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

Summary

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.

Abstract

The skin is the body’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.

Introduction

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’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’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’s immune system after approximately 1 week³. To overcome rejection by the patient’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 medicine^12,13,14. 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.

Protocol

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

1. Medium preparation
   NOTE: Store all medium at 4 °C in a dark environment for up to 3 months. Filter all medium using a 0.22 μm polyethersulfone filter system before use for sterilization. All medium was available in a total volume of 500 mL.
   1. Prepare KDM1 (keratinocyte differentiation medium 1). Mix Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 medium (3:1) with 2% fetal bovine serum (FBS), 0.3 mmol/L L-ascorbic acid, 5 μg/mL insulin, and 24 µg/mL adenine.
   2. 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 μg/mL insulin, and 10 μg/mL adenine.
      NOTE: Defined keratinocyte serum-free medium is optimized to support the growth and expansion of keratinocytes.
   3. 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.
   4. Prepare FDM1 (fibroblast differentiation medium 1). Mix DMEM/F12 medium (3:1) with 5% FBS, 5 μg/mL insulin, 0.18 mM adenine, and 10 ng/mL epidermal growth factor (EGF).
   5. Prepare FDM2 (fibroblast differentiation medium 2). Mix DMEM/F12 medium (1:1) with 5% FBS and 1% nonessential amino acids.
   6. Prepare EP1 (epithelial medium 1). Mix DMEM/F12 (3:1) with 4 mM L-glutamine, 40 μM adenine, 10 μg/mL transferrin, 10 μg/mL insulin, and 0.1% FBS.
   7. Prepare EP2 (epithelial medium 2). Mix EP1 and 1.8 mM calcium chloride.
   8. Prepare EP3 (epithelial medium 3, cornification medium). Mix F12 medium with 4 mM L-glutamine, 40 μM adenine, 10 μg/mL transferrin, 10 μg/mL insulin, 2% FBS, and 1.8 mM calcium chloride.
2. Embryonic body generation
   1. Generate CBMC-iPSCs using the protocol shown in a previous study¹².
   2. Coat culture dishes, using vitronectin. Prepare 5 mL to coat a 100 mm dish.
      1. Thaw and resuspend 50 μL of 0.5 mg/mL vitronectin (final concentration: 5 µg/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).
   3. Maintain the CBMC-derived iPSCs to the vitronectin-coated 100 mm plate and change the iPSC medium (E8) daily at 37 °C with 10% CO₂.
   4. 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.
   5. Treat the cells with 1 mL of 1 mM ethylenediaminetetraacetic acid (EDTA). Incubate at 37 °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.
   6. 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.
   7. Resuspend the transferred cells with 2.5 mL of EB formation medium with 10 μM Rho-associated kinase (ROCK) inhibitor. Drop 1 x 10⁴ cells (25 µL/drop) on a noncoated culture plate lid using a 10–100 μL 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.
   8. Incubate the droplets at 37 °C with 5% CO₂ for 1 day.
   9. 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.
3. Differentiation of CBMC-iPSCs into keratinocytes
   NOTE: For a scheme of the keratinocyte differentiation from CBMC-iPSCs, see Figure 1A.
   1. 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 °C with 5% CO₂ for 1 day.
   2. Coat culture dishes, using type IV collagen. Prepare 5 mL of type IV collagen to coat a 100 mm dish.
      1. Thaw and resuspend the type IV collagen solution (final concentration: 50 µg/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.
   3. 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 µM 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.
   4. Between days 0–8, change the medium every other day to KDM1 with 3 µM retinoic acid (RA) and 25 ng/mL each of BMP4 and EGF. Maintain the EBs at 37 °C with 5% CO₂.
   5. Between days 9–12, change the medium every other day to KDM2 with 3 µM RA, 25 ng/mL BMP4, and 20 ng/mL EGF.
   6. Between days 13–30, change the medium every other day to KDM3 with 10 ng/mL BMP4 and 20 ng/mL EGF.
4. Differentiation of CBMC-iPSC into fibroblasts
   NOTE: For a scheme of the fibroblast differentiation from CBMC-iPSCs, see Figure 2A.
   1. Coat culture dishes, using basement membrane matrix. Prepare 5 mL to coat a 100 mm dish.
      1. 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 °C for 30 min. Aspirate the coating material before use (not to dry out).
   2. 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.
   3. Resuspend the EBs using a 1,000 µL pipette in 6 mL of FDM1 with 10 µM ROCK inhibitor. Transfer the EBs (with medium) to a basement membrane matrix-coated 100 mm dish and incubate at 37 °C with 5% CO₂. Refresh the FDM1 every other day for 3 days.
      NOTE: Only add the ROCK inhibitor at the EB attachment stage.
   4. Add 0.5 nM bone morphogenetic protein 4 (BMP 4) to the FDM1 between days 4 and 6.
   5. At day 7, change the medium to FDM2 every other day for 1 week.
   6. At day 14, add 1 mL of 1 mM EDTA and incubate at 37 °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.
   7. 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 °C with 5% CO₂ and change the medium every other day.
   8. Coat culture dishes, using type I collagen. Prepare 5 mL to coat a 100 mm dish. Dilute type I collagen solution (final concentration: 50 µg/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.
   9. On day 21, add 1 mL of 1 mM EDTA and incubate at 37 °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 °C with 5% CO₂ and change the medium every other day.
   10. On day 28, add 1 mL of 1 mM EDTA and incubate at 37 °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 °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.

2. Application of hiPSC-derived differentiated cells

1. Generation of 3D skin organoid
   1. Prepare neutralized type I collagen on ice, following the manufacturer’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.
   2. Add 1 mL of EDTA to the iPSC-derived fibroblasts from step 1.4.10 and incubate at 37 °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.
   3. 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.
   4. 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 °C with 5% CO₂ for 5-7 days, until the gelation is complete and no longer contracts.
   5. 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 °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.
   6. Remove the supernatant and resuspend 1 x 10⁶ cells in 50-100 µL of low calcium epithelial medium 1 (EP1).
   7. 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 °C with 5% CO₂ for 30 min.
      NOTE: Do not move the plate and do not add any medium for the attachment of keratinocyte.
   8. Add 2 mL of EP1 to the top of the insert and 3 mL of EP1 to the bottom of the well.
   9. After 2 days, aspirate all medium in the membrane insert plate and change the medium to normal calcium EP2 for 2 days.
   10. After 2 days, aspirate all medium and add 3 mL of the cornification medium only to the bottom to generate an air-liquid interface.
   11. Maintain the 3D skin organoid for up to 14 days at 37 °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.
2. Skin graft
   1. 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’s dorsal skin.
   2. Remove a 1 cm x 2 cm section of the mouse’s skin, using curved scissors with forceps.
   3. Place the CBMC-iPSC-derived 3D skin organoid onto the defect site and suture using a tie-over dressing method with silk sutures.
   4. Observe the mice for 2 weeks and sacrifice them for histological analysis. The staining protocol was verified in previous studies¹⁶.

Results

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...

Discussion

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 transplantation^18,19. The use of patient-derived iPSCs can also overcome problems regarding primary cells, a lack of adequate cell numbers, and immune reactions^5,

Materials

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