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pluripotent stem cells (PSC) - reprogramming
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pluripotent stem cells - reprogramming


In a now seminal experiment that challenged the concept of unidirectionality of developmental differentiation, Shinya Yamanaka and Kazutoshi Takahashi demonstrated that an adult somatic cell could be epigenetically reprogrammed to pluripotency with a quartet of key transcription factors known to be expressed in early preimplantation embryo development.   Reprogramming to pluripotency is now an established technology and routinely used in basic research, disease modeling and regenerative medicine. iPSC generation from individual patient cells may be acquired by noninvasive means such as collection of epithelial cells exfoliated from the kidneys in urine or the shedding of hair follicles to isolate dermal papilla cells, or by conventional methods like cell biopsy of dermal fibroblasts and phlebotomy to collect peripheral blood mononuclear cells (PBMCs).  A variety of avenues exist to generate iPSCs though, vehicles and biotypes vary considerably in efficiencies. Viral-mediated transduction robustly supports dedifferentiation to pluripotency through retroviral or DNA-viral routes but carries the onus of random insertional gene inactivation. Additionally, epigenetic reprogramming by enforced expression of OSKM through DNA routes exists such as plasmid DNA, minicircles, transposons, episomes and targeted integration into host chromosomes to express reprogramming genes has also been demonstrated; however, these methods suffer from the burden to potentially alter the recipient genome as they are intentionally integrated or may become integrated during the reprogramming process and would not be satisfactory for clinical translation. While protein-mediated transduction supports reprogramming adult cells to pluripotency, the method is cumbersome and requires recombinant protein expression and purification expertise, and reprograms albeit at very low frequencies (Kim et al., 2009). A major obstacle of using mRNA for reprogramming is its lability, short half-life and that single-stranded RNA biotypes trigger innate antiviral defense pathways such as interferon and NF-kB-dependent pathways. To achieve clinically relevant human iPSCs, a survey to compare non-integrating methods for pluripotency induction demonstrated that Sendai viral RNA was efficient and highly reliable (Schlaeger et al 2015¹). An overarching goal of scientists is to replace current reprogramming methods containing transgenes with small molecule compounds to induce pluripotency by chemical induction. These small molecules should be cell permeable, reversible and easily titrated to manipulate cell fates. Recent advances in cellular reprogramming to pluripotency by chemical induction alone is possible in some strains of mice with a seven-compound cocktail (VPA, CHIR99021, 616452, tranylcypromine, forskolin, AM580, and EPZ004777). However, efforts to translate these protocols to human cells to induce pluripotency by only small molecules has not been achieved. 

At NovoHelix, we offer a flexible pipeline to generate clinical-grade human iPS cells for modeling human genetic disorders or for translational applications such as screening FDA-approved chemical libraries for therapeutic targets.  We have developed effective strategies to generate human iPSC lines without animal products and xenogens, and use chemically defined media and extracellular substrates like recombinant human laminin or vitronectin to support feeder-free human iPSC culture.  For mouse ES and iPS cells, we provide a range of solutions for maintaining naïve pluripotency with defined culture systems such as N2B27 media containing LIF and 2i inhibitors.   
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 Tetraploid embryo/ ES cell Aggregation
CMPR001

 Tetraploid embryo/ ES or iPSC cell aggregation includes amplification and freezing of clonal stocks, chromosome counting, mycoplasma testing, 2-cell embryo collection, electrofusion of 2-cell embryos to tetraploid embryos, overnight culture, removal of zona pellucida, aggregation with targeted ES cells (2 times per clone) or 2 iPSC clones, overnight culture, transfer of the ES cells/tetraploid aggregated blastocysts to pseudopregnant female.  A minimum of 2 chimeras are almost completely ES cell derived will be produced.  C57BL6 and 129 Svev mice will be provided for germline transmission tests.  Additional injection will be charged at $3500 per session. Note use of ES cells or iPSCs have varying potentials to produce chimeras and/or confer germline transmission.

 iPSC Generation Service (episomal)

 CMPR002
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 iPSC Generation Service (retrovirus)

CMPR003
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 iPSC Generation Service (mRNA)

CMPR004
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 Embryoid Body (EB) Formation and Characterization Service
CMPR005
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 Human ES/iPS Cell Pluripotency Characterization Service
CMPR006
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 Human ES/iPS Cell Karyotyping Service (G-banding) 
CMPR007
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 Teratoma Formation Analysis

CMPR008
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references
Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 2010 Nov 5;7(5):618-30. doi: 10.1016/j.stem.2010.08.012. Epub 2010 Sep 30. PubMed PMID: 20888316; PubMed Central PMCID: PMC3656821.

Zhao Y. Chemically induced cell fate reprogramming and the acquisition of plasticity in somatic cells. Curr Opin Chem Biol. 2019 Aug;51:146-153. doi: 10.1016/j.cbpa.2019.04.025. Epub 2019 May 30. Review. PubMed PMID: 31153758.

Shakiba N, Fahmy A, Jayakumaran G, McGibbon S, David L, Trcka D, Elbaz J, Puri MC, Nagy A, van der Kooy D, Goyal S, Wrana JL, Zandstra PW. Cell competition during reprogramming gives rise to dominant clones. Science. 2019 Apr 26;364(6438). pii: eaan0925. doi: 10.1126/science.aan0925. Epub 2019 Mar 21. PubMed PMID: 30898844.

Warren L, Lin C. mRNA-Based Genetic Reprogramming. Mol Ther. 2019 Apr 10;27(4):729-734. doi: 10.1016/j.ymthe.2018.12.009. Epub 2018 Dec 14. Review. PubMed PMID: 30598301; PubMed Central PMCID: PMC6453511.

Mandal PK, Rossi DJ. Reprogramming human fibroblasts to pluripotency using modified mRNA. Nat Protoc. 2013 Mar;8(3):568-82. doi: 10.1038/nprot.2013.019. Epub 2013 Feb 21. PubMed PMID: 23429718.

Draper JM, Vivian JL. Reprogramming of Primary Human Cells to Induced Pluripotent Stem Cells Using Sendai Virus. Methods Mol Biol. 2020;2066:217-234. doi: 10.1007/978-1-4939-9837-1_18. PubMed PMID: 31512220.

Mahmoudi S, Mancini E, Xu L, Moore A, Jahanbani F, Hebestreit K, Srinivasan R, Li X, Devarajan K, Prélot L, Ang CE, Shibuya Y, Benayoun BA, Chang ALS, Wernig M, Wysocka J, Longaker MT, Snyder MP, Brunet A. Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing. Nature. 2019 Oct;574(7779):553-558. doi: 10.1038/s41586-019-1658-5. Epub 2019 Oct 23. PubMed PMID: 31645721.

Hotta A, Cheung AY, Farra N, Garcha K, Chang WY, Pasceri P, Stanford WL, Ellis J. EOS lentiviral vector selection system for human induced pluripotent stem cells. Nat Protoc. 2009;4(12):1828-44. doi: 10.1038/nprot.2009.201. PubMed PMID: 20010937.

Kuo HH, Gao X, DeKeyser JM, Fetterman KA, Pinheiro EA, Weddle CJ, Fonoudi H, Orman MV, Romero-Tejeda M, Jouni M, Blancard M, Magdy T, Epting CL, George AL Jr, Burridge PW. Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture. Stem Cell Reports. 2020 Feb 11;14(2):256-270. doi: 10.1016/j.stemcr.2019.12.007. Epub 2020 Jan 9. PubMed PMID: 31928950; PubMed Central PMCID: PMC7013200.

Bi Y, Tu Z, Zhang Y, Yang P, Guo M, Zhu X, Zhao C, Zhou J, Wang H, Wang Y, Gao S. Identification of ALPPL2 as a Naive Pluripotent State-Specific Surface Protein Essential for Human Naive Pluripotency Regulation. Cell Rep. 2020 Mar 17;30(11):3917-3931.e5. doi: 10.1016/j.celrep.2020.02.090. PubMed PMID: 32187559.

Theunissen TW, Friedli M, He Y, Planet E, O'Neil RC, Markoulaki S, Pontis J, Wang H, Iouranova A, Imbeault M, Duc J, Cohen MA, Wert KJ, Castanon R, Zhang Z, Huang Y, Nery JR, Drotar J, Lungjangwa T, Trono D, Ecker JR, Jaenisch R. Molecular Criteria for Defining the Naive Human Pluripotent State. Cell Stem Cell. 2016 Oct 6;19(4):502-515. doi: 10.1016/j.stem.2016.06.011. Epub 2016 Jul 14. PubMed PMID: 27424783; PubMed Central PMCID: PMC5065525.
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