Advances in Thyroid Gland Regeneration: The Integrated Approach of Cell Biology and Bioengineering

Primary thyroid organoids

The generation of thyroid organoids from primary thyroid cells has been reported in several studies involving human, mouse, and rat tissue. This process typically involves mechanical and enzymatic digestion of native tissue to obtain a homogenous cell suspension, which is then cultured on basement membrane extracts (BMEs) using selective cell culture media. It was presented that the self-organization of thyroid cells into follicle-like structures can be enhanced through various methods. Early studies highlighted the importance of supplementing culture media with keratinocyte growth factor and the beneficial effects of a microgravity environment. ¹⁹ Current protocols often involve highly complex, heterogeneous culture media supplemented with diverse growth factors, such as epidermal growth factor (EGF) and fibroblast growth factor (FGF), as well as proteins and signaling molecules like Noggin, R-spondin1, and TSH. ²⁰ Analyses of these organoids provided evidence of the proliferative capabilities of thyrocytes both in terms of building of the organoid itself and in terms of the demonstration of self-renewal properties. Moreover, organoids cells exhibit expression profiles of key molecular markers analogous to the native thyroid cells. Functional assessments indicate that these organoids are capable of hormone production and can respond to changes in TSH concentration in a manner similar to native thyroid glands.^20–23 In addition, the long-term stability of organoid cultures—over periods exceeding 1 year—has been demonstrated. ²⁰ Taking into consideration potential long-term stability, proliferative properties, and retained in vitro physiological functions, primary thyroid organoids are presented both as model to study thyroid gland biology and as a potential source of material for thyroid gland regeneration. To date, only two in vivo studies have evaluated the implantation of primary thyroid organoids in animal models. Both experiments used mice with induced hypothyroidism. Moreover, the implantation of organoids was performed in the same location, namely, beneath the kidney capsule. The first study results demonstrated the successful implantation of murine organoids under the kidney capsule, as confirmed after period of 4 weeks through the utilization of hematoxylin and eosin staining and immunofluorescence as well as augmented ¹²⁵I uptake in the area of organoid implantation. Unfortunately, no functional studies were conducted. ²⁴ The second study provided evidence of successful transplantation of 600,000 human and murine organoids. In the course of long-term observations, scientists have demonstrated time-dependent increase in the number and size of thyroid follicles in the histological samples, as well as an increase in the expression of key thyroid proteins. Functional testing showed a moderate increase in the levels of T4 that authors attributed to low quantity of implanted organoids as well as the absence of neovasculogenesis at the implantation site. ²⁵

Stem cells-derived organoids

Compared with primary thyroid organoids, stem cell-derived organoids are relatively better characterized, with greater number of organoids’ generation protocols developed and greater number of in vivo studies conducted. This discrepancy may be attributed to the augmented potential for practical applications in regenerative medicine as well as enhanced accessibility of diverse stem cell types possessing almost infinite proliferative capacity.

Thyroid organoids derived from stem cells can be achieved through the utilization of two stem cell types, iPSCs and ESCs. Numerous studies involving human and murine iPSCs have demonstrated their ability to differentiate into thyroid follicular cells via a multistep process. This typically includes the overexpression of PAX8 and NKX2-1, followed by endodermal induction and final differentiation into follicular cells—commonly cultured on growth factor-reduced BMEs. ²⁶ Murine-differentiated iPSCs demonstrated significant increase in the expression of thyroid-specific proteins (SLC5A5 -NIS, TSHR, Tg, and thyroid peroxidase [TPO]), functional response to TSH, and ability to form follicles by approximately 50% of cells. ²⁷ Research conducted on human iPSCs has yielded similar outcomes to mouse counterpart. However, a higher degree of efficiency has been demonstrated in the follicles formation, and the expression of sodium/iodide symporter protein encoded by SLC5A5 gene (NIS) was only detectable in limited population of studied cells. The in vivo analysis of murine iPSCs-derived thyroid organoids was limited to observation of follicles formation and thyroglobulin (TG) deposition inside them after 4 weeks of implantation. In case of differentiated human iPSCs, the results demonstrated that following transplantation after approximately 4 months, between 63% and 74% of the implanted cells still exhibited coexpression of PAX8 and NKX2-1 and formed follicular structures filled with TG-positive colloid. It is important to note that neovasculature development was observed in the graft. Notwithstanding the fact that the presence of vasculature was a highly positive indicator of successful engraftment, there was no increase in T4, and decrease in TSH levels was recorded. The absence of hormonal activity was attributed to the low expression profile of SLC5A5 in organoid cells, indicating necessity for the incorporation of additional maturation steps into the protocol.^28,29

ESC-derived thyroid organoids have been developed using two principal strategies. The first one, known as forward programming, involves direct induction of key transcription factors (TFs) overexpression through the utilization of genetic engineering tools. The second approach, called directed differentiation, revolves around obtaining the final phenotype by series of changes in cell culture medium composition and introduction of various TFs, proteins, and small particles to replicate natural process of differentiation.^30,31 A number of protocols for generation and enrichment at various stages of the process have been developed, presenting different levels of differentiation efficiency oscillating around 50%.^32–35 All organoids displayed expression of key thyroid-specific proteins, follicles’ formative abilities on BME, TG accumulation in spaces corresponding to lumen of native thyroid follicles, and polarization of hormone transport-related proteins similar to native thyrocytes. Studies conducted on ESCs-derived thyroid organoids frequently presented protein expression level that was analogous to or even superior to those observed in native thyrocytes from the respective species. ³⁵ With regard to their functional properties, organoids displayed active iodine organification.^35–37 Notwithstanding the considerable amount of molecular and morphological similarities to native thyroid grand only in three instances, authors reported expression of T4 by the generated organoids.^38–40 It was also posited that T4-producing organoids could be obtained from fetal human thyroid cells. ⁴¹

In vivo studies have shown that murine ESC-derived organoids implanted under the kidney capsule could partially restore thyroid function in hypothyroid mice. Then organoids exhibited partially retained histological structure. It was also noted that a neovascularization process was taking place, as evidenced by the formation of angiofollicular units—the fundamental functional thyroid units. In addition to structural and molecular similarities to native thyroid gland, organoid grafts were found to be capable of assuming physiological functions in a mouse model of hypothyroidism. Despite the constrained timeframe for observation, studies demonstrated potential normalization of T4, T3, and TSH levels in subject animals 4–8 weeks after implantation. In addition, the capacity of grafts to regulate accordingly T4 levels based on artificially changed TSH levels was demonstrated. The restoration of hormonal levels by means of organoid grafts resulted in the normalization of subject’s body temperature, thereby providing evidence of successful hypothyroidism rescue. The longest reported period of observation has shown successful normalization of hormone level for 29 weeks.^36,39 To date, there has been only one available study on the subject of in vivo tests of human ESC-derived thyroid organoid. In a manner consistent with the findings of other studies, organoids were grafted beneath the kidney capsule in a mouse hypothyroidism model. Following a period of 5 weeks, histological analysis of the graft revealed the presence of multiple small thyroid follicles with network of small blood vessels in close proximity, indicating formation of angiofollicular units. In addition, observable differences in cell morphology indicated that the production of hormones was occurring in a subset of follicles. Gene expression analysis demonstrated expression profile consistent with that of mature thyrocytes, with approximately 58% of cells producing T4. The biochemical analysis provided data confirming iodine uptake by graft area, normalization of serum T3 levels, and increase of T4 level. The subphysiological levels of T4 observed were attributed to the relatively brief observation period, with notable weekly tendency for T4 increase. Furthermore, the capacity of grafts to physiologically regulate hormonal response in accordance with the TSH stimulation has been demonstrated. ³⁹

Despite significant advances in understanding thyroid organogenesis and the development of complex stem cell-derived organoids, in vivo testing remains limited. These findings underscore the major challenge of translating in vitro constructs into functionally integrated tissues capable of long-term survival and physiological regulation at the implantation site.^17,42–50

Adult stem cells in thyroid regeneration

Recent studies have provided compelling evidence for the presence of a population of progenitor cells within the adult thyroid gland—a possibility that had long been suspected.^47,51,52 In an experimental model of severe thyroid injury induced by tamoxifen and diphtheria toxin, researchers observed a rapid recovery of thyroid function within 8 weeks of damage induction. This recovery was attributed to the activation of dormant cells expressing stem cell markers such as Oct3/4, NANOG, and SOX2, as well as progenitor cells characterized by PAX8 expression and the absence of TG expression. It was established that these cells tend to accumulate in close proximity to newly forming follicles. ⁵³ In another study in mice that have undergone partial thyroid lobectomy, a population of thyroid progenitor cells was discovered to form new primitive follicles with present mitotic activity and low levels of TG in the lumen. It has been demonstrated that those primitive follicles exhibited capacity to divide into multiple smaller follicles, which underwent final maturation. This process resulted in the presentation of morphology and expression profile of adult normal thyroid follicle on day 21. ⁵⁴ Hidden regenerative potential of thyroid follicular cells was also indicated in the study conducted on thyroid cancer metastases, were highly differentiated thyrocytes gained tissue-context-dependent high proliferative capacity independent from TSH presence. This may suggest that this cell population may be the population responsible for metastasis regeneration rather than small number of thyroid cancer stem cells. ⁵⁵ It is evident that progenitor thyroid cells and residual stem cells are not the only candidates for cellular component of biomimetic thyroid. In 2025, a study was published exploring the application of bone marrow-derived mesenchymal stem cells (BMSCs) in the treatment of Hashimoto’s thyroiditis. The results presented indicated the immunomodulatory potential of BMSCs in reducing inflammation as well as their ability to increase the concentration of growth factors in their immediate proximity and their potential to differentiate into fibroblasts or thyrocytes. ⁵⁶

Following their recent identification and characterization, dormant adult thyroid stem cells and progenitor cells may represent a viable alternative to primary cells, iPSCs, and ESCs in generation of functional thyroid implants. This potential can be attributed to their higher level of differentiation, ease of accessibility, and possible personalization of the implants based on them. The presence of adult stem cells is known to be critical in maintaining both physiological function and structural integrity in almost every organ throughout the human body’s lifespan. ⁵⁷ Incorporating such elements into biomimetic thyroid constructs could enhance the differentiation potential of organoids, improve functional performance, and replace apoptotic cells—thereby increasing the longevity and stability of engineered implants.

Whole thyroid gland production in the bioreactor

Blastocyst complementation is a powerful chimeric organ engineering strategy that enables the in vivo generation of various organs and specialized cell types.^58–62 The technique involves the microinjection of stem cells into embryos in the blastocyst-stage embryos that have been genetically modified to lack the ability to form a specific organ or to undergo normal organogenesis altogether.^63,64 These modified embryos function as biological bioreactors, allowing the development of chimeric organs based on the provided stem cells. There are documented attempts to utilize this method in the fabrication of thyroid tissue and follicular cells (Fig. 4). Two distinct mouse models were generated: one with depletion of FGF10 and the other with NKX2-1 deletion. In the first model, complementation with murine ESCs resulted in development of normal and fully functional thyroid tissue. The majority of generated tissue was comprised of cell derived from the introduced ESCs cells. Although part of the cells, they were originating from the host blastocyst. In the second model, results were similar, with development of thyroid tissue almost entirely derived from injected stem cells.^65,66 Despite the substantial promise of blastocyst complementation as a method for thyroid tissue engineering, several critical challenges remain. Chief among them is the necessity to achieve complete purification of the regenerated thyroid gland from host-derived cells in order to prevent immunological rejection during future transplantation. Another major concern relates to the ethical implications of creating interspecies chimeras, as donor-derived cells may integrate not only into the targeted organ but also into other parts of the host organism.

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FIG. 4.

Blastocyst complementation technique in regeneration of thyroid gland. Created with Biorender.com.

Advancements in C-cells regeneration

In the context of thyroid gland regeneration, it is important to acknowledge that the gland is physiologically responsible not only for the production of TG, T3, and T4 but also calcitonin. While calcitonin deficiency is typically compensated by parathyroid hormone and vitamin D, in rare cases, the absence of calcitonin—produced by parafollicular cells (C-cells)—can lead to increased bone resorption. This may result in osteopenia and elevate the risk of bone fractures. ⁶⁷ In order to create a fully functional artificial thyroid, it is essential that it possess all of the necessary functionalities. To the best of our knowledge, currently only one study tackled the subject of C-cells regeneration. The utilization of human ESCs has enabled the development of the protocols for both two-dimensional (2D) and 3D differentiation into C-like cells. The subjected cells were differentiated through the implementation of forward programming approach and were split into two parts. The first was oriented in similar fashion to thyroid organoids on generation of definitive endoderm phenotype with activin A and low fetal bovine serum (FBS) concentrations. The second one, the final differentiation into C-cell like cells, was performed by addition of 10% FBS, retinoic acid, and insulin-like growth factor-1. Cells differentiated through this protocol were capable of synthesizing calcitonin and exhibited a functional response to changing calcium concentrations in the culture medium. Interestingly, while physiologically being scattered across all of the thyroid gland in the in vitro environment, 3D model presented higher expression of calcitonin in comparison with plain 2D cells. ⁶⁸

Implantation site: optimal environment for graft integration

Given the thyroid glands’ dense internal vasculature, multiple arterial blood supplies, and its intrinsic endocrine function, which by definition depends on the circulatory system, it is imperative not only to restore the functionality of arteriofollicular units but also to ensure sufficient vascularization to support the high metabolic activity of a thyroid implant. ⁶⁹ It is therefore crucial to determine optimal implantation site. Over the years, researchers have explored several potential locations for thyroid autotransplantation. In human subjects, these procedures have been performed predominantly within various muscles, including sternocleidomastoid, rectus femoris, or the forearm muscles. ⁷⁰ Although initially very promising, thyroid autotransplantation frequently failed to result in hypothyroidism rescue or did so just for a brief period of time. ⁷¹ This phenomenon may be partially attributed to the insufficient vasculature and regenerative potential of muscle tissue impeding the integration process. In recent years, in vivo testing of thyroid organoids has been consistently performed by implanting them into the subcapsular space of the kidney. ²⁶ This approach is associated with several advantages over intramuscular implantation. The primary one revolves around high vascularization of the kidney capsule, providing some degree of protection for the graft as well as facilitates easy access for the operator. ⁷² A novel potential implantation site has recently been proposed for thyroid regeneration: the spleen. In a mouse model of thyroid autotransplantation, intrasplenic transplantation was shown to support both the growth and function of thyroid follicular cell. Engraftment of 1.5 g of mouse thyroid minced into small blocks of tissue suspended in the hyaluronic acid resulted in complete integration of implanted tissue with spleen stroma. Within just 2 days posttransplantation, restoration of arteriofollicular units was obtained. Furthermore, this massive blood vessels proliferation was not accompanied by the inflammatory response. Physiological hormonal balance was restored within 4 weeks, along with measurable improvements in cardiac and hepatic function. Following the observation period, the data indicated a potential increase in the thyroid tissue mass in the spleen compared with its initial weight. This finding suggests that the spleen environment may possess proliferative stimulating properties. A comparative study demonstrated the superiority or intrasplenic implantation compared with intramuscular as evidenced by the occurrence of necrosis, and decline in the number and volume of thyroid follicles was observed. ⁷³

Biomaterials in thyroid gland regeneration

The extracellular matrix (ECM) is a complex network of proteins and glycans that forms the scaffold surrounding and supporting nearly all cells in the human body. ⁷⁴ In addition to providing tissues with physical structure and mechanical integrity, the ECM plays a key role in regulating critical cellular processes, including proliferation, differentiation, and migration.^74,75 The complex composition of the ECM provides surrounding cells with a stable environment, thereby enabling them to retain their phenotype and proper physiological functioning. The gold standard for culturing stem cells and developing 3D cellular structures, both spheroids and organoids, is the use of commercially available ECM substitutes. These are typically BMEs presented as specialized hydrogel solutions, with Matrigel being the most widely used example. ⁷⁶ All of the aforementioned principles were utilized as the foundation for the development of both primary and stem cells-derived thyroid gland organoids’ development. ⁷⁷ In recent years, there have been significant advancements in integrating thyroid organoids into lab-on-a-chip platforms designed for in vitro studies of thyroid pathophysiology and pharmacological testing.^78–81 The development of these devices was based on the microfluidic technology, a sophisticated system that enables precise manipulation of small fluid volumes and simulates dynamic physiological conditions. ⁸² The incorporation of a dense, predominantly collagenous ECM substitute as a support for organoids constituted a significant technical challenge. However, this notion can be surpassed by conventional enzymatic extraction of organoids from the BMEs and subsequent suspension in cell culture medium. This approach was unfortunately found to be inefficient due to the fact that organoids deprived of the contact with ECM substitute tend to degenerate and lose their organized follicular architecture. ⁷⁸ Taken into consideration results of studies on microfluidics devices, there is growing body of evidence that the role of interaction between cells and the ECM in thyroid regeneration may be more significant than was previously anticipated, and the absence of collagenous support may negatively impact results of in vivo studies.

The growing recognition of the ECM as a key component in the bioengineering of functional organs has led researchers to search for safe and renewable sources of ECM suitable for clinical use. Many currently available BMEs are derived from tumorigenic cell lines, raising safety concerns for their use in implantable grafts. One potential answer to this challenge may be the development of a range of techniques for decellularization of various tissues and organs.^83–88 Decellularization is defined as the process of removing cellular content from a desired tissue without damaging the native architecture of ECM. ⁸⁹ Meticulous processing, including mechanical, enzymatic end chemical methods, provides scaffolding biomimetic material that is primed for recellularization. Based on the hypothesis that the incorporation of thyroid ECM into thyroid grafts may positively impact cellular component of the graft, several protocols for thyroid tissue decellularization have been developed. The first report on the generation of a decellularized rat thyroid was published in 2012. However, there is a paucity of data concerning the outcome of the decellularization and subsequent recellularization. ⁹⁰ A more comprehensive dataset emerged in 2019, describing a flow-based chemical decellularization protocol for an entire rat thyroid lobe. The biomaterial obtained in this study met all of the canonical decellularization criteria. In addition, it was found to retain the mechanical properties and vasculature bed of the native material. In the functional studies, the authors using mesenchymal stem cells and FRTL-5 rat epithelial thyroid cells demonstrated lack of cytotoxic effect of the scaffold on the cells. Furthermore, it has been demonstrated that to some extent scaffold promoted the growth of stem cells. Flow recellularization of the scaffold with FRTL-5 cells using the same blood vessels that were used for decellularization was found to be successful. Histological analysis revealed formation of cellular spherical structures around former follicles’ ECM. In addition, cells retained the expression of crucial thyroid genes, TPO and TG. ⁹¹ In the same year, another group conducted an extensive mass spectrometry analysis of the decellularized rat thyroid matrix. They developed a reproducible protocol to assess the retention of collagen types I through V after the decellularization procedure, which may be used in the future for the standardization and quality control of mass-produced scaffolds. ⁹² The subsequent approach to this subject was published in the 2021 and was concentrated on the rabbit decellularized thyroid. The authors provided detailed characteristics of the scaffold and for the first time demonstrated that after the decellularization, a significant amount of key growth factors, transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), FGF, platelet-derived growth factor (PDGF), and hepatocyte growth factor, were to some extent preserved in the scaffold. This finding further substantiates the viability of this type of scaffold for thyroid bioengineering. The cellular test showed that the scaffold promotes the proliferation of the human follicular cells. However, a decline in expression of TPO after 7 days was observed, which was attributed to lack of hormonal stimulation during the cell culture. ⁹³ In 2024, the first protocol for the decellularization of human thyroid tissue was published. In this study, authors provided comprehensive comparison between the results of various approaches on the efficiency of decellularization process in this type of material. Cell seeding experiments were conducted using human thyroid cancer cell line and provided evidence of cytocompatibility of obtained scaffolds with human cells. ⁹⁴ Later in the same year, another study introduced a novel application of decellularized ECM (dECM) by developing injectable hydrogels derived from porcine thyroid tissue. These bioinks retained glycosaminoglycans and growth factors from the native ECM and exhibited concentration-dependent capabilities to promote cell proliferation and hormone production. ⁹⁵ All abovementioned advancements in thyroid dECM research were summarized in Table 1.

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Table 1.

Summary of Advancement in Thyroid dECM Research

Research	dECM source species	Notable advancements	Reference
Strusi et al.	Rat	First reported case of thyroid decellularization	90
Pan et al.	Rat	Frist fully documented protocol of decellularization and first recellularization attempt	91
Alifieri et al.	Rat	Mass spectrometry analysis of decellularized thyroid composition	92
Weng et al.	Rabbit	Providing evidence of significant growth factors retention including TGF-β, VEGF, FGF, PDGF	93
Karabıyık Acar et al.	Human	First protocol of human thyroid gland decellularization	94
Zhang et al.	Pig	First injectable dECM-based hydrogel for thyroid regeneration	95

dECM, decellularized extracellular matrix; FGF, fibroblast growth factor.

It has been established that the vast majority of cells possess the capacity to synthesize a minimal amount of ECM in their immediate surroundings. This process facilitates cell-to-cell adhesion and allows formation of 3D cellular structures. ⁹⁶ This fact was instrumental in the development of the cell sheet technique, were in vitro cultured cells form cohesive, dense cellular unit that can be transferred to the desired location, thereby providing high number of cells. ⁹⁷ The potential of this technology in the regeneration of the thyroid gland was also the subject of the study. Two-dimensional culturing of primary rat thyroid follicular cells on thermo-responsive plates has been shown to result in the formation of thick, cohesive cell sheets containing approximately 1 × 10⁷ cells. Histological analysis of the sheets’ morphology unveiled the presence of small central follicles containing TG colloid, and immunohistochemical staining demonstrated the retention of NKX2-1 expression. The subcutaneous implantation of thyroid cell sheet in athymic mice resulted in the appearance of detectable levels of both T3 and T4, which did not reach the physiological levels. These findings suggest that this form of bioengineered thyroid tissue may, with further refinement, evolve into a clinically viable therapy. ⁹⁸

Another promising approach to thyroid bioengineering involves the use of microencapsulation technologies to generate individual hormone-secreting capsules, each functioning as an independent thyroid unit. In the study, porcine primary thyroid cells were encapsulated in the alginate and poly-l-ornithine hydrochloride capsules and subsequently analyzed in terms of morphology, cell viability, and hormone release. It has been demonstrated that, over time, the observation of encapsulated cells revealed gradual organization, with the cells forming 3D structures similar to organoids. Encapsulated cells displayed limited proliferative abilities in comparison with the control cells but were more resistant to the cellular death and produced more thyroid hormones. ⁹⁹ This encapsulation strategy may pave the way for the development of methods dependent on allo- or even xenotransplantation. This is due to the capsule, which provides a partially effective barrier between the endocrine cells and the surrounding environment, resulting in the diminished need for immunosuppression.

Despite being in the early stages of development, scaffold-based techniques show considerable promise in the field of thyroid bioengineering—not only for their ability to enhance the mechanical integrity of engineered grafts, but also for introducing additional layers of molecular regulation that support and modulate the behavior of embedded cellular components.

3D bioprinting

To the best of our knowledge, as of today, the topic of thyroid bioengineering has been discussed in the context of 3D bioprinting on only a single occasion. This limited attention may stem from the insufficient resolution of current 3D bioprinting technologies and the highly complex structure of the arteriofollicular unit, which remains challenging to reproduce artificially. The objective of the referenced study was to apply 3D bioprinting technique to create a functional, vascularized mouse thyroid construct, utilizing a technique similar to the now widely recognized Kenzan method. This method revolves around the precise deposition of cellular spheroids, which form the primary architecture of the tissue, followed by a maturation period during which the spheroids fuse and reorganize into a final tissue-like structure. ¹⁰⁰ To replicate native architecture of the thyroid, the authors used two types of spheroids: one derived from embryonic thyroid explants and the other from the allantois. Based on the prepared in silico mathematical model and the visualization of spheroid fusion, the scientists designed the construct and fabricated it using a specialized 3D bioprinter. After a 4-day maturation period, the constructs exhibited early signs of endothelial cell differentiation and the formation of primitive vascular structures. In addition, the formation of thyroid follicles was also noted. When the bioprinted grafts were implanted under the kidney capsule in mice, an 8-week follow-up revealed a significant increase in circulating T4 levels and normalization of body temperature, indicating a partial reversal of hypothyroidism. ¹⁰¹ These findings demonstrate that 3D bioprinting holds considerable promise as a tool for the future development of bioengineered thyroid glands, potentially enabling the precise reconstruction of complex glandular architecture and vascular systems necessary for functional endocrine integration.