Figura 1 - Polarised human follicles producing T4 transplanted into immunodeficient mice. NKX2-1 (red), E-Cadherin (Green), T4 (light blue) and DNA (blue). Scale bar 20um.
Organoid technology can be considered one of the most significant scientific advances of the last decade. Organoids are tiny, self-organized three-dimensional versions of an organ that are produced in vitro. Moreover, human organoids offer unique opportunities to model the development, physiology, and diseases of human tissues/organs while complementing animal models and reducing the need for animal testing 1.
The thyroid gland is responsible for the synthesis and release of thyroid hormones (THs), T3 and T4, which play a critical role in organ development and homeostasis and control processes such as body growth and energy expenditure 2. Hypothyroidism is a very common disease with a prevalence of 1% to 5% worldwide. It results from inadequate production of thyroid hormones (TH) due to autoimmune damage, toxicity, genetic defects at birth (congenital hypothyroidism, CH) or after surgical/radioactive thyroidectomy 3. Despite well-established TH replacement therapy, it is estimated that up to one-third of patients do not receive adequate treatment, while a significant proportion have impaired health-related quality of life 4,5. Given the lack of a functional in vitro thyroid model to further explore various aspects related to thyroid development and disease, the ability to generate TH -producing human follicles from stem cells would open new perspectives for the human thyroid research field.
In 2012, our laboratory demonstrated for the first time the feasibility of generating 3D-organized thyroid follicles in vitro from mouse embryonic stem cells (mESCs). The resulting thyroid organoids were able to recapitulate thyroid developmental stages, produce THs, and restore hypothyroidism when transplanted into mice with thyroid ablation 6. Despite the progress made in recent years in mouse ESC -derived in vitro thyroid models, generating a functional human ESC -derived thyroid in vitro has been a major challenge.
After 10 years of intensive research and several attempts and failures, our laboratory has achieved the goal of generating a functional human thyroid in vitro from ESCs (Preprint Romitti, Biorxv, 2021) 7. Following the human in vivo thyroid developmental stages, we first differentiated human ESCs into endoderm cells. Transient overexpression of the key thyroid transcription factors Nkx2-1 and Pax8 then allowed us to obtain thyroid progenitor cells that expanded under specific culture conditions, self-organized into 3D follicular structures, and produced THs. Remarkably, these in vitro-grown follicles were able to maintain histological organization, promote vascular formation, and synthesize and release THs when transplanted into the renal capsule of mice whose thyroid gland had been previously destroyed by 131I treatment. The transplanted mice exhibited a marked increase in T4 serological levels compared with non-transplanted hypothyroid animals. Given the challenges associated with inducing thyroid ablation and transplanting human cells into immunodeficient (highly radiosensitive) mice, we present in this study a proof-of-concept experiment demonstrating that human functional thyroid tissue generated from pluripotent stem cells can be transplanted and retain functionality in vivo by producing THs and increasing T4 levels in hypothyroid animals.
In perspective, this model could be used as a new alternative to study aspects related to human thyroid such as: 1. screening tool for toxic compounds such as endocrine disruptors. 2. diagnostic tool for studying the mechanisms leading to congenital hypothyroidism, as several new potential candidate genes have been proposed in recent years that are associated with CH. By combining screening tools such as next generation sequencing (NGS) and exome/genome, newly identified mutations could be reproduced in hESCs using technologies such as CRISPR-Cas9 and their effects on thyroid development could be explored. 3. reproducibility of the protocol using patient induced pluripotent stem cells (iPSCs) to circumvent graft rejection. 4. decipher the mechanisms involved in early thyroid formation using transcriptomic tools aimed at directly differentiating hESC/iPSC without forced expression of genes and enabling a clean model for therapeutic applicability. 5. modeling human thyroid carcinogenesis, decipher early mechanisms and as screening tool for new drugs for thyroid cancer therapy.
Finally, organoids have a considerable advantage for researchers from different fields and are now a reality as a great tool to better understand thyroid mechanisms. Growing tiny thyroid glands in a dish able of producing THs opens new perspectives in the field of thyroid research, partially replacing animal models, and constituting a powerful weapon in the fight for personalized treatments for malignant thyroid diseases.