Transplantable human thyroid organoids generated from embryonic stem cells to rescue hypothyroidism

Mírian Romitti, Barbara de Faria da Fonsecaa, Gilles Doumont, Pierre Gillotay, Adrien Tourneur, Sema Elif Eski, Gaetan Van Simaeys, Laura Chomette, Helene Lasolle, Olivier Monestier, Dominika Figini Kasprzyk, Vincent Detours, Sumeet Pal Singh, Serge Goldman, Samuel Refetoff, Sabine Costagliola

Preprint posted on 1 December 2021 https://www.biorxiv.org/content/10.1101/2021.12.01.470729v1?ct=

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-022-34776-7

Build-it-yourself human thyroid for regenerative therapy

Selected by Anna Meier

Categories: cell biology, developmental biology

Updated 24 January 2023 with a postLight by Anna Meier

This preprint was published in Nature Communications in November 2022, roughly one year after being posted on bioRxiv. The published version of the article shows that the authors performed several new experiments to strengthen their claims (for details, see the ‘Peer review’ file provided by the journal).

The authors expanded their characterization of the steps of thyroid organoid differentiation and provided an additional single-cell RNA-seq analysis of organoids at day 58, which better demonstrated the maturation of organoids over time (Fig. 3a-c). The authors also made efforts to compare their organoids to the equivalent adult tissue in terms of expression profile and morphology, something that has quickly become standard in the field of organoid technology. This revealed a promising degree of similarity between adult thyroid tissue and organoids having undergone maturation through in vivo transplantation in mice (Fig. 4d,e).

Arguably the most interesting addition was the further functional validation of the effects of organoid transplantation after thyroid ablation. Along with higher plasma T4 levels compared to non-transplanted animals, the authors could show increased T3 and decreased thyroid-stimulating hormone (TSH), consistent with the negative regulation of TSH by thyroid hormones (Fig. 4h,i). The liver of transplanted animals also contained higher levels of Dio1 mRNA, which is known to be regulated by thyroid status (Fig. 4j). These systemic effects illustrate the remarkable potential of thyroid organoids for future cell replacement applications.

Background

The thyroid gland uses iodine to produce thyroid hormones (TH) that regulate the body’s growth and metabolism. Insufficient TH production leads to hypothyroidism, a common disorder that causes symptoms ranging from fatigue and weight gain to impaired cardiac function. TH replacement therapy for hypothyroidism was established more than a century ago and is currently based on supplementation with the synthetic hormone levothyroxine¹. However, there is mounting evidence that it inadequately treats up to one-third of patients and new therapeutic approaches are needed².

In the last decade, stem cell-derived self-organized microtissues called organoids have become powerful tools for disease modeling and drug discovery³. Another ambitious but so far lesser-explored application of organoids is to use them as a source of tissue for regenerative medicine. Highly functional thyroid organoids generated from mouse pluripotent stem cells have already been reported, but it remains challenging to translate the technology to human pluripotent stem cells. Organoids obtained from human adult thyroid cells, on the other hand, have shown limited in vivo functionality⁴. In this preprint, the authors used forward programming to establish human embryonic stem cell-derived thyroid organoids that recapitulate key features of thyroid morphology and function in vitro and in vivo.

Key findings

The authors first engineered a human embryonic stem cell (ESC) line allowing doxycycline-inducible expression of the transcription factors NKX2-1 and PAX8, which they had previously shown to drive thyroid differentiation of mouse ESCs⁵. Indeed, after endoderm induction of embryoid bodies, the transient overexpression of NKX2-1 and PAX8 activated an endogenous thyroid differentiation program. The composition of the culture media was then optimized to promote maturation, resulting in monolayer-organized thyroid follicles with well-delimited lumen around day 45 of differentiation. Single-cell RNA sequencing (scRNA-Seq) of these structures allowed the characterization of three populations of thyroid cells – progenitors, immature thyrocytes, and mature thyrocytes – showing progressive upregulation of mature thyroid markers such as TG, TSHR, and TPO. Extending the culture time by another two weeks further increased the expression of these markers and culminated in TH hormone synthesis, as shown by the accumulation of the hormone thyroxine (T4) in the lumen of the follicles (Fig. 1).

Figure 1. Human ESC-derived thyroid follicular structures. (a-d) Immunofluorescence staining for NKX2-1, E-cadherin, TG, TPO, and T4 at day 58 of differentiation. Adapted from Figure 2 of this preprint.

To test the in vivo functionality of the organoids, the authors then transplanted them under the kidney capsule of immunodeficient NOD-SCID mice which had undergone thyroid ablation. After 4 weeks, SPECT-CT imaging showed that the implanted follicles could efficiently take up the radioactive iodine isotope ¹²³I. Importantly, there was also a notable increase in plasma T4 levels in transplanted animals compared to non-transplanted controls.

In conclusion, the organoids established by Romitti and colleagues not only offer a platform to study various facets of thyroid biology in vitro but could also pave the way towards human stem cell-based regenerative therapy for hypothyroidism.

Why I chose this preprint

Remarkable progress has been made in the generation of human organoids mimicking various systems of the body. However, compared to the explosion of their use in various in vitro applications, organoid-based regenerative medicine is still in its infancy. I was therefore quite fascinated by the achievements presented in this study, which suggest that implanted organoids can have a significant impact at the systemic level.

References

Wiersinga, W. M. Thyroid hormone replacement therapy. Horm. Res. Paediatr. 56, 74–81 (2001).
Dew, R. et al. Clinical, behavioural and pharmacogenomic factors influencing the response to levothyroxine therapy in patients with primary hypothyroidism—protocol for a systematic review. Syst. Rev. 6, (2017).
Kim, J., Koo, B.-K. & Knoblich, J. A. Human organoids: model systems for human biology and medicine. Nat. Rev. Mol. Cell Biol. 21, 571–584 (2020).
Ogundipe, V. M. L. et al. Generation and Differentiation of Adult Tissue-Derived Human Thyroid Organoids. Stem Cell Reports 16, 913–925 (2021).
Antonica, F. et al. Generation of functional thyroid from embryonic stem cells. Nature 491, 66–71 (2012).

Tags: hypothyroidism, organoids, pluripotent stem cells, thyroid

Posted on: 10 January 2022 , updated on: 25 January 2023

doi: https://doi.org/10.1242/prelights.31254

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Author's response

Sabine Costagliola shared

What do you expect to be the biggest challenges in bringing organoid transplantation to the clinic (for hypothyroidism or other diseases)?

Challenges to overcome are of different categories: technical, physiological and regulatory. Technical, to develop isogenic organoids from the patient’s iPS cells in order to circumvent rejection. Physiological, to demonstrate that transplanted human organoids function normally and respond to regulatory feedback controls, as appears to be the case with mouse derived organoids. Regulatory, to satisfy FDA requirements regarding safety in terms of long term survival and limited propensity of neoplastic transformation. Another potential rod block is the pressure from the pharmaceutical industry, considering that the second most commonly prescribed medication is TH.

As you mentioned in the article, a constant exogenous supply of TH is not an ideal solution for treating hypothyroidism. Can you speculate on how transplanted thyroid organoids could adapt to variations in TH demand during a patient’s life?

Congenital TH deficiency is the most common endocrine newborn defect. While Insufficient TH affects the metabolism and other body functions throughout life, insufficient TH supply in early life results in irreversible intellectual and developmental retardation. Further, the amount of TH required varies during periods of growth, puberty and pregnancy. Ensuring proper exogenous supply of TH to satisfy varying demands is not easy, dues to timed adjustment made by physicians and the follow up of instructions by parents and later by rebellious teenagers. A normally regulated functional thyroid organoid will adjust TH supply according to physiological demand, avoiding the irreversible consequences of poorly coordinated supply of TH. Finally, the supply of the biologically active TH, triiodothyronine (T3), varies according to tissue and cell type. Some cells are able to generate T3 from the dominant form of secreted hormone thyroxine (T4) while others require the direst supply of T3. Thus, it remains controversial whether both T4 and T3 should be supplied and if so in what proportion. The normal thyroid gland secretes both in different proportions depending on various environmental conditions. The transplanted thyroid organoid is expected to preserve this function.

1 comment

1 year

Sanjay Pillay

when will a “cure” for hypothyroidism be made available to the public? I was rendered hypothyroid through radioactive iodine for my graves disease..can this be reveresed through stem cells?

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Transplantable human thyroid organoids generated from embryonic stem cells to rescue hypothyroidism

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