Differentiation of Human Adipose-Derived Stem Cells into Parathyroid Hormone-Secreting Cells

Abstract


Introduction
The most common causes of acquired hypoparathyroidism involve complications arising from neck surgery.Hypocalcemia, induced by hypoparathyroidism, is characterized by various clinical symptoms ranging from numbness and tingling of ngers or toes to life-threatening bronchospasm, seizure, and congestive heart failure 1 .The incidence of permanent hypoparathyroidism ranges from 20-25% 2,3 .
Currently, autograft is the best procedure to prevent post-surgical hypoparathyroidism, with a success rate of 50-95%.However, there is no opportunity to perform an autograft if parathyroid damage is not detected during surgery 4 .
Parathyroid hormone (PTH) regulates serum calcium and vitamin D levels through its action on bones, kidneys, and gastrointestinal tract.Theoretically, supplementation of PTH is the best treatment for hypoparathyroidism; administration of full-sequence PTH (1-84) for 24 months reduces the requirement for calcium and 1,25-dihydroxyvitamin D 5 .In 2015, Natpara (PTH 1-84) was approved by the Food and Drug Administration for hypoparathyroidism treatment.While PTH has a short half-life of 4 min and PTH secretion exhibits diurnal variation, injection of PTH (1-34) once or twice daily shows considerable e cacy in decreasing uctuation in serum calcium levels and excretion of urine calcium 6,7 .However, its high cost and need for daily injections remain a lifelong burden.Therefore, the commonly used treatment for hypoparathyroidism is supplementation with high doses of calcium and vitamin D; however, hypercalciuria, renal impairment, vitamin D intoxication, and ectopic calci cation may be potential side effects 8 .Hence, researchers continue to explore alternative treatments for hypoparathyroidism.
The parathyroid is a relatively simple gland as each cell performs the organ function; normal function of the gland is possible even with a few cells and no structural arrangement of cells is required for its function 9 .Recent studies have achieved parathyroid gland differentiation from embryonic stem cells, thymic epithelial cells, and tonsil-derived stem cells [10][11][12] .Unlike human embryonic stem cells, adiposederived stem cells (ADSCs) are relatively free from ethical concerns and can be obtained less invasively than other cell lineages.Therefore, to the extent of our knowledge, this study describes a method to differentiate parathyroid glands from human ADSCs for the rst time.

ADSC donors and donor medical history
ADSCs were isolated from subcutaneous abdominal adipose tissues that were obtained from three male donors (mean age, 40 years) during plastic surgery.They exhibited normal serum calcium, phosphorus, and PTH levels.They had received no medications that in uenced calcium levels, including calcium and vitamin D supplements, and anti-resorptive agents.Informed written consent was obtained from all participants in this study, and the study protocol was approved by the Institutional Review Board of the Pusan National University Hospital (IRB number 2013-8).All research was performed in accordance with the guidelines.

Differentiation of human ADSCs into parathyroid-like cells
Knife biopsies of adipose tissue were immediately put in minimum essential medium-alpha (MEM α, Gibco, Life Technologies Corp., Grand Island, NY, USA) which was containing 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco).Samples were transported to the laboratory and processed in less than 30 min outside body.Under aseptic condition, the tissues were chopped and digested with 0.075% type I collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 ℃ with vigorous shaking.Then, 25 mL MEM α supplemented with 10% fetal bovine serum (FBS) was added to neutralize the collagenase, and then centrifuged at 3000 rpm (1614 ×g) for 10 min.Digested tissue was subjected to ltration through a 70-µm nylon cell strainer (BD Biosciences, San Diego, CA, USA), after which the cells were washed with phosphate-buffered saline (PBS), and centrifuged again at 1600 rpm (459 ×g) for 10 min.The ADSCs obtained from sediment were expanded in ADSC culture medium of MEM α containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C and 5% CO 2 .The medium was changed twice per week.

Western blot analysis
Cell proteins were isolated using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea).Protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories Inc., Hercules, CA, USA).PTH protein was separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene uoride membrane (Amersham Biosciences, Little Chalfont, UK).The membrane was blocked using 5% skim milk and incubated with PTH antibody (1:1,000 dilution; Abcam, Cambridge, UK) and GAPDH antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4 °C overnight each and subsequently with horseradish peroxidase-linked secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h.Immunoreactive bands were detected using the ECL Western Blotting detection system (AB Frontier, Seoul, Korea).

Transmission Electron Microscopy (TEM)
The ADSC was xed with 2.5% glutaraldehyde (4 °C, PBS, pH 7.2) for 24 h and then, was xed with 1% osmium tetroxide in the PBS for 1 h at room temperature.The sample was dehydrated with a serial graded ethyl alcohol and embedded in epoxy resin (Epon 812 mixture).Thick sections (1 µm) were stained with 1% toluidine blue for light microscopy.Thin sections (50~60 nm) were prepared using an ultramicrotome (EM UC7, Leica Microsystems, Wetzlar, Germany) and were double stained with uranyl acetate and lead citrate.Thin sections were examined with a transmission electron microscope (JEM-1200 EXII, JEOL Ltd., Tokyo, Japan).We referred to the previous research method 13 .

Results
Alterations in cell morphology were observed during parathyroid-like cell differentiation from ADSCs.As observed under light microscopy, at and vacuolated ADSCs differentiated into secretory parathyroid gland-like nodules (Fig. 1).Transmission electron micrographs showed that the ultrastructure of ADSCs was also altered after differentiation.Compared with the control, distinct dense spots were observed in the lumen of differentiated ADSCs and this observation is typical in many secretory protein-releasing cells such as parathyroid cells (Fig. 2).RT-qPCR analysis revealed a considerablyincrease in mRNA expression of the parathyroid markers PTH, GCM2, and CCL21 over time.Compared with the β-actin housekeeping gene, PTH, GCM-2, and CCL21 were upregulated 13.59-fold, 8.10-fold, and 3.43-fold, respectively, on the seventh day of differentiation and 33.69-fold, 24.42-fold, and 6.09-fold, respectively, on the 21st day of differentiation (p < 0.05) (Fig. 3).Western blot analysis also revealed a signi cant amount of PTH protein on day 7, which increased over time (Fig. 4).

Discussion
In this study, we differentiated ADSC to parathyroid hormone-secreting cells.These cells have proven to be parathyroid gland cells by their expression of parathyroid cell markers, such as PTH, GCM-2, and CCL21, and by their secretion of PTH proteins.
Conventional therapy for hypoparathyroidism includes high doses of calcium and vitamin D. Due to low PTH, this may result in nephrolithiasis by hypercalciuria and extraskeletal calci cation (kidney, lens, and basal ganglia) by calcium phosphate products 1,8 .PTH has anti-calciuric effects that induce renal calcium resorption in the distal convoluted tubule and anti-phosphorus resorption effects that inhibit renal phosphorus resorption in both the proximal and distal convoluted tubules 14 .Thiazide diuretics may help calcium resorption in the distal convoluted tubule, and are thus used to prevent hypercalciuria.Therefore, PTH replacement is a risk-free alternative to the side effects of conventional treatment of hypoparathyroidism.
As the N-terminal is the bioactive fragment of PTH, there are two type of recombinant parathyroid hormones: PTH 1 − 84 and PTH 1 − 34 .As the polypeptide is broken down when administrated orally, recombinant PTH needs to be injected subcutaneously 15 .Injection of recombinant PTH once a day causes uctuation of calcium levels, which results in hypercalcemia in the rst 6-8 h following injection.
Twice a day injection causes less uctuation, but stimulates an above normal bone turnover rate and decreases bone mineral density, as PTH may increase cortical porosity 16 .Continuous PTH infusion with a pump results in less uctuation of serum calcium and moderate stimulation of bone turnover 17 .
However, continuous PTH infusion also causes inconvenience in a patient's daily life and needs regular blood sampling for estimation of serum calcium titers.Furthermore, there is a lack of data for estimating the optimal dose of PTH in children with autosomal dominant or autoimmune hypoparathyroidism.
Another solution to prevent acquired hypoparathyroidism during neck surgery is autografting.PTH produced by grafted parathyroid tissue allows patients to be free from painful daily injections and regular blood sampling for serum calcium titer as the grafted parathyroid tissue is a calcium sensing receptor itself.It takes 6-10 weeks for parathyroid function to normalize after an autograft.However, there is no opportunity to perform an autograft if gland damage is not detected during surgery.
Stem cell and tissue engineering is one of the ultimate elds in biomedicine.Human embryonic stem cells differentiate relatively easily compared with other cells, but its use sparks ethical controversies.Conversely, postnatal adult stem cells have no ethical implications and exhibit restricted differentiation potential.Adipocytes are differentiated from mesenchymal cells, and thus, adult mesenchymal stem cells can be derived from adipose tissues.ADSCs are multipotent with chondrogenic, neurogenic, and osteogenic abilities [18][19][20] .ADSCs may be differentiated into parathyroid cells using the Bingham protocol with Sonic hedgehog (Shh) 9 .In the study by Zhang et al. (2020), which demonstrates differentiation of rat ADSCs into parathyroid-like cells, it was seen that the higher the concentration of activin A, the greater the differentiation into parathyroid-like cells 21 .The differentiation method used in the present study involved 100 ng/mL activin A. To verify that the differentiated cells are parathyroid cells, many studies detect the levels of parathyroid gene expression markers CCL21, GCM2, PTH, and calcium sensing receptors using RT-qPCR 10,11,22 ; PTH protein levels have also been detected using enzyme-linked immunosorbent assays 9,11,12 .Notably, CCL21 and GCM2 23 are markers of parathyroid precursor cells.
The differentiation time of thymic stromal cells into parathyroid-like cells is long, i.e., approximately 10 weeks 10 , and may cause problems in tissue preservation.In contrast, tonsil-derived mesenchymal stem cells differentiate maximally within 7 d 12 , which is a relatively short time for differentiation; however, tonsil tissue cannot be obtained from in ammatory organs in limited cases such as chronic tonsillitis.In this aspect, ADSCs are technically more useful and show rapid differentiation within 7 d.Abundant and intact adipose tissue can be obtained effectively from all parts of subcutaneous as well as mesenchymal fat tissue by liposuction 24 .In case of liposuction, the procedure for harvesting adipose tissue is easy and safe, and involves ne mincing of tissues, with the stromal fraction enriched for ADSCs.Furthermore, even if the differentiation environment is not supplied, ADSCs survive adverse conditions of low glucose, glutamine, and oxygen concentrations 25 .Consequently, ADSCs may be used for native PTH production for the treatment of osteoporosis.Previous reports have shown that recombinant PTH is less active than the native protein 26 .Furthermore, PTH produced by differentiated tonsil-derived mesenchymal stem cells has a hundred times higher osteogenic capacity than that of recombinant human PTH 12 .In conclusion,

Figures
Figure 1 Light microscopy of differentiated adipose-derived stem cells (ADSCs).Morphologies of ADSC observed by light microscopy during differentiation (×100 and ×200 magni cation).Arrow, nodule-like shape representing possible parathyroid gland structure.Figure 1 Light microscopy of differentiated adipose-derived stem cells (ADSCs).Morphologies of ADSC observed by light microscopy during differentiation (×100 and ×200 magni cation).Arrow, nodule-like shape representing possible parathyroid gland structure.Western blot analysis of parathyroid hormone (PTH) protein secreted by differentiated adipose-derived stem cells (ADSC).Western blot analysis reveals that a signi cant amount of PTH protein is secreted by differentiated ADSC after day 7 and increases over time.
Western blot analysis of parathyroid hormone (PTH) protein secreted by differentiated adipose-derived stem cells (ADSC).Western blot analysis reveals that a signi cant amount of PTH protein is secreted by differentiated ADSC after day 7 and increases over time.

Figure 2 Transmission
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