Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Mar;66(2):239-50.
doi: 10.1007/s10616-013-9561-7. Epub 2013 Apr 4.

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells

R Kumar  1 , S P S Ahlawat, M Sharma, O P Verma, G Sai Kumar, G Taru Sharma
Affiliations

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells

R Kumar et al. Cytotechnology. 2014 Mar.

Abstract

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2-5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
a Bright field micrographs of in vitro produced buffalo blastocysts hatched on day nine of in vitro fertilization showing hatched blastocysts (HdBs), empty zona pellucida (EZ), inner cell mass (ICM) and trophoectoderm (TE) (×200). b Morphology of a mitomycin-C treated fully confluent buffalo fetal fibroblast monolayer (×100)
Fig. 2
Fig. 2
Bright field micrographs showing morphology of hatched blastocysts and inner cell masses used for derivation of buESCs. Blastocysts with a large, b small, and c indistinguishable inner cell masses (×400). d Manually and e enzymatically isolated inner cell mass cells and f Intact hatched blastocyst on mitomycin-C treated confluent buffalo fetal fibroblast monolayer (×200). Primary buESC colonies (g–i) on buffalo fetal fibroblast monolayer on day five of culture (×600)
Fig. 3
Fig. 3
Alkaline phosphatase and immunocytochemical staining of buESCs for the expression analysis of pluripotency related markers. a Unstained buESC colony, b the buESCs colony showing (stained red) alkaline phosphatase activity, c A buESC colony under bright light d epifluorescence exhibiting expression of SSEA-4, e A buESC colony under bright light (f) epifluorescence exhibiting expression of TRA-1-81 (×200). (Color figure online)
Fig. 4
Fig. 4
RT–PCR analysis of β-actin, OCT-4, SOX-2 and NANOG gene transcripts in primary buESCs and karyotypic analysis of primary buESCs. a Lane M-100 base pair DNA ladder, Lane 1-OCT-4 gene transcript in primary buESC colonies, Lane 2-β-actin gene transcript in primary buESC colony, Lane 3-Fibroblast feeder cells (negative control). b Lane M-100 base pair DNA ladder, Lane 1-SOX-2 gene transcript in primary buESC colonies, Lane 2-β-actin gene transcript in primary buESC colony, Lane 3- Fibroblast feeder cells (negative control). c Lane M-100 base pair DNA ladder, Lane 1-NANOG gene transcript in primary buESC colonies, Lane 2-β-actin gene transcript in primary buESC colony, Lane 3- Fibroblast feeder cells (negative control). d Metaphase spread showing euploid number of chromosomes in primary buESCs (×1000)
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Anand T, Kumar D, Singh MK, Shah RA, Chauhan MS, Manik RS, Singla SK, Palta P. Buffalo (Bubalus bubalis) embryonic stem cell-like cells and preimplantation embryos exhibit comparable expression of pluripotency-related antigens. Reprod Domest Anim. 2011;46:50–58. doi: 10.1111/j.1439-0531.2009.01564.x. - DOI - PubMed
    1. Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 2003;17:126–140. doi: 10.1101/gad.224503. - DOI - PMC - PubMed
    1. Bao L, He L, Chen J, Wu Z, Liao J, Rao L, Ren J, Li H, Zhu H, Qian L, Gu Y, Dai H, Xu X, Zhou J, Wang W, Cui C, Xiao L. Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors. Cell Res. 2011;21:600–608. doi: 10.1038/cr.2011.6. - DOI - PMC - PubMed
    1. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. 2005;122:947–956. doi: 10.1016/j.cell.2005.08.020. - DOI - PMC - PubMed
    1. Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 2003;113:643–655. doi: 10.1016/S0092-8674(03)00392-1. - DOI - PubMed

LinkOut - more resources