How to generate induced pluripotent stem cells

HOW TO GENERATE INDUCED PLURIPOTENT STEM CELLS
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Introduction
Having the self-renewal potential and the ability to differentiate into different types of cells, stem cells, especially the embryonic stem cells (ESC), have gained a lot of attention over the past few years. Just as ESC studies have played a significant role in understanding the underlying reproduction mechanisms, cell differentiation, and de-differentiation, as well as their development, important efforts have also been made in the biomedical research of the ESC in the past few years. One of the main areas of research has been the production of the iPSC. There are many ways of generating iPSC, but this paper looks at the process of generating induced pluripotent stem cells by reprogramming fibroblasts in humans with the aid of stemgent human TF lentivirus set.
Discussion
The process begins with the reprogramming the human ES/iPS cells. This process takes approximately seven days. Reprogramming is then followed by the preparation of the MEF conditioned medium ADDIN CSL_CITATION { “citationItems” : [ { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.3791/1553”, “ISBN” : “1940-087X (Electronic)”, “ISSN” : “1940-087X”, “PMID” : “19997062”, “abstract” : “In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.(1) The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.

(2) Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.(3) iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells. Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.”, “author” : [ { “dropping-particle” : “”, “family” : “Wu”, “given” : “Dongmei”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Hamilton”, “given” : “Brad”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Martin”, “given” : “Charles”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Gao”, “given” : “Yan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Ye”, “given” : “Mike”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yao”, “given” : “Shuyuan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } ], “container-title” : “Journal of visualized experiments : JoVE”, “id” : “ITEM-1”, “issue” : “34”, “issued” : { “date-parts” : [ [ “2009” ] ] }, “page” : “e1553”, “title” : “Generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human TF lentivirus set.”, “type” : “article-journal” }, “uris” : [ “http://www.mendeley.com/documents/?uuid=d4f4bddf-f9f1-471c-a4aa-82e87bc90044” ] } ], “mendeley” : { “formattedCitation” : “(Wu <i>et al.</i>, 2009)”, “plainTextFormattedCitation” : “(Wu et al., 2009)”, “previouslyFormattedCitation” : “(Wu <i>et al.</i>, 2009)” }, “properties” : { }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Wu, D. et al. 2009, 1553). This process takes four days to complete. The third stage involves the selection and passaging of the iPS. This process involves the selection of the ES-like colony and re-seeding it in the human ES/iPS culture mediumADDIN CSL_CITATION { “citationItems” : [ { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1016/j.stem.2009.05.005.Generation”, “ISBN” : “1934-5909”, “ISSN” : “19345909”, “PMID” : “19481515”, “abstract” : “To-date, all methods to generate induced pluripotent stem (iPS) cells require the use of genetic materials and/or potentially mutagenic molecules. Here we report the generation of stable iPS cells from human fibroblasts by directly delivering four reprogramming proteins (Oct4, Sox2, Klf4, and c-Myc) fused with a cell penetrating peptide (CPP). These protein-induced human iPS (p-hiPS) cells exhibited similarity to human embryonic stem (hES) cells in morphology, proliferation, and expression of characteristic pluripotency markers. p-hiPS cell lines produced with these recombinant proteins were successfully maintained for more than 35 passages and differentiated into derivatives of all three embryonic germ layers both in vitro and in teratomas. This system eliminates the potential risks associated with the use of viruses, DNA transfection, and potentially harmful chemicals, and in the future could potentially provide a safe source of patient-specific cells for regenerative medicine.”, “author” : [ { “dropping-particle” : “”, “family” : “Kim”, “given” : “Dohoon”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kim”, “given” : “Chun-Hyung”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Moon”, “given” : “Jung-Il”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Chung”, “given” : “Young-Gie”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Chang”, “given” : “Mi-Yoon”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Han”, “given” : “Baek-Soo”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Ko”, “given” : “Sangheyeok”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yang”, “given” : “Eungi”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Cha”, “given” : “Kwang Yul”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Robert”, “given” : “Lanza”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “KChang”, “given” : “M-Y”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Han”, “given” : “B-S”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Ko”, “given” : “S”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yang”, “given” : “E”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Cha”, “given” : “K Y”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Lanza”, “given” : “R”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kim”, “given” : “K-S”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } ], “container-title” : “Cell Stem Cell”, “id” : “ITEM-1”, “issue” : “6”, “issued” : { “date-parts” : [ [ “2009” ] ] }, “page” : “472-476”, “title” : “Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins”, “type” : “article-journal”, “volume” : “4” }, “uris” : [ “http://www.mendeley.com/documents/?uuid=7fc771ca-f6f8-4a58-815b-71ee4a8c85fa” ] } ], “mendeley” : { “formattedCitation” : “(Kim <i>et al.</i>, 2009)”, “plainTextFormattedCitation” : “(Kim et al., 2009)”, “previouslyFormattedCitation” : “(Kim <i>et al.</i>, 2009)” }, “properties” : { }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” } (Kim, D. et al. 2009, 475). The next process is that of immunocytochemical examination of the pluripotency markers. This process involves a series of washing the cells in the PBS, and other immunocytochemical processes, and ends by the analysis of the cells under the microscope. The last process is that of representing the realized results. This process involves the analysis of the morphological features of the iPSC and their expressed pluripotency markersADDIN CSL_CITATION { “citationItems” : [ { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1038/cr.2011.107”, “ISBN” : “1748-7838 (Electronic)\r1001-0602 (Linking)”, “ISSN” : “10010602”, “PMID” : “21709693”, “abstract” : “Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.”, “author” : [ { “dropping-particle” : “”, “family” : “Moon”, “given” : “Jai Hee”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Heo”, “given” : “June Seok”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kim”, “given” : “Jun Sung”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Jun”, “given” : “Eun Kyoung”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Lee”, “given” : “Jung Han”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kim”, “given” : “Aeree”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kim”, “given” : “Jonggun”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Whang”, “given” : “Kwang Youn”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kang”, “given” : “Yong Kook”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yeo”, “given” : “Seungeun”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Lim”, “given” : “Hee Joung”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Han”, “given” : “Dong Wook”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kim”, “given” : “Dong Wook”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Oh”, “given” : “Sejong”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yoon”, “given” : “Byung Sun”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Schu00f6ler”, “given” : “Hans R.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “You”, “given” : “Seungkwon”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } ], “container-title” : “Cell Research”, “id” : “ITEM-1”, “issue” : “9”, “issued” : { “date-parts” : [ [ “2011” ] ] }, “page” : “1305-1315”, “title” : “Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1”, “type” : “article-journal”, “volume” : “21” }, “uris” : [ “http://www.mendeley.com/documents/?uuid=e50d2a11-8aba-4980-b0ee-4570ed9416ed” ] } ], “mendeley” : { “formattedCitation” : “(Moon <i>et al.</i>, 2011)”, “plainTextFormattedCitation” : “(Moon et al., 2011)”, “previouslyFormattedCitation” : “(Moon <i>et al.</i>, 2011)” }, “properties” : { }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” } (Moon, J. H. et al. 2011, 1310).
Conclusion
In summary, the process of generating iPSC involves five stages namely reprogramming, the process of preparing the MEF conditioned media, the selection and the passage of the iPS, the process of immunocytochemical examination of the pluripotency markers, and the presentation of the results.
References
ADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY Kim, D. et al. (2009) ‘Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins,’ Cell Stem Cell, 4(6), pp. 472–476. doi: 10.1016/j.stem.2009.05.005.Generation.
Moon, J. H. et al. (2011) ‘Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1’, Cell Research, 21(9), pp. 1305–1315. doi: 10.1038/cr.2011.107.
Wu, D. et al. (2009) ‘Generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human TF lentivirus set.’, Journal of visualized experiments : JoVE, (34), p. e1553. doi: 10.3791/1553.