This work was supported by a grant from the Korea Healthcare Technology R&#38;D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (H16C2177, H18C1178).

All procedures involving animals were performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experimentation provided by the Institutional Animal Care and Use Committee (IACUC) of the School of Medicine of The Catholic University of Korea. The study protocol was approved by the Institutional Review Board of The Catholic University of Korea (CUMC-2018-0191-01). The IACUC and the Department of Laboratory Animals (DOLA) of the Catholic University of Korea, Songeui Campus accredited the Korea Excellence Animal laboratory facility of the Korea Food and Drug Administration in 2017 and acquired Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) International full accreditation in 2018.
1. Skin cell differentiation from induced pluripotent stem cells
<ol>
	<li>Medium preparation 
	NOTE: Store all medium at 4 &#176;C in a dark environment for up to 3 months. Filter all medium using a 0.22 &#956;m polyethersulfone filter system before use for sterilization. All medium was available in a total volume of 500 mL.
	<ol>
		<li>Prepare KDM1 (keratinocyte differentiation medium 1). Mix Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/F12 medium (3:1) with 2% fetal bovine serum (FBS), 0.3 mmol/L L-ascorbic acid, 5 &#956;g/mL insulin, and 24 &#181;g/mL adenine.</li>
		<li>Prepare KDM2 (keratinocyte differentiation medium 2). Mix defined keratinocyte serum-free medium (see the Table of Materials) with 0.3 mmol/l L-ascorbic acid, 5 &#956;g/mL insulin, and 10 &#956;g/mL adenine. 
		NOTE: Defined keratinocyte serum-free medium is optimized to support the growth and expansion of keratinocytes.</li>
		<li>Prepare KDM3 (keratinocyte differentiation medium 3). Mix defined keratinocyte serum-free medium and keratinocyte serum-free medium (1:1) See the Table of Materials for details. 
		NOTE: Keratinocyte serum-free medium is optimized for the growth and maintenance of keratinocytes.</li>
		<li>Prepare FDM1 (fibroblast differentiation medium 1). Mix DMEM/F12 medium (3:1) with 5% FBS, 5 &#956;g/mL insulin, 0.18 mM adenine, and 10 ng/mL epidermal growth factor (EGF).</li>
		<li>Prepare FDM2 (fibroblast differentiation medium 2). Mix DMEM/F12 medium (1:1) with 5% FBS and 1% nonessential amino acids.</li>
		<li>Prepare EP1 (epithelial medium 1). Mix DMEM/F12 (3:1) with 4 mM L-glutamine, 40 &#956;M adenine, 10 &#956;g/mL transferrin, 10 &#956;g/mL insulin, and 0.1% FBS.</li>
		<li>Prepare EP2 (epithelial medium 2). Mix EP1 and 1.8 mM calcium chloride.</li>
		<li>Prepare EP3 (epithelial medium 3, cornification medium). Mix F12 medium with 4 mM L-glutamine, 40 &#956;M adenine, 10 &#956;g/mL transferrin, 10 &#956;g/mL insulin, 2% FBS, and 1.8 mM calcium chloride.</li>
	</ol>
	</li>
	<li>Embryonic body generation
	<ol>
		<li>Generate CBMC-iPSCs using the protocol shown in a previous study12.</li>
		<li>Coat culture dishes, using vitronectin. Prepare 5 mL to coat a 100 mm dish.
		<ol>
			<li>Thaw and resuspend 50 &#956;L of 0.5 mg/mL vitronectin (final concentration: 5 &#181;g/mL) with 5 mL of sterile phosphate-buffered saline (PBS). Add the solution to the dishes and incubate at room temperature (RT) for 1 h. Aspirate the coating material before use (not to dry out).</li>
		</ol>
		</li>
		<li>Maintain the CBMC-derived iPSCs to the vitronectin-coated 100 mm plate and change the iPSC medium (E8) daily at 37 &#176;C with 10% CO2.</li>
		<li>Generate embryonic bodies (EBs) using the protocol shown in a previous study15 (described briefly as follows). Expand iPSCs by changing the medium until the cells have reached 80% confluence. At 80% confluence, remove the medium and wash with PBS.</li>
		<li>Treat the cells with 1 mL of 1 mM ethylenediaminetetraacetic acid (EDTA). Incubate at 37 &#176;C with 5% CO2 for 2 min and harvest the cells using 3 mL of E8 medium. Centrifuge the cells at 250 x g for 2 min.</li>
		<li>Aspirate the supernatant and apply 5 mL of E8 medium to the cells. Count the cells using a hemocytometer and transfer 1 x 106 cells to a new 15 mL conical tube. Centrifuge the cells at 250 x g for 2 min.</li>
		<li>Resuspend the transferred cells with 2.5 mL of EB formation medium with 10 &#956;M Rho-associated kinase (ROCK) inhibitor. Drop 1 x 104 cells (25 &#181;L/drop) on a noncoated culture plate lid using a 10&#8211;100 &#956;L multichannel pipette. Form 100 EBs from 1 x 106 cells (1 x 104 cells/1 EB). Turn over the dish and hang on the droplet to the lid. 
		NOTE: ROCK inhibitor is needed during the attachment step in the maintenance and differentiation process. Add the ROCK inhibitor only at the EB aggregation stage.</li>
		<li>Incubate the droplets at 37 &#176;C with 5% CO2 for 1 day.</li>
		<li>The next day, harvest the 100 EBs and use them for differentiation. Wash out the lid of plate with iPSC medium (E8 medium) or PBS and harvest its contents to a 50 mL conical tube. Maintain the EBs at RT for 1 min to settle them down. Aspirate the supernatant, resuspend the EBs with E8 medium, and maintain them in a 90 mm Petri dish until the differentiation.</li>
	</ol>
	</li>
	<li>Differentiation of CBMC-iPSCs into keratinocytes 
	NOTE: For a scheme of the keratinocyte differentiation from CBMC-iPSCs, see Figure 1A.
	<ol>
		<li>Harvest the 100 EBs to a 50 mL conical tube with iPSC medium or PBS. Maintain at RT for 1 min to settle down the EBs. Make sure they settle at the bottom of the conical tube. Aspirate the supernatant and resuspend the EBs with E8 medium with 1 ng/mL bone morphogenetic protein 4 (BMP4). Transfer the EBs to a 90 mm Petri dish and maintain them at 37 &#176;C with 5% CO2 for 1 day.</li>
		<li>Coat culture dishes, using type IV collagen. Prepare 5 mL of type IV collagen to coat a 100 mm dish.
		<ol>
			<li>Thaw and resuspend the type IV collagen solution (final concentration: 50 &#181;g/mL) with 0.05 N HCl. Add the solution to the dishes and incubate at RT for 1 h. Aspirate the coating material before use (not to dry out). 
			NOTE: Before using the plates, wash the dishes 3x with PBS to remove any acid.</li>
		</ol>
		</li>
		<li>Harvest the EBs (step 1.3.1) to a 50 mL conical tube and maintain them at RT for 1 min to settle them down. Make sure they settle at the bottom of the conical tube, aspirate the supernatant, and resuspend the EBs in 6 mL of KDM1 with 10 &#181;M ROCK inhibitor. Transfer the EBs to the type IV collagen-coated 100 mm dish. 
		NOTE: Add the ROCK inhibitor only at the EB attachment stage.</li>
		<li>Between days 0&#8211;8, change the medium every other day to KDM1 with 3 &#181;M retinoic acid (RA) and 25 ng/mL each of BMP4 and EGF. Maintain the EBs at 37 &#176;C with 5% CO2.</li>
		<li>Between days 9&#8211;12, change the medium every other day to KDM2 with 3 &#181;M RA, 25 ng/mL BMP4, and 20 ng/mL EGF.</li>
		<li>Between days 13&#8211;30, change the medium every other day to KDM3 with 10 ng/mL BMP4 and 20 ng/mL EGF.</li>
	</ol>
	</li>
	<li>Differentiation of CBMC-iPSC into fibroblasts 
	NOTE: For a scheme of the fibroblast differentiation from CBMC-iPSCs, see Figure 2A.
	<ol>
		<li>Coat culture dishes, using basement membrane matrix. Prepare 5 mL to coat a 100 mm dish.
		<ol>
			<li>Thaw basement membrane matrix (final concentration: 600 ng/mL) and dilute it with DMEM/F12 medium. Add the solution to the dishes and incubate at 37 &#176;C for 30 min. Aspirate the coating material before use (not to dry out).</li>
		</ol>
		</li>
		<li>Harvest the 100 EBs to a 50 mL conical tube using a pipette with iPSC medium or PBS. Maintain at RT for 1 min to settle down the EBs. Ensure they settle at the bottom of the conical tube. Remove the supernatant.</li>
		<li>Resuspend the EBs using a 1,000 &#181;L pipette in 6 mL of FDM1 with 10 &#181;M ROCK inhibitor. Transfer the EBs (with medium) to a basement membrane matrix-coated 100 mm dish and incubate at 37 &#176;C with 5% CO2. Refresh the FDM1 every other day for 3 days. 
		NOTE: Only add the ROCK inhibitor at the EB attachment stage.</li>
		<li>Add 0.5 nM bone morphogenetic protein 4 (BMP 4) to the FDM1 between days 4 and 6.</li>
		<li>At day 7, change the medium to FDM2 every other day for 1 week.</li>
		<li>At day 14, add 1 mL of 1 mM EDTA and incubate at 37 &#176;C with 5% CO2 for 2 min. Harvest the cells with 3 mL of FDM2 and centrifuge at 250 x g for 2 min. Remove the supernatant and resuspend the cells in 5 mL of FDM1.</li>
		<li>Count the cells using a hemocytometer, resuspend 2 x 106 cells with FDM1 medium, and transfer the cells to the noncoated dish. Maintain the cells at 37 &#176;C with 5% CO2 and change the medium every other day.</li>
		<li>Coat culture dishes, using type I collagen. Prepare 5 mL to coat a 100 mm dish. Dilute type I collagen solution (final concentration: 50 &#181;g/mL) in 0.02 N acetic acid. Add the solution to the dishes and incubate at RT for 1 h. Aspirate the coating material before use (not to dry out). 
		NOTE: Before using the plates, wash the dishes 3x with PBS to remove the acid.</li>
		<li>On day 21, add 1 mL of 1 mM EDTA and incubate at 37 &#176;C with 5% CO2 for 2 min. Harvest the cells with 3 mL of FDM1 and centrifuge at 250 x g for 2 min. Remove the supernatant and resuspend the cells in 5 mL of FDM1. Count the cells using a hemocytometer and transfer 2 x 106 cells to the type I collagen-coated 100 mm dish with FDM1 medium. Maintain the cells at 37 &#176;C with 5% CO2 and change the medium every other day.</li>
		<li>On day 28, add 1 mL of 1 mM EDTA and incubate at 37 &#176;C with 5% CO2 for 2 min. Harvest the cells with 3 mL of FDM1 and centrifuge at 250 x g for 2 min. Remove the supernatant and resuspend the cells in 5 mL of FDM1. Count the cells using a hemocytometer and transfer 2 x 106 cells to a noncoated dish with FDM1 medium. Maintain the cell at 37 &#176;C with 5% CO2 and change the medium every other day. 
		NOTE: iPSC-derived fibroblasts proliferate like a primary fibroblast cell line and passage up to 10 passages. In this study, we used iPSC-derived fibroblasts of two to five passages for further analysis.</li>
	</ol>
	</li>
</ol>
2. Application of hiPSC-derived differentiated cells
<ol>
	<li>Generation of 3D skin organoid
	<ol>
		<li>Prepare neutralized type I collagen on ice, following the manufacturer&#8217;s recommendations. As final concentration, use 3 mg/mL for type I collagen (stock concentration of type I collagen is 3.47 mg/mL), and make sure the final volume of the mixture is 5 mL. Calculate the volume of 10x PBS (final volume/10 = 0.5 mL). Calculate the volume of type I collagen to be used (final volume x final collagen concentration / stock collagen concentration = 5 mL x 3 mg/mL / 3.47 mg/mL = 4.32 mL). Calculate the volume of 1 N NaOH (volume of collagen to be used x 0.023 mL = 0.1 mL). Calculate the volume of dH2O (final volume - volume of collagen - volume of 10x PBS - volume of 1 N NaOH = 5 mL - 4.32 mL - 0.5mL - 0.1 mL = 0.08 mL). Mix the contents of the tube and keep it on ice until ready to use.</li>
		<li>Add 1 mL of EDTA to the iPSC-derived fibroblasts from step 1.4.10 and incubate at 37 &#176;C with 5% CO2 for 2 min. Harvest the detached cells, count the cells using a hemocytometer, and transfer 2 x 105 cells to a new 15 mL conical tube. Centrifuge at 250 x g for 2 min and remove the supernatant. Resuspend the cells of the iPSC-derived fibroblasts in 1.5 mL of FDM1 and neutralized the type I collagen solution (1:1). 
		NOTE: Mix the solution gently to avoid bubbles.</li>
		<li>Place the membrane insert on a 6-well microplate, transfer the mixture to the insert, and incubate at RT for 30 min. 
		NOTE: Do not move the plates.</li>
		<li>After confirming the gelation, add 2 mL of medium to the top of the insert and 3 mL to the bottom of the well. Incubate the matrix of fibroblasts and collagen at 37 &#176;C with 5% CO2 for 5-7 days, until the gelation is complete and no longer contracts.</li>
		<li>After the complete gelation, detach the iPSC-derived keratinocytes (from step 1.3.6) using EDTA. Add 1 mL of EDTA and incubate at 37 &#176;C with 5% CO2 for 2 min. Harvest the detached cells, count them using a hemocytometer, and transfer 1 x 106 cells to a new 15 mL conical tube. Centrifuge at 250 x g for 2 min.</li>
		<li>Remove the supernatant and resuspend 1 x 106 cells in 50-100 &#181;L of low calcium epithelial medium 1 (EP1).</li>
		<li>Aspirate all medium in the matrix (see step 2.1.5) and seed 1 x 106 cells of the iPSC-derived keratinocytes onto each fibroblast layer. Incubate the plate at 37 &#176;C with 5% CO2 for 30 min. 
		NOTE: Do not move the plate and do not add any medium for the attachment of keratinocyte.</li>
		<li>Add 2 mL of EP1 to the top of the insert and 3 mL of EP1 to the bottom of the well.</li>
		<li>After 2 days, aspirate all medium in the membrane insert plate and change the medium to normal calcium EP2 for 2 days.</li>
		<li>After 2 days, aspirate all medium and add 3 mL of the cornification medium only to the bottom to generate an air-liquid interface.</li>
		<li>Maintain the 3D skin organoid for up to 14 days at 37 &#176;C with 5% CO2 and change the medium every other day. Harvest the 3D skin organoid by cutting the edge of the insert, and use it for a further study of staining and skin graft.</li>
	</ol>
	</li>
	<li>Skin graft
	<ol>
		<li>Perform inhalation anesthesia on NOD/scid mice (male, 6 weeks old), using a standard, institutionally approved method. For skin graft, shave the fur of each mouse&#8217;s dorsal skin.</li>
		<li>Remove a 1 cm x 2 cm section of the mouse&#8217;s skin, using curved scissors with forceps.</li>
		<li>Place the CBMC-iPSC-derived 3D skin organoid onto the defect site and suture using a tie-over dressing method with silk sutures.</li>
		<li>Observe the mice for 2 weeks and sacrifice them for histological analysis. The staining protocol was verified in previous studies16.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Vincent, J. F., Bogatyreva, O. A., Bogatyrev, N. R., Bowyer, A., Pahl, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetics:+its+practice+and+theory.">Biomimetics: its practice and theory.</a> Journal of The Royal Society Interface. 3 (9), 471-482 (2006).</li><li>Madison, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+function+of+the+skin:+"la+raison+d'etre"+of+the+epidermis.">Barrier function of the skin: "la raison d'etre" of the epidermis.</a> Journal of Investigative Dermatology. 121 (2), 231-241 (2003).</li><li>Chen, M., Przyborowski, M., Berthiaume, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+for+skin+tissue+engineering+and+wound+healing.">Stem cells for skin tissue engineering and wound healing.</a> Critical Reviews in Biomedical Engineering. 37 (4-5), 399-421 (2009).</li><li>Dixit, 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=Immunological+challenges+associated+with+artificial+skin+grafts:+available+solutions+and+stem+cells+in+future+design+of+synthetic+skin.">Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin.</a> Journal of Biological Engineering. 11, 49 (2017).</li><li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+past,+present,+and+future.">Induced pluripotent stem cells: past, present, and future.</a> Cell Stem Cell. 10 (6), 678-684 (2012).</li><li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotency+and+nuclear+reprogramming.">Pluripotency and nuclear reprogramming.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 363 (1500), 2079-2087 (2008).</li><li>Scheiner, Z. S., Talib, S., Feigal, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+for+immunogenicity+of+autologous+induced+pluripotent+stem+cell-derived+therapies.">The potential for immunogenicity of autologous induced pluripotent stem cell-derived therapies.</a> Journal of Biological Chemistry. 289 (8), 4571-4577 (2014).</li><li>Zimmermann, A., Preynat-Seauve, O., Tiercy, J. M., Krause, K. H., Villard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haplotype-based+banking+of+human+pluripotent+stem+cells+for+transplantation:+potential+and+limitations.">Haplotype-based banking of human pluripotent stem cells for transplantation: potential and limitations.</a> Stem Cells and Development. 21 (13), 2364-2373 (2012).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Terasaki, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+history+of+HLA.">A brief history of HLA.</a> Immunologic Research. 38 (1-3), 139-148 (2007).</li><li>Haase, 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=Generation+of+induced+pluripotent+stem+cells+from+human+cord+blood.">Generation of induced pluripotent stem cells from human cord blood.</a> Cell Stem Cell. 5 (4), 434-441 (2009).</li><li>Rim, Y. 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=Recent+progress+of+national+banking+project+on+homozygous+HLA-typed+induced+pluripotent+stem+cells+in+South+Korea.">Recent progress of national banking project on homozygous HLA-typed induced pluripotent stem cells in South Korea.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (3), 1531-1536 (2018).</li><li>Nakatsuji, N., Nakajima, F., Tokunaga, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-haplotype+banking+and+iPS+cells.">HLA-haplotype banking and iPS cells.</a> Nature Biotechnology. 26 (7), 739-740 (2008).</li><li>Pappas, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proceedings:+human+leukocyte+antigen+haplo-homozygous+induced+pluripotent+stem+cell+haplobank+modeled+after+the+california+population:+evaluating+matching+in+a+multiethnic+and+admixed+population.">Proceedings: human leukocyte antigen haplo-homozygous induced pluripotent stem cell haplobank modeled after the california population: evaluating matching in a multiethnic and admixed population.</a> Stem Cells Translational Medicine. 4 (5), 413-418 (2015).</li><li>Embryoid body formation from human pluripotent stem cells in chemically defined E8 media. StemBook Available from: <a target="_blank" href="https://www.stembook.org/node/6632">https://www.stembook.org/node/6632</a> (2008)</li><li>Kim, 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=Establishment+of+a+complex+skin+structure+via+layered+co-culture+of+keratinocytes+and+fibroblasts+derived+from+induced+pluripotent+stem+cells.">Establishment of a complex skin structure via layered co-culture of keratinocytes and fibroblasts derived from induced pluripotent stem cells.</a> Stem Cell Research &amp; Therapy. 9 (1), 217 (2018).</li><li>Diecke, S., Jung, S. M., Lee, J., Ju, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+technological+updates+and+clinical+applications+of+induced+pluripotent+stem+cells.">Recent technological updates and clinical applications of induced pluripotent stem cells.</a> The Korean Journal of Internal Medicine. 29 (5), 547-557 (2014).</li><li>Shi, Y., Inoue, H., Wu, J. C., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+technology:+a+decade+of+progress.">Induced pluripotent stem cell technology: a decade of progress.</a> Nature Reviews Drug Discovery. 16 (2), 115-130 (2017).</li><li>Yoshida, Y., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+stem+cell+advances:+induced+pluripotent+stem+cells+for+disease+modeling+and+stem+cell-based+regeneration.">Recent stem cell advances: induced pluripotent stem cells for disease modeling and stem cell-based regeneration.</a> Circulation. 122 (1), 80-87 (2010).</li><li>Pham, T. L., Nguyen, T. T., Van Bui, A., Nguyen, M. T., Van Pham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+heart+extract+facilitates+the+differentiation+of+human+umbilical+cord+blood-derived+mesenchymal+stem+cells+into+heart+muscle+precursor+cells.">Fetal heart extract facilitates the differentiation of human umbilical cord blood-derived mesenchymal stem cells into heart muscle precursor cells.</a> Cytotechnology. 68 (4), 645-658 (2016).</li><li>Stecklum, 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=Cell+differentiation+mediated+by+co-culture+of+human+umbilical+cord+blood+stem+cells+with+murine+hepatic+cells.">Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.</a> In Vitro Cellular &amp; Developmental Biology - Animal. 51 (2), 183-191 (2015).</li><li>Nam, Y., Rim, Y. A., Ju, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+Pellet+Formation+from+Cord+Blood-derived+Induced+Pluripotent+Stem+Cells.">Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells.</a> Journal of Visualized Experiments. (124), e55988 (2017).</li><li>Rim, Y. A., Nam, Y., Ju, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Cord+Blood+and+Cord+Blood-derived+Induced+Pluripotent+Stem+Cells+for+Cartilage+Regeneration.">Application of Cord Blood and Cord Blood-derived Induced Pluripotent Stem Cells for Cartilage Regeneration.</a> Cell Transplantation. , (2018).</li><li>Shevde, N. K., Mael, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+embryoid+body+formation+from+human+pluripotent+stem+cells.">Techniques in embryoid body formation from human pluripotent stem cells.</a> Methods in Molecular Biology. 946, 535-546 (2013).</li><li>Shamis, 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=iPSC-derived+fibroblasts+demonstrate+augmented+production+and+assembly+of+extracellular+matrix+proteins.">iPSC-derived fibroblasts demonstrate augmented production and assembly of extracellular matrix proteins.</a> In Vitro Cellular &amp; Developmental Biology - Animal. 48 (2), 112-122 (2012).</li><li>Bikle, D. D., Xie, Z., Tu, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+regulation+of+keratinocyte+differentiation.">Calcium regulation of keratinocyte differentiation.</a> Expert Review of Endocrinology &amp; Metabolism. 7 (4), 461-472 (2012).</li><li>Bernstam, L. I., Vaughan, F. L., Bernstein, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocytes+grown+at+the+air-liquid+interface.">Keratinocytes grown at the air-liquid interface.</a> In Vitro Cellular &amp; Developmental Biology. 22 (12), 695-705 (1986).</li><li>Prunieras, M., Regnier, M., Woodley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+cultivation+of+keratinocytes+with+an+air-liquid+interface.">Methods for cultivation of keratinocytes with an air-liquid interface.</a> Journal of Investigative Dermatology. 81, 28-33 (1983).</li><li>Steven, A. C., Bisher, M. E., Roop, D. R., Steinert, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthetic+pathways+of+filaggrin+and+loricrin--two+major+proteins+expressed+by+terminally+differentiated+epidermal+keratinocytes.">Biosynthetic pathways of filaggrin and loricrin--two major proteins expressed by terminally differentiated epidermal keratinocytes.</a> Journal of Structural Biology. 104 (1-3), 150-162 (1990).</li><li>Hohl, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+loricrin.+Structure+and+function+of+a+new+class+of+epidermal+cell+envelope+proteins.">Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins.</a> Journal of Biological Chemistry. 266 (10), 6626-6636 (1991).</li><li>Bern, 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=Original+and+modified+technique+of+tie-over+dressing:+Method+and+application+in+burn+patients.">Original and modified technique of tie-over dressing: Method and application in burn patients.</a> Burns. 44 (5), 1357-1360 (2018).</li><li>Joyce, C. W., Joyce, K. M., Kennedy, A. M., Kelly, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Running+Barbed+Tie-over+Dressing.">The Running Barbed Tie-over Dressing.</a> Plastic and Reconstructive Surgery - Global Open. 2 (4), 137 (2014).</li><li>Wang, C. K., Nelson, C. F., Brinkman, A. M., Miller, A. C., Hoeffler, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+cell+sorting+of+fibroblasts+and+keratinocytes+creates+an+organotypic+human+skin+equivalent.">Spontaneous cell sorting of fibroblasts and keratinocytes creates an organotypic human skin equivalent.</a> Journal of Investigative Dermatology. 114 (4), 674-680 (2000).</li><li>Yang, 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=Generation+of+folliculogenic+human+epithelial+stem+cells+from+induced+pluripotent+stem+cells.">Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells.</a> Nature Communications. 5, 3071 (2014).</li></ol>

The authors have nothing to disclose.

Human iPSCs have been suggested as a new alternative for personalized regenerative medicine17. Patient-derived personalized iPSCs reflect patient characteristics that can be used for disease modeling, drug screening, and autologous transplantation18,19. The use of patient-derived iPSCs can also overcome problems regarding primary cells, a lack of adequate cell numbers, and immune reactions5,<sup class="xr...

Skin covers the outermost surface of the body and protects internal organs. The skin has various functions, including protecting against pathogens, absorbing and storing water, regulating body temperature, and excreting body waste2. Skin grafts can be classified depending on the skin source; grafts using skin from another donor are termed allografts, and grafts using the patient&#8217;s own skin are autografts. Although an autograft is the preferred treatment due to its low rejection risk, skin biopsies are difficult to perform on patients with severe lesions or an insufficient number of skin cells. In patients with severe burns, three times the number of skin cells are necessary to cover large areas. The limited availability of skin cells from a patient&#8217;s body results in situations where allogenous transplantation is necessary. An allograft is temporarily used until autologous transplantation can be performed since it is usually rejected by the host&#8217;s immune system after approximately 1 week3. To overcome rejection by the patient&#8217;s immune system, grafts must come from a source with the same immune identity as the patient4.
Human iPSCs are an emerging source of cells for stem cell therapy5. Human iPSCs are generated from somatic cells, using reprogramming factors such as OCT4, SOX2, Klf4, and c-Myc6. Using human iPSCs overcomes the ethical and immunological issues of embryonic stem cells (ESCs)7,8. Human iPSCs have pluripotency and can differentiate into three germ layers9. The presence of HLA, a critical factor in regenerative medicine, determines the immune response and the possibility of rejection10. The use of patient-derived iPSCs resolves the problems of cell-source limitation and immune system rejection. CBMCs have also emerged as an alternative cell source for regenerative medicine11. Mandatory HLA typing, which occurs during CBMC banking, can easily be used for research and transplantation. Further, homozygous HLA-type iPSCs can widely apply to various patients12. A CBMC-iPSC bank is a novel and efficient strategy for cell therapy and allogenic regenerative medicine12,13,14. In this study, we use CBMC-iPSCs, differentiated into keratinocytes and fibroblasts, and generate stratified 3D skin layers. Results from this study suggest that a CBMC-iPSC-derived 3D skin organoid is a novel tool for in vitro and in vivo dermatologic research.

<table><tbody><tr><td>Adenine</td><td>Sigma</td><td>A2786</td><td>Component of differentiation medium for fibroblast</td></tr><tr><td>AggreWell Medium (EB formation medium)</td><td>STEMCELL</td><td>05893</td><td>EB formation</td></tr><tr><td>Anti-Fibronectin antibody</td><td>abcam</td><td>ab23750</td><td>Fibroblast marker</td></tr><tr><td>Anti-KRT14 antibody</td><td>abcam</td><td>ab7800</td><td>Keratinocyte marker</td></tr><tr><td>Anti-Loricrin antibody</td><td>abcam</td><td>ab85679</td><td>Stratum corneum marker</td></tr><tr><td>Anti-p63 antibody</td><td>abcam</td><td>ab124762</td><td>Keratinocyte marker</td></tr><tr><td>Anti-Vimentin antibody</td><td>Santa cruz</td><td>sc-7558</td><td>Fibroblast marker</td></tr><tr><td>BAND AID FLEXIBLE FABRIC</td><td>Johnson &amp;amp; Johnson</td><td>-</td><td>Bandage</td></tr><tr><td>Basement membrane matrix (Matrigel)</td><td>BD</td><td>354277</td><td>Component of differentiation medium for fibroblast</td></tr><tr><td>BLACK SILK suture</td><td>AILEEE</td><td>SK617</td><td>Skin graft</td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C5670</td><td>Component of epithelial medium for 3D skin organoid</td></tr><tr><td>Collagen type I</td><td>BD</td><td>354236</td><td>3D skin organoid</td></tr><tr><td>Collagen type IV</td><td>Santa-cruz</td><td>sc-29010</td><td>Component of differentiation medium for keratinocyte</td></tr><tr><td>Defined keratinocyte-Serum Free Medium</td><td>Gibco</td><td>10744-019</td><td>Component of differentiation medium for keratinocyte</td></tr><tr><td>DMEM, high glucose</td><td>Gibco</td><td>11995065</td><td>Component of differentiation medium</td></tr><tr><td>DMEM/F12 Medium</td><td>Gibco</td><td>11330-032</td><td>Component of differentiation medium</td></tr><tr><td>Essential 8 medium</td><td>Gibco</td><td>A1517001</td><td>iPSC medium</td></tr><tr><td>FBS, Qualified</td><td>Corning</td><td>35-015-CV</td><td>Component of differentiation medium for fibroblast and keratinocyte</td></tr><tr><td>Glutamax Supplement&amp;nbsp;</td><td>Gibco</td><td>35050061</td><td>Component of differentiation medium for fibroblast</td></tr><tr><td>Insulin</td><td>Invtrogen</td><td>12585-014</td><td>Component of differentiation medium for fibroblast and keratinocyte</td></tr><tr><td>Iris standard curved scissor</td><td>Professional</td><td>PC-02.10</td><td>Surgical instrument</td></tr><tr><td>Keratinocyte Serum Free Medium</td><td>Gibco</td><td>17005-042</td><td>Component of differentiation medium for keratinocyte</td></tr><tr><td>L-ascorbic acid 2-phosphata sesquimagnesium salt hydrate</td><td>Sigma</td><td>A8960</td><td>Component of differentiation medium for keratinocyte</td></tr><tr><td>MEM Non-Essential Amino Acid</td><td>Gibco</td><td>1140050</td><td>Component of differentiation medium for fibroblast</td></tr><tr><td>Meriam Forceps Thumb 16 cm</td><td>HIROSE</td><td>HC 2265-1</td><td>Surgical instrument</td></tr><tr><td>NOD.CB17-Prkdc SCID/J</td><td>The Jackson Laboratory</td><td>001303</td><td>Mice strain for skin graft</td></tr><tr><td>Petri dish 90 mm</td><td>Hyundai Micro</td><td>H10090</td><td>Plastic ware</td></tr><tr><td>Recombinant Human BMP-4</td><td>R&amp;amp;D</td><td>314-BP</td><td>Component of differentiation medium for keratinocyte</td></tr><tr><td>Recombinant human EGF protein</td><td>R&amp;amp;D</td><td>236-EG</td><td>Component of differentiation medium for keratinocyte</td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td>Component of differentiation medium for keratinocyte</td></tr><tr><td>T/C Petridish 100 mm, 240/bx</td><td>TPP</td><td>93100</td><td>Plastic ware</td></tr><tr><td>Transferrin</td><td>Sigma</td><td>T3705</td><td>Component of epithelial medium for 3D skin organoid</td></tr><tr><td>Transwell-COL collagen-coated membrane inserts&amp;nbsp;</td><td>Corning</td><td>CLS3492</td><td>Plastic ware for 3D skin organoid&amp;nbsp;</td></tr><tr><td>Vitronectin</td><td>Life technologies</td><td>A14700</td><td>iPSC culture</td></tr><tr><td>Y-27632 Dihydrochloride</td><td>peprotech</td><td>1293823</td><td>iPSC culture</td></tr></tbody></table>

generation of 3d skin organoid from cord blood-derived induced pluripotent stem cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

Skin is composed, for the most part, of the epidermis and the dermis. Keratinocytes are the main cell type of the epidermis, and fibroblasts are the main cell type of the dermis. The scheme of keratinocyte differentiation is shown in Figure 1A. CBMC-iPCSc were maintained in a vitronectin-coated dish (Figure 1B). In this study, we differentiated CBMC-iPSCs into keratinocytes and fibroblasts using EB formation. We generated EBs us...

Watch this Scientific Journal Video about Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells at JoVE.com

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

generation-3d-skin-organoid-from-cord-blood-derived-induced

Research

JoVE Journal

Developmental Biology

13.4K Views.  The Catholic University of Korea. Navelstrengbloed mononucleaire cellen, of CBMC, zijn naar voren gekomen als een potentiële cel bron voor regeneratieve geneeskunde, omdat menselijke leukocyten antigeen typen is essentieel voor de cel banksysteem. Door CBMC geïnduceerde pluripotente stamcellen, of CMBC-iPSCs, kunnen worden onderscheiden in keratinocyten en fibroblasten. Om 3D-huidorganoïde te genereren, gebruiken we dermatologisch onderzoek.Begin met het kweken van CBMC-iPSC op vitronectine-gecoate 100-milliliter pl...

Video: Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Using Induced Pluripotent Stem Cell Suspension for Kidney Organoid Production

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

Author Spotlight: Optimizing iPSC Differentiation for Efficient Production to Generate Kidney Organoids 

This protocol presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells (iPSCs) using suspension culture conditions. The primary emphasis of this study lies in the determination of the initial cell density and the WNT agonist concentration, thereby benefiting investigators interested in kidney organoid research.

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Bioengineering

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 ...

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

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

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

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

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

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Medicine

Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids

Source: Koch, L. S., et al. <a href="https://app.jove.com/v/64586">Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research.</a> J. Vis. Exp. (2023).
This video demonstrates an assay to generate cerebral organoids from induced pluripotent stem cells (iPSCs). Human iPSCs are induced to form embryoid bodies (EBs). The EBs are cultured over a polymer scaffold using an induction medium, promoting differentiation into neural stem cells, progenitor cells, and immature neurons. Finally, the EBs are embedded in a matrix and transferred to a maintenance medium for cerebral organoid formation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Figure 1 illustrates a schematic ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video