Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture

Stem Cells. 2004;22(5):790-7. doi: 10.1634/stemcells.22-5-790.


Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5-7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Age Factors
  • Animals
  • Antigens, Surface / immunology
  • Blastocyst / cytology
  • Blastocyst / physiology*
  • Cell Culture Techniques / methods
  • Cell Differentiation / drug effects
  • Cell Differentiation / genetics
  • Cell Line
  • Cell Proliferation / drug effects
  • Cell Separation / methods
  • Cell Transformation, Neoplastic / genetics
  • Cell Transformation, Neoplastic / immunology
  • Culture Media, Conditioned / pharmacology
  • Female
  • Genetic Markers / genetics
  • Humans
  • Karyotyping
  • Male
  • Mice
  • Mice, SCID
  • Neoplastic Stem Cells / drug effects
  • Neoplastic Stem Cells / metabolism
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / drug effects
  • Pluripotent Stem Cells / physiology*
  • Rats
  • Stem Cell Transplantation / methods*
  • Teratoma / genetics
  • Teratoma / immunology


  • Antigens, Surface
  • Culture Media, Conditioned
  • Genetic Markers