
==== Front
Endocrinol Metab (Seoul)
Endocrinol Metab (Seoul)
ENM
Endocrinology and Metabolism
2093-596X
2093-5978
Korean Endocrine Society

38853617
10.3803/EnM.2024.1989
enm-2024-1989
Review Article
Calcium & Bone Metabolism
Parathyroid Gland Generation from Pluripotent Stem Cells
http://orcid.org/0000-0002-2761-0461
Kano Mayuko
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
8 2024
10 6 2024
39 4 552558
25 3 2024
17 4 2024
7 5 2024
Copyright © 2024 Korean Endocrine Society
2024
https://creativecommons.org/licenses/by-nc/4.0/ 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.
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.

Parathyroid glands
Pluripotent stem cells
Regenerative medicine
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pmcINTRODUCTION

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.

PARATHYROID GLAND DEVELOPMENT

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].

CONCLUSIONS

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.

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.

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.

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.

CONFLICTS OF INTEREST

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

1 Mitchell DM Regan S Cooley MR Lauter KB Vrla MC Becker CB Long-term follow-up of patients with hypoparathyroidism J Clin Endocrinol Metab 2012 97 4507 14 23043192
2 Zorn AM Wells JM Vertebrate endoderm development and organ formation Annu Rev Cell Dev Biol 2009 25 221 51 19575677
3 Graham A Development of the pharyngeal arches Am J Med Genet A 2003 119A 251 6 12784288
4 Grevellec A Tucker AS The pharyngeal pouches and clefts: development, evolution, structure and derivatives Semin Cell Dev Biol 2010 21 325 32 20144910
5 Jerome LA Papaioannou VE DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1 Nat Genet 2001 27 286 91 11242110
6 Gunther T Chen ZF Kim J Priemel M Rueger JM Amling M Genetic ablation of parathyroid glands reveals another source of parathyroid hormone Nature 2000 406 199 203 10910362
7 Liu Z Yu S Manley NR Gcm2 is required for the differentiation and survival of parathyroid precursor cells in the parathyroid/thymus primordia Dev Biol 2007 305 333 46 17382312
8 Su D Ellis S Napier A Lee K Manley NR Hoxa3 and pax1 regulate epithelial cell death and proliferation during thymus and parathyroid organogenesis Dev Biol 2001 236 316 29 11476574
9 Peters H Neubuser A Kratochwil K Balling R Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities Genes Dev 1998 12 2735 47 9732271
10 Green MD Chen A Nostro MC d’Souza SL Schaniel C Lemischka IR Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells Nat Biotechnol 2011 29 267 72 21358635
11 Moore-Scott BA Manley NR Differential expression of Sonic hedgehog along the anterior-posterior axis regulates patterning of pharyngeal pouch endoderm and pharyngeal endoderm-derived organs Dev Biol 2005 278 323 35 15680353
12 Gordon J Manley NR Mechanisms of thymus organogenesis and morphogenesis Development 2011 138 3865 78 21862553
13 Lawton BR Martineau C Sosa JA Roman S Gibson CE Levine MA Differentiation of PTH-expressing cells from human pluripotent stem cells Endocrinology 2020 161 bqaa141 32810225
14 Nakatsuka R Kato T Zhang R Uemura Y Sasaki Y Matsuoka Y The induction of parathyroid cell differentiation from human induced pluripotent stem cells promoted via TGF-α/EGFR signaling Stem Cells Dev 2023 32 670 80 37639359
15 D’Amour KA Bang AG Eliazer S Kelly OG Agulnick AD Smart NG Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells Nat Biotechnol 2006 24 1392 401 17053790
16 Huang SX Green MD de Carvalho AT Mumau M Chen YW D’Souza SL The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells Nat Protoc 2015 10 413 25 25654758
17 Patel SR Gordon J Mahbub F Blackburn CC Manley NR Bmp4 and Noggin expression during early thymus and parathyroid organogenesis Gene Expr Patterns 2006 6 794 9 16517216
18 Cozzolino M Lu Y Sato T Yang J Suarez IG Brancaccio D A critical role for enhanced TGF-alpha and EGFR expression in the initiation of parathyroid hyperplasia in experimental kidney disease Am J Physiol Renal Physiol 2005 289 F1096 102 15998841
19 Kano M Mizutani E Homma S Masaki H Nakauchi H Xenotransplantation and interspecies organogenesis: current status and issues Front Endocrinol (Lausanne) 2022 13 963282 35992127
20 Chen J Lansford R Stewart V Young F Alt FW RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development Proc Natl Acad Sci U S A 1993 90 4528 32 8506294
21 Kobayashi T Yamaguchi T Hamanaka S Kato-Itoh M Yamazaki Y Ibata M Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells Cell 2010 142 787 99 20813264
22 Yamaguchi T Sato H Kato-Itoh M Goto T Hara H Sanbo M Interspecies organogenesis generates autologous functional islets Nature 2017 542 191 6 28117444
23 Chang AN Liang Z Dai HQ Chapdelaine-Williams AM Andrews N Bronson RT Neural blastocyst complementation enables mouse forebrain organogenesis Nature 2018 563 126 30 30305734
24 Usui J Kobayashi T Yamaguchi T Knisely AS Nishinakamura R Nakauchi H Generation of kidney from pluripotent stem cells via blastocyst complementation Am J Pathol 2012 180 2417 26 22507837
25 Goto T Hara H Sanbo M Masaki H Sato H Yamaguchi T Generation of pluripotent stem cell-derived mouse kidneys in Sall1-targeted anephric rats Nat Commun 2019 10 451 30723213
26 Kobayashi T Goto T Oikawa M Sanbo M Yoshida F Terada R Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats Nat Commun 2021 12 1328 33637711
27 Isotani A Hatayama H Kaseda K Ikawa M Okabe M Formation of a thymus from rat ES cells in xenogeneic nude mouse↔rat ES chimeras Genes Cells 2011 16 397 405 21401810
28 Hamanaka S Umino A Sato H Hayama T Yanagida A Mizuno N Generation of vascular endothelial cells and hematopoietic cells by blastocyst complementation Stem Cell Reports 2018 11 988 97 30245211
29 Das S Koyano-Nakagawa N Gafni O Maeng G Singh BN Rasmussen T Generation of human endothelium in pig embryos deficient in ETV2 Nat Biotechnol 2020 38 297 302 32094659
30 Mori M Furuhashi K Danielsson JA Hirata Y Kakiuchi M Lin CS Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells Nat Med 2019 25 1691 8 31700187
31 Kano M Mizuno N Sato H Kimura T Hirochika R Iwasaki Y Functional calcium-responsive parathyroid glands generated using single-step blastocyst complementation Proc Natl Acad Sci U S A 2023 120 e2216564120 37379351
32 Akiyama Y Hosoya T Poole AM Hotta Y The gcm-motif: a novel DNA-binding motif conserved in Drosophila and mammals Proc Natl Acad Sci U S A 1996 93 14912 6 8962155
33 Masaki H Kato-Itoh M Takahashi Y Umino A Sato H Ito K Inhibition of apoptosis overcomes stage-related compatibility barriers to chimera formation in mouse embryos Cell Stem Cell 2016 19 587 92 27814480
34 Nishimura T Suchy FP Bhadury J Igarashi KJ Charlesworth CT Nakauchi H Generation of functional organs using a cell-competitive niche in intra- and inter-species rodent chimeras Cell Stem Cell 2021 28 141 9 33373620
35 Tan T Wu J Si C Dai S Zhang Y Sun N Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo Cell 2021 184 2020 32 33861963
