Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system

Abstract

The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 microl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs.

Effect of Oxygen Tension on the Amino Acid Utilisation of Human Embryonic Stem Cells

“…Shef3 (supplied by the UK Stem Cell Bank) [32] and Hues-7 (D. Melton, Howard Hughes Medical Institute/Harvard University) [33] hESCs were cultured at 20% oxygen in Knockout DMEM (Invitrogen) supplemented with 15% knockout serum replacement (Invitrogen), 100 µg/ml penicillin/streptomycin (Invitrogen), 1 mM L-glutamine (Invitrogen), 1 x non-essential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol and 10 ng/ml FGF2 (Peprotech) on γ-irradiated mouse embryonic fibroblasts (MEFs; a primary source derived in institutional facilities following University of Southampton ethical review committee approval and in accordance with UK Home Office regulations). hESCs were then transferred to Matrigel (BD Biosciences) coated plates and cultured in MEF-conditioned medium at both 20% and 5% oxygen.…”

Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes

“…Normal human tissue RNA samples were purchased from Applied Biosystems. SHEF-6 human embryonic stem cell DNA and RNA was a kind gift from Dr. David Hay (Center for Regenerative Medicine, Edinburgh, UK) (Aflatoonian et al 2010). Clinical specimens of normal human breast tissue were obtained through the Edinburgh Experimental Cancer Medicine Center.…”

Heterogeneity in Human Embryonic Stem Cells May Prevent Endodermal Guided Differentiation

“…The cells showed a high level of pluripotent markers despite being cultured in a pointedly differentiation inducing medium. The cells showed low CFU-c or clonogenic potential and the cells show resistance to differentiation into the desired cell phenotype although other ESC lines such as H1, H7, KCL002-WT4, Shef-6 and others do [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55].…”