimide hydrochloride</td><td>Sigma-Aldrich</td><td>E7750</td><td>Specification: Chemical&lt;br/&gt; 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&lt;br/&gt; Abbreviation: TCP</td></tr><tr><td>TCP dish</td><td>Corning</td><td>353001</td><td>Specification: 60 mm&lt;br/&gt; 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 &amp;amp; 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 &amp;ndash; conjugated Goat anti-Mouse antibody</td><td>Invitrogen</td><td>A-21422</td><td>Specification: Antibody</td></tr><tr><td>Alexa Fluor 488 &amp;ndash; 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 ...

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

Research

JoVE Journal

Bioengineering

9.2K Views.  National Central University. Een protocol is bedoeld om te bereiden polyvinyl alcohol-co-itacon zuur hydrogels met verschillende stijfheid, die waren geënt met en zonder oligopeptides, te onderzoeken van het effect van de stijfheid van biomaterialen op de differentiatie en de verspreiding van de cellen van de stam. De stijfheid van de hydrogels werd gecontroleerd door het crosslinking tijd.

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

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Developmental Biology

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

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 ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Matrix Comparison for Efficient Human Intestinal Organoid Generation from iPSCs

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

Author Spotlight: Advancing Organoid Generation for Drug Development Using iPSCs

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Biology

Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent development of procedures describing the generation of human cerebral organoids, derived from human induced pluripotent stem cells (iPSCs), has the potential to fundamentally transform the ability to model and understand the aging of the human brain and related pathogenic processes. Here, an optimized protocol for generating, maintaining, aging, and characterizing human iPSC-derived cerebral organoids is presented. This protocol can be implemented to generate brain organoids in a reproducible manner and serves as a step-by-step guide, incorporating the latest techniques that result in improved organoid maturation and aging in culture. Specific issues related to organoid maturation, necrosis, variability, and batch effects are being addressed. Taken together, these technological advances will allow the modeling of brain aging in organoids derived from a variety of young and aged human donors, as well as individuals afflicted with age-related brain disorders, allowing the identification of physiologic and pathogenic mechanisms of human brain aging.

The present protocol provides a step-by-step procedure for the reproducible generation, maintenance, and aging of cerebral organoids derived from human-induced pluripotent stem cells (iPSCs). This method enables culturing and maturing cerebral organoids for extended periods, which facilitates the modeling of processes involved in brain aging and age-related pathogenesis.

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent ...

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

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