Complex human skin organoids

By Shivani Rajhansa

Organoids, which are three-dimensional, self-organised tissue cultures derived from stem cells, are one of the most cutting-edge innovations in the biosciences today. From studying development and diseases of human tissues to personalised medical treatment, organoids have a scope of application that may not even be completely understood (Organoids: what are they & how do they help regenerative medicine, 2020). Earlier this year, a paper describing the in-vitro development of functional, hair-bearing human skin was published (Lee et al., 2020). This proof-of-concept study may not have generated a product that is ready for application, but with a bit of work this concept could have immense potential. 

Jiyoon Lee and colleagues managed to use the rather weak knowledge pool regarding skin induction from pluripotent stem cells to develop a protocol that generated skin with hair follicles and other complex skin structures (Lee et al., 2020). To understand how truly amazing the findings of this study are, one needs to understand the structure of the skin.  The skin is composed of two main layers: the epidermis and the dermis. The epidermis is the outermost layer and is formed by cells that grow from the basement membrane and migrate upwards, flattening as they do so. This makes the epidermis a stratified structure consisting of multiple layers. Below the epidermis is the dermis which is home to pilosebaceous units – hair follicles and the appendages around it, such as sebaceous glands, sweat glands, the arrector pili muscle and hair root (Hasudungan, 2019).

Based on previous studies Lee et al. treated their pluripotent cell aggregates with two factors to induce epidermis formation. One induced the ectoderm germ layer (which gives rise to epidermal skin cells and nervous system); the other promoted the surface ectoderm while inhibiting the neuroectoderm. This produced epidermal cysts in culture, but they never developed higher order skin morphology like stratified layers. Predicting that this was due to a lack of dermis, the researchers decided to induce fibroblasts which would produce the extracellular matrix of the dermis. One of the embryonic origins of facial dermal fibroblasts are cranial neural crest (CNC) cells. Therefore, two more factors were added to the culture to induce differentiation of these cell types. After around 10 days they again observed epidermal cysts, but this time enveloped by transient CNC-like cells. It helps to think of this structure as ‘inside-out’: the traditionally outer, epidermal layer was on the inside, the soon-to-be dermis on the outside. The CNC-like cells could be divided in two subtypes expressing either a mesenchyme-associated marker or a neuroglia-associated marker. Mesenchymal cells are multipotent stem cells and are a precursor of fibroblasts. After this, the organoids formed two distinct poles: one pole, the head, contained the epidermal cyst and the other pole, the tail, contained an opaque, cartilage-rich cell mass which consisted entirely of off-target cells (Lee et al., 2020). 

After managing to induce these organoids, the team began to record some very exciting observations. After 50 days in culture, immunostaining revealed three distinct layers in the epidermis: basal, intermediate and peridermal layers. This was the first indication of higher-level skin morphogenesis. After 70 days, hair-germ-like buds had started emerging and extending. Presence of dermal papilla cells, merkel cells and melanocytes was also noted at the bottom of the hair follicles. After more than 100 days of development, melanocytes and pigmented hairs were detected. The team also examined the organoids for neural networks due the presence of neural progenitors in early development. Much to their delight, a neural network was found in every single organoid they examined. Most neurones assumed a pseudo-unipolar morphology similar to mechanoreceptors and proprioceptors in the skin. Neurones also formed bundles which associated with Schwann-like cells and satellite glia-like cells. Furthermore, the way the axons were interwoven between hair follicles was very similar to actual human fetuses. Overall timing of development in the organoids was quite consistent with actual human fetal development (Lee et al., 2020). 

Finally, after sufficient development, Lee et al. attempted to implant the organoids onto the skin of nude mice. The organoids did unfurl from their cyst-like structure and integrated well into planar skin. Only 27 xenografts were implanted; 55% of these had 2-5 mm long outgrown hair. The rest of the grafts had either ingrown hair (22%) or failed due to technical problems. A major relief was that there was no tumour-like growth observed in any of the xenografts. Sebaceous glands and bulge stem cells, being hallmarks of mature pilosebaceous units, were also examined. All xenografted follicles contained sebaceous glands that had multiple lobes. Furthermore, in the bulge region there were stem cells similar to epithelial stem cells found in fetal hair follicles. The presence of these two factors suggested the pilosebaceous units had matured (Lee et al., 2020). 

Although translating any mouse study into a human study is a long road, this paper has established an important foundation in the field. Lee et al. generated skin organoids containing many functional appendages found in human skin, at the appropriate location and developing at an appropriate time. Long term observation would be required to see whether the follicles would keep developing in vivo. Furthermore, there was a lack of sweat glands and no development of the arrector pili muscle. There was also a generation of off-target cell lineages as observed in the ‘tail’ pole which had to be cut off before implantation. Both these factors would have to be addressed to create a more accurate representation of the skin. Regardless of these limitations, these findings could enable us to accurately model genetic skin disorders and cancers to aid drug discovery. Maybe with some additional work, these skin organoids could even be used as personalised, appendage-bearing skin grafts for patients with skin burns or wounds. 

References:

Eurostemcell.org. 2020. Organoids: What Are They & How Do They Help Regenerative Medicine? | Eurostemcell. [online] Available at: <https://www.eurostemcell.org/organoids-what-are-they-how-do-they-help-regenerative-medicine&gt; [Accessed 23 November 2020].

Hasudungan, A., 2019. Introduction To Skin Anatomy And Physiology. Available at: <https://www.youtube.com/watch?v=xUW3E6eDbzU&t=366s&gt; [Accessed 23 November 2020].

Lee, J., Rabbani, C., Gao, H., Steinhart, M., Woodruff, B., Pflum, Z., Kim, A., Heller, S., Liu, Y., Shipchandler, T. and Koehler, K., 2020. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature, [online] 582(7812), pp.399-404. Available at: <https://www.nature.com/articles/s41586-020-2352-3&gt;.

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