title: Suppression of transforming growth factor β signaling promotes ground state pluripotency from single blastomeres.
abstract: OBJECTIVE
  Can transforming growth factor β (TGFβ) inhibition promote ground state pluripotency of embryonic stem cells (ESCs) from single blastomeres (SBs) of cleavage embryos in different mouse stains?
  
  
  CONCLUSIONS
  Small molecule suppression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and TGFβ signaling (designated as R2i) can enhance the generation of mouse ESCs from SBs of different cleavage stage embryos compared with the dual suppression of ERK1/2 and glycogen synthase kinase 3 (GSK3), designated as 2i, regardless of the strain of mouce.
  
  
  BACKGROUND
  It is known that chemical inhibition of TGFβ promotes ground state pluripotency in the generation and sustenance of naïve ES cells from mouse blastocysts compared with the well-known 2i condition. However, the positive effect of this inhibition on mouse ESCs from early embryonic SBs remains obscure.
  
  
  METHODS
  We used 155 cleavage-stage mouse embryos to optimize the culture conditions for blastocyst development. Then, to assess the effects of R2i and 2i on ESC generation from SBs, we cultured isolated SBs in 2i and R2i for 10 days. SBs were replated under the same conditions to produce ESCs. In total, 46 embryos and 321 SBs from two- to eight-cell stages were recovered from NMRI and BALB/c mouse strains and used in this study.
  
  
  METHODS
  Blastomeres from 2- to 8-cell stage mouse embryos were dispersed and individually seeded into a 96-well plates that included mitotically inactivated feeder cells. ESCs were generated in B27N2 deﬁned medium supplemented with R2i or 2i. Randomly selected ESC lines, generated from SBs of each stage, were assessed for pluripotency and germ-line transmission.
  
  
  RESULTS
  We demonstrated that dual inhibition of ERK1/2 and TGFβ (R2i) enhanced efficient blastocyst development and efficient establishment of ESCs from SB of 2- to 8-cell stage mouse embryos compared with the dual inhibition of ERK1/2 and GSK3 (2i) regardless of the embryonic stage and strain of mice. The proportions of SBs that produced ESC were 50-60 versus 20-30%.
  
  
  CONCLUSIONS
  This study was done with mouse embryos, it is not known whether these findings are transferable to humans.
  
  
  CONCLUSIONS
  These findings resulted in an increased efficiency of ESC generation from one biopsied blastomere for autogeneic or allogeneic matched pluripotent cells without the need to destroy viable embryos. The results also provided information about the developmental capacity of early embryonic blastomeres.
  
  
  BACKGROUND
  This study was funded by grants provided from Royan Institute, the Iranian Council of Stem Cell Research and Technology and the Iran National Science Foundation. The authors have no conﬂict of interest to declare.
authors: Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, PO Box 19395-4644, Tehran, Iran Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran.
authors: Seyedeh-Nafiseh
authors: SN
authors: Hassani
authors: Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, PO Box 19395-4644, Tehran, Iran.
authors: Mohammad
authors: M
authors: Pakzad
authors: Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, PO Box 19395-4644, Tehran, Iran.
authors: Behrouz
authors: B
authors: Asgari
authors: Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, PO Box 19395-4644, Tehran, Iran.
authors: Adeleh
authors: A
authors: Taei
authors: Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, PO Box 19395-4644, Tehran, Iran Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran baharvand@royaninstitute.org.
authors: Hossein
authors: H
authors: Baharvand
mesh_terms: D000818:Animals; D001757:Blastomeres / Q000187:drug effects* / Q000378:metabolism; D002454:Cell Differentiation; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects*; D047108:Embryonic Development / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects; D005260:Female; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors*
chemical_list: D016212:Transforming Growth Factor beta; D038362:Glycogen Synthase Kinase 3
keywords: ERK inhibition; TGFβ inhibition; ground state pluripotency; mouse embryonic stem cell derivation; single blastomeres