Development and application of primary rat parathyroid cells for transplantation in hypoparathyroidism

Introduction

Hypoparathyroidism (HPO) is a clinical syndrome characterized by hypocalcemia and hyperphosphatemia, resulting from an insufficiency in parathyroid hormone (PTH) secretion. Clinically, HPO primarily manifests as paresthesia of the extremities, muscle cramps, and seizures, with severe cases potentially progressing to fatal outcomes (1). The most prevalent cause of HPO is inadvertent damage to or removal of the parathyroid glands during cervical surgery (1). Due to the anatomical proximity of the parathyroid glands to the thyroid and their often variable and concealed locations, the incidence of HPO following thyroidectomy ranges from 5.2% to 21.6% (2,3). The probability of postoperative HPO increases in proportion to the extent of thyroid surgery (4,5). It is estimated that approximately 1/4 of patients who experience parathyroid injury or accidental removal develop permanent HPO, which requires lifelong replacement therapy and close monitoring to prevent potentially life-threatening hypocalcaemia (6).

The prevailing therapeutic strategies for HPO are primarily based on high-dose vitamin D and calcium supplementation. However, these strategies do not address the underlying deficiency and are associated with various short- and long-term complications (7,8). Prolonged use of high-dose vitamin D and calcium heightens the risk of nephrolithiasis and irreversible renal impairment (8). While recombinant human full-length PTH 1-84 (rhPTH 1-84) replacement therapy initially demonstrated potential in reducing the reliance on calcium and vitamin D analogs and improving patient quality of life, it is no longer produced by any manufacturer despite previous approval (8). Recently, Yorvipath^® [1-34], a synthetic PTH fragment, received Food and Drug Administration (FDA) approval in August 2024 for use in adults with HPO, representing a new treatment option (9). However, both therapies require daily self-injection, posing challenges for long-term patient compliance, and the long-term safety profile of such treatments continues to require further investigation. Parathyroid transplantation is considered the ideal treatment for HPO, yet obstacles such as donor shortages, difficulties in determining appropriate graft volume, and the risk of immune rejection continue to impede progress (10,11). Consequently, the exploration of alternative sources of parathyroid tissue or cells is crucial for advancing parathyroid transplantation.

The absence of an effective animal model for HPO has significantly limited progress in related fundamental research. While Tu et al. previously developed an HPO model using CaSR-deficient mice, the model exhibited limitations, including poor survival rates and reproductive challenges, and it failed to adequately mimic surgically induced HPO (12). It is therefore imperative that a reliable surgical animal model be developed in order to advance basic research on HPO.

The scarcity of parathyroid tissue and the highly differentiated nature of its cells present significant challenges in establishing cell lines and maintaining functional primary parathyroid cells in long-term culture. Recent research on parathyroid cells and organoids has focused on the in vitro differentiation of pluripotent stem cells (PSCs) into parathyroid-like cells (13-15). Karabiyik Acar et al. utilized Activin A and Sonic Hedgehog (Shh) to induce the differentiation of cells isolated from the thyroid, leveraging their shared embryological origin and close anatomical relationship in the adult human body, into Parathyroid-like cells (16). Lawton et al. reported the consistent differentiation of PSCs into parathyroid-like cells through the addition of the cyclin-dependent kinase (CDK) inhibitor PD0332991 to the culture medium (CM) (15). Although these studies achieved some success, with the detection of PTH in culture supernatant and confirmation of PTH mRNA expression, the induced cells were not directly derived from the parathyroid gland, raising concerns about potential dedifferentiation, tumorigenesis, and other unknown risks. Importantly, Parathyroid chief cells express CaSR, encoded by Casr, on their surface, enabling them to respond to minute changes in extracellular Ca²⁺ concentrations. This calcium-sensing function is crucial for the precise secretion of PTH, which in turn maintains plasma Ca²⁺ concentration within a narrow range. However, these studies did not assess the response of differentiated cells to changes in calcium ion concentration (17). These findings suggest that the initial stages of in vitro parathyroid cell culture limit the applicability of these cells, underscoring the need for further exploration of culture conditions that preserve both PTH secretion and calcium-sensing functions.

Parathyroid glands, particularly in model animals such as rats and mice, are challenging to identify macroscopically. Fortunately, 5-aminolevulinic acid (5-ALA) has been reported to be effective in visualizing parathyroid glands (18). Leveraging this method, we conducted parathyroidectomy in rats to develop an HPO model, enabling the effective acquisition of rat parathyroid tissue. Through the assessment of blood calcium and intact-PTH (iPTH) levels, we confirmed the effective establishment of the rat HPO model. By dissociating rat parathyroid tissue and sorting CaSR⁺/EpCAM⁺ cells for culture, we optimized CM and successfully generated functional parathyroid cells with calcium-sensing capabilities under long-term culture conditions. This study presents an effective surgical model of HPO in rats and establishes a functional primary rat parathyroid cell line, rPT-1, which may serve as a valuable experimental model for future research on HPO and parathyroid diseases, offering new experimental evidence and therapeutic avenues for HPO and related conditions. We present this article in accordance with the ARRIVE reporting checklist (available at https://gs.amegroups.com/article/view/10.21037/gs-24-411/rc).

Methods

Animal model and parathyroidectomy

Sprague Dawley (SD) rats were purchased from Hunan SJA Laboratory Animal Co., Ltd. [Animal Qualification Number No. SCXK(Hunan)2021-0002]. All experiments were conducted under project license (No. CQU-IACUC-RE-202412-002) granted by the Ethics Committee of Chongqing University, in compliance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Healthy adult male SD rats weighing between 250–300 g were selected. The rats were acclimatized for one week in a controlled environment with a 12-hour light-dark cycle and free access to food and water. The feeding environment was specific pathogen free (SPF) grade. Randomization was used to allocate the rats to control and treatment groups. The rats were randomly selected and assigned using a simple randomization method to ensure unbiased distribution across experimental groups. One hour after intraperitoneal injection of 5-ALA (#A800001, Macklin, Shanghai, China; 500 mg/kg), the rats were anesthetized with isoflurane (#R510-22-10, REWARD, Shenzhen, China) and subjected to surgery under sterile conditions. The rats were placed in a supine position, the neck was shaved, and the area was disinfected and draped. A midline incision was made in the neck skin, followed by separation of neck muscles to expose the thyroid and parathyroid glands. A 405 nm light source was used to visualize the parathyroid glands. Fine dissection of the surrounding tissues was performed, and the parathyroid blood supply was ligated with 4-0 absorbable sutures before excising the parathyroid tissue. The wound was closed in layers, and postoperative care included antibiotic treatment to prevent infection.

Construction and CM screening for rat parathyroid cells

The parathyroid tissue was washed, minced, and digested with Collagenase Type I (#A417694, Sangon Biotech, Shanghai, China; 2 mg/mL) at 37 ℃ for 20 minutes. To terminate the digestion process, an equal volume of 1640 medium (#11875085, Gibco, Waltham, USA) supplemented with 10% fetal bovine serum (FBS) (#16000-044, Gibco) was added. The resulting cell suspension was filtered through a 70 µm filter (#431751, Corning) and centrifuged at 1,000 rpm for 5 minutes at 4 ℃. The cell pellet was resuspended in 1640 medium (#11875085, Gibco) containing 10% FBS (#16000-044, Gibco) and a triple-antibiotic mixture (penicillin, streptomycin, and amphotericin B; # P7630, Solarbio, Beijing, China) and then seeded at a density of 5×10⁴ cells per well in 12-well plates (#354474, Corning). The cultures were maintained at 37 ℃ in a humidified incubator with 5% CO₂. Subsequently, the cells were transferred into four distinct CM after achieving stable attachment to the wall. The following is an overview the four different CM formulations used: DMEM-F12 (#12660012, Gibco) complete medium was used for all CM.

CM1 composition

• 2% B-27 supplement (#17504-044, Gibco);
• 1% insulin (#P3376, Beyotime, Shanghai, China);
• 1% hydrocortisone (#A610506, Sangon Biotech);
• 1% penicillin/streptomycin/amphotericin B (#P7630, Solarbio);
• 10% FBS (#16000-044, Gibco).

CM2 composition

• 2% B-27 supplement (#17504-044, Gibco);
• 50 ng/mL epithelial growth factor (EGF) (#HY-P7092, MedChemExpress, New Jersey, USA);
• 1% insulin (#P3376, Beyotime);
• 1% hydrocortisone (#A610506, Sangon Biotech);
• 1% penicillin/streptomycin/amphotericin B (#P7630, Solarbio);
• 10% FBS (#16000-044, Gibco).

CM3 composition

• 2% B-27 supplement (#17504-044, Gibco);
• 50 ng/mL EGF (#HY-P7092, MedChemExpress);
• 2 µM A83-01 (#HY-10432, MedChemExpress);
• 1% insulin (#P3376, Beyotime);
• 1% hydrocortisone (#A610506, Sangon Biotech);
• 1% penicillin/streptomycin/amphotericin B (#P7630, Solarbio);
• 10% FBS (#16000-044, Gibco).

CM4 composition

• 2% B-27 supplement (#17504-044, Gibco);
• 50 ng/mL Shh (#R1831r, EIAab, Wuhan, China);
• 50 ng/mL Activin A (#HY-P70311, MedChemExpress);
• 50 ng/mL EGF (#HY-P7092, MedChemExpress);
• 2 µM A83-01 (#HY-10432, MedChemExpress);
• 1% insulin (#P3376, Beyotime);
• 1% hydrocortisone (#A610506, Sangon Biotech);
• 1% penicillin/streptomycin/amphotericin B (#P7630, Solarbio);
• 10% FBS (#16000-044, Gibco).

rPT-1 cells were seeded at an initial density of 5×10⁴ cells per well in 12-well plates (#354474, Corning) and cultured in different CM. The CM was replaced every 2 days. Cells were monitored daily using phase-contrast microscopy to assess adhesion, morphology, and confluence. Once cultures reached approximately 80% confluence, they were passaged. To do this, the spent medium was discarded, and the cells were gently rinsed once with phosphate buffered saline (PBS). An appropriate amount of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (#25300120, Gibco) was then added and incubated for 1–2 minutes until the cells detached. After detachment, serum-containing medium was added to neutralize the trypsin. The cell suspension was transferred to a centrifuge tube and spun at 1,000 rpm for 5 minutes. Following centrifugation, the supernatant was discarded, and the cell pellet was resuspended in different CM. The cells were then reseeded into a new 12-well plates (#354474, Corning) at approximately 5×10⁴ cells per well. Each instance of enzymatic detachment and reseeding after cells had reached confluence was defined as one passage, with each passage typically occurring every 2–3 days.

Cell growth, viability, and PTH secretion assays

Cell growth and viability were assessed using the Cell Counting Kit-8 (CCK-8; #C0005, TargetMol, Boston, USA) assay. rPT-1 were seeded in 96-well plates at an initial density of 5×10³ cells per well and cultured in four CM (CM1, CM2, CM3, and CM4) under identical conditions. At 24, 48, and 72 hours, 10 µL of CCK-8 reagent was added to each well and incubated for 2 hours at 37 ℃. Absorbance at 450 nm was measured using a microplate reader (Invitrogen, Waltham, USA). For the PTH measurement in different CM, rPT-1 cells were seeded in 12-well plates at an initial density of 5×10⁴ cells per well and cultured in different CM under identical conditions. Cells from the 3rd and 5th passages were cultured in various CM for 48 hours. After the incubation period, the supernatants were collected for PTH analysis. For the PTH measurement in passaged cells cultured in CM4, each generation was subcultured at a density of 1×10⁶ cells per T25 flask (#430168, Corning) and cultured in CM4 for 48 hours. After the incubation, the culture supernatant was collected for PTH measurement. PTH levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Rat Intact-PTH ELISA Kit; #MM-0346R2, Meimian Industrial, Nanjing, China), with a standard curve ranging from 31.25 to 1,000 pg/mL, and absorbance was read at 450 nm using a microplate reader (Invitrogen).

Fluorescence-activated cell sorting (FACS)

The primary isolated parathyroid cells, obtained as single-cell suspensions after enzymatic digestion and totaling approximately 1×10⁶ cells, as well as the 3rd and 5th passage cultured rPT-1 cells in different CM, each numbering approximately 5×10⁵ cells, were subjected to flow cytometry analysis and sorting to identify and isolate functional parathyroid cells. The cells were incubated with biotin-labeled anti-EpCAM antibody (#bs-1513R-Biotin, Bioss, Beijing, China; dilution: 1:100) at 25 ℃ for 30 minutes, then the cells were washed with PBS buffer and incubated with APC Streptavidin (#405207, BioLegend, San Diego, USA; dilution: 1:100) in the dark at 25 ℃ for 30 minutes. After incubation, the fluorescein isothiocyanate (FITC)-labeled anti-CaSR (#CaSR-FITC, Fabgennix, Arlington, USA; dilution: 1:100) in the dark at 25 ℃ for 30 minutes. Following three times of PBS wash, the cells were analyzed using a flow cytometer (Beckman Coulte, MoFlo XDP, California, USA), and double-positive cells (CaSR⁺/EpCAM⁺) were sorted for in vitro culture.

Immunofluorescence staining

Cells were fixed with paraformaldehyde (PFA, 4%), permeabilized, and blocked. They were then incubated overnight at 4 ℃ with anti-PTH (rabbit monoclonal anti-PTH; #A9704, Abclonal, Wuhan, China; dilution: 1:200), anti-CaSR (mouse monoclonal anti-CaSR; #MA1-934, Invitrogen; dilution: 1:200), and anti-GCM2 (mouse monoclonal anti-GCM2; #sc-390603, Santa Cruz Biotechnology, Santa Cruz, USA; dilution: 1:200). The next day, the cells were washed with PBS and incubated with goat anti-mouse (#A32728, Invitrogen; dilution: 1:500)/rabbit (#A32733, Invitrogen; dilution: 1:500) IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 647 at room temperature in the dark for 1 hour. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (#P0131, Beyotime), and the slides were mounted for observation under fluorescence microscope (ZEISS, Oberkochen, Germany, Axio Imager 2).

In vitro analysis of rPT-1 function

rPT-1 cells were seeded in 12-well plates and cultured in CM4. When the cell density reached 70–80% (approximately 1×10⁶ cells per well), the medium was replaced with DMEM/F-12 medium containing ionized Ca²⁺ (0.4, 1, 2.5 mM), supplemented with 0.1% FBS, and incubated for 30 minutes at 37 ℃. Ionized calcium levels in the medium were determined using a Mindray BS-460 Chemistry Analyzer (Mindray Medical International Limited, Shenzhen, China). After incubation, the supernatant was collected and stored at −80 ℃ until the PTH assay. PTH levels were measured using an ELISA kit (Rat Intact-PTH ELISA Kit; #MM-0346R2, Meimian Industrial), with a standard curve ranging from 31.25 to 1,000 pg/mL, and absorbance was read at 450 nm using a microplate reader (Invitrogen).

Cell transplantation

One week after undergoing parathyroidectomy, rats with confirmed HPO were prepared for transplantation under isoflurane anesthesia. The establishment of the HPO model was validated by measuring serum calcium and PTH levels. Experimental group: rPT-1 cells (passage 4), cultured in CM4 medium, were suspended in PBS at a concentration of 5×10⁵ cells/mL, and 100 µL of the cell suspension (containing 5×10⁴ cells) was transplanted under the renal capsule of each rat. A total of 5 rats were included in this group, of which 4 rats survived until the end of the study. Sham group: normal rats without parathyroidectomy were injected with 100 µL PBS under the renal capsule to serve as controls. A total of 6 rats were included in this group, all rats survived until the end of the study. HPO group: rats with confirmed HPO were injected with 100 µL PBS under the renal capsule to evaluate the effects of PBS injection alone in the absence of rPT-1 cells. A total of 6 rats were included in this group, of which 3 rats survived until the end of the study. All rats were housed under identical environmental conditions and provided with a standard diet throughout the experiment to ensure consistency across groups. Postoperatively, penicillin G (#ST2555, Beyotime; 20,000 U/kg, once daily) was administered via intraperitoneal injection to all rats to prevent infection. Two weeks post-transplantation, blood samples were collected from all rats under isoflurane anesthesia via retro-orbital sampling. The collected blood was allowed to clot at room temperature and subsequently centrifuged at 3,000 rpm for 10 minutes at 4 ℃ to obtain serum. Serum calcium and PTH levels were measured for all groups. Blood calcium levels were reduced by intraperitoneal injection of 8.4% NaHCO₃ (17), and the iPTH concentration and calcium levels were measured at 15, 30, 60, and 90 minutes to assess the functional recovery capacity of the transplanted cells. PTH and ionized calcium levels were measured using the same method as described above.

Hematoxylin and eosin (H&E) staining and immunohistochemistry

H&E staining was performed as previously described (19). For immunohistochemical analysis, kidney tissue sections from SD rats [sham surgery/transplantation group (TRANS group)] were processed as follows: 4% PFA-fixed, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was conducted by heating the sections in citrate buffer (pH 6.0) using a microwave oven. To block endogenous peroxidase activity, the sections were incubated with 3% hydrogen peroxide (#PV-6002, ZSGB-BIO, Beijing, China). The tissue sections were then incubated overnight at 4 ℃ with primary antibodies, including an anti-CaSR monoclonal antibody (#MA1-934, Invitrogen; dilution: 1:200) and an anti-PTH monoclonal antibody (#A9704, Abclonal; dilution: 1:200). Following primary antibody incubation, the sections were treated with biotinylated goat anti-mouse (#PV-6002, ZSGB-BIO)/rabbit IgG secondary antibodies (#PV-6001, ZSGB-BIO). Immunoreactivity was visualized using a horseradish peroxidase detection kit (#ZL9018, ZSGB-BIO). The sections were counterstained with hematoxylin, dehydrated through an ethanol series, and mounted using a neutral resin. Images were captured using a Leica DM3000 microscope (Leica, Wetzlar, Germany) to assess marker expression.

Statistical analysis

All experimental data were presented as mean ± standard deviation. Statistical analysis was performed using Prism 9.5.1 software (https://www.graphpad.com).

Results

Development of rat HPO model and acquisition of parathyroid tissue

Due to the small size of parathyroid tissue, it is challenging to identify it accurately with the naked eye, making surgical models of mammalian HPO rare. Previous research suggested that 5-ALA is an effective tracer for rat parathyroid glands, emitting visible red light at 635 nm when exposed to 405 nm excitation light (18). To develop a rat HPO model, we selected SD rats, administered 5-ALA intraperitoneally, and performed parathyroidectomy one hour later. The surgical procedures are as follows: (I) anaesthesia, neck preparation; (II) sterilisation of sheets; (III) incision of neck skin and exposure of salivary glands (Figure 1A); (IV) blunt dissection of the sternohyoid muscle to expose the thyroid gland (Figure 1B); (V) gentle retraction of the thyroid gland and dissection of surrounding tissues; (VI) visualization of the parathyroid gland as a red structure under 405 nm light (Figure 1C); (VII) fine dissection of surrounding tissues and ligation of parathyroid blood supply (Figure 1D); (VIII) careful removal of parathyroid tissue along the ligated area; (IX) closure of the incision in layers (Figure 1E). The parathyroid tissue was approximately 1 mm × 1 mm in size (Figure 1F). One week postoperatively, we assessed blood calcium and iPTH levels in the rats (Figure 1G,1H) and confirmed the effective establishment of the rat HPO model (20).

Figure 1 Development of rat hypoparathyroidism model and parathyroid tissue acquisition. (A-F) Representative images of the surgical procedure for HPO in rats: (A) incision of skin to the platysma; (B) dissection of muscle to expose one side of the thyroid (areas outlined by dotted lines); (C) 405 nm ultraviolet imaging (the parathyroid tissue is encircled by dotted lines); (D) ligation of periparathyroid blood vessels (the parathyroid tissue is encircled by dotted lines); (E) bilateral parathyroidectomy followed by layer-by-layer suturing; (F) excision of parathyroid gland (the parathyroid tissue is encircled by dotted lines); (G) the serum concentration of Ca²⁺ in both the control and HPO rats, one week following surgery. CTRL rats, n=6; HPO rats n=6; (H) the serum concentration of iPTH in both the control and HPO rats, one week following surgery. CTRL rats, n=6; HPO rats, n=6. ****, P<0.0001. CTRL, control; HPO, hypoparathyroidism; iPTH, intact parathyroid hormone.

Extraction and culture of rPT-1

To obtain rPT-1 cells, we isolated parathyroid tissue, digested it into single cells (Figure 2A). The cells were then subjected to FACS based on CaSR and EpCAM markers, with CaSR⁺/EpCAM⁺ rat parathyroid cells being selectively enriched for ex vivo culture (Figure 2B). There are three key factors influencing the growth of primary endocrine epithelial cells: (I) the significant role of EGF in epithelial growth (21); (II) the inhibition of epithelial-mesenchymal transition (EMT) (22); and (III) the addition of specific growth factors such as Shh and Activin A to maintain parathyroid function (16). Based on these factors, we used four different CM formulations to culture primary parathyroid cells. The results indicated that CM4 supported the growth of primary cells, with the formulation containing Shh, EGF, and Activin A showing the least cellular debris (Figure 2C) and a more distinct epithelial morphology. Cell viability assays revealed that cells grew faster in media CM3 and CM4 (Figure 2D). Additionally, ELISA analysis demonstrated that all conditioned media supported varying levels of iPTH secretion, with significantly higher iPTH concentrations observed in the supernatant collected from passage 3 (P3) cells cultured in CM4 (Figure 2E). Furthermore, we assessed the percentage of EpCAM⁺/CaSR⁺ cells and the corresponding levels of iPTH in the supernatant of rPT-1 cells at P3, P5 under various CM conditions (Figure S1, supporting information). We found that the abundance of EpCAM⁺/CaSR⁺ cells cultured in CM4 was greater, and iPTH secretion was further elevated (Figure 2F). These results indicate that CM4 is the most effective CM for supporting the growth, epithelial morphology, and functional secretion of iPTH in rPT-1 cells.

Figure 2 Isolation and culture of rPT-1 cells. (A) Primary cell extraction process. The schematic diagram illustrates the process of primary cell extraction, sorting and culture; (B) flow cytometric analysis of CaSR and EpCAM expression in rat parathyroid glands; (C) BF microscopy of rPT-1 (P3) cultured in different CM after 48 h. Scale bars represent 50 µm in the top panel and 20 µm in the bottom panel; (D) relative proliferation rates of rPT-1 cells (P3) cultured for 24, 48, and 72 in various CM were assessed using the CCK-8 assay. The relative proliferation rate was calculated relative to rPT-1 cells cultured in CM1 for 24 h. CM1, n=6; CM2, n=6; C3, n=6; CM4, n=6; (E) the different CM supernatant of iPTH in rPT-1 (P3). CM1, n=3; CM2, n=3; CM3, n=3; CM4, n=3; (F) correlation between the percentage of EpCAM⁺/CaSR⁺ cells among all cells (P5), detected by flow cytometric analysis under various CM conditions, and the iPTH levels measured by ELISA in the corresponding CM. CM1, n=3; CM2, n=3; CM3, n=3; CM4, n=3. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. BF, bright field; CaSR, calcium-sensing receptor; CCK-8, Cell Counting Kit-8; CM, culture medium; ELISA, enzyme-linked immunosorbent assay; EpCAM, epithelial cell adhesion molecule; iPTH, intact parathyroid hormone; ns, not significant; P3, passage 3; P5, passage 5.

Calcium-sensing function of rPT-1

PTH is a critical hormone for maintaining calcium ion homeostasis, and calcium sensing is essential for the precise secretion of PTH by the parathyroid gland. To confirm that rPT-1 are functional parathyroid cells, we performed immunofluorescence staining for PTH, CaSR, and GCM2, demonstrating high levels of PTH, CaSR, and GCM2 expression in rPT-1 cells (Figure 3A). Further ELISA analysis confirmed that the passaged rPT-1 cells cultured in CM4 remained capable of secreting high levels of iPTH (Figure 3B). Furthermore, the fourth generation of rPT-1 cells was stimulated using low-calcium (0.4 mM), normal (1.0 mM) and high-calcium (2.5 mM) media, and iPTH levels in the culture supernatant were measured using ELISA. It was observed that iPTH levels in the medium were significantly elevated after low-calcium stimulation, indicating that the in vitro-cultured rPT-1 cells possess a calcium-sensing function. Under high-calcium conditions, while the reduction in iPTH secretion was not statistically significant, a downward trend was noted (Figure 3C). These findings demonstrate that rPT-1 cells retain essential parathyroid characteristics and exhibit responsive behavior to calcium levels in vitro, including the cessation of iPTH secretion under high-calcium conditions.

Figure 3 Calcium-sensing functionality of rPT-1 cells. (A) Representative immunofluorescence images of rPT-1 culture in CM4, showing parathyroid-specific markers. Scale bars, 40 µm. Markers indicated at the top are shown as a red fluorescent signal. Nuclei are shown as a blue fluorescent signal; (B) measurement of iPTH secretion from successive passages of rPT-1 (P0–P4) cultured in CM4 via ELISA. Each passage, n=3; (C) measurement of iPTH secretion from parathyroid cells under low, CTRL and high calcium conditions via ELISA. CTRL, n=3; low-Ca²⁺, n=3; high-Ca²⁺, n=3. **, P<0.01; ***, P<0.001. CaSR, calcium-sensing receptor; CM, culture medium; CTRL, control; ELISA, enzyme-linked immunosorbent assay; iPTH, intact parathyroid hormone; ns, not significant; P0–P4, passage 0–4.

rPT-1 effectively treat rat HPO

Subcapsular renal transplantation is regarded as a preferential site for transplanting organs such as islets, as it effectively prevents instant blood-mediated inflammatory reactions (IBMIR) and provides a supportive environment for graft growth (23). Although the normal kidney expresses the CaSR, its expression within the renal subcapsular space is suboptimal, and PTH expression in the kidney is insufficient (Figure S2). Consequently, we selected the renal subcapsular space as the transplantation site for rPT-1 cultured in CM4 (Figure 4A). Histological examination of the kidney two weeks after subcapsular transplantation revealed favorable graft growth, as evidenced by elevated PTH and CaSR expression (Figure 4B). Serum analysis revealed that, compared to HPO rats, the transplanted rats exhibited significantly higher serum calcium and iPTH levels (Figure 4C,4D). Importantly, after intraperitoneal injection of NaHCO₃ to reduce blood calcium, we measured serum calcium and iPTH levels at 0, 15, 30, 60, and 90 minutes, observing a more pronounced recovery of Ca²⁺ and iPTH levels in the TRANS group rats compared to the HPO group (Figure 4E,4F). By 90 minutes, calcium levels had returned to near-normal, comparable to the sham-operated group (Figure 4E). These findings indicate that the cultured rPT-1 cells are functional parathyroid cells with calcium-sensing capabilities and can effectively treat rat HPO.

Figure 4 Therapeutic efficacy of rPT-1 cells in treating rat hypoparathyroidism. (A) Representative images of rat kidney subcapsular graft (the transplanted rPT-1 is outlined by the dotted line); (B) representative images of H&E staining (left) and immunohistochemical staining for PTH (middle) and CaSR (right) in kidney subcapsular grafts. Scale bars represent 50 µm in the top panel and 10 µm in the bottom panel; (C) measurement of serum Ca²⁺ ion from two weeks after transplantation. TRANS rats, n=3; HPO rats, n=3; sham-operated rats, n=3; (D) measurement of iPTH secretion from two weeks after transplantation via ELISA. TRANS rats, n=3; HPO rats, n=3; sham-operated rats, n=3; (E) changes in serum ionized Ca²⁺ upon sodium bicarbonate infusion test in rats. TRANS rats, n=2; HPO rats, n=3; sham-operated rats, n=3; (F) changes in serum iPTH concentration upon sodium bicarbonate infusion test in rats. TRANS rats, n=2; HPO rats, n=3; sham-operated rats, n=3. *, P<0.05; ****, P<0.0001. CaSR, calcium-sensing receptor; ELISA, enzyme-linked immunosorbent assay; H&E, hematoxylin and eosin; HPO, hypoparathyroidism; iPTH, intact parathyroid hormone; ns, not significant; TRANS, transplantation.

Discussion

PTH plays a critical role in regulating calcium and phosphate homeostasis in the bloodstream (24). The CaSR is integral to this process, as it mediates the synthesis and release of PTH and governs the proliferation of parathyroid cells through various signal transduction pathways. HPO is predominantly caused by defects in PTH secretion, and parathyroid transplantation is widely regarded as the ideal therapeutic approach for HPO. However, this treatment faces significant challenges, including the determination of appropriate graft volume, donor scarcity, and the risk of immune rejection (25).

Recent research has focused on inducing PSCs to differentiate into Parathyroid-like cells (13-15). Although these studies efficient detected PTH in the culture supernatant and confirmed the expression of PTH mRNA, the induced cells were not directly isolated from parathyroid glands, which may limit their ability to fully replicate the functions of native parathyroid cells. Additionally, these studies did not evaluate the response of these cells to fluctuations in calcium ion concentration. For instance, primary monolayer cultures of bovine parathyroid cells typically lose their calcium responsiveness within a few days and are rapidly overgrown by fibroblasts (26). Fabbri et al. advanced this field by developing a rat cell line, PTH-C1, which consistently expresses parathyroid phenotypes, including PTH and CaSR genes, and maintains responsiveness to calcium concentration changes (27). Similarly, our research involved the direct isolation of cells from parathyroid tissue, followed by validated long-term culture under optimized conditions that preserved calcium-sensing functionality, offering a more accurate in vitro simulation of parathyroid cell function. The discovery of functional primary parathyroid cells has led to new considerations regarding their potential application in cell transplantation therapy for HPO models.

The development of suitable in vivo and in vitro models has been limited (28), restricting the ability to assess the long-term regulatory mechanisms of calcium on parathyroid cell physiology and hindering transplantation studies. In this study, we employed 5-ALA as a tracer for rat parathyroid glands, which significantly enhanced the visibility of parathyroid tissue and addressed the challenge of identifying small parathyroid glands. Through meticulous surgical techniques, we established and validated a stable and reliable rat model of HPO. Furthermore, we isolated CaSR⁺/EpCAM⁺cells from rat parathyroid tissue and maintained their functionality in long-term in vitro culture by optimizing the CM, with a particular focus on preserving their calcium-sensing capabilities. These cells not only exhibited high levels of PTH secretion during culture but also demonstrated a robust response to low-calcium stimulation, effectively mimicking the function of in vivo parathyroid cells. Finally, we demonstrated that transplantation of our cultured rPT-1 cells, which possess preserved calcium-sensing functionality, effectively ameliorates HPO in rats.

Our research introduces a novel method for the long-term culture of parathyroid cells, addressing the critical challenge of maintaining their function in vitro, and provides experimental evidence supporting the potential of cell replacement therapy for HPO. Future studies should focus on further optimizing cell culture and transplantation protocols, as well as exploring their clinical applications. Additionally, our findings offer a new experimental model and theoretical foundation for the basic research and treatment of HPO and other parathyroid-related disorders.

Conclusions

This study established a method for the long-term culture of primary rat parathyroid cells that retain their hormone-secreting and calcium-sensing functionalities. By isolating CaSR⁺/EpCAM⁺ cells and optimizing the culture medium with Shh, Activin A, and EMT inhibitors, we maintained functional parathyroid cells in vitro. Transplantation of these cultured cells into hypoparathyroid rats restored serum calcium and PTH levels, effectively alleviating hypocalcemic symptoms. These results demonstrate the potential of using cultured primary parathyroid cells as a viable cell-based therapy for HPO, offering a promising alternative to conventional treatments and providing a foundation for future clinical applications.

Acknowledgments

Special thanks to Dr. Wang Yang (Department of Gastroenterology, Chongqing General Hospital, Chongqing University, Chongqing, China) and Dr. Jianwu Wang (Department of Urology, The Affiliated Lihuili Hospital, Ningbo University, Ningbo, China) for guidance in data analysis and preparation of the manuscript.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://gs.amegroups.com/article/view/10.21037/gs-24-411/rc

Data Sharing Statement: Available at https://gs.amegroups.com/article/view/10.21037/gs-24-411/dss

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Funding: This work was supported by the Chongqing Technology Innovation and Application Development Special Social Development Field Key Projects (grant No. CSTB2022TIAD-KPX0177).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://gs.amegroups.com/article/view/10.21037/gs-24-411/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All experiments were conducted under project license (No. CQU-IACUC-RE-202412-002) granted by the Ethics Committee of Chongqing University, in compliance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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