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Embryonic Chimeras with Human Pluripotent Stem Cells

  • Alejandro De Los AngelesEmail author
  • Masahiro Sakurai
  • Jun WuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2005)

Abstract

Human pluripotent stem (PS) cells can be isolated from preimplantation embryos or by reprogramming of somatic cells or germline progenitors. Human PS cells are considered the “holy grail” of regenerative medicine because they have the potential to form all cell types of the adult body. Because of their similarity to humans, nonhuman primate (NHP) PS cells are also important models for studying human biology and disease, as well as for developing therapeutic strategies and test bed for cell replacement therapy. This chapter describes adjusted methods for cultivation of PS cells from different primate species, including African green monkey, rhesus monkey, chimpanzee, and human. Supplementation of E8 medium and inhibitors of the Tankyrase and GSK3 kinases to various primate PS cell media reduce line-dependent predisposition for spontaneous differentiation in conventional PS cell cultures. We provide methods for basic characterization of primate PS cell lines, which include immunostaining for pluripotency markers such as OCT4 and TRA-1-60, as well as in vivo teratoma formation assay. We provide methods for generating alternative PS cells including region-selective primed PS cells, two different versions of naïve-like cells, and recently reported extended pluripotent stem (EPS) cells. These derivations are achieved by acclimation of conventional PS cells to target media, episomal reprogramming of somatic cells, or resetting conventional PS cells to a naïve-like state by overexpression of KLF2 and NANOG. We also provide methods for isolation of PS cells from human blastocysts. We describe how to generate interspecies primate-mouse chimeras at the blastocyst and postimplantation embryo stages. Systematic evaluation of the chimeric competency of human and primate PS cells will aid in efforts to overcome species barriers and achieve higher grade chimerism in postimplantation conceptuses that could enable organ-specific enrichment of human xenogeneic PS cell derivatives in large animals such as pigs and sheep.

Key words

Pluripotent stem cells Primed pluripotent stem cells Embryonic stem cell Induced pluripotent stem cell Extended pluripotent stem cells Nonhuman primates Primates Region-selective Interspecies chimeras FGF WNT GSK3 Tankyrase TNKS1/2 Naïve-like pluripotent stem cells LCDM 5iLAF t2iL KLF2 NANOG Reprogramming Human pluripotent stem cells Monkey pluripotent stem cells Primate pluripotent stem cells OCT4 SOX2 KLF4 LMYC LIN28 p53 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of PsychiatryYale University School of MedicineNew HavenUSA
  2. 2.Department of Molecular BiologyUniversity of Texas Southwestern Medical CenterDallasUSA
  3. 3.Hamon Center for Regenerative Science and MedicineUniversity of Texas Southwestern Medical CenterDallasUSA

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