Method for culturing differentiation-promoting and -sustaining spheroid form of tonsil-derived stem cells

Abstract

The present invention relates to a method for producing a spheroid form of tonsil-derived stem cells, the method enhances growth and differentiation efficiency of the tonsil-derived stem cells. The present method also produces a spheroid form of para-thyroid hormones. The method for producing a spheroid form of tonsil-derived stem cells enhances the proliferation rate of the stem cells per se and significantly increase differentiation potency into parathyroid cells.

Claims

1. A method for producing a spheroid form of parathyroid cells from tonsil-derived stem cells, the method comprising: (a) transferring a suspension of undifferentiated tonsil-derived stem cells into a hemispherical microwell that is formed in such a way that at least one liquid polymer is hardened in a form of hemispherical microwell, wherein the liquid polymer is selected from among polydimethylsiloxane, silicon polymer, polybutadiene, polyisobutylene and polyurethane and forms a meniscus due to surface tension; (b) producing a spheroid form of undifferentiated tonsil-derived stem cells by culturing the undifferentiated tonsil-derived stem cells prepared by the step (a) in the hemispherical microwell; and (c) adding activin A and sonic hedgehog into the spheroid form of undifferentiated tonsil-derived stem cells of the step (b), thereby differentiating the stem cells into parathyroid cells.

2. The method of claim 1, wherein the microwell is formed in such a way that polydimethylsiloxane is hardened.

3. The method of claim 1, wherein the culture of the step (b) is performed for 1 to 20 days.

4. The method of claim 3, wherein the culture of the step (b) is performed for 1 to 14 days.

5. The method of claim 1, wherein activin A is contained at a concentration of 50 to 300 ng/ml and sonic hedgehog is contained at a concentration of 50 to 300 ng/ml.

6. The method of claim 1, further comprising (d) separating the produced spheroid form of parathyroid cells, wherein the separated form of parathyroid cells is formulated as a pharmaceutical composition for treating hypoparathyroidism or osteoporosis.

7. The method of claim 6, wherein the pharmaceutical composition of the spheroid form of parathyroid cells is administered to subject with hypoparathyroidism or osteoporosis for treating hypoparathyroidism or osteoporosis.

8. The method of claim 1, wherein the parathyroid cell is capable of producing or secreting parathyroid hormone.

9. A method for producing parathyroid hormone from tonsil-derived stem cells, the method comprising: (a) transferring a suspension of undifferentiated tonsil-derived stem cells into a hemispherical microwell that is formed in such a way that at least one liquid polymer is hardened in a form of hemispherical microwell, wherein the liquid polymer is selected from among polydimethylsiloxane, silicon polymer, polybutadiene, polyisobutylene and polyurethane and forms a meniscus due to surface tension; (b) producing a spheroid form of undifferentiated tonsil-derived stem cells by culturing the undifferentiated tonsil-derived stem cells prepared by the step (a) in the hemispherical microwell containing a culture medium; (c) adding activin A and sonic hedgehog into the spheroid form of undifferentiated tonsil-derived stem cells of the step (b), thereby differentiating the stem cells into parathyroid cells; and (d) separating produced parathyroid hormone from the culture medium or a cell lysis solution.

10. The method of claim 9, the method further comprising: allowing parathyroid hormone to be secreted out of cells by adjusting a calcium concentration of the culture medium after the step (c).

11. The method of claim 9, wherein the culture is performed for 1 to 14 days.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 depicts a diagram illustrating a process of producing a spheroid form of cells, using a multiple-concave mold that is formed in such a way that polydimethylsiloxane (PDMS) is hardened.

(2) FIG. 2 depicts two- and three-dimensional culture process periods for producing a spheroid form of cells.

(3) FIG. 3 depicts results of observing changes in a shape, size and survival of SP1 spheroid form of cells during producing the same.

(4) FIG. 4 depicts results of observing changes in a shape, size and survival of an SP2 spheroid form of cells during producing the same.

(5) FIG. 5 depicts results of observing changes in a shape, size and survival of an SP3 spheroid form of cells during producing the same.

(6) FIG. 6 depicts results of observing changes in a gene expression levels of parathyroid hormone from tonsil-derived stem cells and differentiated cells therefrom via two-dimensional culture and three-dimensional culture of spheroid forms.

(7) FIG. 7 depicts results of observing expression levels and distribution of parathyroid hormone (PTH) and a chromogranin A (CHGA) protein on a spheroid surface or inside of SP1, SP2 and SP3 spheroid forms of cells.

(8) FIG. 8 depicts results of observing changes in a concentration of parathyroid hormone secreted from tonsil-derived stem cells and differentiated cells therefrom via two-dimensional culture and three-dimensional culture of spheroid forms.

(9) FIG. 9 depicts results of observing changes in survival rates of a spheroid form of cells-administered parathyroidectomized rats SP3 for 3 months.

(10) FIG. 10 depicts results of observing changes in a behavior and appearance of a spheroid form of cells-administered parathyroidectomized rats SP3 after 3 months.

DETAILED DESCRIPTION OF THE INVENTION

(11) Hereinafter, embodiments and manufacturing examples will be suggested for better understanding of the present invention. However, the following examples and manufacturing examples are provided only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.

Example 1

(12) Culture of Tonsil-Derived Stem Cells

(13) Tonsillectomy was performed on six patients aged less than ten (five boys and one girl with an average age of 7.2 years) out of those who are patients in the department of otolaryngology head & neck surgery of Ewha Womans Univ. Mokdong Hospital. This study has passed a review of the Clinical Ethics Committee (ECT 11-53-02). Two thirds of enucleated tonsil tissues were used for a clinical biopsy, while the remaining one thirds of enucleated tonsil tissues was used for this experiment.

(14) A collected tonsil tissue was washed in physiological saline water, after which a connective tissue within the tonsil tissue was comminuted in a cell culture medium RPMI-1640 (Roswell Park Memorial Institute medium 1640, Invitrogen Corporation, Carlsbad, Calif.), in which a collagenase type I (Invitrogen) of 210 U/ml of an enzyme-mixed solution is mixed with 10 g/ml of DNAse (Sigma-Aldrich, St. Louise, Mo.), under the condition of 37 C. for 30 minutes. A suspended tissue was centrifuged and filtered out to obtain cells. The obtained cells were washed twice in RPMI 1640/20% NHS (normal human serum) (PAA Laboratories, GmbH, Paching, Austria) and once with RPMI 1640/10% NHS. The washed cells were applied to gradient centrifugation using Ficoll-Paque (GE Healthcare, Little Chalfont, Buckinghamshire, UK), such that mononuclear cells were obtained. The 10.sup.8 mononuclear cells were inoculated into DMEM-HG (Dulbecco's modified Eagle's medium-High Glucose) (Invitrogen) medium containing 10% FBS (Invitrogen), 100 g/ml of streptomycin and 100 U/ml of ampicillin, and cultured for 48 hours. After culture, non-attached cells were removed while attached cells, considered as tonsil-derived mesenchymal stem cells (T-MSC), were subcultured up to three to five generations for four weeks, such that resulting cells were used in this experiment.

Example 2

(15) Three-Dimensional Spheroid Culture of Tonsil-Derived Mesenchymal Stem Cells (TMSC) Using Concave Mold

(16) A method for three-dimensional culture of tonsil-derived stem cells obtained from the Example 1, was performed in a mold having a structure of FIG. 1. The mold used for inducing cells to form a three-dimensional spheroid structure is one which includes a microwell with 500 m width and a depth of 300 m and has a concave structure manufactured from polydimethylsiloxane (PDMS). This mold is one having a microwell width and depth optimal to the cell growth by forming a cell aggregate with a spheroid form structure, and has a merit that hundreds to thousands of spheroid-formed cells can be produced, if the mold is formed as a multiple-concave one.

(17) Specifically, as described in Korean Patent Registration No. 10-1282926, this mold was manufactured in such a way that wells could be arranged at a density of 100 wells per a unit cm.sup.2 by using soft lithography techniques and a meniscus of a polydimethysiloxane (PDMS) prepolymer, and then was used in an experiment after being coated with 3% (w/v) bovine serum albumin (BSA) in order to prevent cell attachment.

(18) Using the concave mold, a spheroid form of cells were produced in such a way that tonsil-derived stem cells in a undifferentiated state or parathyroid cells differentiated from tonsil-derived stem cells were classified into the followings three types on the same condition as described in FIG. 2.

(19) A first spheroid form of cells (SP1) was produced in such a way that cells in an undifferentiated state were cultured in a two-dimensional state for seven days under the condition of culturing tonsil-derived stem cells in the Example 1, after which the cells were removed with trypsin, transferred into a concave mold for inducing cells in a three-dimensional spheroid form, and further cultured for 14 days.

(20) A second spheroid form of cells (SP2) was produced in such a way that tonsil-derived stem cells were cultured in a two-dimensional state for seven days in DMEM medium containing 5% FBS, activin A (100 ng/ml, R&D System, Inc. Minneapolis) and sonic hedgehog (Shh, 100 ng/ml, R&D Systems), so as to induce a differentiation thereof into parathyroid cells, after which the tonsil-derived cells were removed with trypsin, transferred into a concave mold for inducing cells in a three-dimensional spheroid formulation, and further cultured for 14 days.

(21) A third spheroid form of cells (SP3) was produced in such a way that tonsil-derived stem cells were cultured in a two-dimensional state for seven days under the condition of culturing tonsil-derived stem cells in the Example 1, after which the tonsil-derived stem cells were removed with trypsin, transferred into a concave mold for producing the cells in a three-dimensional spheroid formulation, cultured for seven days under the condition of same medium without differentiation condition, so as to induce the cells to have a preferred orientation in the process of forming a spheroid form. After checking the spheroid shape has been formed, they were cultured for additional seven days in DMEM differentiation medium containing 5% FBS, activin A (100 ng/ml, R&D System, Inc. Minneapolis) and sonic hedgehog (Shh, 100 ng/ml, R&D Systems), which is differentiation medium.

(22) In a process of producing a spheroid form of cells, 110.sup.3 cells were seeded in one concave well, and undifferentiation or differentiation culture medium was replaced with new one every 2 to 3 days.

Example 3

(23) Observation of Changes of a Shape, Size and Survival Rate of a Spheroid Form of Cells

(24) A shape and size of a spheroid form of cells, produced in the three types of SP1, SP2 and SP3, were observed with a microscope for appropriate culture period, and a size of each spheroid form of cells was measured in contrast by means of a scale bar in microscopy.

(25) Also, in order to determining there is any change in cell survival, a survival state of the spheroid form of cells produced in three types of SP1, SP2 and SP3 was observed. Using LIVE/DEADassays kit (Invitrogen), live cells (green) and dead cells (red) during a process of producing spheroids were observed. This is based on a principle of promptly distinguishing between dead and live cells by simultaneously carrying out a green-fluorescent calcein-AM stain that reflects an presence of an intercellular esterase activity in live cells as well as a red-fluorescent ethidium homodimer-1 stain that indicates a loss of integrity of a plasma membrane, sensed from dead cells.

(26) Results of the above-mentioned experiment were shown in FIGS. 3 to 5.

(27) FIG. 3 shows results of experimenting on an SP1 spheroid form of cells, FIG. 4 does an SP2 spheroid form of cells and FIG. 5 does an SP3 spheroid form of cells.

(28) FIG. 3 indicates results of experimenting on the SP1 spheroid form of cells.

(29) FIG. 3A indicates a shape of a spheroid form of cells on first, third and seventh days during a process of culturing, while FIG. 3B does a size of cells. It was identified that the SP1 spheroid form of cells formed a spheroid sized at 237.44 m at first and a small and condensed spheroid form of cells sized at 138.17 m were produced on a seventh day. Also, FIG. 3C shows changes in a survival rate during a process of producing a spheroid form of cells. As shown in FIG. 3C, there was a very high frequency of green-stained live cells until a seventh day, while there were few red-stained dead cells until that day.

(30) FIG. 4 shows results of experimentation on SP2 spheroid form of cells.

(31) Changes in an SP2 spheroid shape were such as that shown in FIG. 4A, while changes in a size thereof were shown in FIG. 4B. It was identified that the SP2 formed a spheroid sized at 278.88 m, somewhat larger than the SP1 at first, after which a size thereof was reduced gradually down to 177.88 m on a seventh day. In other words, it was identified that the SP2 formed a larger and less-condensed spheroid shape than the SP1. Furthermore, unlike the SP1, it was identified that the SP2 showed a phenomenon, in which cells present within a spheroid spread out around the spheroid of the SP2, suggesting that this phenomenon is highly associated with an in vivo engraftment of the SP2 spheroid form of cells. FIG. 4C showed a viability of the SP2 spheroid form of cells, thus identifying that the SP2 showed a high survival rate of sustaining most live cells stained in green even until a seventh day.

(32) FIG. 5 shows results of experimenting on an SP3 spheroid form of cells.

(33) Changes in a shape of the SP3 spheroid were such as that shown in FIG. 5A, while changes in a size thereof were such as that shown in FIG. 5B. On a first day, a size of cells was 255.78 m, in a middle between SP1 and SP2 spheroids. After period of spheroid forming, cells was condensed in size of 152.89 m on a seventh day. A movement phenomenon, in which cells spread out around the SP3 spheroid, was identified more frequently than the SP2. FIG. 5C indicates viability of SP3 tonsil-derived stem cells, and it was identified that the cells showed a high survival rate even until a seventh day of culture.

Example 4

(34) Observation of Differentiation of a Spheroid Form of Tonsil-Derived Stem Cells into Parathyroid Cells

(35) After producing a spheroid form cells, gene expression level of parathyroid hormone (PTH) was observed in which tonsil-derived stem cell group and differentiated parathyroid cell group with two-dimensional culture without spheroid formation was used as a control.

(36) Using a trizol reagent, total RNA in cells were extracted and then gene expression pattern was measured by real time PCR (quantitative real time polymerase chain reaction, qRT-PCR) method using primers for a human parathyroid hormone gene. At the same time, glyceraldehyde-3-phosphate (GAPDH) was used as a control and its primers are as follows:

(37) TABLE-US-00001 ParathyroidHormone Forward: (SEQIDNO:1) 5-GAGTAGAATGGCTGCGTAAGAAG-3 Reverse: (SEQIDNO:2) 5-TTCATGGCTCTCAACCAAGAC-3 GAPDH Forward: (SEQIDNO:3) 5-GGAGCGAGATCCCTCCAAAAT-3 Reverse: (SEQIDNO:4) 5-GGCTGTTGTCATACTTCTCATGG-3

(38) Results of the above experiment were shown in FIG. 6.

(39) As shown in FIG. 6, as a result of determining expression level of parathyroid hormone compared to an expression level of parathyroid hormone in undifferentiated tonsil-derived stem cells, an experiment group of tonsil-derived stem cells differentiated in a two-dimensional culture, an SP2 group and an SP3 group showed mRNA expression levels which were increased respectively by 2.3, 5.2 and 4.3 times more than an expression of parathyroid hormone in undifferentiated tonsil-derived stem cells. In particular, it was also identified that the SP2 and the SP3 showed mRNA expression levels, which were increased respectively by 2.2 and 1.9 times more than even an expression level of cells differentiated in a two-dimensional culture. In other words, it was identified that a culture into a spheroid form has a remarkable effect in comparison with the two-dimensional culture in terms of differentiation efficiency.

(40) Based on the results, it was concluded that an expression of parathyroid hormone genes was induced more effectively in the three-dimensional culture method than in the two-dimensional culture one. Accordingly, it was suggested that a culture method under the condition of a microwell of a concave structure, which is formed in such a way that a liquid polymer is hardened, might be used as a physical culture environment in inducing an effect on expression of an identical gene, such that a period of using a differentiation inducing agent can be remarkably reduced, a period of pre-differentiation in a two-dimensional aspect can be further unnecessary, and a method for inducing expression and secretion of parathyroid hormone can be efficiently simplified.

Example 5

(41) Observation of Differentiation of a Spheroid Form of Tonsil-Derived Stem Cells into Parathyroid Cells

(42) After a spheroid was completely formed for each culture period during a process of forming the spheroid form of cells SP1, SP2 and SP3, it was investigated by means of a confocal laser scanning microscope (LSM-5 Pascal EXCITER, Carl Zeiss) whether parathyroid hormone (PTH) and a chromogranin A (CHGA), associated to secretion of the parathyroid hormone (PTH), are expressed or not in cells present on a surface of tonsil-derived stem cells produced in a spheroid form as well as inside the spheroid during a differentiation into a parathyroid tissue.

(43) After the spheroid is completely formed, a surface of the spheroid form of cells was washed in PBS, after which the cells were fixed with a 10% formalin solution. In the fixed cells, non-specific proteins were inactivated with a 2% bovine serum albumin (BSA) solution, after which each cell was treated with the 2% BSA solution containing a first antibody (PTH, CHGA and Cyr61) and reacted at 37 C. for four hours. After the reaction with the first antibody, a non-reacted antibody was washed twice in PBS and then reacted at 37 C. for one hour with the 2% BSA solution containing a second antibody (mouse IgG-FITC, rabbit-IgC-Rhodamine, Invitrogen) having a fluorescence corresponding to the first antibody. At this time, a nucleus-specific DAPI (4,6-Diamidino-2-Phenylindole, Dihydrochloride) (Molecular Probes) stain was added therein, such that a nucleus in each cell was stained at the same time. After an end of reaction with the second antibody, the surface of the cell was washed twice again in PBS, after which a cover glass attached with cells was removed and put on a glass for a fluorescence microscope, and attached onto the glass with a mounting solution, such that an expression level of an antibody and a distribution thereof within the cell were investigated on the fluorescence microscope.

(44) Results were shown in FIG. 7.

(45) As shown in FIG. 7, a presence of parathyroid hormone (PTH) produced in cells present on a surface and inside of a spheroid form of cells was identified by using three kinds of cells, which formed a spheroid form of cells. As a result of checking a presence of CHGA, a secretory granule protein of parathyroid hormone and the parathyroid hormone in SP1, SP2 and Sp3, by means of an immunostaining chemical method using an antibody specific to parathyroid hormone, SP1 did not show any remarkable distribution of expressions of PTH and CHGA on a surface or inside of the spheroid. On the other hand, it was concluded that the SP2 exhibited a significantly high expression both on a surface and inside of the spheroid than SP1. The SP3 indicated a remarkably high expression both on a surface and than SP2. Based on the experiment results, it was identified that the SP3 spheroid form of cells was the most advantageous culture condition in terms of PTH and CHGA expression. In particular, even if differentiation was induced just for seven days, it was identified that the SP3 showed a remarkable expression of PTH and CHGA. Such period was remarkably short in comparison with an existing known method, which took at least three weeks to induce differentiation.

Example 6

(46) Observation of Differentiation of Spheroid Form of Tonsil-Derived Stem Cells into Parathyroid Cells

(47) During a process of forming a spheroid form of cells SP1, SP2 and SP3, whenever being replaced with a cell culture medium containing a differentiation inducing agent for each culture period, differentiated or undifferentiated media were collected to analyze a concentration of parathyroid hormone secreted outside cells. The cell culture medium collected for each period was filtered with a syringe filter of 0.45 m, so as to remove cell floating matters, cell debris or the like, after which a filtered cell culture medium was freeze-dried and concentrated by using a freeze dryer (Operon).

(48) A sample of a freeze-dried powder type was suspended again with phosphate buffered saline (pH 7.4), so as to investigate PTH concentration discharged by means of an electro-chemiluminescence immunoassay (ECLIA) method using avidity with an antibody to PTH protein.

(49) Based on the above PTH concentration experiment, the results of a comparative experiment between SP3 and control group in which two-dimensional culture was performed with treatment of differentiation inducing agents.

(50) It was concluded that the SP3 showed a remarkably higher PTH secretion concentration range of 18.0-35.6 pg/ml than a range of 16.1-59.8 pg/ml. In case of the SP3, even if an induced differentiation was performed just for seven days, the SP3 showed a remarkable PTH expression concentration, which was remarkably shortened in comparison with an existing known method, which took at least three weeks to induce differentiation, thereby exhibiting an effect of the present invention by showing a high level of PTH secretion.

Example 7

(51) Observation of Differentiation of Spheroid Form of Tonsil-Derived Stem Cells into Parathyroid Cells

(52) Parathyroidectomized rats (PTX-rata), an animal model with a parathyroid resected, were used to investigate in vivo activity of a spheroid form of cells of SP3.

(53) The PTX animal model was established in such a way that a rat was administered with 5-aminolevulinic acid hydrochloride (ALA) that is a fluorescent stain specific to parathyroid tissue, a neck region thereof was opened after two hours, and then parathyroid tissue was detected and surgically removed out.

(54) An experimental animal group is one that an SP3 spheroid form of cells was administered into the PTX rat model, and was named PTX-SP3, out of which 10 animals were used. An SP3 spheroid form of cells was percutaneously administered once by 1000 cells per rat, after which a survival of rats was checked and a behavioral ecology of rats was investigated periodically for three months. Out of a group of rats intraperitoneally administered with PTH drug at a normal physiological concentration (PTX-rhPTH) and normal rats without a surgery, ten animals were used respectively as a positive control group.

(55) Results of the experiment were shown in FIGS. 9 and 10.

(56) FIG. 9 indicates changes in a survival rate in case of administration of a spheroid form of cells. As a result of the experiment, it was identified that a survival rate was increased upon an administration of a spheroid form of cells, though an effect thereof was somewhat less than PTH drug itself used as a positive control. In case of an experiment performed for 90 days, it was also identified that the SP3 showed a remarkable effect in a continuity aspect by maintaining an effect resulting from the administration of cells.

(57) Also, behaviors and appearances of a rat percutaneously administered with spheroid were shown in FIG. 10. It was identified that the rat showed a similar degree of behaviors to the rat of the positive control group in behavioral aspects such as weight loss, fur shape and walking, while also achieving a similar degree of an effect to the positive control group in terms of weight changes.