Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
권호 표지

Differentiation of Human Embryonic Stem Cells into Germ Cell and Culture Condition for Single Embryonic Stem Cells Dissociated by Enzyme

인간 배아줄기세포의 생식세포로의 분화 및 효소에 의해 분리된 단일줄기세포 배양조건

  • 지희준 (미즈메디병원 불임의학연구소) ;
  • 최순영 (미즈메디병원 불임의학연구소) ;
  • 정다연 (미즈메디병원 불임의학연구소)
  • Published : 2010.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

Objective: The present study was carried out to induce differentiation of human embryonic stem cells (hESCs) into germ cells and to establish a culture condition for single hESCs dissociated by enzyme. Methods: Embryonic body (EB) was formed by hanging drop culture for 3 days from hESCs colony. The EBs were cultured in the medium supplemented with retionic acid (RA) or/and bone morphogenetic protein-4 (BMP4) for 14 days to differentiate into germ cells. Germ cell specific markers, c-kit and VASA were used for immunohistochemistry of EB. Human ESCs colonies were dissociated into single cells by Collagenase, Tryple and Accutase, and then colony formation rate of the single cells was examined. Rho-associated kinase inhibitor (ROCK inhibitor, Y27632) was added into the culture medium of single cells to reduce the apoptotic damage during the dissociation. Results: Single cells dissociated with Tryple or Accutase showed higher colony formation rates compared to the cells dissociated with Collagenase. Seeding of $5{\times}10^3$ cells/well (4 well dish) was efficient to obtain high colony formation rate compared to other concentrations of seeding cell. Addition of Y27632 significantly increased the colony formation rate of the single cells dissociated by Tryple. Immunohistochemistry of EB with c-kit and VASA markers showed a weak fluorescence signals compared to the signals from the testicular tissue. Conclusion: Dissociation with Tryple was useful to obtain healthy single cells and addition of Y27632 was beneficial for survival and colony formation of the single cells. Unlike other studies, we just observed a dim fluorescence staining of the germ cell markers, probably caused by the short-term culture for the differentiation of EB compared to other studies.

목 적: 본 연구는 인간 배아줄기세포를 생식세포로의 분화를 유도하고 효소에 의해 분리된 단일 배아줄기세포의 배양조건을 확립하기 위해 수행하였다. 연구방법: Embryonic body (EB)는 배아줄기세포 (hESCs) colony를 떼어내어 3일간 hanging drop culture 방법으로 작성하였고, 이러한 EB를 retionic acid (RA)와 bone morphogenetic protein-4 (BMP4)를 단독 또는 함께 배양액에 첨가하여 14일간 배양함으로써 생식세포로의 분화를 유도하였다. 분화를 유도한 EB는 생식세포 발현유전자인 c-kit과 VASA의 표지인자를 이용한 면역조직형광법으로 분화여부를 조사하였다. 줄기세포는 Collagenase, Tryple 그리고 Accutase 등의 효소로 각각 분리하였고 분리된 단일세포들의 colony formation rate를 조사하였다. 한편 Rho-associated kinase inhibitor (Y-27632)를 단일세포 배양액에 첨가하여 단일세포 분리과정 중에 발생하는 apoptotic damage를 감소시키고자 하였다. 결 과: Tryple 또는 Accutase를 이용하여 분리한 단일세포가 Collagenase에 의해 분리된 세포에 비해 높은 colony formation rate를 나타내었다. 단일세포를 $5{\times}10^3$ cells/well (4 well dish) 농도로 지지세포 위에 seeding하였을 때 다른 농도의 세포를 seeding한 것에 비해 높은 colony formation rate를 확보하는데 효과적이었다. Y27632의 첨가는 단일세포의 colony formation rate를 유의하게 향상시켰으며 특히 Tryple로 분리한 단일세포에 보다 효과적이었다. EB의 분화유도후 c-kit과 VASA의 표지인자를 이용한 면역조직형광염색은 대조군인 정소조직에 비해 약한 형광염색을 나타내었다. 결 론: Tryple을 이용한 단일세포 분리가 건강한 단일세포를 얻는데 가장 효율적이었으며 Y27632 의 첨가는 단일 세포의 생존 및 colony formation에 유익하다는 것을 확인하였다. 다른 연구와는 달리 본 연구에서는 단지 생식세포 표지인자의 희미한 형광염색만을 관찰하였는데 이러한 결과는 본 연구의 분화유도기간이 상대적으로 짧았던 것이 원인이었을 것으로 생각된다.

Keywords

References

  1. Ben-Hur T, Idelson M, Khaner H, Pera M, Reinhartz E, Itzik A, et al. Transplantation of human embryonic stem cellderived neural progenitors improves behavioral deficit in Parkinsonian rats. Stem Cells 2004; 22: 1246-55. https://doi.org/10.1634/stemcells.2004-0094
  2. Mummery C, Ward D, van den Brink CE, Bird SD, Doevendans PA, Opthof T, et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 2003; 107: 2733-4.
  3. Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 2001; 98: 10716-21. https://doi.org/10.1073/pnas.191362598
  4. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001; 50: 1691-7. https://doi.org/10.2337/diabetes.50.8.1691
  5. Chen HF, Kuo HC, Chien CL, Shun CT, Yao YL, Ip PL, et al. Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum Reprod 2007; 22(2): 567-77. https://doi.org/10.1093/humrep/del412
  6. Clark AT, Bodnar MS, Fox M, Rodriquez RT, Abeyta MJ, Firpo MT, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum Mol Genet. 2004; 13(7): 727-39. https://doi.org/10.1093/hmg/ddh088
  7. Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, et al. Derivation of oocytes from mouse embryonic stem cells. Science 2003; 300(5623): 1251-6. https://doi.org/10.1126/science.1083452
  8. Kiatpongsan S. From embryonic stem cells to functioning germ cells: science, clinical and ethical perspectives. J Med Assoc Thai 2007; 90(10): 2233-7.
  9. Qing T, Shi Y, Qin H, Ye X, Wei W, Liu H, et al. Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differentiation 2007; 75(10): 902-11. https://doi.org/10.1111/j.1432-0436.2007.00181.x
  10. Lacham-Kaplan O, Chy H, Trounson A. Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem Cells 2006; 24: 266-73. https://doi.org/10.1634/stemcells.2005-0204
  11. Kerkis A, Fonseca SA, Serafim RC, Lavagnolli TM, Abdelmassih S, Abdelmassih R, et al. In vitro differentiation of male mouse embryonic stem cells into both presumptive sperm cells and oocytes. Cloning Stem Cells 2007; 9(4): 535 -48. https://doi.org/10.1089/clo.2007.0031
  12. Silva C, Wood JR, Salvador L, Zhang Z, Kostetskii I, Williams CJ, et al. Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone. Mol Reprod Dev 2009; 76(1): 11-21. https://doi.org/10.1002/mrd.20925
  13. Toyooka Y, Tsunekawa N, Akasu R, Noce T. Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A 2003; 100(20): 11457-62. https://doi.org/10.1073/pnas.1932826100
  14. Kee K, Gonsalves JM, Clark AT, Reijo Pera RA. Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells. Stem Cells Dev 2006; 15(6): 831-7. https://doi.org/10.1089/scd.2006.15.831
  15. Teramura T, Takehara T, Kawata N, Fujinami N, Mitani T, Takenoshita M, et al. Primate embryonic stem cells proceed to early gametogenesis in vitro. Cloning Stem Cells 2007; 9(2): 144-56. https://doi.org/10.1089/clo.2006.0070
  16. Richards M, Fong CY, Bongso A. Comparative evaluation of different in vitro systems that stimulate germ cell differentiation in human embryonic stem cells. Fertil Steril 2010; 93(3): 986 -94. https://doi.org/10.1016/j.fertnstert.2008.10.030
  17. Novak I, Lightfoot DA, Wang H, Eriksson A, Mahdy E, Hoog C. Mouse embryonic stem cells form follicle-like ovarian structures but do not progress through meiosis. Stem Cells 2006; 24(8): 1931-6. https://doi.org/10.1634/stemcells.2005-0520
  18. Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 2009; 462; 222-5. https://doi.org/10.1038/nature08562
  19. Hasegawa K, Fujioka T, Nakamura Y, Nakatsuji N, Suemori H. A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation. Stem Cells 2006; 24: 2649-60. https://doi.org/10.1634/stemcells.2005-0657
  20. Nicholas CR, Gaur M, Wang S, Reijo Pera RA, Leavitt AD. A method for single-cell sorting and expansion of generally modified human embryonic stem cells. Stem Cells Dev 2007; 16: 109-17. https://doi.org/10.1089/scd.2006.0059
  21. Ellerstrom C, Strehl R, Noaksson K, Hyllner J, Semb H. Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation. Stem Cells 2007; 25: 1690 -6. https://doi.org/10.1634/stemcells.2006-0607
  22. Li X, Krawetz R, Liu S, Meng G, Rancourt DE. ROCK inhibitor improves survival of cryopreserved serum/feederfree single human embryonic stem cells. Hum Reprod 2009; 24(3): 580-9.
  23. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology 2007; 25(6): 681-6. https://doi.org/10.1038/nbt1310
  24. Nayernia K, Nolte J, Michelmann HW, Lee JH, Rathsack K, Drusenheimer N, et al. In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev Cell 2006; 11(1): 125-32. https://doi.org/10.1016/j.devcel.2006.05.010
  25. Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004; 427(6970): 148-54. https://doi.org/10.1038/nature02247
  26. Pyle AD, Lock LF, Donovan PJ. Neurotropins mediate human embryonic stem cell survival. Nat Biotechnol 2006; 24: 344 -50. https://doi.org/10.1038/nbt1189