CN113215081B

CN113215081B - Method for inducing stem cells to differentiate into parathyroid cells and composition thereof

Info

Publication number: CN113215081B
Application number: CN202110611017.0A
Authority: CN
Inventors: 李兴睿; 杜雅莹; 王戈; 杨志芳; 沈文状; 董鸿
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2021-06-01
Filing date: 2021-06-01
Publication date: 2023-04-14
Anticipated expiration: 2041-06-01
Also published as: CN113215081A

Abstract

The invention discloses a method for inducing stem cells to differentiate into parathyroid cells and a composition thereof, wherein the method comprises the steps of firstly, placing the stem cells in a first-stage culture medium for culturing for a plurality of days to obtain deterministic endoderm cells; then placing the definitive endoderm cells in a second-stage culture medium to culture for multiple days to obtain foregut front-end endoderm cells; culturing the foregut front-end endoderm in a third-stage culture medium to obtain a pharyngeal pouch endoderm; and finally, culturing the pharyngeal pouch endoderm cells in a fourth-stage culture medium to obtain the parathyroid gland cells. The present invention can make stem cell differentiate into parathyroid cell in high yield in short time by simulating the development process of in vivo embryo, and when the differentiated parathyroid cell is transplanted into body, it can play the role of stabilizing blood calcium in body, so as to attain the goal of preventing or curing hypoparathyroidism.

Description

Method for inducing stem cells to differentiate into parathyroid cells and composition thereof

Technical Field

The invention relates to the field of stem cell induced differentiation, in particular to a method for inducing stem cells to differentiate into parathyroid cells and a composition thereof.

Background

Hypoparathyroidism is an endocrine disease caused by hyposecretion of parathyroid hormone (PTH) and characterized by hypocalcemia and hyperphosphatemia, the most common cause being parathyroid gland damage in cervical surgery.

According to the newly published cancer statistics, the incidence of thyroid cancer has been continuously rising in recent decades, and thyroid surgery has been increasing. Because the parathyroid gland and the thyroid gland are adjacent, the anatomical position has more variation and is relatively hidden, and the identification and protection of two pairs of parathyroid glands are difficult to realize in the operation process, so that more and more patients with transient or persistent hypoparathyroidism after the operation are available. The symptoms of the disease are caused by the increase of the excitation of nerve muscles caused by hypocalcemia, including hand and foot stabbing pain, muscle spasm, epileptic seizure and the like, and serious patients can die.

Current treatment regimens for hypoparathyroidism mainly involve high dose vitamin D and calcium supplementation, PTH replacement therapy, and parathyroid auto-or allograft transplantation, however none of these regimens can treat the disease radically and can bring about various short and long term complications, such as:

1. the supplement of vitamin D and calcium agent with high dose can cause lithangiuria, abnormal calcification focus of brain and irreversible renal function damage;

PTH replacement therapy can lead to osteosarcoma morbidity to increase, long-term use complication is unknown, use and is greatly limited on the clinic too;

3. parathyroid autografts or allotransplantation also face the dilemma of uncertainty in selection of graft volume, unknown survival time of graft, possible secondary surgery, immune rejection.

In conclusion, the incidence of hypoparathyroidism is increasing, long-term effective treatment means are lacked, patients have poor quality of life and heavy social burden, and new treatment methods are urgently needed.

At present, schemes and technologies for inducing pluripotent stem cells to directionally differentiate and develop into terminal cells such as nerve cells, liver cells, islet cells, skeletal muscle cells and the like in vitro are mature, particularly, the stem cell technology is widely applied to the research of diabetes mechanism and the research of cell transplantation treatment, and we see eosin for treating the loss-of-function diseases.

Hypoparathyroidism, another type of disease of the endocrine system, may also be treated by stem cells. In the research on the induction of human pluripotent stem cells to differentiate into parathyroid cells, bingham et al have conducted preliminary studies, but do not simulate the process of embryonic development, and thus have low differentiation efficiency. Betty r. et al also tried to induce differentiation, but failed to induce cells to secrete parathyroid hormone.

Disclosure of Invention

The present invention has been made in an effort to overcome the disadvantages of the prior art, and an object of the present invention is to provide a method for inducing stem cells to differentiate into parathyroid cells, which simulates the process of human parathyroid embryo development without gene implantation or co-culture with various tissue cells, and a composition thereof, by inducing human stem cells to differentiate into parathyroid cells in high yield only through the sequential use of small molecule inhibitors and proteins, by overcoming the disadvantages of the prior art.

The parathyroid cell obtained by differentiation of the method can monitor the calcium ion concentration in blood plasma through a surface calcium ion receptor (CaSR), and further regulate the release of parathyroid hormone synthesized in cells to the outside of cells, so that the purpose of treatment can be achieved without reconstructing the histological structure of parathyroid; therefore, the differentiated parathyroid cells are implanted into a body to stably play a role in regulating blood calcium, and a new selection scheme is provided for treating the hypoparathyroidism-related diseases in the future.

To achieve the above object, the present invention provides a method for inducing differentiation of stem cells into parathyroid cells, comprising the following steps:

1) Culturing the stem cells in a first-stage culture medium for multiple days to obtain deterministic endoderm cells;

2) Placing the definitive endoderm cells in a second-stage culture medium to culture for multiple days to obtain foregut front-end endoderm cells;

3) Culturing foregut front-end endoderm cells in a third-stage culture medium to obtain pharyngeal pouch endoderm cells;

4) Culturing the pharyngeal pouch endoderm cells in a fourth stage culture medium to obtain the parathyroid gland cells.

Further, in the step 1), the stem cells include Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs).

Still further, in the step 1), the first stage culture time is 3 days, and the culture conditions are different every day:

the first day: the RPMI culture medium contains 90-110 ng/ml Activin A and 2-4 uM CHIR99021;

the following day: the RPMI culture medium contains 90-110 ng/ml Activin A and 0.1-0.3% (v/v) Fetal Bovine Serum (FBS);

and on the third day: RPMI medium contains 90-110 ng/ml Activin A and 1-3% (v/v) Fetal Bovine Serum (FBS).

the first day: RPMI medium contains 100ng/ml Activin A and 3uM CHIR99021;

the next day: RPMI medium contains 100ng/ml Activin A and 0.2% (v/v) Fetal Bovine Serum (FBS);

and (3) on the third day: RPMI medium contained 100ng/ml Activin A and 2% (v/v) Fetal Bovine Serum (FBS).

Still further, in the step 2), the second stage culture time is 5 days, and the 5 days of culture conditions are as follows: 40-60 mg/ml ascorbic acid (L-ascorbyl acid), 450-550 ug/ml thioglycerol (monothioglycerol), 0.5-1.5% (v/v) B27 supplement, 0.5-1.5% (v/v) N2 supplement, 150-250 ng/ml Noggin and 8-12 uM SB431542 are added into DMEM/F12 medium.

Still further, in the step 2), the second stage culture time is 5 days, and the 5 days of culture conditions are all as follows: DMEM/F12 medium was supplemented with 50mg/ml ascorbic acid (L-ascorbyl acid), 500ug/ml thioglycerol (monothioglycerol), 1% (v/v) supplement of B27, 1% (v/v) supplement of N2, 200ng/ml Noggin and 10uM SB431542.

Still further, in the step 3), the culture time of the third stage is 7 days, and the culture conditions of the 7 days are that 40-60 mg/ml ascorbic acid (L-ascorbyl acid), 450-550 ug/ml thioglycerol (monothioglycerol), 0.5-1.5% (v/v) B27 supplement, 0.5-1.5% (v/v) N2 supplement, 8-12 uM SB431542, 48-52 ng/ml FGF8B, 0.08-0.12 uM RA, 46-52 ng/ml BMP4 are added into DMEM/F12 culture medium;

in the step 4), the fourth stage culture time is 7 days, and the culture conditions of the 7 days are that 40-60 mg/ml ascorbic acid (L-ascorbyl acid), 450-550 ug/ml thioglycerol (monothioglycerol), 0.5-1.5% (v/v) B27 supplement, 0.5-1.5% (v/v) N2 supplement, 7-13 uM SB431542, 45-55 ng/ml FGF8B, 0.08-0.12 uM RA, 90-110 ng/ml shh, and 180-220 ng/ml Noggin are added into DMEM/F12 culture medium.

Still further, in the step 3), the third stage culture time is 7 days, and the culture conditions of the 7 days are DMEM/F12 medium supplemented with 50mg/ml ascorbic acid (L-ascorbyl acid), 500ug/ml thioglycerol (monothioglycerol), 1% (v/v) supplement B27, 1% (v/v) supplement N2, 10uM SB431542, 50ng/ml FGF8B,0.1uM RA,50ng/ml BMP4;

in the step 4), the fourth stage culture time is 7 days, and the culture conditions of the 7 days are that 50mg/ml ascorbic acid (L-ascorbyl acid), 500ug/ml thioglycerol (monothioglycerol), 1% (v/v) B27 supplement, 1% (v/v) N2 supplement, 10uM SB431542, 50ng/ml FGF8B,0.1uM RA,100ng/ml shh and 200ng/ml Noggin are added into DMEM/F12 culture medium.

The present invention also provides a composition for treating a hypoparathyroidism-related disease comprising the parathyroid cell differentiated according to claim 1, and matrigel which maintains cellular viability.

Preferably, the treatment for hypoparathyroidism-related disorders comprises postoperative hypoparathyroidism, congenital parathyroid dysplasia.

Biomarkers to identify whether the above differentiated parathyroid cells successfully differentiated:

identifying whether the cells are successfully differentiated through the mRNA and protein expression levels of characteristic genes, wherein the characteristic genes of the definitive endoderm cells, the foregut front-end endoderm cells, the pharyngeal pouch endoderm cells and the parathyroid gland cells are as follows:

1) The characteristic genes of definitive endoderm cells are selected from the group consisting of a combination of at least two genes selected from Foxa2, sox17, CXCR4, C-kit, epCAM;

2) The characteristic gene of foregut endoderm cells is selected from Foxa2, sox2;

3) A combination of at least two genes selected from Hoxa3, EYA1, tbx1, pax9 and Six1, characteristic of pharyngeal cystic endoderm cells;

4) The characteristic gene of parathyroid cell is the combination of at least two genes of PTH, caSR, GCM2, CHGA and MAFB.

The invention has the beneficial effects that:

the present invention enables stem cells to differentiate into parathyroid cells in a short time with high yield by simulating the process of in vivo embryonic development. The differentiated parathyroid cells can secrete parathyroid hormone, and when transplanted into a body, the parathyroid cells can play a role in stabilizing blood calcium in the body, so that the aim of preventing or treating hypoparathyroidism is fulfilled. The invention perfects and innovates the short plate in the previous research results, provides a new differentiation scheme and a treatment composition which are more suitable for clinical transformation, and provides a new treatment selection for patients with hypoparathyroidism.

Drawings

FIG. 1 is a graph identifying the efficiency of differentiation of stem cells into definitive endoderm cells (DE);

in the figure, fig. 1A is a graph for detecting the difference expression of FOXA2 gene in mRNA in stem cells and DE cells by PCR, fig. 1B is a graph for detecting the difference expression of SOX17 gene in mRNA in stem cells and DE cells by PCR, fig. 1C is a graph for detecting the difference expression of CXCR4 gene in mRNA in stem cells and DE cells by PCR, fig. 1D is a graph for detecting the difference expression of SOX2 gene in mRNA in stem cells and DE cells by PCR, and fig. 1E and 1F are graphs for detecting the difference expression of FOXA2 protein and SOX17 protein in stem cells and DE cells by immunofluorescence;

FIG. 2 is a graph depicting the identification of the efficiency of differentiation from definitive endoderm cells (DE) to foregut anterior endoderm cells (AFE);

in the figure, fig. 2A is a PCR detection of mRNA differential expression of FOXA2 gene in DE cells and AFE cells, fig. 2B is a PCR detection of mRNA differential expression of SOX2 gene in DE cells and AFE cells, and fig. 2C and fig. 2D are immunofluorescence detection of FOXA2 protein and SOX2 protein differential expression in DE cells and AFE cells;

FIG. 3 is a graph depicting the identification of the efficiency of differentiation from foregut anterior endoderm cells (AFE) to pharyngeal pouch endoderm cells (PE);

in the figure, fig. 3A shows the PCR detection of the differential expression of the EYA1 gene, HOXA3 gene, PAX1 gene, PAX9 gene, SIX1 gene, TBX1 gene in mRNA in stem cells and PE cells, fig. 3B shows the immunofluorescence detection of the expression of HOXA3 protein in PE cells, and fig. 3C shows the immunofluorescence detection of the expression of EYA1 protein in PE cells;

FIG. 4 is a graph showing the identification of the efficiency of differentiation from pharyngeal pouch endoderm cells (PE) to parathyroid gland cells;

in the figure, FIG. 4A is PCR detection of the difference expression of mRNA in stem cells and parathyroid cells of PTH gene, GCM2 gene, MAFB gene, GATA3 gene, GATA4 gene and CaSR gene, and FIG. 4B is immunofluorescence detection of the expression of PTH protein and GCM2 protein in parathyroid cells;

FIG. 5 is a graph showing evaluation of implantation of parathyroid cells of differentiated origin into model animals and treatment effect thereof

In the figure, fig. 5A shows that bilateral parathyroid glands show purple red under ultraviolet light with the wavelength of 405nm after rats are injected with 5-aminolevulinic acid (5-ALA) solution in the abdominal cavity for 1 hour, fig. 5B shows that surgery-excised tissue is parathyroid glands by HE staining to prove that model animals are successfully constructed, fig. 5C shows that the concentration changes of serum parathyroid hormone (PTH) of rats in an ELISA detection negative control group, a parathyroidectomy group and a stem cell composition treatment group are detected, and fig. 5D shows that the concentration changes of serum calcium ions of rats in the negative control group, the parathyroidectomy group and the stem cell composition treatment group are detected by an electrochemiluminescence method.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

EXAMPLE 1 culture of Stem cells

1. Species of Stem cell

Stem cell types human embryonic stem cells (hESC) lines H9 (WA 09, normal karyotype XX) and H7 (WA 07, normal karyotype XX) and the human Induced Pluripotent Stem Cell (iPSC) line iPS-1 (normal karyotype XY) were selected, all purchased from WiCell Research Institute (Madison, wis., USA).

2. Stem cell culture

Human embryonic stem cells and induced pluripotent stem cells are maintained and propagated in a feeder-free system, and the cells are replenished daily using stem Technologies mTeSR 1 or mTeSR PLUS to maintain the karyotypic stability and multipotent differentiation potential of the embryonic stem cells and induced pluripotent stem cells. The medium can be used in combination with Corning Matrigel matrix to maintain high quality hESC and iPSC cells, or with Vitronectin XF to ensure the defined composition of the whole culture system.

The stem cells are cultured for 5-7 days, and the density reaches 70% -80% for passage, and large cell clones are dispersed into a plurality of small aggregates by adopting an enzyme method or an enzyme-free method, and are re-plated on Matrigel or Vitronectin XF.

During stem cell passaging, immunochemical staining was performed at regular time intervals with OCT4 and SSEA4 antigens characteristic of undifferentiated stem cells to monitor the degree of differentiation. And (3) monitoring whether the stem cells have karyotypic abnormality by periodically adopting a human pluripotent stem cell gene detection kit. Mycoplasma detection kits are used periodically to monitor stem cell cultures for the presence of mycoplasma contaminants that can adversely affect stem cell differentiation.

Example 2

Inducing human stem cells to differentiate into parathyroid cells in vitro, wherein the stem cells are embryonic stem cells (hESCs) or Induced Pluripotent Stem Cells (iPSCs), and the differentiation method of the two stem cells has the same steps and comprises the following steps:

1. differentiation from stem cells to Definitive Endoderm (DE) cells

Inducing human stem Cell differentiation by adopting a single Cell differentiation scheme, dispersing stem cells into single cells by using accutase enzyme or Gentle Cell differentiation Reagent, suspending the cells by using mTeSR culture medium with final concentration of 10uM Y-27632, and suspending the cells according to 2x10^5cells/cm ² (iii) cell seed has been plated on a Matrigel or Vitronectin XF well plate to make the cell fineThe cells reached a confluent state of 90% -100% density the next day.

After the stem cells adhere to the wall and reach a fusion state, adding a culture medium capable of inducing differentiation to the definitive endoderm cells to culture for three days: the first day: RPMI,100ng/ml Activin A,3uM CHIR99021. The next day: RPMI,100ng/ml Activin A,0.2% (v/v) Fetal Bovine Serum (FBS). And on the third day: RPMI,100ng/ml Activin A,2% (v/v) Fetal Bovine Serum (FBS). Before the medium was changed, the cells were rinsed with DMEM/F12 medium and the dead cells were washed.

As shown in fig. 1, after differentiation at this stage is finished, real-time quantitative PCR and immunofluorescence experiments are used to identify the differentiation efficiency of cells, FOXA2, SOX17 and CXCR4 are differentiation markers of DE, SOX2 is a marker of stem cells, and the differentiation efficiency from stem cells to definitive endoderm is greater than 90%.

2. Differentiation from definitive endoderm cells (DE) to foregut anterior endoderm cells (AFE)

The definitive endoderm cells described above were cultured for 5 days in the provided medium (DMEM/F12, 50mg/ml L-ascorbic acid,500ug/ml monothioglycerol,1% (v/v) B27 supplement, 1% (v/v) N2 supplement, 200ng/ml Noggin,10uM SB431542) to induce differentiation to foregut anterior endoderm cells, thereby producing foregut anterior endoderm cells.

As shown in fig. 2, after differentiation at this stage is finished, real-time quantitative PCR and immunofluorescence experiments are used to identify the differentiation efficiency of the cells, co-expression of FOXA2 and SOX2 is a marker of AFE differentiation, and the differentiation efficiency from the definitive endoderm to the foregut endoderm is greater than 90%;

3. differentiation of foregut anterior endoderm cells (AFE) to pharyngeal pouch endoderm cells (PE)

The foregut anterior endoderm cells described above were cultured in the provided medium (DMEM/F12, 50mg/ml L-ascorbic acid,500ug/ml moneoglycerol, 1% (v/v) B27 supplement, 1% (v/v) N2 supplement, 10uM SB431542, 50ng/ml FGF8B,0.1uM RA,50ng/ml BMP 4) for 7 days to induce differentiation to pharyngeal-vesicular endoderm cells, thereby producing pharyngeal-vesicular endoderm cells.

As shown in fig. 3, after differentiation at this stage is finished, real-time quantitative PCR and immunofluorescence experiments are used to identify the differentiation efficiency of the cells, EYA1, HOXA3, PAX1, PAX9, SIX1, and TBX1 are markers of pharyngeal vesicle differentiation, and the differentiation efficiency from foregut anterior endoderm cells to pharyngeal vesicle endoderm cells is greater than 50%;

4. differentiation of pharyngeal bursa endoderm cells (PE) into parathyroid cells

The above pharyngeal vesicle endoderm cells were cultured for 7 days in the provided medium (DMEM/F12, 50mg/ml L-ascorbic acid,500ug/ml monothioglycerol,1% (v/v) B27 supplement, 1% (v/v) N2 supplement, 10uM SB431542, 50ng/ml FGF8B,0.1uM RA,100ng/ml shh,200ng/ml Noggin) for inducing differentiation to parathyroid cells, thereby producing parathyroid cells.

As shown in FIG. 4, after differentiation at this stage is finished, real-time quantitative PCR and immunofluorescence experiments are adopted to identify the differentiation efficiency of the cells, PTH, GCM2, MAFB, caSR, GATA3 and GATA4 are markers of parathyroid cell differentiation, and the differentiation efficiency from pharyngeal pouch endoderm cells to parathyroid cells is more than 50%.

The above markers associated with cell differentiation were determined as follows:

RT-PCR detection of mRNA expression of cell differentiation-associated markers

In order to examine whether each stage was successfully differentiated, RT-PCR experiments were performed at each stage of differentiation induction. Total RNA of cells was extracted with Trizol, RNA was quantified by 260/280nm wavelength, RNA was reverse transcribed into cDNA using reverse transcription kit, real-time quantitative PCR was performed in Bio-Rad CFX96 PCR instrument. Primers were all designed on the National Center for Biotechnology Information (NCBI) website. Relative quantification of RNA expression Using 2 ^-ΔΔCt And (4) calculating by using the method.

Primer design of differentiation related markers at each stage:

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2. immunofluorescence staining identification of cell differentiation related markers

Immunofluorescence experiments were used to identify the expression of marker proteins in the various differentiation stages. Sterile cell slide is paved in a 24-well plate in advance, matrigel 300 ul/well is added, differentiation is carried out according to the differentiation method, and cells differentiated at each stage are selected as identification objects. At the end of each differentiation stage, the cell slide was carefully removed with forceps, washed 2 times with PBS solution, fixed for 20-30 minutes by adding 4% (v/v) paraformaldehyde solution, and washed 3 times with PBS solution for 5 minutes each. Blocking solution (10% (v/v) donkey serum +0.4% (v/v) Triton + PBS) was prepared, 50-100 ul/slide was added, and incubation was performed at room temperature for 1 hour. Prepare primary antibody dilution (5% (v/v) donkey serum +0.2% (v/v) Triton + PBS), add primary antibody, 50-100 ul/slide, 4 degrees wet box overnight. Washing with PBS solution for 3 times, adding secondary antibody (5% (v/v) donkey serum + PBS + secondary antibody in proper proportion), incubating at room temperature for 45 min, dyeing nuclei with DAPI, washing with PBS, sealing with anti-fluorescence quencher, air drying, storing at 4 deg.C in dark place, and taking pictures as soon as possible.

EXAMPLE 3 Parathyroid cell engraftment and evaluation of treatment for the implanted cells

1. Establishment of hypoparathyroidism model

The animal experiment is carried out by selecting female SD rats with weight of 300-350g, all animal experiment operations are carried out according to the ethical standard of animal ethical committee, and bilateral Parathyroidectomy (PTX) is carried out on the rats to establish a hypoparathyroidism model. Firstly, 5-aminolevulinic acid (5-ALA) is injected into abdominal cavity of a rat according to the dosage of 500mg/kg, after 1 hour, the rat is anesthetized by 6% (v/v) chloral hydrate and is fixed on an operating table in a horizontal supine position, a vertical incision with the length of 1.5cm is made at the midline of the neck after skin preparation, subcutaneous tissues and muscles are sequentially cut open, an air outlet pipe and the thyroid are exposed, ultraviolet rays with the wavelength of 405nm are adopted to irradiate the neck to search parathyroid glands, and the parathyroid glands show purple due to the accumulation of 5-ALA under the irradiation of the ultraviolet rays (as shown in figure 5A). Bilateral parathyroid glands were excised and the muscle, muscle fascia and skin were sutured layer by layer. Fixing the cut bilateral parathyroid glands with paraformaldehyde, embedding paraffin, and performing HE staining identification on the section to determine that the bilateral parathyroid glands are cut (as shown in FIG. 5B).

Before and after bilateral parathyroidectomy of rats, subclavian vein blood of the rats is collected in heparin anticoagulation tubes, the specimens are centrifuged for 15 minutes at 3000 rpm of a 4-degree centrifuge within 30 minutes after collection, and supernatant, namely blood plasma, is collected and is subpackaged at-80 ℃ for subsequent parathyroid hormone and blood calcium detection. After surgery, venous blood plasma was collected and separated every 3 days as described above, and the body weight of the rat was weighed to observe whether the rat exhibited symptoms of low calcium convulsion.

A parathyroid hormone ELISA kit of IBL company is selected to detect parathyroid hormone in blood plasma, and an ELISA experiment is carried out according to the steps recommended by the instruction. As shown in fig. 5C, at 3 days post-surgery, a decrease in blood parathyroid hormone (PTH) occurred in rats in the parathyroidectomy group compared to the non-operated negative control group. The blood calcium concentration in the blood plasma is detected by using a blood calcium detection kit of BD company, and the experiment is carried out according to the steps recommended by the instruction. As shown in fig. 5D, at 3 days post-surgery, serum calcium was significantly reduced in the parathyroidectomy group compared to the negative control group, and rats developed significant symptoms of low calcium convulsions.

2. Formation and implantation of stem cell therapeutic compositions

Parathyroid cells from which stem cells are differentiated are mixed with matrigel in a1 to 1 volume ratio to form a composition that is likely to exert a therapeutic effect. The composition is transplanted into the muscle of the above-mentioned hypoparathyroidism model animal, and the effect of the treatment is observed. All animal experimental procedures were performed according to ethical standards of the animal ethics committee. Before cell transplantation, the parathyroidectomized rat is injected with cyclosporine A subcutaneously to reduce the immunity of the rat and prevent rejection of human-derived cells. Parathyroid cells differentiated from stem cells according to the present invention were isolated with accutase cell isolation solution and suspended in sterile PBS (D-PBS, invitrogen) solution, the cell suspension was placed on ice, and matrigel was added in a volume ratio of 1 to 1 while being cooled, thereby forming a single cell suspension having a density of 6 to 10 × 104 cells/ul, i.e., a stem cell therapeutic composition. After anaesthetising the PTX model rats, 10 μ L of the cell suspension was injected into the right forelimb of the rats by means of a 10 μ L syringe equipped with a 35 gauge needle (WPI). The negative control group rats were injected with the same dose of PBS solution only.

3. Evaluation of graft treatment Effect

14 days after the composition was transplanted into the animal, venous blood from rats in the cell-free transplanted PTX group and the stem cell composition-treated group was drawn, and plasma was obtained by centrifugation according to the above method and stored in a-80 ℃ refrigerator. Note that the rat low calcium tic symptoms were observed, and the body weight of the rat was measured every 7 days.

A parathyroid hormone ELISA kit of IBL company is selected to detect parathyroid hormone in blood plasma, and ELISA experiments are carried out according to the steps recommended by the instruction. As shown in fig. 5C, the rat blood parathyroid hormone in the stem cell composition treated group was elevated compared to the untreated Parathyroidectomy (PTX) group, demonstrating that the transplanted parathyroid cells survive and secrete parathyroid hormone in the rat.

The blood calcium concentration in the blood plasma is detected by using a blood calcium detection kit of BD company, and the experiment is carried out according to the steps recommended by the instruction. As shown in fig. 5D, the rats in the stem cell composition-treated group had elevated blood calcium and reduced low calcium tic symptoms compared to the untreated Parathyroidectomy (PTX) group, demonstrating that the transplanted parathyroid cells survive and function in the rats.

In summary, the present invention discloses a method for inducing stem cells to differentiate into parathyroid cells in vitro and a composition thereof, wherein the differentiated parathyroid cells can play a role in stabilizing blood calcium in vivo when transplanted into a body, thereby achieving the purpose of preventing or treating hypoparathyroidism.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. A method of inducing stem cells to differentiate into parathyroid cells, comprising: the method comprises the following steps:

1) Placing the embryonic stem cells and the induced pluripotent stem cells in a first-stage culture medium to culture for 3 days to obtain definitive endoderm cells; wherein the culture conditions are different every day:

the first day: the RPMI culture medium contains 90-110 ng/ml of Activin A and 2-4 uM CHIR99021;

the following day: the RPMI culture medium contains 90-110 ng/ml Activin A and 0.1% -0.3% fetal calf serum;

and on the third day: the RPMI culture medium contains 90-110 ng/ml Activin A and 1-3% fetal calf serum;

2) Placing the definitive endoderm cells in a second-stage culture medium to culture for 5 days to obtain foregut front-end endoderm cells; wherein, the culture conditions of the 5 days are as follows: adding 40-60 mg/ml ascorbic acid, 450-550 ug/ml thioglycerol, 0.5-1.5% B27 supplement, 0.5-1.5% N2 supplement, 150-250 ng/ml Noggin and 8-12 uM SB431542 into a DMEM/F12 culture medium;

3) Culturing foregut front-end endoderm cells in a third-stage culture medium for 7 days to obtain pharyngeal pouch endoderm cells; wherein, the culture conditions of the 7 days are that 40-60 mg/ml ascorbic acid, 450-550 ug/ml thioglycerol, 0.5-1.5% B27 supplement, 0.5-1.5% N2 supplement, 8-12 uM SB431542, 48-52 ng/ml FGF8B, 0.08-0.12 uM RA and 46-52 ng/ml BMP4 are added into a DMEM/F12 culture medium;

4) And (2) placing the pharyngeal pouch endoderm cells in a fourth-stage culture medium to culture for 7 days to obtain parathyroid cells, wherein the culture conditions of the 7 days are DMEM/F12 culture medium added with 40-60 mg/ml of ascorbic acid, 450-550 ug/ml of thioglycerol, 0.5-1.5% of B27 supplement, 0.5-1.5% of N2 supplement, 7-13 uM of SB431542, 45-55 ng/ml of FGF8B, 0.08-0.12 uM of RA, 90-110 ng/ml of shh and 180-220 ng/ml of Noggin.

2. The method of inducing differentiation of stem cells into parathyroid cells according to claim 1, wherein: in the step 1), the first stage culture time is 3 days, and the culture conditions are all different every day:

the first day: RPMI medium contains 100ng/ml Activin A and 3uM CHIR99021;

the next day: RPMI medium contains 100ng/ml Activin A and 0.2% fetal calf serum;

and on the third day: RPMI medium contains 100ng/ml Activin A and 2% fetal bovine serum.

3. The method of inducing differentiation of stem cells into parathyroid cells according to claim 1, wherein: in the step 2), the second-stage culture time is 5 days, and the 5-day culture conditions are as follows: DMEM/F12 medium was supplemented with 50mg/ml ascorbic acid,500ug/ml thioglycerol,1% B27 supplement, 1% N2 supplement, 200ng/ml Noggin and 10uM SB431542.

4. The method for inducing differentiation of stem cells into parathyroid cells according to claim 1, wherein: in the step 3), the culture time of the third stage is 7 days, and the culture conditions of the 7 days are that DMEM/F12 culture medium is added with 50mg/ml ascorbic acid,500ug/ml thioglycerol,1% B27 supplement, 1% N2 supplement, 10uM SB431542, 50ng/ml FGF8B,0.1uM RA and 50ng/ml BMP4;

in the step 4), the fourth stage culture time is 7 days, and the culture conditions of the 7 days are that DMEM/F12 medium is added with 50mg/ml ascorbic acid,500ug/ml thioglycerol,1% B27 supplement, 1% N2 supplement, 10uM SB431542, 50ng/ml FGF8B,0.1uM RA,100ng/ml shh and 200ng/ml Noggin.