Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals

TY - JOUR

T1 - Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals

AU - Secher, Jan

AU - Liu, Ying

AU - Petkov, Stoyan

AU - Luo, Yonglun

AU - Li, Dong

AU - Hall, Vanessa Jane

AU - Schmidt, Mette

AU - Callesen, Henrik

AU - Bentzon, Jacob Fog

AU - Sørensen, Charlotte Brandt

AU - Freude, Karla Kristine

AU - Hyttel, Poul

PY - 2017/3

Y1 - 2017/3

N2 - Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells.

AB - Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells.

KW - Embryonic germ cell

KW - Handmade cloning

KW - Induced pluripotent stem cells

KW - Pluripotency

KW - Somatic cell nuclear transfer

KW - Transgenic pigs

KW - Cell Line

KW - Green Fluorescent Proteins

KW - Fibroblasts/physiology

KW - Animals, Genetically Modified

KW - Swine/genetics

KW - Male

KW - Embryonic Development

KW - Cellular Reprogramming

KW - Embryo Transfer/veterinary

KW - Pregnancy

KW - Animals

KW - Cloning, Organism/methods

KW - Embryo, Mammalian/physiology

KW - Female

KW - Pluripotent Stem Cells/physiology

KW - Nuclear Transfer Techniques/veterinary

UR - http://www.scopus.com/inward/record.url?scp=85009956038&partnerID=8YFLogxK

U2 - 10.1016/j.anireprosci.2017.01.007

DO - 10.1016/j.anireprosci.2017.01.007

M3 - Journal article

C2 - 28126267

SN - 0378-4320

VL - 178

SP - 40

EP - 49

JO - Animal Reproduction Science

JF - Animal Reproduction Science

ER -