Assessment of different stages of parthenogenetic embryos for production of embryonic stem cell like colonies

JUHI PATHAK; S D KHARCHE; ANJANA GOEL; A K S SIKARWAR; SONIA SARASWAT; RAVI RANJAN; CHETNA GANGWAR; S P SINGH; A K GOEL; M S CHAUHAN

doi:10.56093/ijans.v90i5.104614

Authors

JUHI PATHAK ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India
S D KHARCHE ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India
ANJANA GOEL ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India
A K S SIKARWAR ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India
SONIA SARASWAT ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India
RAVI RANJAN ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India
CHETNA GANGWAR ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India
S P SINGH ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India
A K GOEL ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India
M S CHAUHAN ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 India

https://doi.org/10.56093/ijans.v90i5.104614

Keywords:

Embryonic stem cells, Hatched blastocyst, LIF, Parthenogenesis, Stem cell culture

Abstract

Parthenogenetic activation of oocytes has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. In this study, we described the production of goat parthenogenetic ES like cells from different stages of parthenogenetic embryos. We compared the source material on the pESCs outgrowth and culture. 8-16 cell stage embryos, morula, blastocysts and ICM (inner cell mass) from hatched blastocysts produced from chemical activation were cultured on goat fetal fibroblast monolayer in stem cell culture media. Colonies were passaged when signs of differentiation were visible. ESC like colonies were cultured on feeder cells in the presence of hLIF however, some ESC like colonies were also cultured in absence of hLIF for random differentiation. In our study, ESC like colony formation with ICM of hatched blastocyst was comparatively higher as compared to blastocysts while ESC like colony formation with blastocysts and ICM of hatched blastocyst was significantly higher as compared 8-16 cells and morula. It is observed that inner cell mass source is an important criterion for the ES like cells derivation in goats. Also, in comparison to the expanded blastocysts (80.10%), hatched blastocysts showed higher (86.06%) attachment rate and primary colony formation rate. ESC like colonies in absence of hLIF differentiated in vitro into epithelial like and neuronal like cells. Undifferentiated ESC like colonies stained positive for alkaline phosphatase, SSEA-3 and OCT-4.

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References

Hou Z, An L, Han J, Yuan Y, Chen D and Tian J. 2018. Revolutionize livestock breeding in the future: an animal embryo-stem cell breeding system in a dish. Journal of Animal Science Biotechnology 9: 90. DOI: https://doi.org/10.1186/s40104-018-0304-7

Kharche S D, Pathak J, Agarwal S, Kushwah B and Sikarwar A K S. 2016. Effect of Ca ionophore on blastocyst production following intracytoplasmic sperm injection in caprine oocytes. Reproduction in Domestic Animals 51: 611–17. DOI: https://doi.org/10.1111/rda.12701

Mitalipova M, Beyhan Z and First N L. 2001. Pluripotency of bovine embryonic cell line derived from precompacting embryos. Cloning 3: 59–67. DOI: https://doi.org/10.1089/15204550152475563

Mulas C, Kalkan T, Meyenn F, Leitch H G, Nichols J and Smith A. 2019. Defined conditions for propagation and manipulation of mouse embryonic stem cells. Development 146: 1–13. DOI: https://doi.org/10.1242/dev.173146

Renard J P, Maruotti J, Jouneau A and Vignon X. 2007. Nuclear reprogramming and pluripotency of embryonic cells: Application to the isolation of embryonic stem cells in farm animals. Theriogenology 68: 196–205. DOI: https://doi.org/10.1016/j.theriogenology.2007.05.060

Revazova E S, Turovets N A and Kochetkova O D. 2008. HLA homozygous stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 10: 11–24. DOI: https://doi.org/10.1089/clo.2007.0063

Saito S, Sawai K, Ugai H, Moriyasu S, Minmihashi A, Yamamoto Y, Hiroki H, Soichi Y, Kageyama S, Pan J, Murata T, Kobayashi Y, Obata Y and Yokoyama K K. 2003. Generation of cloned calves and transgenic chimeric embyos from bovine embryonic stemlike cells. Biochemical and Biophysical Research Communications 309: 104–13. DOI: https://doi.org/10.1016/S0006-291X(03)01536-5

Verma V, Gautam S K, Singh B, Manik R S, Palta P, Singla S K, Goswami S L and Chauhan M S. 2007. Isolation and characterization of embryonic stem cell-like cells from in vitroproduced buffalo (Bubalus bubalis) embryos. Molecular Reproduction and Development 74: 520–29. DOI: https://doi.org/10.1002/mrd.20645

Wang L, Duan E, Sung L Y, Jeong B S, Yang X and Tian X C. 2005. Generation and characterization of pluripotent stem cells from cloned bovine embryos. Biology of Reproduction 73: 149–55. DOI: https://doi.org/10.1095/biolreprod.104.037150

Zakrzewski W, Dobrzyñski M, Szymonowicz M and Rybak Z. 2019. Stem cells: past, present, and future. Stem Cell Research and Therapy 10: 68. DOI: https://doi.org/10.1186/s13287-019-1165-5