Derivation of Human Embryonic Stem Cells from Day‐8 Blastocysts Recovered after Three‐Step In Vitro Culture

Abstract

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.

Activation of p53 by nutlin leads to rapid differentiation of human embryonic stem cells

“…Cell lines, their maintenance and treatment with low-molecular weight compounds Human ESCs were grown on mitotically inactivated mouse embryonic fibroblasts and passaged essentially as described in Stojkovic et al (2004). Few passages before experiments, human ESCs were transferred to Matrigel-coated plates with feeder-conditioned media as previously described in Stojkovic et al (2004); Hyslop et al (2005a).…”

“…Few passages before experiments, human ESCs were transferred to Matrigel-coated plates with feeder-conditioned media as previously described in Stojkovic et al (2004); Hyslop et al (2005a). Cells were treated with 10 and 30 mM nutlin-3, 2 mM sodium butyrate, 10 and 30 mM retinoic acid (all from Sigma, Dorset, UK) or 1 mM 1a,25-dihydroxyvitamin D3 (Biomol International, Exeter, UK).…”

“…Cells were treated with 10 and 30 mM nutlin-3, 2 mM sodium butyrate, 10 and 30 mM retinoic acid (all from Sigma, Dorset, UK) or 1 mM 1a,25-dihydroxyvitamin D3 (Biomol International, Exeter, UK). H9, H1 (both from WiCell Inc., Madison, WI, USA) and hES-NCL1 (Stojkovic et al, 2004) were used in this study.…”

Derivation of buffalo embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells

“…leading to an increased inclination for BuES-like cell derivation from hatched blastocysts compared to expanded blastocysts. In earlier studies too, the isolation of ICMs either from morula, early stage of blastocyst or late stage of blastocyst gave an unsatisfactory result as compared to hatched blastocyst in various species [16,17,35,36]. In our earlier study from our laboratory [17], the comparison of ICM isolation method on buffalo ES cell generation was evaluated.…”

Xeno-free derivation and culture of human embryonic stem cells: current status, problems and challenges

“…Morula-stage embryos [9] or late-stage blastocysts (7-8 days) [10] may also be used to create hESC lines. Although all the hESC lines derived worldwide share the expression of characteristic pluripotency markers [11,12], many differences are emerging between lines that may be more associated with the wide range of culture conditions in current use than with the inherent genetic variations of the embryos from which hESC were derived [13].…”