integrative-protocol-for-generation-ipsc-derived-3d-human-hepatic

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

University of Chicago

Deiodinases modify the biological activity of thyroid hormone (TH) molecules, ie, they may activate thyroxine (T4) to 3,5,3'-triiodothyronine (T3), or they may inactivate T3 to 3,3'-diiodo-L-thyronine (T2) or T4 to reverse triiodothyronine (rT3). Although evidence of deiodination of T4 to T3 has been available since the 1950s, objective evidence of TH metabolism was not established until the 1970s. The modern paradigm considers that the deiodinases not only play a role in the homeostasis of circulating T3, but they also provide dynamic control of TH signaling: cells that express the activating type 2 deiodinase (D2) have enhanced TH signaling due to intracellular build-up of T3; the opposite is seen in cells that express type 3 deiodinase (D3), the inactivating deiodinase. D2 and D3 are expressed in metabolically relevant tissues such as brown adipose tissue, skeletal muscle and liver, and their roles have been investigated using cell, animal, and human models. During development, D2 and D3 expression customize for each tissue/organ the timing and intensity of TH signaling. In adult cells, D2 is induced by cyclic adenosine monophosphate (cAMP), and its expression is invariably associated with enhanced T3 signaling, expression of PGC1 and accelerated energy expenditure. In contrast, D3 expression is induced by hypoxia-inducible factor 1α (HIF-1a), dampening T3 signaling and the metabolic rate. The coordinated expression of these enzymes adjusts TH signaling in a time- and tissue-specific fashion, affecting metabolic pathways in health and disease states.

Deiodinases and the Metabolic Code for Thyroid Hormone Action.

We report a patient with congenital hypothyroidism due to athyreosis complicated by a heterozygous thyroid hormone receptor beta ( gene mutation (R320L), resulting in a severe resistance to thyroid hormone beta phenotype. The proband inherited the mutant allele from his father, presenting a very mild phenotype. While the precise reason for this discrepancy remains unknown, we postulate the possibility of  mutation and mosaicism in the father. Correlating thyrotropin (TSH) with free thyroxine (fT4) allowed us to predict the amount of fT4 required to normalize the proband's TSH, which supported the treatment with high dose of levothyroxine.

Severe Resistance to Thyroid Hormone Beta in a Patient with Athyreosis.

The development of the selective RET inhibitors selpercatinib and pralsetinib has revolutionized the treatment of metastatic progressive RET-mutant medullary thyroid carcinoma (MTC) and other RET-driven cancers, given their more favorable side-effect profile. The aim of this study is to investigate the mechanisms of selpercatinib-induced thyroid dysfunction in athyreotic patients with RET-mutant MTC and in patients with RET-mutant non-small-cell lung cancer (NSCLC) who had a functional thyroid.

Selpercatinib-Induced Hypothyroidism Through Off-Target Inhibition of Type 2 Iodothyronine Deiodinase.

Analyze the cost contributors and their impact on the drug cost avoidance (DCA) resulting from cancer clinical trials over the period of 2015-2020 in a tertiary-level hospital in Spain (HCUVA).

Drug cost avoidance analysis of cancer clinical trials in Spain: a study on cost contributors and their impact.

The clinical availability of tissue-specific biomarkers of thyroid hormone (TH) action constitutes a "holy grail" for the field. Scientists have investigated several TH-dependent markers, including the tissue content of triiodothyronine (T3)-the active form of TH. The study of animal models and humans indicates that the T3 content varies among different tissues, mostly due to the presence of low-affinity, high-capacity cytoplasmic T3 binding proteins. Nonetheless, given that T3 levels in the plasma and tissues are in equilibrium, T3 signaling is defined by the intracellular free T3 levels. The available techniques to assess tissue T3 are invasive and not clinically applicable. However, the tracer kinetic studies revealed that serum T3 levels can accurately predict tissue T3 content and T3 signaling in most tissues, except for the brain and pituitary gland. This is true not only for normal individuals but also for patients with hypo or hyperthyroidism-but not for patients with non-thyroidal illness syndrome. Given this direct relationship between serum and tissue T3 contents and T3 signaling in most tissues, clinicians managing patients with hypothyroidism could refocus attention on monitoring serum T3 levels. Future clinical trials should aim at correlating clinical outcomes with serum T3 levels.

T3 levels and thyroid hormone signaling.

 Iodine is required for the synthesis of thyroid hormone (TH), but its natural availability is limited. Dehalogenase1 (Dehal1) recycles iodine from mono- and diiodotyrosines (MIT, DIT) to sustain TH synthesis when iodine supplies are scarce, but its role in the dynamics of storage and conservation of iodine is unknown.  -knockout (KO) mice were generated by gene trapping. The timing of expression and distribution was investigated by X-Gal staining and immunofluorescence using recombinant Dehal1-beta-galactosidase protein produced in fetuses and adult mice. Adult KO and wild-type () animals were fed normal and iodine-deficient diets for 1 month, and plasma, urine, and tissues were isolated for analyses. TH status was monitored, including thyroxine, triiodothyronine, MIT, DIT, and urinary iodine concentration (UIC) using a novel liquid chromatography with tandem mass spectrometry method and the Sandell-Kolthoff (S-K) technique throughout the experimental period.  Dehal1 is highly expressed in the thyroid and is also present in the kidneys, liver, and, unexpectedly, the choroid plexus.  transcription of  was induced by iodine deficiency only in the thyroid tissue. Under normal iodine intake, KO mice were euthyroid, but they showed negative iodine balance due to a continuous loss of iodotyrosines in the urine. Counterintuitively, the UIC of KO mice is twofold higher than that of  mice, indicating that S-K measures both inorganic and organic iodine. Under iodine restriction, KO mice rapidly develop profound hypothyroidism, while  mice remain euthyroid, suggesting reduced retention of iodine in the thyroids of KO mice. Urinary and plasma iodotyrosines were continually elevated throughout the life cycles of KO mice, including the neonatal period, when pups were still euthyroid.  Plasma and urine iodotyrosine elevation occurs in Dehal1-deficient mice throughout life. Therefore, measurement of iodotyrosines predicts an eventual iodine shortage and development of hypothyroidism in the preclinical phase. The prompt establishment of hypothyroidism upon the start of iodine restriction suggests that KO mice have low iodine reserves in their thyroid glands, pointing to defective capacity for iodine storage.

Iodotyrosines Are Biomarkers for Preclinical Stages of Iodine-Deficient Hypothyroidism in -Knockout Mice.

Pregnant women with mutations in the thyroid hormone receptor beta (THRB) gene expose their fetuses to high thyroid hormone (TH) levels shown to be detrimental to a normal fetus (NlFe) but not to an affected fetus (AfFe). However, no information is available about differences in placental TH regulators.

Effect of the Fetal THRB Genotype on the Placenta.

The development of the brain, as well as mood and cognitive functions, are affected by thyroid hormone (TH) signaling. Neurons are the critical cellular target for TH action, with T3 regulating the expression of important neuronal gene sets. However, the steps involved in T3 signaling remain poorly known given that neurons express high levels of type 3 deiodinase (D3), which inactivates both T4 and T3. To investigate this mechanism, we used a compartmentalized microfluid device and identified a novel neuronal pathway of T3 transport and action that involves axonal T3 uptake into clathrin-dependent, endosomal/non-degradative lysosomes (NDLs). NDLs-containing T3 are retrogradely transported via microtubules, delivering T3 to the cell nucleus, and doubling the expression of a T3-responsive reporter gene. The NDLs also contain the monocarboxylate transporter 8 (Mct8) and D3, which transport and inactivate T3, respectively. Notwithstanding, T3 gets away from degradation because D3's active center is in the cytosol. Moreover, we used a unique mouse system to show that T3 implanted in specific brain areas can trigger selective signaling in distant locations, as far as the contralateral hemisphere. These findings provide a pathway for L-T3 to reach neurons and resolve the paradox of T3 signaling in the brain amid high D3 activity.

Axonal T3 uptake and transport can trigger thyroid hormone signaling in the brain.

Graves' disease is an autoimmune disease associated with high levels of circulating thyroid hormones (THs). Resistance to thyroid hormone beta (RTHβ) caused by mutations in the thyroid hormone receptor beta () gene also can lead to high TH levels. Here, we describe 2 related cases, one of a woman with Graves' disease, and her newborn with RTHβ.

Resistance to Thyroid Hormone Beta in a Patient Born to a Mother With Undiagnosed Graves' Disease.

The Relationship Between Fetal  Genotype and Maternal Thyroid Function.

Mutations and single nucleotide polymorphisms (SNPs) in the genes encoding the network of proteins involved in thyroid hormone signaling (TH) may have implications for the effectiveness of the treatment of hypothyroidism with LT4. It is conceivable that loss-of-function mutations or SNPs impair the ability of LT4 to be activated to T3, reach its targets, and ultimately resolve symptoms of hypothyroidism. Some of these patients do benefit from therapy containing LT4 and LT3.

Gene polymorphisms and thyroid hormone signaling: implication for the treatment of hypothyroidism.

Thyroid hormone (TH) levels are low during development, and the deiodinases control TH signaling through tissue-specific activation or inactivation of TH. Here, we studied human induced pluripotent stem cell-derived (iPSC-derived) hepatic organoids and identified a robust induction of DIO2 expression (the deiodinase that activates T4 to T3) that occurs in hepatoblasts. The surge in DIO2-T3 (the deiodinase that activates thyroxine [T4] to triiodothyronine [T3]) persists until the hepatoblasts differentiate into hepatocyte- or cholangiocyte-like cells, neither of which expresses DIO2. Preventing the induction of the DIO2-T3 signaling modified the expression of key transcription factors, decreased the number of hepatocyte-like cells by ~60%, and increased the number of cholangiocyte-like cells by ~55% without affecting the growth or the size of the mature liver organoid. Physiological levels of T3 could not fully restore the transition from hepatoblasts to mature cells. This indicates that the timed surge in DIO2-T3 signaling critically determines the fate of developing human hepatoblasts and the transcriptome of the maturing hepatocytes, with physiological and clinical implications for how the liver handles energy substrates.

Localized T3 production modifies the transcriptome and promotes the hepatocyte-like lineage in iPSC-derived hepatic organoids.

The regulation of thyroid activity and thyroid hormone (TH) secretion is based on feedback mechanisms that involve the anterior pituitary TSH and medial basal hypothalamus TSH-releasing hormone. Plasma T3 levels can be "sensed" directly by the anterior pituitary and medial basal hypothalamus; plasma T4 levels require local conversion of T4 to T3, which is mediated by the type 2 deiodinase (D2). To study D2-mediated T4 to T3 conversion and T3 production in the anterior pituitary gland, we used mouse pituitary explants incubated with 125I-T4 for 48 hours to measure T3 production at different concentrations of free T4. The results were compared with cultures of D1- or D2-expressing cells, as well as freshly isolated mouse tissue. These studies revealed a unique regulation of the D2 pathway in the anterior pituitary gland, distinct from that observed in nonpituitary tissues. In the anterior pituitary, increasing T4 levels reduced D2 activity slightly but caused a direct increase in T3 production. However, the same changes in T4 levels decreased T3 production in human HSkM cells and murine C2C12 cells (both skeletal muscle) and mouse bone marrow tissue, which reached zero at 50 pM free T4. In contrast, the increase in T4 levels caused the pig kidney LLC-PK1 cells and kidney fragments to proportionally increase T3 production. These findings have important implications for both physiology and clinical practice because they clarify the mechanism by which fluctuations in plasma T4 levels are transduced in the anterior pituitary gland to mediate the TSH feedback mechanism.

Sustained Pituitary T3 Production Explains the T4-mediated TSH Feedback Mechanism.

Patients with mutations in the thyroid hormone (TH) cell transporter monocarboxylate transporter 8 (MCT8) gene develop severe neuropsychomotor retardation known as Allan-Herndon-Dudley syndrome (AHDS). It is assumed that this is caused by a reduction in TH signaling in the developing brain during both intrauterine and postnatal developmental stages, and treatment remains understandably challenging. Given species differences in brain TH transporters and the limitations of studies in mice, we generated cerebral organoids (COs) using human induced pluripotent stem cells (iPSCs) from MCT8-deficient patients. MCT8-deficient COs exhibited (i) altered early neurodevelopment, resulting in smaller neural rosettes with thinner cortical units, (ii) impaired triiodothyronine (T3) transport in developing neural cells, as assessed through deiodinase-3-mediated T3 catabolism, (iii) reduced expression of genes involved in cerebral cortex development, and (iv) reduced T3 inducibility of TH-regulated genes. In contrast, the TH analogs 3,5-diiodothyropropionic acid and 3,3',5-triiodothyroacetic acid triggered normal responses (induction/repression of T3-responsive genes) in MCT8-deficient COs, constituting proof of concept that lack of T3 transport underlies the pathophysiology of AHDS and demonstrating the clinical potential for TH analogs to be used in treating patients with AHDS. MCT8-deficient COs represent a species-specific relevant preclinical model that can be utilized to screen drugs with potential benefits as personalized therapeutics for patients with AHDS.

Impaired T3 uptake and action in MCT8-deficient cerebral organoids underlie Allan-Herndon-Dudley syndrome.

 Normal brain development, mood, and cognitive functions depend on thyroid hormone (TH) action. However, little is known about how TH mediates its actions in the human brain. This is due to limited access to human brains deprived of TH during fetal and early postnatal life, as well as from adults with altered thyroid status. One way to partially bypass these limitations is by using magnetic resonance imaging and spectroscopy, two neuroimaging techniques that provide detailed, noninvasive information on human brain structure and function. Another way is using human-induced pluripotent stem cell (hiPSCs)-derived three-dimensional  systems, known as brain organoids, which allow for the study of fundamental aspects of the early stages of human brain development.  This narrative review focuses on neuroimaging and brain organoid studies. Neuroimaging of human brains performed in individuals with different thyroid conditions provides information on the volume, myelination, blood flow, neural activity, and connectivity of different areas. Such studies show that suboptimal thyroid status can impact human brain development and its normal function throughout life. This is true not only for patients with sporadic congenital hypothyroidism, during pregnancy or early after birth, but also for adult patients with hypo- or hyperthyroidism, patients carrying mutations that manifest as impaired sensitivity to TH, and even for normal individuals during aging. Studies using brain organoids generated from hiPSCs of healthy individuals or patients with thyroid genetic conditions provide insights into how TH can impact the early development of the human cerebral cortex.  The developmental alterations in children born to mothers with different degrees of gestational hypothyroidism or who developed hypothyroidism early in life are remarkable, affecting multiple brain regions and pathways, including the cerebral cortex, hippocampus, cerebellum, interhemispheric and corticospinal tracts, and associative nuclei. The data connecting such changes to poor neurological outcomes in adult patients with hypothyroidism represent an objective link between thyroid-specific functional brain alterations and behavior. Growing brain organoids require TH, which is critical for human neurogenesis and oligodendrogenesis. These models have proven useful in screening drugs with potential therapeutic effects for patients with genetic thyroid diseases.

Mapping Thyroid Hormone Action in the Human Brain.

The identification of critical factors that can worsen the mechanisms contributing to the pathophysiology of Alzheimer disease is of paramount importance. Thyroid hormones (TH) fit this criterion. Epidemiological studies have identified an association between altered circulating TH levels and Alzheimer disease. The study of human and animal models indicates that TH can affect all the main cellular, molecular, and genetic mechanisms known as hallmarks of Alzheimer disease. This is true not only for the excessive production in the brain of protein aggregates leading to amyloid plaques and neurofibrillary tangles but also for the clearance of these molecules from the brain parenchyma via the blood-brain barrier and for the escalated process of neuroinflammation-and even for the effects of carrying Alzheimer-associated genetic variants. Suboptimal TH levels result in a greater accumulation of protein aggregates in the brain. The direct TH regulation of critical genes involved in amyloid beta production and clearance is remarkable, affecting the expression of multiple genes, including APP (related to amyloid beta production), APOE, LRP1, TREM2, AQP4, and ABCB1 (related to amyloid beta clearance). TH also affects microglia by increasing their migration and function and directly regulating the immunosuppressor gene CD73, impacting the immune response of these cells. Studies aiming to understand the mechanisms that could explain how changes in TH levels can contribute to the brain alterations seen in patients with Alzheimer disease are ongoing. These studies have potential implications for the management of patients with Alzheimer disease and ultimately can contribute to devising new interventions for these conditions.

Article	Year
Deiodinases and the Metabolic Code for Thyroid Hormone Action. Endocrinology\| PubMed ID: 33720335	2021
Severe Resistance to Thyroid Hormone Beta in a Patient with Athyreosis. Thyroid : official journal of the American Thyroid Association\| PubMed ID: 34969265	2022
Selpercatinib-Induced Hypothyroidism Through Off-Target Inhibition of Type 2 Iodothyronine Deiodinase. JCO precision oncology\| PubMed ID: 35704797	2022
Drug cost avoidance analysis of cancer clinical trials in Spain: a study on cost contributors and their impact. BMC health services research\| PubMed ID: 35883128	2022
T3 levels and thyroid hormone signaling. Frontiers in endocrinology\| PubMed ID: 36387853	2022
Iodotyrosines Are Biomarkers for Preclinical Stages of Iodine-Deficient Hypothyroidism in -Knockout Mice. Thyroid : official journal of the American Thyroid Association\| PubMed ID: 36879468	2023
Effect of the Fetal THRB Genotype on the Placenta. The Journal of clinical endocrinology and metabolism\| PubMed ID: 37149816	2023
Axonal T3 uptake and transport can trigger thyroid hormone signaling in the brain. eLife\| PubMed ID: 37204837	2023
Resistance to Thyroid Hormone Beta in a Patient Born to a Mother With Undiagnosed Graves' Disease. AACE clinical case reports\| PubMed ID: 37251972	2023
The Relationship Between Fetal Genotype and Maternal Thyroid Function. Thyroid : official journal of the American Thyroid Association\| PubMed ID: 37597194	2023
Gene polymorphisms and thyroid hormone signaling: implication for the treatment of hypothyroidism. Endocrine\| PubMed ID: 37740833	2024
Localized T3 production modifies the transcriptome and promotes the hepatocyte-like lineage in iPSC-derived hepatic organoids. JCI insight\| PubMed ID: 37856222	2023
Sustained Pituitary T3 Production Explains the T4-mediated TSH Feedback Mechanism. Endocrinology\| PubMed ID: 37864846	2023
Impaired T3 uptake and action in MCT8-deficient cerebral organoids underlie Allan-Herndon-Dudley syndrome. JCI insight\| PubMed ID: 38376950	2024
Mapping Thyroid Hormone Action in the Human Brain. Thyroid : official journal of the American Thyroid Association\| PubMed ID: 38757586	2024
Thyroid Hormone and Alzheimer Disease: Bridging Epidemiology to Mechanism. Endocrinology\| PubMed ID: 39276028	2024

Federico Salas-Lucia

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