Journal: Nature protocols

Article Title: Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

doi: 10.1038/s41596-022-00681-y

Figure Lengend Snippet: a, A timeline of in vitro skin organogenesis in the skin organoid model. hPSCs form aggregates on day 0 of differentiation. These aggregates are treated with TGF-β inhibitor (SB), BMP4, and a low concentration of bFGF, giving rise to surface ectoderm by day 3. The aggregates are then treated with BMP inhibitor (LDN) and a high concentration of bFGF. This day 3 treatment induces the development of non-epithelial cell populations consisting, in part, of cranial neural crest (CNC) cells, which further differentiate into diverse mesenchymal and neuro-glial cell populations contributing to the dermis layer of the organoid. Although rare, hair germs can be seen as early as day 56 of differentiation, with more mature hair pegs and hair follicles arising around day 70 through day 130 of differentiation. The fully mature skin organoid includes appendages, such as hair follicles, adipocytes, melanocytes, sebaceous glands, and sensory neurons. b, A schematic of neurulation in vivo. Neurulation occurs around 3–4 weeks of development, corresponding to days 6–18 of organoid culture. The ectoderm folds and pinches inward, resulting in the formation of the neural tube and, in the cranial region, delaminating CNC cells. CNC cells give rise to diverse cell lineages, such as chondrocytes, myocytes, fibroblasts, neurons, and Schwann cells. c, A timeline of in vivo skin development. Following fertilization, the zygote undergoes many rounds of rapid division. By day 12, the epiblast (amniotic cavity) and hypoblast (yolk sac) compose the bilaminar disk. The epiblast gives rise to the definitive germ layers, the ectoderm, mesoderm, and endoderm. In relation to the skin, the ectoderm gives rise to keratinocyte precursors (epidermis layer of skin), while the dermal fibroblasts (dermis layer) are derived primarily from the mesoderm in the body and CNCs in the face. By 6 weeks, melanocyte precursors, sensory neuron progenitors, and other diverse cell progenitors appear. Hair germs, which develop into hair pegs and finally into hair follicles, start to form around 12 weeks of gestation. Fully stratified skin with erupted lanugo hair is reached around week 18 of gestation. The skin further matures and develops diverse appendages, such as blood vessels, sweat glands, sebaceous glands, and a network of sensory neurons (thermoreceptors, mechanoreceptors, and nociceptors).

Article Snippet: LDN (BMP inhibitor) , Stemgent , 04-0074-02 , 10 mM , 1 μM (200 nM X5) , 0.5 μL.

Techniques: In Vitro, Concentration Assay, In Vivo, Derivative Assay

Journal: Nature protocols

Article Title: Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

doi: 10.1038/s41596-022-00681-y

Figure Lengend Snippet: Briefly, on day (−2) of differentiation, the hPSCs cultured as colonies are dissociated into single cells and aggregated at a concentration of 3,500 cells in 100 μL of E8 + Y per well in a 96-well U-bottom plate. The E8 contains Y to inhibit apoptosis of cells when they become single-celled. After centrifugation, the single cells concentrate at the bottom of the 96-well U-bottom plate, forming a circular cell cluster layer. The single cells migrate and tightly bind each other, becoming a small sphere aggregate in the center of each well by day (−1). On day (−1), additional E8 is added to dilute Y and provide a better proliferative environment. During the process of introducing additional E8, any dead cells adhered to the aggregates get released so that the surface of the aggregates becomes clean. Starting on day 0, the differentiation initiates. All aggregates from the 96-well U-bottom plates are collected and washed thoroughly to remove any residues of E8 that may interfere with differentiation. The aggregates are individually transferred to each well of 96-well U-bottom plates in E6 medium containing SB, bFGF, and BMP4. SB is a TGF-β inhibitor that promotes ectoderm induction. BMP4 induces surface ectoderm formation. The combination of SB, BMP4, and bFGF (low concentration) at an optimal concentration and timing guides the outer layer of cells on the aggregate to differentiate purely into the surface ectoderm. By day 3 of differentiation, the aggregate will form a thin, bright, transparent-like epithelium surrounding the aggregate. Depending on the cell lines, the epithelium may appear wavy or linear and will be visible between days 3 and 5. Inhibition of the BMP signaling pathways with LDN and activation of FGF signaling with a high concentration of bFGF on day 3 induces the formation of NC cells. By day 6 of differentiation, the aggregate becomes more cystic, containing dark core and radial traces of migrating mesenchymal cells from the core to the epithelium. Fresh E6 is added to provide nutrition. On day 9 of differentiation, half the volume of spent medium is replenished with fresh E6, providing nutrition. The aggregate continues to grow larger, and the mesenchymal cells populate more on the surface of the aggregate. Around day 12 of differentiation, the mesenchymal cells typically start to concentrate on one pole of the aggregate, leaving the other pole more cystic. All aggregates are collected and washed thoroughly on day 12 of differentiation to remove any residual E6 differentiation medium. The individual aggregates are transferred to each well of the 24-well plate in OMM1%M and placed on an orbital shaker to provide a floating environment, where the aggregates self-organize further. The OMM is regularly replenished to provide sufficient nutrition during development. See Extended Data Fig. 1 and Supplementary information in ref. 25 for additional optimization details. Representative images are taken at 40X (4X microscope objective × 10X eyepiece), or 100X (10X microscope objective × 10X eyepiece) magnifications as noted in each image. Scale bars, 200 μm.

Article Snippet: LDN (BMP inhibitor) , Stemgent , 04-0074-02 , 10 mM , 1 μM (200 nM X5) , 0.5 μL.

Techniques: Cell Culture, Concentration Assay, Centrifugation, Inhibition, Activation Assay, Microscopy