Comparative Evaluation of Various Human Feeders for Prolonged Undifferentiated Growth of Human Embryonic Stem Cells

Abstract

Human embryonic stem (hES) cells are typically derived and serially propagated on inactivated murine embryonic fibroblast (MEF) feeders. The use of MEFs and other components of animal origin in the culture media for hES cell support substantially elevates the risk of contaminating these cell lines with infectious agents of animal origin thereby severely limiting their potential for clinical application. We have previously shown that it is possible to derive and establish new hES cell lines in a xeno-free culture system using human fetal muscle fibroblast feeders. In this report, we have comparatively evaluated a panel of 11 different human adult, fetal, and neonatal feeders for hES cell support and have ranked them as supportive and non-supportive. We report that two adult skin fibroblast cell lines established in-house from abdominal skin biopsies supported prolonged undifferentiated hES cell growth for over 30 weekly passages in culture. Furthermore, hES cell lines cultured on adult skin fibroblast feeders retain hES cell morphology and remain pluripotent. Also, differences in feeder support exist between human cell types and sources. The use of human adult skin feeders is convenient for hES cell support given the ease of obtaining skin biopsies.

Toward xeno-free culture of human embryonic stem cells

“…Various human cell lines have been examined and compared by Richards, Fong, Chan, Wong, and Bongso (2002) and Richards et al (2003) for the ability to support thehESClines, HES-3 and HES-4 (ES03 and ES04). Initially they compared fetal muscle, fetal skin and adult fallopian tube epithelial cells, all of which were found to support undifferentiated hESC culture through 20 passages (Richards et al, 2002).…”

“…At this time, human serum was substituted for fetal bovine serum (FBS) in the fetal fibroblast culture medium to further reduce potential contamination by animal products. However, it was later found that increasing differentiation of hESCs was observed beyond 10 passages when fibroblasts were maintained in human serum (Richards et al, 2003). The growth medium was, therefore, switched to standard growth medium supplemented with 20% knockout serum replacer (KSR) or FBS.…”

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

“…In case of the farm animals, ES cell-like cells are also commonly cultured on feeder cells because the molecular pathways and key molecules required in maintaining pluripotency in these species are still unknown [14]. As stated earlier in various reports, homologous feeder cell layers supported self renewal and maintained pluripotent characteristics of human and monkey ES cells, with characteristics similar to those of ES cells cultured on mouse fetal fibroblast (MEF) [9,15]. Recently, buffalo embryonic stem cell-like cells have been generated on buffalo fetal fibroblasts [16,17]; although the buffalo ES (BuES) cell-like cells grew on homologous feeder cell layers but cell colonies did not maintain an undifferentiated state over a long period of time.…”

“…Recently feeder-free system has been adapted especially for culturing hES cells to eliminate the potential issues of infection and cross-species contamination [6,7]. Some feeder-free systems support the culture of undifferentiated hES cells more efficiently than others [8,9], however, porcine and bovine epiblast and ICM cells cultured on a feeder free system did not grow continuously and started senescence or differentiation [10][11][12][13]. In case of the farm animals, ES cell-like cells are also commonly cultured on feeder cells because the molecular pathways and key molecules required in maintaining pluripotency in these species are still unknown [14].…”

Koreans admit disguising stem-cell lines

“…Human ES cells (hESCs) can be maintained and propagated on mouse or human fibroblast feeders for extended periods in media containing basic fibroblast growth factor (bFGF) [1][2][3][4] while retaining the ability to differentiate into ectoderm, endoderm and mesoderm as well as trophoectoderm and germ cells. Gene expression in hESC has been investigated by a variety of techniques including massively parallel signature sequencing (MPSS), serial analysis of gene expression (SAGE), expressed sequence tag (EST) scan, large scale microarrays, focused cDNA microarrays, and immunocytochemistry [5][6][7].…”