161 Establishment of bovine induced pluripotent stem cells
Y. Su A , L. Wang A , Z. Fan B , Y. Liu B , J. Zhu A , D. Kaback C , J. Oudiz A , T. Patrick B , S. P. Yee C , X. Tian A , I. Polejaeva B and Y. Tang AA Department of Animal Science, Institute of Systems Genetics, University of Connecticut, Storrs, CT, USA
B Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT, USA
C Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA
Reproduction, Fertility and Development 34(2) 318-319 https://doi.org/10.1071/RDv34n2Ab161
Published: 7 December 2021
© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Pluripotent stem cells (PSCs) are great cell resources for early embryonic development and cell differentiation, drug screening, and genome-editing studies due to their indefinite self-renewing capacity and the ability to differentiate into any cell types of an animal. However, generating bovine induced pluripotent stem cells (biPSCs) has been challenging. Here we report the successful generation of biPSCs from bovine somatic cells using retroviral human OCT4, SOX2, KLF4, MYC, NANOG, and LIN28-based transduction system. The established biPSCs exhibited prolonged self-renewal capacity over 70 passages. The expression of transgenes from four established biPSC lines was silenced at both early (P10-P17) and later passages (P25-P43) by quantitative reverse transcription-PCR (qRT-PCR) test. The biPSCs have flat, primed-like PSC colony morphology with high expression of endogenous pluripotent genes (OCT4, NANOG, and SOX2) as verified by qRT-PCR and immunostaining, and are refractory to single cell colonisation by trypsin treatment. However, when switched to 2i medium, the biPSCs developed dome-shaped, naïve-like PSC colony morphology with single cell colonisation capacity. These cells have a normal karyotype and exhibit proliferation rates comparable to the reported primed- or naïve-state human PSCs, 30.4 ± 1.1 h and 23.2 ± 4.6 h, respectively. The biPSCs are capable of forming embryoid bodies (EBs) in vitro, which give rise to three-germ layers with the detection of the endoderm (AFP), mesoderm (SMA), and ectoderm (TUJ1) markers by immunostaining. RNA-seq transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues, which was verified by PCR using bovine- and retroviral vector-specific primers. Finally, nuclear transfer (NT) has been widely applied to generate cloned animals. Using two lines of naïve-like biPSCs with high passage numbers as nuclear donors, we demonstrated that biPSCs could achieve a cloning efficiency of 24.6%, which is comparable to that of bovine adult fibroblasts. One-way ANOVA with Tukey’s multiple comparison test or Student’s t-test was used for data analysis. Our report represents a significant advance in the establishment of bona fide bovine PSCs.
