Coordinated Development of Immune Cell Populations in Vascularized Skin Organoids from Human Induced Pluripotent Stem Cells

Abstract

Human skin is a highly vascularized organ, where blood vessels perform essential roles in maintaining skin homeostasis and participate in thermal regulation. Currently, there is an unmet challenge to generate vascularized, functional skin models. In this study, human fetal placental endothelial colony forming cells (ECFC) are incorporated into human induced pluripotent stem cell (hiPSC)-derived skin organoids (SKO), forming capillary-like structures and generating endothelialized SKOs. However, this approach is limited by the inability of ECFCs to establish a complete vascular network, and impeding full epidermal stratification and hair follicle morphogenesis. In independent experiments, hiPSC-derived vascular organoids (VO), are incorporated into the SKO, forming complex vascular structures and generating fully vascularized skin organoids (VSKO) with resident immune cell populations. Immunofluorescence microscopy and flow cytometric analyses reveal the transfer and integration of endothelial, mural, hematopoietic, and mesenchymal cells from VOs into the skin components of VSKOs. This study pioneers the establishment of VSKOs as a transformative platform for studying human skin biology, and immune-skin interactions with applications in investigating inflammatory and other immune-mediated skin disorders.

Keywords: biofabrication; disease modeling; hair follicle; regenerative medicine; wound healing.

Figure 1

Development of Endothelialized Skin Organoids. a) Overview of the co‐culture of one skin organoid (SKO) with green fluorescent protein (GFP)‐tagged endothelial colony forming cells (ECFC). Created with BioRender.com. b) Representative brightfield microscopy images of P111 human induced pluripotent stem cell (hiPSC)‐derived SKOs with all experimental conditions at day 18, 46, 90, and 115 of SKO differentiation. Scale bars are 1 mm. Yellow colored arrows indicate hair follicles. Yellow color circles indicate SKOs. Blue color circles indicate the Growth Factor Reduced Basement Membrane Matrigel® Matrix (GFR‐M) droplet. c) Representative immunofluorescence microscopy images of P111 hiPSC‐derived SKOs at day 115 of differentiation stained with cleaved caspase‐3 (CC3, magenta color) and cytokeratin 10 (K10, yellow color) antibodies. Scale bars are 1 mm. Magnified image scale bars are 500 µm. Yellow color boxes indicate regions of interest. Cell nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue color). Organoid maturation medium (OMM). Vascular endothelial growth factor (VEGF).

Figure 2

Skin Organoids Contain Endothelial Cells. a) Representative immunofluorescence microscopy images of day 115 P111 human induced pluripotent stem cell (hiPSC)‐derived skin organoids (SKO) with all experimental conditions. The presence of cluster of differentiation (CD)31⁺ and CD34⁺ staining (both magenta color) confirmed the formation of capillary‐like structures in the SKO + green fluorescent protein (GFP)‐endothelial colony formin cells (ECFC) group, which overlapped with GFP⁺ expression. In contrast, no CD31⁺ areas were detected in the SKO Alone or SKO + Matrigel groups. Cytokeratin 10⁺ (K10, yellow color) staining demonstrated the maturation of ketatinocytes in both the SKO Alone and SKO + Matrigel groups, further confirmed by positive expression of epithelial cadherin (ECAD, yellow color) in the epithelial layer. Notably, hair follicles are present only in the SKO Alone group, and stained positively for platelet‐derived growth factor receptor α (PDGFRα, magenta color). Immunofluorescence microscopy of neural/glial antigen 2 (NG2), a key marker for mesenchymal cells and perciytes, revealed abundant cartilage, as well as NG2⁺ capillary‐like structures in the SKO + GFP‐ECFC group. Scale bars are 1 mm. Magnified image scale bars are 500 µm. b) Representative immunoflourescence microscopy images of day 115 P111 SKO + GFP‐ECFC condition with the GFP channel only, confirms the presence of GFP‐tagged ECFCs around the SKO. Scale bars are 1 mm. Magnified image scale bars are 500 µm. c) Representative immunoflourescence microscopy images of all experimental conditions at day 115 of SKO differentiation. SRY‐box transcription factor 2⁺ (SOX2, yellow color) cells, a key marker for hair follicles, were detected only in the SKO Alone group. The presence of hair follicles were further confirmed by the co‐expression of K14 and cytokeratin 17 (K17). Cytokeratin 15 (K15) and K17 expression were observed in both the hair follicle regions and epithelial layer of the SKO Alone and SKO + Matrigel groups, suggestive of keratinization in these experimental groups. No SOX2⁺, K14⁺, K15⁺, or K17⁺ cells were detected in the basal or suprabasal layer of SKO + GFP‐ECFC group, indicating the inhibitory effect of ECFCs on hair follicle development in this setting. Scale bars are 1 mm. Magnified image scale bars are 500 µm. Cell nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue color). Yellow colored arrows indicate hair follicles. Yellow color boxes indicate regions of interest.

Figure 3

Development of Vascular Organoids. a) Overview of the differentiation protocol of human induced pluripotent stem cells (hiPSC) into vascular organoids (VO). Created with BioRender.com. b) Representative brightfield microscopy images of P111 hiPSC‐derived VOs at key differentiation timepoints. Green color circles indicate VOs. Blue color circles indicate the Growth Factor Reduced Basement Membrane Matrigel® Matrix droplet (GFR‐M) droplet. Scale bars are 500 µm. c) Representative brightfield microscopy images of day 21 P111 hiPSC‐derived VOs with and without GFR‐M droplets. Scale bars are 1 mm. d) Representative brightfield microscopy images of day 21 C32 hiPSC‐derived VOs with and without GFR‐M droplets. Scale bars are 1 mm. e) Representative immunofluorescence microscopy images of day 7 P111 hiPSC‐derived VOs (before the addition of a GFR‐M droplet), and day 21 P111 hiPSC‐derived VOs. While cluster of differentiation (CD)31⁺ (yellow color) and neural/glial antigen 2⁺ (NG2, magenta color) cells are localized at the periphery, the platelet‐derived growth factor receptor α⁺ (PDGFRα, magenta color) cells are present throughout the organoids. Scale bars are 200 µm. Yellow color boxes indicate regions of interest. Cell nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue color). EGM‐2 MV Microvascular Endothelial Cell Growth Medium‐2 Bulletkit (EGM‐2MV). Bone morphogenetic protein 4 (BMP‐4). Fibroblast growth factor 2 (FGF‐2). Organoid maturation medium (OMM). Transforming growth factor β inhibitor (TGF‐β_i). StemMACS Y27632 (ROCKi, rho‐associated kinase (ROCK) inhibitor). Vascular endothelial growth factor (VEGF).

Figure 4

Vascular Organoids Recapitulate a Human Blood Vessel. a, b) Representative flow cytometry plots of day 42 C32 human induced pluripotent stem cell (hiPSC)‐derived vascular organoids (VO). VO are stained with platelet‐derived growth factor receptor β (PDGFRβ) (pericyte marker), PDGFRα, cluster of differentiation (CD)31 (endothelial cell marker), CD45 (hematopoietic cell marker), and CD90 (mesenchymal cell marker) antibodies. The CD31⁻/CD45⁻ population is gated for the expression of PDGFRβ, and PDGFRα markers. Percentage of positive cells (VO Composition %) are determined using fluorescence minus one (FMO) of day 42 C32 hiPSC‐derived VOs c). d) Representative immunofluorescence microscopy images of day 21 P111 hiPSC‐derived VO stained for CD31 (yellow color) and neural/glial antigen 2 (NG2, magenta colour). Scale bar is 50 µm. Magnified image scale bar is 10 µm. e) Representative immunofluorescence microscopy images of day 21 P111 hiPSC‐derived VO demonstrating arterial differentiation with delta‐like protein 4⁺ (DLL4, magenta color) cells. f) Representative immunofluorescence microscopy images of day 21 P111 hiPSC‐derived VO demonstrating α‐smooth muscle actin⁺ (α‐SMA, magenta color) cells. g) Representative immunofluorescence microscopy images of day 21 P111 hiPSC‐derived VO demonstrating venous differentiation with endomucin⁺ (EMCN, magenta color) cells. Cell nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue color). Scale bars are 200 µm for parts e‐g. Yellow color boxes indicate regions of interest.

Figure 5

Development of Vascularized Human Skin Organoids. a) Overview of the co‐culture of one skin organoid (SKO) with two vascular organoids (VO) on an air‐liquid interface (ALI). Original figure created with BioRender.com. b) Representative brightfield microscopy images of P111 human induced pluripotent (hiPSC)‐derived SKO Alone and SKO + Matrigel control groups, and the vascularized skin organoid (VSKO) group at day 115 of SKO differentiation. Scale bars are 1 mm. c) Representative brightfield microscopy images of two green fluorescent protein (GFP)‐tagged C32 VOs co‐cultured with one GFP⁻ C32 SKO during week 1, week 2, and week 14 of co‐culture. Blue color circles indicate SKOs. Green color circles indicate VOs. Yellow color arrows indicate hair follicles. Scale bars are 1 mm. d) Representative immunofluorescence microscopy images of day 115 VSKOs‐derived from C32 and P111 hiPSCs stained with cleaved caspase‐3 (CC3, magenta color), and cytokeratin 10 (K10, yellow color) antibodies indicate the absence of necrotic cores, whilst highlighting the formation of a well‐developed, thick epidermal layer within VSKOs. Green color represents GFP⁺ vascular cells migrating from the VO to the skin layers and hair follicles of the VSKO. Scale bars are 1 mm. Magnified image scale bars are 500 µm. Yellow boxes indicate regions of interest. Cell nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue color). Growth Factor Reduced Basement Membrane Matrigel® Matrix (GFR‐M). Organoid maturation medium (OMM). Vascular endothelial growth factor (VEGF).

Figure 6

Characterization of Vascularized Human Skin Organoids. a) Representative immunofluorescence microscopy images of day 115 P111 and C32 human induced pluripotent stem cell (hiPSC)‐derived vascularized skin organoids (VSKO) with control groups. Neural/glial antigen 2 (NG2, yellow color) antibody characterizes pericyte capillary formation when stained with cluster of differentiation (CD)31 (magenta color), which shows endothelial capillary formation, as well as CD34 (magenta color). Terminally differentiated keratinocytes are cytokeratin 10⁺ (K10, yellow color), epithelium is epithelial cadherin⁺ (ECAD, yellow color), dermal region is platelet‐derived growth factor receptor α⁺ (PDGFRα, magenta color). The P111 VSKO contains two green fluorescent protein (GFP)⁻ vascular organoids (VO) co‐cultured with one GFP⁻ skin organoid (SKO), while the C32 VSKO group includes two GFP‐tagged VOs co‐cultured with one GFP⁻ SKO. Scale bars are 500 µm. Magnified image scale bars are 200 µm. b) Representative immunofluorescence microscopy images of day 115 P111 and C32 hiPSC‐derived VSKOs with control groups. The P111 VSKO contains two GFP⁻ VOs co‐cultured with one GFP⁻ SKO, while the C32 VSKO group includes two GFP‐tagged VOs co‐cultured with one GFP⁻ SKO. All delta‐like protein 4⁺ (DLL4, magenta color) capillaries are also GFP⁺, showing arterial differentiation from the VOs. c) Representative immunofluorescence microscopy images of day 115 P111 and C32 hiPSC‐derived VSKOs with control groups. All endomucin⁺ (EMCN, magenta color) capillaries are also GFP⁺, highlighting venous differentiation from the VOs. All α‐smooth muscle actin⁺ (α‐SMA, yellow color) capillaries are also GFP⁺, demonstrating the differentiation of vascular smooth muscle cell‐derived capillaries from the VOs. Scale bars are 500 µm. Magnified image scale bars are 200 µm. Yellow colored arrows indicate hair follicles. Yellow color boxes indicate regions of interest. Cell nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue color).

Figure 7

Hair Follicle Characterization of Vascularized Human Skin Organoids. a) Representative hematoxylin & eosin staining images of paraffin‐embedded sections of day 115 P111 human induced pluripotent stem cell (hiPSC)‐derived vascularized skin organoid (VSKO). Scale bar is 200 µm. Magnified image scale bar is 100 µm. Black color box indicates the region of interest. Black color arrow highlights a hair follicle. b) Representative immunofluorescence microscopy images of day 115 C32 hiPSC‐derived VSKOs. Two green fluorescent protein (GFP)‐tagged vascular organoids (VO) are co‐cultured with one GFP⁻ skin organoid (SKO) and stained for cluster of differentiation (CD)34 (magenta color), demonstrating perifollicular vascularization. Scale bar is 500 µm. Magnified image scale bar is 200 µm. c) Representative immunofluorescence microscopy images of day 115 P111 and C32 hiPSC‐derived VSKOs with control groups. The P111 VSKO contains two GFP⁻ VOs co‐cultured with one GFP⁻ SKO, while the C32 VSKO group includes two GFP‐tagged VOs co‐cultured with one GFP⁻ SKO. SKOs and VSKOs were stained with the following antibodies: SRY‐box transcription factor 2 (SOX2, yellow color), CD31 (magenta color), cytokeratin 14 (K14, yellow color), cytokeratin 15 (K15, magenta color), and cytokeratin 17 (K17, magenta color). Scale bars are 500 µm. Magnified image scale bars are 200 µm. Cell nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue color). Yellow color boxes indicate regions of interest. Yellow colored arrows indicate hair follicles. Dermal papillae (DP).

Figure 8

Vascular Organoid‐Derived Immune Cells Migrate into Skin Organoids. a) Representative hematoxylin & eosin staining images of paraffin‐embedded sections of day 40 P111 human induced pluripotent stem cell (hiPSC)‐derived vascular organoid (VO) shows the development of vascular‐like structures containing immune‐like cells. Scale bar is 200 µm. Magnified image scale bar is 50 µm. Black color box indicates the region of interest. b) Representative immunofluorescence microscopy images of day 7 and day 21 P111 hiPSC‐derived VOs. While cluster of differentiation (CD)45⁺ cells (yellow color) are not present at day 7, their numbers increase, and they disperse throughout the VOs by day 21. Notably, some CD45⁺ cells are also positive for CD34 (magenta color). Scale bar is 200 µm. c) Representative immunofluorescence microscopy images of day 115 C32 hiPSC‐derived vascularized skin organoid (VSKO). The VSKO contains two green fluorescent protein (GFP)‐tagged VOs co‐cultured with one GFP⁻ SKO. These images highlight the presence of CD45⁺ (magenta color) and CD68⁺ (yellow color) cells within the dermal layer of the VSKO. Scale bar is 1 mm. Magnified image scale bar is 200 µm. d) Representative immunofluorescence microscopy images of day 115 C32 hiPSC‐derived VSKO show the presence of CD207⁺ (magenta color) cells within the dermal layer next to a hair follicle. Scale bars are 200 µm. e) Representative immunofluorescence microscopy images of day 115 C32 hiPSC‐derived VSKO demonstrate the presence of CD66b⁺ (magenta color) cells within the dermal layer. Scale bars are 200 µm. Cell nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue color). Yellow color boxes indicate regions of interest. Yellow color arrows indicate hair follicles.