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 th....

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.......

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