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Review Article
Calcium & bone metabolism Parathyroid Gland Generation from Pluripotent Stem Cells
Keypoint
Generating parathyroid glands, or PTGs, from a patient’s own induced pluripotent stem cells, or PSCs, with transplantation of these PTGs, would be an effective treatment option. Multiple methods for generating PTGs from PSCs have been reported. One major trend is in vitro differentiation of PSCs into PTGs. Another is in vivo generation of PSC-derived PTGs by injecting PSCs into PTG-deficient embryos. This article reviews the current achievements and future challenges in regenerative medicine focusing on the parathyroid gland.
Mayuko Kanoorcid
Endocrinology and Metabolism 2024;39(4):552-558.
DOI: https://doi.org/10.3803/EnM.2024.1989
Published online: June 10, 2024

Department of Metabolism and Endocrinology, St. Marianna University School of Medicine, Kawasaki, Japan

Corresponding author: Mayuko Kano. Department of Metabolism and Endocrinology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan Tel: +81-44-977-8111, Fax: +81-44-976-8941, E-mail: kano@marianna-u.ac.jp
• Received: March 25, 2024   • Revised: April 17, 2024   • Accepted: May 7, 2024

Copyright © 2024 Korean Endocrine Society

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • Patients with permanent hypoparathyroidism require lifelong treatment. Current replacement therapies sometimes have adverse effects (e.g., hypercalciuria and chronic kidney disease). Generating parathyroid glands (PTGs) from the patient’s own induced pluripotent stem cells (PSCs), with transplantation of these PTGs, would be an effective treatment option. Multiple methods for generating PTGs from PSCs have been reported. One major trend is in vitro differentiation of PSCs into PTGs. Another is in vivo generation of PSC-derived PTGs by injecting PSCs into PTG-deficient embryos. This review discusses current achievements and challenges in present and future PTG regenerative medicine.
  • Keywords: Parathyroid glands; Pluripotent stem cells; Regenerative medicine
The parathyroid glands (PTGs) are crucial in regulating calcium (Ca) homeostasis. PTG chief cells express the Ca-sensing receptor (CaSR) on their surface and release parathyroid hormone (PTH) in response to minute changes in the extracellular Ca concentration ([Ca]). The absence of PTG function can be congenital or acquired. The most common cause is neck surgery (e.g., for thyroid cancer). Patients with permanent hypoparathyroidism need lifelong replacement therapy to prevent life-threatening hypocalcemia. The current treatment for hypoparathyroidism consists of oral Ca supplements and vitamin D analogs. However, it is sometimes difficult to keep [Ca] in the low-normal range. With current replacement therapy, patients are still at high risk of hypercalciuria and chronic kidney disease [1].
The PTGs are well suited as a regenerative-medicine target organ. Their structure, consisting mostly of PTH-secreting chief cells, is simple. Like pancreatic islet cells, these function independently, free from interrelationships among other endocrine organs. Safe and easy subcutaneous PTG transplantation is well-established. Therefore, it could be clinically significant to generate functional PTGs from a patient’s own induced pluripotent stem cells (PSCs), followed by PTG transplantation. This article describes current attempts to generate PTGs from PSCs using two different approaches: in vitro and in vivo.
Pluripotent epiblast cells, arising from the inner cell mass in the mammalian blastocyst, differentiate into the three germ layers—ectoderm, mesoderm, and definitive endoderm (DE). The DE gives rise to the respiratory and digestive systems and to the thyroid glands, PTGs, thymus, liver, and pancreas. After gastrulation, the DE forms a primitive gut tube [2]. This is segmented into foregut, midgut, and hindgut and has an anterior-posterior axis. The foregut gives rise to the thyroid glands, PTGs, esophagus, trachea, stomach, lungs, liver, biliary system, and pancreas. The midgut forms the small intestine, and the hindgut forms the large intestine. The most rostral foregut endoderm is the anterior foregut endoderm (AFE). The caudal AFE gives rise to the lungs and trachea. The more rostral AFE forms the pharyngeal endoderm (PE). The pharyngeal arches appear in embryonic day (E) 8.5 to 10 in mice. The PE lines the internal surface of the pharyngeal arches [3]. The internal pockets between arches are the pharyngeal pouches. The PTGs, with the thymus, develop from the third pharyngeal pouch in both humans and rodents [4]. Most mammals, including humans, have four PTGs. These occur as superior and inferior pairs that adjoin the lateral borders of the thyroid gland. The superior PTGs arise from the fourth pharyngeal pouch.
Several genes are important in PTG development (Table 1). Tbox transcription factor 1 (Tbx1) is expressed in the endoderm, ectoderm, and mesoderm of the pharyngeal arches during early mouse development (E8.5‒10.5). Deletion of Tbx1 recapitulated human 22q11.2 deletion syndrome (also known as DiGeorge syndrome), with craniofacial and cardiovascular malformations and the absence of the thymus and PTGs [5]. Glial cells missing 2 (Gcm2) is a crucial and specific gene for PTG development. In mice, Gcm2 is first expressed on E9.5 in PTG precursors within the third pharyngeal pouch. Gunther et al. [6] have demonstrated that Gcm2 knockout (KO) mice lack PTGs completely. In Gcm2-/- mice, parathyroid primordium derived from the third pharyngeal pouch appeared normal before E12.5, but soon underwent programmed cell death [7]. Paired box 1 (Pax1) and Pax9 are broadly expressed in the endoderm of the pharyngeal pouches as soon as E9.5. In Pax1-/- mice, PTGs are reduced [8]. Pax9-/- mice lack a thymus, PTGs, and ultimobranchial bodies, all of which are derived from the pharyngeal pouches [9].

IN VITRO GENERATION OF PTGs FROM HUMAN PSCs

To generate PTG cells from PSCs in vitro means recapitulating early mammalian PTG development in a dish (Fig. 1). Green et al. [10] have proposed a method for inducing AFE from PSCs. Undifferentiated human PSCs were first differentiated into DE with high concentrations of activin A (days 2 to 5), then into AFE with a combination of noggin, a physiological inhibitor of bone morphogenetic protein (BMP) signaling, and SB-431542, a pharmacological inhibitor of activin A/nodal and transforming growth factor (TGF)-β signaling (days 5 to 7). These noggin/SB431542-treated cultured cells expressed several AFE markers, such as forkhead box A2 (FOXA2), SRY-box transcription factor 2 (SOX2), Tbx1, and Pax9. On day 7 of culture, these AFE cells were cultured in the presence of Wnt family member 3A (WNT3a), keratinocyte growth factor, fibroblast growth factor (FGF) 10, BMP4, and epidermal growth factor (all factors, WKFBE) for further differentiation (days 7 to 10). This culture induced expression of NK2 homeobox 1 (NKX2-1), which marks lung and thyroid gland progenitors, and Pax1, a pharyngeal pouch marker. This indicates that exposure of noggin/SB-431542-induced AFE to WKFBE results in ventral differentiation. In mouse PTG development, sonic hedgehog (SHH) and FGF8 play an important role [11,12]. Adding SHH or FGF8 to ventral AFE cultures (noggin/SB-431542-induced AFE to WKFBE) induced the PTG-specific marker Gcm2 (days 11 to 19). Expression of PTH or CASR was not assessed. Therefore, it is unclear whether differentiation into more mature PTG cells had occurred.
Two methods of differentiating parathyroid-like cells from human PSCs have been published [13,14]. The protocol of Lawton et al. [13] is unique in that the cyclin-dependent kinase (CDK) inhibitor PD-0332991 was added to both DE and AFE stages. DE was induced from PSCs by using high concentrations of activin A, Wnt3a [15], and PD-0332991 (days 2 to 5). The addition of PD-0332991 was continued until day 9 of differentiation, the end of the AFE stage. AFE was induced based on established protocols [10,16]. On days 6 and 7, cultures were treated with noggin/SB-431542. On days 8 and 9, these noggin/SB-431542-treated cultures were supplemented with IWP2, which inhibits endogenously produced Wnts. After AFE differentiation, the expression of ISL LIM homeobox 1 (ISL1; necessary for the growth and patterning of the pharyngal arches) and NKX2-3 (important for development of pharyngeal pouch endoderm) was higher than in undifferentiated PSCs. AFE cultures were subsequently cultured until day 23 in the presence of LY-364947 (an inhibitor of TGF-β signaling), all-transretinoic acid (ATRA), FGF10, cyclopamine (an inhibitor of SHH signaling), and BMP4. The opposed expression of Bmp4 and Noggin is important for thymus and PTG patterning [17]. Therefore, after day 24, BMP4 was inhibited with noggin. Lawton et al. [13] achieved upregulation of PTH, Gcm2, and CASR mRNA expression throughout the 37-day culture. However, they did not address PTH expression at the protein level.
In the protocol of Nakatsuka et al. [14], human PSCs differentiated into DE and AFE under treatment with high concentrations of activin A and CHIR-99021 and, subsequently, with LDN-193189, a BMP receptor inhibitor (days 1 to 3) [17]. Cultured AFE cells were then stimulated with ATRA and IWRl-endo (Wnt inhibitors) to induce differentiation into PE (days 6 to 10). Finally, treatment with SHH and activin A enabled cultured PE cells to differentiate into parathyroid cells expressing parathyroid markers, including PTH and Gcm2. Some cells formed PTG-like clusters on Matrigel-coated culture plates. Immunofluorescence staining revealed PTH and Gcm2 expression. Because upregulated TGF-α/epidermal growth factor receptor (EGFR) signaling promotes parathyroid hyperplasia [18], Nakatsuka et al. [14] next examined whether TGF-α/EGFR signaling induced the differentiation of parathyroid cells from undifferentiated human PSCs. Flow cytometry analysis demonstrated that differentiated parathyroid cells from human PSCs were concentrated in the cell fraction expressing CASR and the epithelial cell adhesion molecule (EpCAM) cell fraction. The number of parathyroid cells expressing both CASR and EpCAM increased after TGF-α treatment. In contrast, treatment with erlotinib, an EGFR tyrosine kinase inhibitor, significantly reduced the number of such cells. It was accordingly inferred that TGF-α/EGFR signaling may promote parathyroid cell differentiation from human PSCs.

IN VIVO GENERATION OF PTGs FROM RODENT PSCs

In another organ generation method, blastocyst complementation (BC), PSCs are injected into blastocysts of animals deficient in the targeted organ. This organ then is present in the fetus or live-born animal, but is constituted almost entirely of descendants of donor PSCs. Vascular endothelial cells and mesenchymal cells in organs are chimeric between cells derived from donor PSCs and from host blastocysts. With humans, BC-derived organs generated in animals thus are largely derived from patient PSCs and are predicted to be rejection-exempt [19]. BC was first reported in B- and T-lymphocyte complementation by using recombination-activating gene 2 (Rag-2) KO mouse embryos [20] to permit the evaluation of gene function in lymphocytes. Kobayashi et al. [21] developed BC for whole-organ generation, succeeding initially with insulin-secreting β-cells: Mouse wildtype PSCs (mPSCs) were injected into pancreatic and duodenal homeobox 1 (Pdx1)-/- pancreas-deficient mouse blastocysts, generating a pancreas that was almost wholly PSC-derived and exhibited the ability to synthesize insulin and normalize host blood glucose levels. Interspecific BC also succeeded in generating rat pancreas in Pdx1-/- mice. Rat PSCs differentiated into a functioning pancreas in Pdx1-/- pancreas-deficient mice, which saved hosts from neonatal death [21].
Functioning mPSC-derived pancreas was similarly created in Pdx1-/- pancreas-deficient rats [22]. This generated enough islets to treat insulin-dependent diabetes mellitus model mice via islet transplantation. Transplanted mPSC-derived islets generated in Pdx1-/- pancreas-deficient rats successfully normalized and maintained host blood glucose levels for over 1 year without immunosuppression (excluding the first 5 days after islet transplantation). To date, BC has been used to generate forebrain [23], kidneys [24,25], germ cells [26], thymus [27], vascular endothelial cells [28,29], and lungs and bronchi [30].
Efforts to generate functional Ca-responsive PTGs from mouse embryonic stem cells (mESCs) using BC have recently succeeded (Fig. 2) [31]. Mouse Gcm2 has five exons. Exons 2 and 3 encode the entire DNA-binding domain, the gcm motif [32]. This region of Gcm2 thus was targeted for clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPRassociated protein 9 (Cas9) mediated zygote genome editing to generate parathyroid-deficient Gcm2-/- embryos for BC. mESCs were subsequently injected into Gcm2-/- parathyroid-deficient mouse embryos. The resulting Gcm2-/- mice complemented with mESCs had normal plasma [Ca] and PTH levels. They survived into adulthood without hypercalcemic problems. mESC-derived PTGs secreted PTH in response to hypocalcemia induced by intraperitoneal administration of NaHCO3, indicating functionality and maturity. Finally, mESC-derived PTGs transplanted into post-parathyroidectomy mice ameliorated postoperative hypoparathyroidism, demonstrating the suitability of these glands as allografts.
For clinical use of human PTGs established by BC, generating human PSC-derived interspecies chimeras is crucial. A substantial problem is that chimerism between human PSCs and animal blastocysts is too low to generate human organs in animals [19]. Attempts to solve this are underway. Preventing apoptosis in donor human PSCs [33] or deleting insulin-like growth factor 1 (Igf1r) in host embryos [34] might increase human–animal chimerism. The use of non-human primates evolutionarily near humans is another strategy [35].
For clinical application in humans, PSC-derived PTGs must be able to regulate PTH in response to extracellular Ca variation. While significant progress has been made in generating parathyroid-like cells in vitro, the Ca responsiveness and functionality of grafts following transplantation into diseased-animal models have yet to be fully verified. PTG generation via embryonic development might be more physiological; indeed, mouse data have demonstrated the functionality of PTGs generated by BC [31]. However, the generation of human PTGs in vivo must still overcome low chimerism between human PSCs and animal embryos. Whichever method is chosen, the goal of PTG transplantation medicine using human PSCs is to provide a safe and practicable method of eliminating the suffering caused by irreversible hypoparathyroidism.

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

Acknowledgements
This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant JP21K16337 and 24K19296 (to Mayuko Kano); the Japan Medical Women’s Association (to Mayuko Kano); the Yamaguchi Endocrine Research Foundation (to Mayuko Kano); and the Uehara Memorial Foundation (to Mayuko Kano). BioRender (https://biorender.com) was used in the creation of the figures. Dr A.S. Knisely commented on a version of the manuscript.
Fig. 1.
Schematic diagram of parathyroid gland (PTG) differentiation in vitro. In vitro PTG differentiation recapitulates early mammalian PTG development. Human pluripotent stem cells (PSCs) differentiate into definitive endoderm (DE) under high concentrations of activin A. This process is common to all three methods reviewed herein (upper [10], middle [13], bottom [14]). DE is subsequently induced to become anterior foregut endoderm (AFE). Rostral AFE differentiates into pharyngeal endoderm (PE). Oct4, POU class 5 homeobox 1; Nanog, nanog homeobox; Sox2, SRY-box transcription factor 2; Foxa2, forkhead box A2; Hoxa3, homeobox A3; Tbx1, T-box 1; Pax1, paired box 1; Pth, parathyroid hormone; Gcm2, glial cells missing 2; Casr, Ca-sensing receptor; Nkx2.1, NK2 homeobox 1; BMP4, bone morphogenetic protein 4; bFGF, basic fibroblast growth factor; WNT3a, Wnt family member 3A; KGF, keratinocyte growth factor; FGF10, fibroblast growth factor 10; EGF, epidermal growth factor; SHH, sonic hedgehog; ATRA, all-transretinoic acid.
enm-2024-1989f1.jpg
Fig. 2.
In vivo generation of mouse embryonic stem cell (mESC)-derived parathyroid glands (PTGs) via blastocyst complementation (BC) [31]. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated zygote glial cells missing 2 (Gcm2) knockout (KO) yielded PTG-deficient embryos. Parathyroid hormone (Pth)-tdTomato knock-in mESCs were injected into Gcm2 KO parathyroid-deficient mouse embryos. The resulting chimeric mice had PTGs derived from the injected cells. Immunostaining of mESC-derived PTGs generated in Gcm2-/- mice. tdTomato (magenta), PTH (green). Scale bars: 100 μm. From [31] Fig. 2M.
enm-2024-1989f2.jpg
Table 1.
Genes Related to PTG Development
Gene symbol
 Full gene name Expression
Mouse Human
Casr CASR Calcium sensing receptor PTG
Foxa2 FOXA2 Forkhead box A2 AFE
Gcm2 GCM2 Glial cells missing transcription factor 2 PTG
Nkx2-1 NKX2-1 NK2 homeobox 1 Lung and thyroid gland
Nkx2-3 NKX2-3 NK2 homeobox 3 PE
Pax1 PAX1 Paired box 1 PE
Pax9 PAX9 Paired box 9 PE
Pth PTH Parathyroid hormone PTG
Sox2 SOX2 SRY-box 2 (mouse) AFE
SRY-box transcription factor 2 (human)
Sox17 SOX17 SRY-box 17 (mouse) DE
SRY-box transcription factor 17 (human)
Tbx1 TBX1 T-box 1 (mouse) PE
T-box transcription factor 1 (human)

PTG, parathyroid gland; AFE, anterior foregut endoderm; PE, pharyngeal endoderm; DE, definitive endoderm.

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    Parathyroid Gland Generation from Pluripotent Stem Cells
    Image Image
    Fig. 1. Schematic diagram of parathyroid gland (PTG) differentiation in vitro. In vitro PTG differentiation recapitulates early mammalian PTG development. Human pluripotent stem cells (PSCs) differentiate into definitive endoderm (DE) under high concentrations of activin A. This process is common to all three methods reviewed herein (upper [10], middle [13], bottom [14]). DE is subsequently induced to become anterior foregut endoderm (AFE). Rostral AFE differentiates into pharyngeal endoderm (PE). Oct4, POU class 5 homeobox 1; Nanog, nanog homeobox; Sox2, SRY-box transcription factor 2; Foxa2, forkhead box A2; Hoxa3, homeobox A3; Tbx1, T-box 1; Pax1, paired box 1; Pth, parathyroid hormone; Gcm2, glial cells missing 2; Casr, Ca-sensing receptor; Nkx2.1, NK2 homeobox 1; BMP4, bone morphogenetic protein 4; bFGF, basic fibroblast growth factor; WNT3a, Wnt family member 3A; KGF, keratinocyte growth factor; FGF10, fibroblast growth factor 10; EGF, epidermal growth factor; SHH, sonic hedgehog; ATRA, all-transretinoic acid.
    Fig. 2. In vivo generation of mouse embryonic stem cell (mESC)-derived parathyroid glands (PTGs) via blastocyst complementation (BC) [31]. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated zygote glial cells missing 2 (Gcm2) knockout (KO) yielded PTG-deficient embryos. Parathyroid hormone (Pth)-tdTomato knock-in mESCs were injected into Gcm2 KO parathyroid-deficient mouse embryos. The resulting chimeric mice had PTGs derived from the injected cells. Immunostaining of mESC-derived PTGs generated in Gcm2-/- mice. tdTomato (magenta), PTH (green). Scale bars: 100 μm. From [31] Fig. 2M.

    Fig. 1.

    Fig. 2.

    Parathyroid Gland Generation from Pluripotent Stem Cells
    Gene symbol
    		 Full gene name Expression
    Mouse Human
    Casr CASR Calcium sensing receptor PTG
    Foxa2 FOXA2 Forkhead box A2 AFE
    Gcm2 GCM2 Glial cells missing transcription factor 2 PTG
    Nkx2-1 NKX2-1 NK2 homeobox 1 Lung and thyroid gland
    Nkx2-3 NKX2-3 NK2 homeobox 3 PE
    Pax1 PAX1 Paired box 1 PE
    Pax9 PAX9 Paired box 9 PE
    Pth PTH Parathyroid hormone PTG
    Sox2 SOX2 SRY-box 2 (mouse) AFE
    SRY-box transcription factor 2 (human)
    Sox17 SOX17 SRY-box 17 (mouse) DE
    SRY-box transcription factor 17 (human)
    Tbx1 TBX1 T-box 1 (mouse) PE
    T-box transcription factor 1 (human)
    Table 1. Genes Related to PTG Development

    PTG, parathyroid gland; AFE, anterior foregut endoderm; PE, pharyngeal endoderm; DE, definitive endoderm.

    Table 1.

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