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’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 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 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.
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
2. Application of hiPSC-derived differentiated cells
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 using the hanging drop method to ensure a uniform and controlled differentiation of keratinocytes and fibroblasts (Figure 1C). EBs were attached to type IV collagen-coated plates for keratinocyte differentiation, and the medium was changed daily. CBMC-iPSCs were treated with RA, BMP4, and EGF. CBMC-iPSCs were differentiated to keratinocytes. During the differentiation, the morphology of the CBMC-iPSC-derived keratinocytes changed over time (Supplementary Figure 1).
CBMC-iPSC-derived keratinocytes have morphologies similar to primary keratinocytes (Figure 1D). The gene expression of the pluripotent marker OCT4 was downregulated in CBMC-iPSC-derived keratinocytes. Primer sequences are shown in Table 1. The expression of keratinocyte markers Np63, KRT5, and KRT14 was increased in CBMC-iPSC-derived keratinocytes (Figure 1F). CBMC-iPSC-derived keratinocytes were confirmed by the expression of Np63 and KRT14 by immunohistochemistry (Figure 1E). These results confirmed that CBMC-iPSC-derived keratinocytes have the characteristics of primary keratinocytes.
The scheme of fibroblast differentiation is shown in Figure 2A. We also maintained CBMC-iPSCs in a vitronectin-coated dish and used EB formation for fibroblast differentiation (Figure 2B,C). We attached the EBs to basement membrane matrix-coated plates and changed the medium every other day. Outgrowth cells were transferred to noncoated and type I collagen-coated plates. CBMC-iPSCs were differentiated to fibroblasts. During the differentiation, the morphology of the CBMC-iPSC-derived fibroblasts changed over time (Supplementary Figure 2).
CBMC-iPSC-derived fibroblasts have morphologies similar to primary fibroblasts (Figure 2D). The expression of pluripotent stem cell marker OCT4 was downregulated in CBMC-iPSC-derived fibroblasts. Fibroblast markers of COL1A1, COL1A2, COL3A1, and CD44 were upregulated in CBMC-iPSC-derived fibroblasts (Figure 2F). Primer sequences are shown in Table 1. Also, CBMC-iPSC-derived fibroblasts were confirmed by the expression of vimentin and fibronectin by immunohistochemistry (Figure 2E). These results suggest that CBMC-iPSC-derived fibroblasts are similar to primary fibroblasts.
We generated a 3D skin organoid using the CBMC-iPSC-derived keratinocytes and fibroblasts. The scheme of formation of the 3D skin organoid is shown in Figure 3A. We generated a 3D skin organoid on a membrane insert plate. For the 3D culture, CBMC-iPSC-derived fibroblasts were stratified with type I collagen and overlaid with CBMC-iPSC-derived keratinocytes. After seeding the CBMC-iPSC-derived keratinocytes, the medium was changed to a normal calcium concentration for 2 days. After 2 days, a high calcium concentration medium was added only to the lower chamber for the formation of air-liquid interface culture. The air-liquid interface culture induced the maturation and stratification of the keratinocytes. The thickness of the 3D skin organoid was increased during 3D culture. These results confirmed that the 3D skin organoid was generated from iPSC-derived keratinocytes and fibroblasts by hematoxylin and eosin (H&E) staining (Figure 3C).
Using the CBMC-iPSC-derived 3D skin organoid, we generated a humanized mice model (Figure 3B) by grafting the 3D skin organoid to the mice. A 1 cm x 2 cm defect was induced, and the tie-over method was used for transplantation. After 2 weeks, the transplanted skin was efficiently grafted to the mice, and we confirmed this by H&E and immunocytochemical analysis (Figure 3D). Keratinocyte maturation and the epidermal differentiation markers of loricrin and KRT14 were expressed in the CBMC-iPSC-derived 3D skin organoids (Figure 3E). The CBMC-iPSC-derived 3D skin organoids were functionally differentiated, efficiently grafted onto mice, and effectively healed mice skin defects.
Figure 1: Keratinocyte differentiation of CBMC-iPSCs. (A) Scheme of keratinocyte differentiation from CBMC-iPSCs. (B and C) Morphology of the CBMC-iPSCs (panel B) and iPSC-derived EBs (panel C). (D) Morphology of the CBMC-iPSC-derived keratinocytes. (E) Immunocytochemical analysis of Np63 (red) and KRT14 (green), together with DAPI staining (blue). The scale bars = 100 μm. (F) Gene expression of the pluripotent marker and keratinocyte markers of iPSC-derived keratinocytes (iPSC-Ks). The graphs show the mean with SEM of five independent samples. Differences between groups were examined for statistical significance using Student’s t-test. The t-test was applied to analyze nonparametric quantitative datasets, and the one-tailed p-value was calculated (*p < 0.05, **p < 0.01, ***p < 0.001 indicated statistical significance). Please click here to view a larger version of this figure.
Figure 2: Fibroblast differentiation of CBMC-iPSCs. (A) Scheme of fibroblast differentiation from CBMC-iPSCs. (B and C) Morphology of the CBMC-iPSCs (panel B) and iPSC-derived EBs (panel C). (D) Morphology of the CBMC-iPSC-derived fibroblasts. (E) Immunocytochemical analysis of vimentin (red) and fibronectin (red), together with DAPI staining (blue). The scale bars = 100 μm. (F) Gene expression of the pluripotent marker and fibroblast markers of iPSC-derived fibroblast (iPSC-Fs). The graphs show the mean with SEM of five independent samples. Differences between groups were examined for statistical significance using Student’s t-test. The t-test was applied to analyze nonparametric quantitative datasets, and the one-tailed p-value was calculated (*p < 0.05, **p < 0.01, ***p < 0.001 indicated statistical significance). Please click here to view a larger version of this figure.
Figure 3: Generation of CBMC-iPSC-derived skin organoid and humanized mice model. (A) Schematic diagram of the iPSC-derived skin organoid (iSO) generation process. (B) Transplantation process of the iSO into mice. (C) Histological analysis of the iSO in vitro. (D) Histological analysis of the transplanted iSO in vivo. (E–H) Immunocytochemical analysis of loricrin and KRT14. MOCK control (panel E), transplanted iSO (panel F, loricrin), mice skin (negative control, panel G), transplanted iSO (panel H, KRT14). The scale bars = 200 μm. Please click here to view a larger version of this figure.
Supplementary figure 1: Morphology of iPSC-derived keratinocytes. Please click here to view a larger version of this figure.
Supplementary figure 2: Morphology of iPSC-derived fibroblasts. Please click here to view a larger version of this figure.
Target name | Direction | Primer sequence (5’-3’) | Size (Base pairs) | Refseq_ID | |
OCT4 | Forward Reverse |
ACCCCTGGTGCCGTGAA GGCTGAATACCTTCCCAAATA |
190 | NM_203289.5 | |
PAX6 | Forward Reverse |
GTCCATCTTTGCTTGGGAAA TAGCCAGGTTGCGAAGAACT |
110 | NM_000280.4 | |
SOX1 | Forward Reverse |
CACAACTCGGAGATCAGCAA GGTACTTGTAATCCGGGTGC |
133 | NM_005986.2 | |
Np63 | Forward Reverse |
GGAAAACAATGCCCAGACTC GTGGAATACGTCCAGGTGGC |
294 | NM_001114982.1 | |
KRT5 | Forward Reverse |
ACCGTTCCTGGGTAACAGAGCCAC GCGGGAGACAGACGGGGTGATG |
198 | NM_000424.3 | |
KRT14 | Forward Reverse |
GCAGTCATCCAGAGATGTGACC GGGATCTTCCAGTGGGATCT |
181 | NM_000526.4 | |
CD44 | Forward Reverse |
AAGGTGGAGCAAACACAACC AGCTTTTTCTTCTGCCCACA |
151 | NM_001202556.1 | |
COL1A1 | Forward Reverse |
CCCCTGGAAAGAATGGAGATG TCCAAACCACTGAAACCTCTG |
148 | NM_000088.3 | |
COL1A2 | Forward Reverse |
GGATGAGGAGACTGGCAACC TGCCCTCAGCAACAAGTTCA |
77 | NM_000089.3 | |
COL3A1 | Forward Reverse |
CGCCCTCCTAATGGTCAAGG TTCTGAGGACCAGTAGGGCA |
161 | NM_000090.3 | |
Vimentin | Forward Reverse |
GAGAACTTTGCCGTTGAAGC TCCAGCAGCTTCCTGTAGGT |
170 | NM_003380.5 | |
GAPDH | Forward Reverse |
ACCCACTCCTCCACCTTTGA CTGTTGCTGTAGCCAAATTCGT |
110 | NM_002046.5 |
Table 1: Sequences of primers used for quantitative real-time polymerase chain reaction.
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 reactions5,17,19. However, the generation of personalized iPSCs is not economically feasible due to time, cost, and labor restrictions. HLA-homozygous CBMC-derived iPSCs have emerged as a new possibility. HLA-homozygous iPSCs can be economically valuable and can be applied to a large number of patients8,11,12,13. Furthermore, HLA typing of CBMCs occurs during cell bank storage, thereby making them easy to use for research and transplantation. Protocols to differentiate CBMC-iPSCs into cardiomyocytes, hepatocytes, and chondrocytes have been reported16,20,21,22,23.
Epidermal and dermal layers are components of the skin. The epidermis consists of keratinocytes and the dermis consists of fibroblasts. So, we differentiated CBMC-iPSCs into keratinocytes and fibroblasts, respectively. For the differentiation, uniformed, well-controlled, and optimized EBs were generated by the hanging drop method15,24. Type IV collagen is a major component of the basement membrane. For keratinocyte differentiation, EBs were attached to type IV collagen-coated dishes. CBMC-iPSC-derived keratinocytes had a cobblestone-like morphology (Figure 1D). Keratinocyte markers Np63 and KRT14 were expressed in iPSC-Ks (Figure 1E,F). That result confirmed that RA and BMP4 induced the upregulation of the keratinocyte markers. Furthermore, CBMC-iPSCs were differentiated into keratinocytes similar to primary keratinocytes.
For fibroblast differentiation, EBs were attached to basement membrane matrix-coated plates, and the differentiated cells were serially passaged onto noncoated and type I collagen-coated plates. A serial subculture was induced to specify fibroblast differentiation. Fibroblasts produced an extracellular matrix (ECM) that had migration and adhesion functions. Fibroblasts also produce abundant collagen components25. In CBMC-iPSC-derived fibroblasts, the fibroblast surface marker CD44 was increased. The expression of the collagen was upregulated in iPSC-Fs (Figure 2F). The expression of the fibronectin and vimentin was increased in iPSC-Fs (Figure 2E).
Using the differentiated keratinocytes and fibroblasts, we generated CBMC-iPSC-derived skin organoids (Figure 3A). We used an air-liquid interface culture with a high-calcium medium that induced stratified layers of CBMC-iPSC-derived skin organoids. The high concentration of calcium was necessary for keratinocyte maturation in vivo and in vitro, while the air-liquid interface was used to develop multilayered strata26,27,28. We used this method to mimic real skin, and histological analysis showed that the skin was stratified (Figure 3C). To confirm the wound-healing ability, we transplanted the iSO into mice skin, using the tie-over dressing method (Figure 3D). After transplantation, the skin organoids were efficiently grafted and healed the mice skin adequately. KRT14 was expressed in the basal layer of stratifying squamous and nonsquamous epithelia. Loricrin is a main component of the stratum corneum found in terminally differentiated and keratinized epithelial cells29,30. The epidermal differentiation marker of loricrin was expressed in transplanted skin. The expression of KRT14 and loricrin confirmed that the skin organoid was fully mature, and differentiation was demonstrated by immunohistochemical staining (Figure 3E).
In this study, we developed a protocol to differentiate CBMC-iPSCs into keratinocytes and fibroblasts, the main cell types of human skin. We confirmed that the CBMC-iPSC-derived keratinocytes and fibroblasts showed phenotypes similar to primary cell lines. Using these differentiated cells, we generated a 3D skin organoid and grafted it into NOD/scid mice using the tie-over dressing method. This original technique was first described in 1929 by Blair and Brown and has been commonly used for skin grafting31,32. This method prevented the graft from moving, favored a good adhesion to the wound, and thus accelerated tissue healing. Histological analysis confirmed that the 3D skin organoid mimicked a human skin phenotype that successfully stratified and matured over 2 weeks. Skin grafting is generally performed using single cells of keratinocytes and fibroblasts by silicon bubble chamber33,34. This system is easy to graft but we needed more time for observed to transplantation efficiency after graft. The plastic or silicon chamber functions as a barrier against the mice’s skin. The 3D skin organoid system-derived iPSCs do not use a plastic or silicon chamber. In this system, transplantation was efficient; however, it was difficult to block the natural healing process of mice. So, the mice’s skin covered many parts of the iSO for a long time after the transplantation. This is a part of the method presented here that must be improved.
In conclusion, CBMC-iPSCs are a potential cell source for skin grafts. Using these protocols, CBMC-iPSC-derived keratinocytes, fibroblasts, and a 3D skin organoid can be used in studies related to dermatology, drug and cosmetic screening, and regenerative medicine.
The authors have nothing to disclose.
This work was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (H16C2177, H18C1178).
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 |