Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder

Abstract

Derivation of human embryonic stem cell lines has been a remarkable scientific achievement during the last decade. Human embryonic stem cells are regarded as an unlimited cell source for replacement therapy in regenerative medicine. Clearly, the scientific community requires proper derivation, characterization, and registration with the purpose of making them available for research and future medical applications worldwide. In this paper, we report our derivation work as the Valencian Node of the Spanish Stem Cell Bank in the generation, characterization, and registration of VAL-3, -4, -5, -6M, -7, -8, and 9 (www.isciii/htdocs/terapia/terapia_bancocelular.jsp). The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout Dulbecco's modified Eagle's medium supplemented with knockout serum replacement and basic fibroblast growth factor. Fingerprinting of the cell lines was performed to allow their identification and traceability. All lines were expressed at the mRNA and specific protein markers for undifferentiation and were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All lines displayed high levels of telomerase activity and were shown to successfully overcome cryopreservation and thawing. Finally, we demonstrated the potential to differentiate in vitro (embryoid body formation) and in vivo (teratoma formation) into cell types from all three germ layers. Teratoma derived from all human embryonic stem cell lines present similar morphological features except VAL-8 that display more aggressive tumor behavior with a larger proportion of solid tissues, as opposed to cyst formation in the other cell lines.

Reconstruction of Endometrium from Human Endometrial Side Population Cell Lines

“…Importantly a normal karyotype corresponding to 46XX was confirmed in ICEp and ICEs at passages 8 to 9 (Figure 2B). The pattern of telomerase activity in these lines indicated it was intermediate between the human embryonic stem cell line (hESC) VAL-9 [29] and human foreskin fibroblast (Figure 2C). …”

“…Human embryonic stem cell (hESC) line VAL-9 [29] was used as a positive control for undifferentiated markers and diverse tissues (heart, kidney, etc.) were used to test mesodermal origin.…”

Human foreskin fibroblast produces interleukin-6 to support derivation and self-renewal of mouse embryonic stem cells

“…The HFF system can be widely applied in the large-scale expansion of the ESCs in vitro . Human ESCs were previously reported to be successfully derived on human feeders [32,33]. HFF can therefore be used as feeder cells in the human ESC culture to eliminate contaminations of animal origin.…”

Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

“…For replacing animal cells, various alternative human feeder layers such as fetal foreskin, fetal muscle, and skin (Richards et al, 2002; Amit et al, 2003), have been validated for culture of undifferentiated hESCs. Human foreskin fibroblasts (HFFs) feeder cells have been used to establish the new hESCs lines (Aguilar-Gallardo et al, 2010). Moreover, MEFs and HFFs differed in their capacity to support the pluripotency and non-differentiation of hESCs.…”