Replacement of mouse embryonic fibroblasts with bone marrow stromal cells for use in establishing and maintaining embryonic stem cells in mice

Lee, Chae Hyun; Park, Junhong; Lee, Jae Hee; Ahn, Ji Yeon; Park, Jong Heum; Lee, Bo‐Ram; Kim, Dae Yong; Lim, Jeong Mook

doi:10.1042/cbi20110395

Cited by 7 publications

(5 citation statements)

References 15 publications

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“…hESCs grown over MEF and hHFDC layers were maintained under the same culture conditions and hHFDCs displayed a fibroblast-like morphology similar to MEF, as described in the literature (14,18). No morphological changes were observed when these cells were inactivated with mitomycin C (data not shown), in agreement with the results of Lee et al (29). In previous studies the morphology of hESCs maintained on human feeder layers differed slightly from hESCs maintained on MEF layers (30,31).…”

Section: Discussionsupporting

confidence: 93%

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: 93%

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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“…8 ESC express LIF receptor and the binding of LIF to these receptors activates JAK-STAT pathways, critical for stem cell maintenance. 18 It is known that purified growth factors and also the conditioned medium of other cell lines can be used in some instances to make fastidious cells independent of the presence of feeder cell layers. The growth factor requirements of the ESC appear to have been elucidated.…”

Section: Mechanism Of Actionmentioning

confidence: 99%

Feeder Layer Cell Actions and Applications

Llames

García-Pérez

Meana

et al. 2015

Tissue Engineering Part B: Reviews

136

110

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Cultures of growth-arrested feeder cells have been used for years to promote cell proliferation, particularly with low-density inocula. Basically, feeder cells consist in a layer of cells unable to divide, which provides extracellular secretions to help another cell to proliferate. It differs from a coculture system because only one cell type is capable to proliferate. It is known that feeder cells support the growth of target cells by releasing growth factors to the culture media, but this is not the only way that feeder cells promote the growth of target cells. In this work, we discuss the different mechanisms of action of feeder cells, tackling questions as to why for some cell cultures the presence of feeder cell layers is mandatory, while in some other cases, the growth of target cells can be achieved with just a conditioned medium. Different treatments to avoid feeder cells to proliferate are revised, not only the classical treatments as mitomycin or γ-irradiation but also the not so common treatments as electric pulses or chemical fixation. Regenerative medicine has been gaining importance in recent years as a discipline that moves biomedical technology from the laboratory to the patients. In this context, human stem and pluripotent cells play an important role, but the presence of feeder cells is necessary for these progenitor cells to grow and differentiate. This review addresses recent specific applications, including those associated to the growth of embryonic and induced pluripotent stem cells. In addition, we have also dealt with safety issues, including feeder cell sources, as major factors of concern for clinical applications.

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“…Similar results with feeder layers points to a protective effect of culture, upon feeder layers or extracellular matrix (ECM) proteins, on karyotype stability (Escobedo-Lucea and Stojkovic 2010; Lee et al 2012) not only for ESC but also in IPSCs (Nakagawa et al 2014;Ghasemi-Dehkordi et al 2015), mESCs cultured on feeder layers made of hUSSCs maintain normal karyotype (Keshel et al 2012). Feeder layer made up of bone marrow MSCs maintained the adequate phenotype in ESCs and absence of aneuploidy (Lee et al 2012). The use of smaller feeder layer like the microdrop culture method have been applied for the generation of bovine ESCs with favorable results regarding pluripotency and the conservation of a normal karyotype (Kim et al 2012a, b).…”

Section: Embryonic Stem Cellsmentioning

confidence: 61%

A Multilevel Approach to the Causes of Genetic Instability in Stem Cells

González

2022

Handbook of Stem Cell Therapy

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This chapter addresses the genetic instability in stem cells, a central feature that is an important determinant for the safety and effectiveness of cell-based therapies. First, DNA damage response mechanism and the gene network that regulates the DNA integrity homeostasis are revisited, focusing on relationship between genetic integrity and stemness maintenance. Also, several factors have been included that influence genetic instability in vivo, i.e., ROS generation and inflammation, and in vitro regarding cell isolation, culture conditions, i.e., oxygen levels and the use of feeder layers and different carriers, splitting procedures, passage number, etc. Telomere shortening, replicative senescence aneuploidy, and the senescence-associated secretory phenotype are different processes involved in deterioration of stem cells abilities for homing, reparability, and paracrine modulation. Moreover, less effective DNA repair upon senescence increases the propensity of developing tumors for stem cells that is one the main concerns related to genetic instability in stem cells. Nuclear organization and their determinants linked to chromosome aberrations and aneuploidy in stem cells and those epigenetic changes affecting stability are addressed. Differences between adipose, embryonic, and induced pluripotent stem cells are analyzed regarding to their ontogeny, changes in culture, and variation in proliferative capacity and stemness. The effect of reprogramming methods in genetic instability and variation in mutagenicity according to genes utilized for pluripotency induction, i.e., Yamanaka's factors and others, is discussed. Donor-related factors, i.e., age, smoking, and alcohol consumption, comorbidities, i.e., obesity, insulin resistance, diabetes, are mentioned for wide evaluation of genetic instability. The next years must witness consensus protocols that will contribute to control the effects of factors that alter stem cell genetic stability to increase the security and applicability of cell-based therapy.

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Replacement of mouse embryonic fibroblasts with bone marrow stromal cells for use in establishing and maintaining embryonic stem cells in mice

Cited by 7 publications

References 15 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

Feeder Layer Cell Actions and Applications

A Multilevel Approach to the Causes of Genetic Instability in Stem Cells

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