Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions

Ilić, Duško; Stephenson, Emma; Wood, Victoria; Jacquet, Laureen; Stevenson, Danielle; Petrova, Anastasia; Kadeva, Neli; Codognotto, Stefano; Patel, Harshad B.; Semple, M J; Cornwell, Glenda; Ogilvie, Caroline Mackie; Braude, Peter

doi:10.3109/14653249.2011.623692

Cited by 77 publications

(97 citation statements)

References 22 publications

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“…They found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells continued proliferation, than bovine embryonic fibroblasts. However, feeder cells represent a potential source of putative pathogens and thus a component we shall not try to employ in our work as others did [4,6,10].…”

Section: Discussionmentioning

confidence: 99%

“…Despite the announcement of the creation of the first clinical-grade hESC line [6], with potential therapeutic applications, there is still the need for more research to develop protocols that are economically viable and repeatable under different laboratory settings and also safe, for clinical use.…”

Section: Discussionmentioning

confidence: 99%

“…These cell lines were generated in presence of human foreskin fibroblast feeders and cultured in defined medium supplemented with human serum [4]. In addition, two independend groups claim to have generated xeno-free clinical grade hESC lines [6,10], also using feeder layers for derivation and thereafter culturing them in feeder-free systems.…”

Section: Introductionmentioning

confidence: 99%

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The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Ruggeri

Watanabe²,

Meirelles³

et al. 2012

J Assist Reprod Genet

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Purposes Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions. Methods Cumulus-oocyte-complexes were in vitro matured and activated using Ca 2+ Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemPro® medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4. Results Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39 % and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker. Conclusion Our data show that Ca 2+ Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Ruggeri

Watanabe²,

Meirelles³

et al. 2012

J Assist Reprod Genet

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“…Excitingly, a recent study has demonstrated the derivation and growth of hESCs that are potentially pure enough to be used in therapies and have deposited these in the UK Stem Cell banks, which will be available to laboratories across Europe (Ilic et al, 2011). Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (Huntington's disease and myotonic dystrophy 1) genomically stable hESC lines, and their adaptation to feeder-free culture, all under completely xeno-free conditions, using human fetal fibroblast extracellular matrix as a growth substrate and TeSR™2, an improved version of mTeSR®1, as a growth medium.…”

Section: Synthetic Ra Analoguesmentioning

confidence: 99%

“…Such diseases/ disorders include Parkinson's disease (Chung et al, 2011;Kriks et al, 2011;Kim et al, 2011b), retinal degeneration (Tucker et al, 2011), spinal chord injury (Nori et al, 2011), hypopigmentation disorders (Nissan et al, 2011), Alzheimer's disease (Bissonnette et al, 2011) and orthopaedic disease (Bilousova et al, 2011), and efficient protocols for derivation of specific cell types from hESC and hiPSCs may lead to the use of such cells to treat human disease. To this end, multiple small molecule drugs that can modulate the differentiation of clinical-grade hESCs (Ilic et al, 2011) or hiPSCs have been discovered and may be used in the future in cGMP-compliant differentiation protocols to produce transplantable cells. Refinements in differentiation protocols, such as the application of such drugs, reducing cell time in culture and starting with a good source of hESCs, may all contribute to providing a source of karyotypically and phenotypically stable cells for transplantation purposes.…”

Section: Pharmacological Control Of Differentiationmentioning

confidence: 99%

Potential for pharmacological manipulation of human embryonic stem cells

Atkinson

Lako

Armstrong

2013

British J Pharmacology

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The therapeutic potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is vast, allowing disease modelling, drug discovery and testing and perhaps most importantly regenerative therapies. However, problems abound; techniques for cultivating self‐renewing hESCs tend to give a heterogeneous population of self‐renewing and partially differentiated cells and general include animal‐derived products that can be cost‐prohibitive for large‐scale production, and effective lineage‐specific differentiation protocols also still remain relatively undefined and are inefficient at producing large amounts of cells for therapeutic use. Furthermore, the mechanisms and signalling pathways that mediate pluripotency and differentiation are still to be fully appreciated. However, over the recent years, the development/discovery of a range of effective small molecule inhibitors/activators has had a huge impact in hESC biology. Large‐scale screening techniques, coupled with greater knowledge of the pathways involved, have generated pharmacological agents that can boost hESC pluripotency/self‐renewal and survival and has greatly increased the efficiency of various differentiation protocols, while also aiding the delineation of several important signalling pathways. Within this review, we hope to describe the current uses of small molecule inhibitors/activators in hESC biology and their potential uses in the future.LINKED ARTICLES This article is part of a themed section on Regenerative Medicine and Pharmacology: A Look to the Future. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue‐2

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