Summary Defective placentation is the underlying cause of various pregnancy complications, such as severe intrauterine growth restriction and preeclampsia. However, studies on human placental development are hampered by the lack of a self-renewing in vitro model that would recapitulate formation of trophoblast progenitors and differentiated subtypes, syncytiotrophoblast (STB) and invasive extravillous trophoblast (EVT), in a 3D orientation. Hence, we established long-term expanding organoid cultures from purified first-trimester cytotrophoblasts (CTBs). Molecular analyses revealed that the CTB organoid cultures (CTB-ORGs) express markers of trophoblast stemness and proliferation and are highly similar to primary CTBs at the level of global gene expression. Whereas CTB-ORGs spontaneously generated STBs, withdrawal of factors for self-renewal induced trophoblast outgrowth, expressing the EVT progenitor marker NOTCH1, and provoked formation of adjacent, distally located HLA-G+ EVTs. In summary, we established human CTB-ORGs that grow and differentiate under defined culture conditions, allowing future human placental disease modeling.

Self-Renewing Trophoblast Organoids Recapitulate the Developmental Program of the Early Human Placenta

The past decade has seen an explosion in the field of in vitro disease modelling, in particular the development of organoids. These self-organizing tissues derived from stem cells provide a unique system to examine mechanisms ranging from organ development to homeostasis and disease. Because organoids develop according to intrinsic developmental programmes, the resultant tissue morphology recapitulates organ architecture with remarkable fidelity. Furthermore, the fact that these tissues can be derived from human progenitors allows for the study of uniquely human processes and disorders. This article and accompanying poster highlight the currently available methods, particularly those aimed at modelling human biology, and provide an overview of their capabilities and limitations. We also speculate on possible future technological advances that have the potential for great strides in both disease modelling and future regenerative strategies.

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Disease modelling in human organoids.

Engineering Stem Cell Self-organization to Build Better Organoids

The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met3–9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4–5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery. Skin organoids generated in vitro from human pluripotent stem cells form complex, multilayered skin tissue with hair follicles, sebaceous glands and neural circuitry, and integrate with endogenous skin when grafted onto immunocompromised mice.

Hair-bearing human skin generated entirely from pluripotent stem cells

Organoid cultures have emerged as an alternative in vitro system to recapitulate tissues in a dish. While mouse models and cell lines have furthered our understanding of liver biology and associated diseases, they suffer in replicating key aspects of human liver tissue, in particular its complex architecture and metabolic functions. Liver organoids have now been established for multiple species from induced pluripotent stem cells, embryonic stem cells, hepatoblasts and adult tissue-derived cells. These represent a promising addition to our toolbox to gain a deeper understanding of this complex organ. In this perspective we will review the advances in the liver organoid field, its limitations and potential for biomedical applications.

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Liver organoids: from basic research to therapeutic applications.

The derivation of human inner ear tissue from pluripotent stem cells would enable in vitro screening of drug candidates for the treatment of hearing and balance dysfunction and may provide a source of cells for cell-based therapies of the inner ear. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and WNT signaling to generate multiple otic-vesicle-like structures from a single stem-cell aggregate. Over 2 months, the vesicles develop into inner ear organoids with sensory epithelia that are innervated by sensory neurons. Additionally, using CRISPR-Cas9, we generate an ATOH1-2A-eGFP cell line to detect hair cell induction and demonstrate that derived hair cells exhibit electrophysiological properties similar to those of native sensory hair cells. Our culture system should facilitate the study of human inner ear development and research on therapies for diseases of the inner ear.

/pdf/generation-of-inner-ear-organoids-containing-functional-hair-4a42rhgl83.pdf

Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells

Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids

The skin is important for regulating bodily fluid retention and temperature, guarding against external stresses, and mediating touch and pain sensation. The skin is also susceptible to damage from burns, diseases, or genetic defects, which affect nearly one billion people worldwide1,2. For the advancement of skin regenerative therapies, it remains challenging to construct new skin with hair follicles and nerves in tissue cultures and in bioengineered skin grafts3–8. Here, we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use step-wise modulation of the TGFβ and FGF signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During 4-5 months incubation, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis, and pigmented hair follicles equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking human touch circuitry. Single-cell RNA sequencing and direct comparison to foetal specimens suggest that skin organoids are equivalent to human facial skin in the second-trimester of development. Moreover, we show that skin organoids produce planar hair-bearing skin when grafted on nude mice. Together, our results demonstrate the self-assembly of nearly complete skin tissue in vitro that can be used to reconstitute skin in vivo. We anticipate that our skin organoid model will be foundational to future studies of human skin development, disease modelling, or reconstructive surgery.

/pdf/hair-bearing-human-skin-generated-entirely-from-pluripotent-4ciaorzvsj.pdf

Jiyoon Lee

Papers

Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells

Hair-bearing human skin generated entirely from pluripotent stem cells

Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids

Hair-bearing human skin generated entirely from pluripotent stem cells

Hair follicle development in mouse pluripotent stem cell-derived skin organoids