悬滴培养法促进鸡胚胎干细胞形成类胚体

doi:10.11843/j.issn.0366-6964.2015.08.008

Abstract

Abstract:

This experiment was conducted to explore the best suspension culture system for embryoid body (EB) formation from chicken embryonic stem cells (ESCs)，and to improve the induction efficiency of chicken ESCs in vitro.Pure clones from the third generation of chicken ESCs was collected，re-suspensed the cells into 3 cell concentrations：1×10⁴，3×10⁴and 6×10⁴ mL^-1 to make suspension culture.Morphology changes of EB were observed，qRT-PCR and immunofluorescence methods were used to detect the marker genes’s expression，self-differentiation and karyotype analysis were made to make full test of EB.The results showed that,the amount of EB with 3×10⁴mL^-1 cell concentration was higher than the other groups，and the number of EB reached 267 in one single view with the same spherical shape.The stem cell marker genes Nanog,Sox2,Oct4 and C-kit remained expression with 48 h of EB formation，and stem cell surface specific antigen (Nanog and SSEA-1) detection was positive.After self-differentiation of EB，3 embryonic germ layers specific antigen (SOX17, SMA and TUJ-1) detection all showed positive.Karyotype analysis showed that the formatted EB maintained normal karyotype.The results indicated that the suitable concentration for chicken ESCs form the EB through suspension culture was 3×10⁴ mL^-1，and the formatted EB had viability to provide experimental foundation for chicken ESCs induction in vitro.

CLC Number:

S831.2

ZHANG Lei，WANG Xiao-yan，ZUO Qi-sheng，LU Zhen-yu，WANG Fei，JI Yan-qin，WANG Ying-jie，ZHANG Ya-ni，LI Bi-chun. Embryoid Body Formation from Chicken Embryonic Stem Cells through Suspension Culture[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(8): 1333-1340.

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Embryoid Body Formation from Chicken Embryonic Stem Cells through Suspension Culture

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