Human embryonic stem cell lines isolation, cultivation, and characterization

Abstract

A large number of human embryonic stem cell (hESC) lines have been derived worldwide since the first hESC line establishment in 1998. Despite many common characteristics, most important of which is the pluripotency, hESC lines vary significantly in their transcriptional profiles, genetic, and epigenetic state. These differences may arise both from individual genetics of the cell lines and from variations in their handling such as isolation and cultivation. In order to minimize the latter differences, the standardized protocols of cultivation and inter-laboratory comprehensive studies should be performed. In this report, we summarized our experience of derivation and characterization of hESC lines as well as of adaptation of hESCs to novel cultivation protocols. We have successfully derived five hESC lines and characterized them by previously established criteria, including expression of specific markers and the capacity to differentiate both in vitro and in vivo. Four of these lines, namely hESM01-04, were initially derived using mouse fibroblasts as a feeder and currently are maintained under feeder-free, serum-free conditions using mTeSR1 and Matrigel. The fifth line, hESMK05 was derived in feeder-free, serum-free conditions using mTeSR1 and Matrigel. Cell lines retain their pluripotent status and normal karyotype for more than 70 passages and are available to the scientific community.

Culture Conditions Affect Cardiac Differentiation Potential of Human Pluripotent Stem Cells

“…Matrigel matrix together with mTeSR1 medium is widely used in different laboratories, and several research groups have reported this culture condition to maintain the undifferentiated growth of hPSCs [8], [9], [26], [27], [38]. In these studies, the pluripotency of hPSCs was confirmed by teratoma and EB formation and analysis of the expression of markers specific to all three germ layers present in these structures.…”

Characteristics of Induced Human Pluripotent Stem Cells Using DNA Microarray Technology

“…Line hiPS-31L was derived from human embryonic fi broblasts (culture MAN1) via induction with retroviruses pMX-Oct4, pMX-Sox2 pMX-Klf4, and pMX-c-Myc [1]. We also used human ESC lines HUES9 [8] and hESM04 [12], cell culture obtained from human amniotic fl uid (culture Amn-Fluid2), and partially reprogrammed amniotic fl uid cell line (AF2-r4v4).…”

In Vitro Histogenesis of Human Embryonic Stem Cells into Retina Components

“…We used three strains of human ESC: hESM01, hESM03, and hESM04 [7,8]. The cells were cultured on gelatincoated 35-mm Petri dishes (Costar) using mouse embryonic fi broblasts as the feeder in KODMEM (Invitrogen) containing 20% serum substitute (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), 1 mM glutamine (Gibco), 1 mM amino acid mixture (Gibco), 4 ng/ml bFGF (Peprotech), and antibiotics.…”

“…Reverse transcription-PCR (RT-PCR) and histoche mical analysis were carried out as described previously [7].…”