Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture.
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.
Stojkovic
Miodrag
M
Institute of Human Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK. miodrag.stojkovic@ncl.ac.uk
Lako
Majlinda
M
Stojkovic
Petra
P
Stewart
Rebecca
R
Przyborski
Stefan
S
Armstrong
Lyle
L
Evans
Jerry
J
Herbert
Mary
M
Hyslop
Louise
L
Ahmad
Sajjad
S
Murdoch
Alison
A
Strachan
Tom
T
D000328:Adult; D000367:Age Factors; D000818:Animals; D000954:Antigens, Surface / Q000276:immunology; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000276:immunology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005260:Female; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D007621:Karyotyping; D008297:Male; D051379:Mice; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D033581:Stem Cell Transplantation / Q000379:methods*; D013724:Teratoma / Q000235:genetics / Q000276:immunology
D000954:Antigens, Surface; D017077:Culture Media, Conditioned; D005819:Genetic Markers