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>

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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. 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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 border="0" cellpadding="0" cellspacing="0" style="width:965px;" width="964">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Adenine</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:136px;">A2786</td>
			<td style="width:401px;">Component of differentiation medium for fibroblast</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AggreWell Medium (EB formation medium)</td>
			<td>STEMCELL</td>
			<td>05893</td>
			<td>EB formation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Fibronectin antibody</td>
			<td>abcam</td>
			<td>ab23750</td>
			<td>Fibroblast marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-KRT14 antibody</td>
			<td>abcam</td>
			<td>ab7800</td>
			<td>Keratinocyte marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Loricrin antibody</td>
			<td>abcam</td>
			<td>ab85679</td>
			<td>Stratum corneum marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-p63 antibody</td>
			<td>abcam</td>
			<td>ab124762</td>
			<td>Keratinocyte marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Vimentin antibody</td>
			<td>Santa cruz</td>
			<td>sc-7558</td>
			<td>Fibroblast marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BAND AID FLEXIBLE FABRIC</td>
			<td>Johnson &#38; Johnson</td>
			<td>-</td>
			<td>Bandage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Basement membrane matrix (Matrigel)</td>
			<td>BD</td>
			<td>354277</td>
			<td>Component of differentiation medium for fibroblast</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BLACK SILK suture</td>
			<td>AILEEE</td>
			<td>SK617</td>
			<td>Skin graft</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CaCl2</td>
			<td>Sigma</td>
			<td>C5670</td>
			<td>Component of epithelial medium for 3D skin organoid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Collagen type I</td>
			<td>BD</td>
			<td>354236</td>
			<td>3D skin organoid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Collagen type IV</td>
			<td>Santa-cruz</td>
			<td>sc-29010</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Defined keratinocyte-Serum Free Medium</td>
			<td>Gibco</td>
			<td>10744-019</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM, high glucose</td>
			<td>Gibco</td>
			<td>11995065</td>
			<td>Component of differentiation medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM/F12 Medium</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>Component of differentiation medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Essential 8 medium</td>
			<td>Gibco</td>
			<td>A1517001</td>
			<td>iPSC medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FBS, Qualified</td>
			<td>Corning</td>
			<td>35-015-CV</td>
			<td>Component of differentiation medium for fibroblast and keratinocyte</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glutamax Supplement&#160;</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Component of differentiation medium for fibroblast</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Insulin</td>
			<td>Invtrogen</td>
			<td>12585-014</td>
			<td>Component of differentiation medium for fibroblast and keratinocyte</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Iris standard curved scissor</td>
			<td>Professional</td>
			<td>PC-02.10</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Keratinocyte Serum Free Medium</td>
			<td>Gibco</td>
			<td>17005-042</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">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 height="40">
			<td height="40" style="height:40px;">MEM Non-Essential Amino Acid</td>
			<td>Gibco</td>
			<td>1140050</td>
			<td>Component of differentiation medium for fibroblast</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Meriam Forceps Thumb 16 cm</td>
			<td>HIROSE</td>
			<td>HC 2265-1</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">NOD.CB17-Prkdc SCID/J</td>
			<td>The Jackson Laboratory</td>
			<td>001303</td>
			<td>Mice strain for skin graft</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Petri dish 90 mm</td>
			<td>Hyundai Micro</td>
			<td>H10090</td>
			<td>Plastic ware</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Recombinant Human BMP-4</td>
			<td>R&#38;D</td>
			<td>314-BP</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant human EGF protein</td>
			<td>R&#38;D</td>
			<td>236-EG</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">T/C Petridish 100 mm, 240/bx</td>
			<td>TPP</td>
			<td>93100</td>
			<td>Plastic ware</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Transferrin</td>
			<td>Sigma</td>
			<td>T3705</td>
			<td>Component of epithelial medium for 3D skin organoid</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Transwell-COL collagen-coated membrane inserts&#160;</td>
			<td>Corning</td>
			<td>CLS3492</td>
			<td>Plastic ware for 3D skin organoid&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Vitronectin</td>
			<td>Life technologies</td>
			<td>A14700</td>
			<td>iPSC culture</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">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

13.2K Views.  The Catholic University of Korea. 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.

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

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

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

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

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

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

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

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

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

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

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.