Cargando…

Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line

PURPOSE: Mouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC pr...

Descripción completa

Detalles Bibliográficos
Autores principales: Desai, Nina, Ludgin, Jennifer, Goldberg, Jeffrey, Falcone, Tommaso
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663960/
https://www.ncbi.nlm.nih.gov/pubmed/23575766
http://dx.doi.org/10.1007/s10815-013-9977-1
_version_ 1782271057087954944
author Desai, Nina
Ludgin, Jennifer
Goldberg, Jeffrey
Falcone, Tommaso
author_facet Desai, Nina
Ludgin, Jennifer
Goldberg, Jeffrey
Falcone, Tommaso
author_sort Desai, Nina
collection PubMed
description PURPOSE: Mouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application. METHODS: A novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2–3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells. RESULTS: ESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation. CONCLUSIONS: Human feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.
format Online
Article
Text
id pubmed-3663960
institution National Center for Biotechnology Information
language English
publishDate 2013
publisher Springer US
record_format MEDLINE/PubMed
spelling pubmed-36639602013-05-28 Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line Desai, Nina Ludgin, Jennifer Goldberg, Jeffrey Falcone, Tommaso J Assist Reprod Genet Stem Cell Biology PURPOSE: Mouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application. METHODS: A novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2–3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells. RESULTS: ESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation. CONCLUSIONS: Human feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging. Springer US 2013-04-11 2013-05 /pmc/articles/PMC3663960/ /pubmed/23575766 http://dx.doi.org/10.1007/s10815-013-9977-1 Text en © The Author(s) 2013 https://creativecommons.org/licenses/by-nc/2.0/ Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
spellingShingle Stem Cell Biology
Desai, Nina
Ludgin, Jennifer
Goldberg, Jeffrey
Falcone, Tommaso
Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line
title Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line
title_full Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line
title_fullStr Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line
title_full_unstemmed Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line
title_short Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line
title_sort development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line
topic Stem Cell Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663960/
https://www.ncbi.nlm.nih.gov/pubmed/23575766
http://dx.doi.org/10.1007/s10815-013-9977-1
work_keys_str_mv AT desainina developmentofaxenofreenoncontactcoculturesystemforderivationandmaintenanceofembryonicstemcellsusinganovelhumanendometrialcellline
AT ludginjennifer developmentofaxenofreenoncontactcoculturesystemforderivationandmaintenanceofembryonicstemcellsusinganovelhumanendometrialcellline
AT goldbergjeffrey developmentofaxenofreenoncontactcoculturesystemforderivationandmaintenanceofembryonicstemcellsusinganovelhumanendometrialcellline
AT falconetommaso developmentofaxenofreenoncontactcoculturesystemforderivationandmaintenanceofembryonicstemcellsusinganovelhumanendometrialcellline