Name: generation of 3d skin organoid from cord blood-derived induced pluripotent stem cells
Uploaded: 2019-04-18T14:30:00
Description: Watch this Scientific Journal Video about Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells at JoVE.com

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...

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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 ...

Cord blood mononuclear cells, or CBMC, have emerged as a potential cell source for regenerative medicine, because human leukocyte antigen typing is essential to the cell banking system. CBMC-induced pluripotent stem cells, or CMBC-iPSCs, can be differentiated into keratinocytes and fibroblasts. To generate 3D skin organoid, we're using dermatologic research.Begin by culturing CBMC-iPSC onto vitronectin-coated 100-milliliter plates at 37 degrees Celsius and 10%carbon dioxide. When the cells have reached 80%confluency, wash the cultures with PBS, and treat them with one milliliter of one-millimolar EDTA per plate for two minutes at 37 degrees Celsius and 5%carbon dioxide. When the cells have detached, stop the reaction with three milliliters of E8 medium per plate, and collect the cells by centrifugation.Re-suspend the pellets in five milliliters of E8 medium for counting, and transfer one-times-10-to-the-sixth cells to a 15-milliliter conical tube for centrifugation. Re-suspend the transferred cells with 2.5-milliliters of embryonic body formation medium, supplemented with 10-micromolar rho-associated kinase, or ROCK inhibitor. Use a pipette to transfer 125-microliter droplets of cells onto an uncoated culture plate lid.When all of the droplets have been placed, turn over the dish to allow the droplets to hang from the lid for 24 hours at 37 degrees Celsius and 5%carbon dioxide. The next day, wash the lid with fresh E8 medium, and transfer the embryonic body solution to a 50-milliliter conical tube. Allow the embryonic bodies to settle for one minute at room temperature, before aspirating the supernatant and re-suspending the embryonic bodies with fresh E8 medium for their culture in a 90-millimeter Petri dish at 37 degrees Celsius and 5%carbon dioxide until their differentiation.For CBMC-iPSC keratinocyte differentiation, replace the E8 medium from the embryonic body culture with fresh E8 medium, supplemented with one nanogram per milliliter of bone morphogenetic protein 4 for a 24-hour incubation at 37 degrees Celsius and 5%carbon dioxide. The next day, harvest the embryonic bodies into a 50-milliliter conical tube as demonstrated, and allow the bodies to settle for one minute at room temperature. Next, replace the supernatant with six milliliters of keratinocyte differentiation medium 1, or KDM1, supplemented with 10 micromolar ROCK inhibitor, and transfer the embryonic bodies to a Type IV Collagen-coated 100-millimeter dish.On days zero through eight of culture, replace the medium every other day with fresh KDM1, supplemented with three-micromolar retinoic acid and 25 nanograms per milliliter each of bone morphogenetic protein 4 and epidermal growth factor. Between days nine through 12, replace the medium every other day with KDM2, supplemented with three-micromolar retinoic acid, 25 nanograms per milliliter of bone morphogenetic protein 4, and 20 nanograms per milliliter of epidermal growth factor. Between days 13 through 30, change the medium every other day to KDM3, supplemented with 10 nanograms per milliliter of bone morphogenetic protein 4, and 20 nanograms per milliliter of epidermal growth factor.For CBMC-iPSC fibroblast differentiation, re-suspend the embryonic body culture in six milliliters of fibroblast differentiation medium 1, supplemented with 10-micromolar ROCK inhibitor, and transfer the embryonic body suspension to a basement membrane matrix-coated 100-millimeter dish. After three days of incubation, treat the culture with 0.5-nanomolar bone morphogenetic protein 4 for four days, before changing the medium to fibroblast differentiation medium 2. On day seven of culture, change the medium to FDM2 every other day for one week.On day 14 of culture, detach the cells with one-millimolar EDTA as demonstrated, and collect the cells in three milliliters of fibroblast differentiation medium 2 for centrifugation. Re-suspend the cells in five milliliters of fibroblast differentiation medium 1 for counting, and transfer two-times-10-to-the-sixth cells to a non-coated dish. On day 21 of culture, transfer two-times-10-to-the-sixth cells from the culture to a Type I Collagen-coated 100-millimeter dish in fresh fibroblast differentiation medium 1.On day 28 of culture, transfer two-times-10-to-the-six of the iPSC-derived fibroblasts to a new non-coated dish. For three-dimensional skin organoid generation, harvest the iPSC-derived fibroblasts from culture, and transfer two-times-10-to-the-fifth cells to a 15-milliliter conical tube for centrifugation. Re-suspend the fibroblast pellet in 1.5 milliliters of fibroblast differentiation medium 1, and 1.5 milliliters of neutralized Type I Collagen solution.Incubate the cells on an insert in a six-well microplate for 30 minutes at room temperature. When the solution has solidified, add two milliliters of medium to the top of the insert, and three milliliters to the bottom of the well. Then place the fibroblast matrix into the incubator for five to seven days, until the gelation is complete, and the matrix no longer contracts.At the end of the incubation, harvest the iPSC-derived keratinocytes, and transfer one-times-10-to-the-sixth cells to a 15-milliliter conical tube for centrifugation. Re-suspend the pellet in 50 to 100 microliters of low calcium epithelial medium 1, and replace the medium over the fibroblast matrix with the keratinocyte cell solution. After 15 minutes at 37 degrees Celsius, replace the medium at the top of the insert with two milliliters of epithelial medium 1, and the medium in the bottom well with three milliliters of the same, and return the plate to the incubator.After two days, replace the medium in the insert and the well with normal calcium epithelial medium 2 for two additional days of culture. On day four of culture, remove all of the medium, and add three milliliters of cornification medium to the bottom well only, to generate an air-liquid interface. Then return the plate to the incubator for up to 14 days before harvesting the 3D skin organoid for downstream analysis.CBMC-iPSC-derived keratinocytes have morphology similar to primary keratinocytes, and the expression of keratinocyte markers is increased in these cells. CBMC-iPSC-derived fibroblasts have morphology similar to primary fibroblasts, and the expression of pluripotent stem cell marker Oct-4 is down-regulated, while fibroblast markers are up-regulated. The thickness of the 3D skin organoid increases during 3D culture, confirming that the 3D skin organoid is generated from iPSC-derived keratinocytes and fibroblasts.After two weeks, a transplanted 3D skin organoid graft efficiently incorporates into a recipient mouse skin, as confirmed by HNE analysis. Further, keratinocyte maturation and epidermal differentiation markers are expressed in the CBMC-iPSC-derived 3D skin organoids, demonstrating a functional differentiation, efficient grafting, and effective healing of the mouse skin defects. We use in the past with the high calcium medium that induces stratified layers of 3D skin organoid to mimic near skin.analysis shows that the skin is stratified. CBMC-iPSCs are a potential cell source for skin grafts, and CBMC-iPSC-derived 3D skin organoids can be used in studies related to dermatology, cosmetic screening, and regenerative medicine.

Research

JoVE Journal

Developmental Biology

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 ...

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, ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

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 ...

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 ...

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 ...

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 ...

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. ...

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. ...

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 ...