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