Human feeder cell line for derivation and culture of hESc/hiPSc

McKay, Tristan R.; Camarasa, María V.; İskender, Banu; Ye, Jinpei; Bates, Nicola; Miller, Duncan C.; Fitzsimmons, Jayne C.; Foxler, Daniel E.; Mee, Maureen; Sharp, Tyson V.; Aplin, John D.; Brison, Daniel R.; Kimber, Susan J.

doi:10.1016/j.scr.2011.04.005

Cited by 18 publications

(19 citation statements)

References 19 publications

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“…The cells were able to differentiate, spontaneously, forming EBs containing the cell types representative of the three germ layer; this is, similar to results found in studies using feeder layers of breast parenchymal cells, endometrial cells and human fibroblast cells (33), placental cells (54), foreskin cells (30), umbilical cord blood cells (32), and umbilical cord stromal cells (40). Following 14 days culture, the expression of pluripotency transcription factors was still detected, suggesting that 14 days was insufficient to induce the differentiation of all EBs.…”

Section: Discussionsupporting

confidence: 85%

Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells

Oliveira

Santos

Vairo

et al. 2017

Experimental and Therapeutic Medicine

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The aim of the present study was to investigate whether feeder layers composed of human hair follicle-derived mesenchymal stem cells (hHFDCs) are able to support human embryonic stem cells (hESCs). hHFDCs and mouse embryonic fibroblasts (MEFs) were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and low-glucose DMEM, respectively. hHFDCs were passaged three times and subsequently characterized. hHFDCs and MEFs were mitotically inactivated with mitomycin C for 3 h prior to co-culture with H9-hESCs. hESCs were initially established on a mouse feeder layer, subsequently transferred onto a human feeder layer and split every 5 days. Cell morphology, expression of specific ‘undifferentiation’ markers and growth factors, and the differentiation capacity of hESCs grown on the hHFDC feeder layer were analyzed. hHFDCs are adherent to plastic, possess the classic mesenchymal stem cell phenotype [they express cluster of differentiation (CD)90, CD73 and CD105] and are able to differentiate into adipocytes, chondroblasts and osteocytes, indicating that these cells are multipotent. Population-doubling time analysis revealed that hHFDCs rapidly proliferate over 34.5 h. As a feeder layer, hHFDC behaved similarly to MEF in maintaining the morphology of hESCs. The results of alkaline phosphatase activity, reverse transcription-quantitative polymerase chain reaction analysis of the expression of pluripotency transcription factors [octamer-binding transcription factor 4 (Oct4), Nanog and sex determining region Y-box 2], and immunofluorescence assays of markers (stage-specific embryonic antigen-4 and Oct4) in hESCs co-cultured over hHFDC, indicated that the undifferentiated state of hESCs was preserved. No change in the level of growth factor transcripts (bone morphogenetic protein 4, fibroblast growth factor-2, vascular endothelial growth factor, Pigment epithelium-derived factor and transforming growth factor-β1) was detected for either feeder layer prior to or following inactivation. Similar phenotypes of embryoid body formation, size and morphology were observed in the hHFDC and MEF feeders. In conclusion, hHFDC maintained hESCs in an undifferentiated state comparable to MEF in standard conditions, which may be an important finding regarding the establishment of stem cell-based translational applications.

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Section: Discussionsupporting

confidence: 85%

Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells

Oliveira

Santos

Vairo

et al. 2017

Experimental and Therapeutic Medicine

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“…We initially used human placental stromal fibroblasts (hPSFs) to replace mouse embryonic fibroblasts (MEFs) as feeder cells with xeno-free knock-out serum (KO-SR ZF) hESC medium [29]. However, hPSFs had to be utilised before passage 4 to establish lines successfully, and we were unable to generate a suitable GMP-grade hPSF line (data not shown).…”

Section: Resultsmentioning

confidence: 99%

High quality clinical grade human embryonic stem cell lines derived from fresh discarded embryos

Bates

Soteriou

et al. 2017

Stem Cell Res Ther

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BackgroundHuman embryonic stem cells (hESCs) hold tremendous promise for cell replacement therapies for a range of degenerative diseases. In order to provide cost-effective treatments affordable by public health systems, HLA-matched allogeneic tissue banks of the highest quality clinical-grade hESCs will be required. However only a small number of existing hESC lines are suitable for clinical use; they are limited by moral and ethical concerns and none of them apply Good Manufacturing Practice (GMP) standards to the earliest and critical stages of gamete and embryo procurement. We thus aimed to derive new clinical grade hESC lines of highest quality from fresh surplus GMP grade human embryos.MethodsA comprehensive screen was performed for suitable combinations of culture media with supporting feeder cells or feeder-free matrix, at different stages, to support expansion of the inner cell mass and to establish new hESC lines.ResultsWe developed a novel two-step and sequential media system of clinical-grade hESC derivation and successfully generated seven new hESC lines of widely varying HLA type, carefully screened for genetic health, from human embryos donated under the highest ethical and moral standards under an integrated GMP system which extends from hESC banking all the way back to gamete and embryo procurement.ConclusionsThe present study, for the first time, reports the successful derivation of highest-quality clinical-grade hESC lines from fresh poor-quality surplus human embryos generated in a GMP-grade IVF laboratory. The availability of hESC lines of this status represents an important step towards more widespread application of regenerative medicine therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0561-y) contains supplementary material, which is available to authorized users.

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“…İEKH'i insan kökenli besleyici hücreleri üzerinde de MEF hücrelerindeki kadar etkin bir şekilde üre-tilmiş olmalarına rağmen, hücre kültür stoklarında dönem dönem gözlemlenen varyasyonlar ve hüc-relerin yaşlanması bu in vitro kültür sisteminde de sorun teşkil etmektedir. Besleyici hücre kültürünün eskimesi ve buna bağlı olarak bu hücrelerin insan embriyonik kök hücre pluripotent özelliğini destekleme yeteneklerini yitirmelerini önlemek amacıy-la bazı gruplar tarafından genetik manipulasyonla ölümsüzleştirilen (immortal) insan sünnet derisi ve plasenta stroma fibroblastları gibi besleyici hücreler de kullanılmıştır [39,40].…”

Section: İnsan Embriyonik Kök Hücre Kültürüunclassified

Human embryonic stem cells and microenvironment

İskender

izgi²,

Şanlıoğlu³

et al. 2014

J Clin Exp Invest

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Human embryonic stem cells (hESCs) possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less laborintensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3): 486-495

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Human feeder cell line for derivation and culture of hESc/hiPSc

Cited by 18 publications

References 19 publications

Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells

Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells

High quality clinical grade human embryonic stem cell lines derived from fresh discarded embryos

Human embryonic stem cells and microenvironment

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