Int J Stem Cells.  2016 Nov;9(2):176-185. 10.15283/ijsc16029.

Induced Pluripotent Stem (iPS) Cells in Dentistry: A Review

• Malhotra N

• KMID: 2362511
• DOI: http://doi.org/10.15283/ijsc16029

Abstract

iPS cells are derived from somatic cells via transduction and expression of selective transcription factors. Both viral-integrating (like retroviral) and non-integrating (like, mRNA or protein-based) techniques are available for the production of iPS cells. In the field of dentistry, iPS cells have been derived from stem cells of apical papilla, dental pulp stem cells, and stem cells from exfoliated deciduous teeth, gingival and periodontal ligament fibroblasts, and buccal mucosa fibroblasts. iPS cells have the potential to differentiate into all derivatives of the 3 primary germ layers i.e. ectoderm, endoderm, and mesoderm. They are autogeneically accessible, and can produce patient-specific or disease-specific cell lines without the issue of ethical controversy. They have been successfully tested to produce mesenchymal stem cells-like cells, neural crest-like cells, ameloblasts-like cells, odontoblasts-like cells, and osteoprogenitor cells. These cells can aid in regeneration of periodontal ligament, alveolar bone, cementum, dentin-pulp complex, as well as possible Biotooth formation. However certain key issues like, epigenetic memory of iPS cells, viral-transduction, tumorgenesis and teratoma formation need to be overcome, before they can be successfully used in clinical practice. The article discusses the sources, pros and cons, and current applications of iPS cells in dentistry with an emphasis on encountered challenges and their solutions.

Keyword

Autogenic; Epigenetic memory; iPS cells; Regenerative dentistry; Teratoma; Transduction

MeSH Terms

Cell Line
Dental Cementum
Dental Papilla
Dentistry*
Ectoderm
Endoderm
Epigenomics
Fibroblasts
Germ Layers
Induced Pluripotent Stem Cells
Memory
Mesoderm
Mouth Mucosa
Periodontal Ligament
Regeneration
RNA, Messenger
Stem Cells
Teratoma
Tooth, Deciduous
Transcription Factors
RNA, Messenger
Transcription Factors

Figure

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  Fig. 1 Illustration showing sources, generation and application of iPS cells in dentistry.

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  Fig. 2 Illustration showing approaches to generate mesenchymal stem cells-like cells (MSLCs) from human iPS cells.

Reference

References

1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126:663–676. DOI: 10.1016/j.cell.2006.07.024. PMID: 16904174.

2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131:861–872. DOI: 10.1016/j.cell.2007.11.019. PMID: 18035408.

3. Ebert AD, Liang P, Wu JC. Induced pluripotent stem cells as a disease modeling and drug screening platform. J Cardiovasc Pharmacol. 2012; 60:408–416. DOI: 10.1097/FJC.0b013e318247f642. PMID: 22240913. PMCID: 3343213.

4. Egusa H, Sonoyama W, Nishimura M, Atsuta I, Akiyama K. Stem cells in dentistry--part I: stem cell sources. J Prosthodont Res. 2012; 56:151–165. DOI: 10.1016/j.jpor.2012.06.001. PMID: 22796367.

5. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318:1917–1920. DOI: 10.1126/science.1151526. PMID: 18029452.

6. Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S. A more efficient method to generate integration-free human iPS cells. Nat Methods. 2011; 8:409–412. DOI: 10.1038/nmeth.1591. PMID: 21460823.

7. Yu J, Chau KF, Vodyanik MA, Jiang J, Jiang Y. Efficient feeder-free episomal reprogramming with small molecules. PLoS One. 2011; 6:e17557. DOI: 10.1371/journal.pone.0017557. PMID: 21390254. PMCID: 3046978.

8. Silva M, Daheron L, Hurley H, Bure K, Barker R, Carr AJ, Williams D, Kim HW, French A, Coffey PJ, Cooper-White JJ, Reeve B, Rao M, Snyder EY, Ng KS, Mead BE, Smith JA, Karp JM, Brindley DA, Wall I. Generating iPSCs: translating cell reprogramming science into scalable and robust biomanufacturing strategies. Cell Stem Cell. 2015; 16:13–17. DOI: 10.1016/j.stem.2014.12.013. PMID: 25575079.

9. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 2008; 26:101–106. DOI: 10.1038/nbt1374.

10. Okita K, Ichisaka T, Yamanaka S. Generation of germ-line-competent induced pluripotent stem cells. Nature. 2007; 448:313–317. DOI: 10.1038/nature05934. PMID: 17554338.

11. Amabile G, Meissner A. Induced pluripotent stem cells: current progress and potential for regenerative medicine. Trends Mol Med. 2009; 15:59–68. DOI: 10.1016/j.molmed.2008.12.003. PMID: 19162546.

12. Otsu K, Kumakami-Sakano M, Fujiwara N, Kikuchi K, Keller L, Lesot H, Harada H. Stem cell sources for tooth regeneration: current status and future prospects. Front Physiol. 2014; 5:36. DOI: 10.3389/fphys.2014.00036. PMID: 24550845. PMCID: 3912331.

13. Hiyama T, Ozeki N, Mogi M, Yamaguchi H, Kawai R, Nakata K, Kondo A, Nakamura H. Matrix metalloproteinase-3 in odontoblastic cells derived from ips cells: unique proliferation response as odontoblastic cells derived from ES cells. PLoS One. 2013; 8:e83563. DOI: 10.1371/journal.pone.0083563. PMID: 24358294. PMCID: 3865184.

14. Egusa H, Kayashima H, Miura J, Uraguchi S, Wang F, Okawa H, Sasaki J, Saeki M, Matsumoto T, Yatani H. Comparative analysis of mouse-induced pluripotent stem cells and mesenchymal stem cells during osteogenic differentiation in vitro. Stem Cells Dev. 2014; 23:2156–2169. DOI: 10.1089/scd.2013.0344. PMID: 24625139. PMCID: 4155416.

15. Yan X, Qin H, Qu C, Tuan RS, Shi S, Huang GT. iPS cells reprogrammed from human mesenchymal-like stem/progenitor cells of dental tissue origin. Stem Cells Dev. 2010; 19:469–480. DOI: 10.1089/scd.2009.0314. PMCID: 2851830.

16. Tamaoki N, Takahashi K, Tanaka T, Ichisaka T, Aoki H, Takeda-Kawaguchi T, Iida K, Kunisada T, Shibata T, Yamanaka S, Tezuka K. Dental pulp cells for induced pluripotent stem cell banking. J Dent Res. 2010; 89:773–778. DOI: 10.1177/0022034510366846. PMID: 20554890.

17. Oda Y, Yoshimura Y, Ohnishi H, Tadokoro M, Katsube Y, Sasao M, Kubo Y, Hattori K, Saito S, Horimoto K, Yuba S, Ohgushi H. Induction of pluripotent stem cells from human third molar mesenchymal stromal cells. J Biol Chem. 2010; 285:29270–29278. DOI: 10.1074/jbc.M109.055889. PMID: 20595386. PMCID: 2937959.

18. Miyoshi K, Tsuji D, Kudoh K, Satomura K, Muto T, Itoh K, Noma T. Generation of human induced pluripotent stem cells from oral mucosa. J Biosci Bioeng. 2010; 110:345–350. DOI: 10.1016/j.jbiosc.2010.03.004. PMID: 20547351.

19. Wada N, Wang B, Lin NH, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts. J Periodontal Res. 2011; 46:438–447. DOI: 10.1111/j.1600-0765.2011.01358.x. PMID: 21443752.

20. Arakaki M, Ishikawa M, Nakamura T, Iwamoto T, Yamada A, Fukumoto E, Saito M, Otsu K, Harada H, Yamada Y, Fukumoto S. Role of epithelial-stem cell interactions during dental cell differentiation. J Biol Chem. 2012; 287:10590–10601. DOI: 10.1074/jbc.M111.285874. PMID: 22298769. PMCID: 3323010.

21. Otsu K, Kishigami R, Oikawa-Sasaki A, Fukumoto S, Yamada A, Fujiwara N, Ishizeki K, Harada H. Differentiation of induced pluripotent stem cells into dental mesenchymal cells. Stem Cells Dev. 2011; 21:1156–1164. DOI: 10.1089/scd.2011.0210. PMID: 22085204.

22. Ono H, Obana A, Usami Y, Sakai M, Nohara K, Egusa H, Sakai T. Regenerating salivary glands in the microenvironment of induced pluripotent stem cells. Biomed Res Int. 2015; 2015:293570. DOI: 10.1155/2015/293570. PMID: 26185754. PMCID: 4491559.

23. Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, Kim J, Aryee MJ, Ji H, Ehrlich LI, Yabuuchi A, Takeuchi A, Cunniff KC, Hongguang H, McKinney-Freeman S, Naveiras O, Yoon TJ, Irizarry RA, Jung N, Seita J, Hanna J, Murakami P, Jaenisch R, Weissleder R, Orkin SH, Weissman IL, Feinberg AP, Daley GQ. Epigenetic memory in induced pluripotent stem cells. Nature. 2010; 467:285–290. DOI: 10.1038/nature09342. PMID: 20644535. PMCID: 3150836.

24. Polo JM, Liu S, Figueroa ME, Kulalert W, Eminli S, Tan KY, Apostolou E, Stadtfeld M, Li Y, Shioda T, Natesan S, Wagers AJ, Melnick A, Evans T, Hochedlinger K. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat Biotechnol. 2010; 28:848–855. DOI: 10.1038/nbt.1667. PMID: 20644536. PMCID: 3148605.

25. Hynes K, Menicanin D, Mrozik K, Gronthos S, Bartold PM. Generation of functional mesenchymal stem cells from different induced pluripotent stem cell lines. Stem Cells Dev. 2014; 23:1084–1096. DOI: 10.1089/scd.2013.0111. PMCID: 4015475.

26. Hynes K, Menichanin D, Bright R, Ivanovski S, Hutmacher DW, Gronthos S, Bartold PM. Induced pluripotent stem cells: a new frontier for stem cells in dentistry. J Dent Res. 2015; 94:1508–1515. DOI: 10.1177/0022034515599769. PMID: 26285811.

27. Beltrão-Braga PC, Pignatari GC, Maiorka PC, Oliveira NA, Lizier NF, Wenceslau CV, Miglino MA, Muotri AR, Kerkis I. Feeder-free derivation of induced pluripotent stem cells from human immature dental pulp stem cells. Cell Transplant. 2011; 20:1707–1719. DOI: 10.3727/096368911X566235. PMID: 21457612.

28. Toriumi T, Takayama N, Murakami M, Sato M, Yuguchi M, Yamazaki Y, Eto K, Otsu M, Nakauchi H, Shirakawa T, Isokawa K, Honda MJ. Characterization of mesenchymal progenitor cells in the crown and root pulp of primary teeth. Biomed Res. 2015; 36:31–45. DOI: 10.2220/biomedres.36.31. PMID: 25749149.

29. Zhang Q, Shi S, Liu Y, Uyanne J, Shi Y, Shi S, Le AD. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis. J Immunol. 2009; 183:7787–7798. DOI: 10.4049/jimmunol.0902318. PMID: 19923445. PMCID: 2881945.

30. Egusa H, Okita K, Kayashima H, Yu G, Fukuyasu S, Saeki M, Matsumoto T, Yamanaka S, Yatani H. Gingival fibroblasts as a promising source of induced pluripotent stem cells. PLoS One. 2010; 5:e12743. DOI: 10.1371/journal.pone.0012743. PMID: 20856871. PMCID: 2939066.

31. Umezaki Y, Hashimoto Y, Nishishita N, Kawamata S, Baba S. Human gingival integration-free iPSCs; a source for MSC-like cells. Int J Mol Sci. 2015; 16:13633–13648. DOI: 10.3390/ijms160613633. PMID: 26084043. PMCID: 4490513.

32. Lian Q, Zhang Y, Zhang J, Zhang HK, Wu X, Zhang Y, Lam FF, Kang S, Xia JC, Lai WH, Au KW, Chow YY, Siu CW, Lee CN, Tse HF. Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Circulation. 2010; 121:1113–1123. DOI: 10.1161/CIRCULATIONAHA.109.898312. PMID: 20176987.

33. Jung Y, Bauer G, Nolta JA. Concise review: induced pluripotent stem cell-derived mesenchymal stem cells: progress toward safe clinical products. Stem Cells. 2012; 30:42–47. DOI: 10.1002/stem.727.

34. Otsu K, Fujiwara N, Harada H. Organ cultures and kidney-capsule grafting of tooth germs. Methods Mol Biol. 2012; 887:59–67. DOI: 10.1007/978-1-61779-860-3_7. PMID: 22566047.

35. Yoshida K, Sato J, Takai R, Uehara O, Kurashige Y, Nishimura M, Chiba I, Saitoh M, Abiko Y. Differentiation of mouse iPS cells into ameloblast-like cells in cultures using medium conditioned by epithelial cell rests of Malassez and gelatin-coated dishes. Med Mol Morphol. 2015; 48:138–145. DOI: 10.1007/s00795-014-0088-6.

36. Liu JS, Cheung M. Neural Crest stem cells/progenitors and their potential applications in disease therapies. Journal of Stem Cell Research & Therapeutics. 2016; 1:00014.

37. Seki D, Takeshita N, Oyanagi T, Sasaki S, Takano I, Hasegawa M, Takano-Yamamoto T. Differentiation of odontoblast-like cells from mouse induced pluripotent stem cells by Pax9 and Bmp4 transfection. Stem Cells Transl Med. 2015; 4:993–997. DOI: 10.5966/sctm.2014-0292. PMID: 26136503. PMCID: 4542869.

38. Ozeki N, Mogi M, Kawai R, Yamaguchi H, Hiyama T, Nakata K, Nakamura H. Mouse-induced pluripotent stem cells differentiate into odontoblast-like cells with induction of altered adhesive and migratory phenotype of integrin. PLoSONE. 2013; 8:e80026. DOI: 10.1371/journal.pone.0080026.

39. Liu L, Liu YF, Zhang J, Duan YZ, Jin Y. Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells. J Tissue Eng Regen Med. 2013; 10:466–474. DOI: 10.1002/term.1742. PMID: 23606575.

40. Duan X, Tu Q, Zhang J, Ye J, Sommer C, Mostoslavsky G, Kaplan D, Yang P, Chen J. Application of induced pluripotent stem (iPS) cells in periodontal tissue regeneration. J Cell Physiol. 2011; 226:150–157. DOI: 10.1002/jcp.22316.

41. Hynes K, Menicanin D, Han J, Marino V, Mrozik K, Gronthos S, Bartold PM. Mesenchymal stem cells from iPS cells facilitate periodontal regeneration. J Dent Res. 2013; 92:833–839. DOI: 10.1177/0022034513498258. PMID: 23884555.

42. Yin X, Li Y, Li J, Li P, Liu Y, Wen J, Luan Q. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells. Biochem Biophys Res Commun. 2016; 473:726–732. DOI: 10.1016/j.bbrc.2015.10.012.

43. Yang H, Aprecio RM, Zhou X, Wang Q, Zhang W, Ding Y, Li Y. Therapeutic effect of TSG-6 engineered iPSC-derived MSCs on experimental periodontitis in rats: a pilot study. PLoS One. 2014; 9:e100285. DOI: 10.1371/journal.pone.0100285. PMID: 24979372. PMCID: 4076279.

44. Cai J, Zhang Y, Liu P, Chen S, Wu X, Sun Y, Li A, Huang K, Luo R, Wang L, Liu Y, Zhou T, Wei S, Pan G, Pei D. Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells. Cell Regen (Lond). 2013; 2:6. DOI: 10.1186/2045-9769-2-6.

45. Ibarretxe G, Alvarez A, Cañavate ML, Hilario E, Aurrekoetxea M, Unda F. Cell reprogramming, IPS limitations, and overcoming strategies in dental bioengineering. Stem Cells Int. 2012; 2012:365932. DOI: 10.1155/2012/365932. PMID: 22690226. PMCID: 3368489.

46. Liu J, Chen W, Zhao Z, Xu HH. Reprogramming of mesenchymal stem cells derived from iPSCs seeded on biofunctionalized calcium phosphate scaffold for bone engineering. Biomaterials. 2013; 34:7862–7872. DOI: 10.1016/j.biomaterials.2013.07.029. PMID: 23891395. PMCID: 3845377.

47. Wang P, Zhao L, Chen W, Liu X, Weir MD, Xu HH. Stem cells and calcium phosphate cement scaffolds for bone regeneration. J Dent Res. 2014; 93:618–625. DOI: 10.1177/0022034514534689. PMID: 24799422. PMCID: 4107550.

48. TheinHan W, Liu J, Tang M, Chen W, Cheng L, Xu HH. Induced pluripotent stem cell-derived mesenchymal stem cell seeding on biofunctionalized calcium phosphate cements. Bone Res. 2013; 4:371–384. DOI: 10.4248/BR201304008. PMID: 24839581. PMCID: 4023512.

49. Tang M, Chen W, Liu J, Weir MD, Cheng L, Xu HH. Human induced pluripotent stem cell-derived mesenchymal stem cell seeding on calcium phosphate scaffold for bone regeneration. Tissue Eng Part A. 2014; 20:1295–1305. DOI: 10.1089/ten.tea.2013.0211. PMCID: 3993076.

50. Bar-Nur O, Russ HA, Efrat S, Benvenisty N. Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells. Cell Stem Cell. 2011; 9:17–23. DOI: 10.1016/j.stem.2011.06.007. PMID: 21726830.

51. Vaskova EA, Stekleneva AE, Medvedev S, Zakian SM. “Epigenetic memory” phenomenon in induced pluripotent stem cells”. Acta Naturae. 2013; 5:15–21.

52. Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, Kim J, Aryee MJ, Ji H, Ehrlich LI, Yabuuchi A, Takeuchi A, Cunniff KC, Hongguang H, McKinney-Freeman S, Naveiras O, Yoon TJ, Irizarry RA, Jung N, Seita J, Hanna J, Murakami P, Jaenisch R, Weissleder R, Orkin SH, Weissman IL, Feinberg AP, Daley GQ. Epigenetic memory in induced pluripotent stem cells. Nature. 2010; 467:285–290. DOI: 10.1038/nature09342. PMID: 20644535. PMCID: 3150836.

53. Kim K, Zhao R, Doi A, Ng K, Unternaehrer J, Cahan P, Huo H, Loh YH, Aryee MJ, Lensch MW, Li H, Collins JJ, Feinberg AP, Daley GQ. Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells. Nat Biotechnol. 2011; 29:1117–1119. DOI: 10.1038/nbt.2052. PMID: 22119740. PMCID: 3357310.

54. Stadtfeld M, Apostolou E, Akutsu H, Fukuda A, Follett P, Natesan S, Kono T, Shioda T, Hochedlinger K. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature. 2010; 465:175–181. DOI: 10.1038/nature09017. PMID: 20418860. PMCID: 3987905.

55. Nishino K, Toyoda M, Yamazaki-Inoue M, Fukawatase Y, Chikazawa E, Sakaguchi H, Akutsu H, Umezawa A. DNA methylation dynamics in human induced pluripotent stem cells over time. PLoS Genet. 2011; 7:e1002085. DOI: 10.1371/journal.pgen.1002085. PMID: 21637780. PMCID: 3102737.

56. Ma H, Morey R, O’Neil RC, He Y, Daughtry B, Schultz MD, Hariharan M, Nery JR, Castanon R, Sabatini K, Thiagarajan RD, Tachibana M, Kang E, Tippner-Hedges R, Ahmed R, Gutierrez NM, Van Dyken CV, Polat A, Sugawara A, Sparman M, Gokhale S, Amato P, Wolf DP, Ecker JR, Laurent LC, Mitalipov S. Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature. 2014; 511:177–183. DOI: 10.1038/nature13551. PMID: 25008523. PMCID: 4898064.

57. Schuldiner M, Itskovitz-Eldor J, Benvenisty N. Selective ablation of human embryonic stem cells expressing a “suicide” gene. Stem Cells. 2003; 21:257–265. DOI: 10.1634/stemcells.21-3-257.

58. Tang C, Lee AS, Volkmer JP, Sahoo D, Nag D, Mosley AR, Inlay MA, Ardehali R, Chavez S, Pera RR, Behr B, Wu JC, Weissman IL, Drukker M. An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells. Nat Biotechnol. 2011; 29:829–834. DOI: 10.1038/nbt.1947. PMID: 21841799. PMCID: 3537836.

59. Lee MO, Moon SH, Jeong HC, Yi JY, Lee TH, Shim SH, Rhee YH, Lee SH, Oh SJ, Lee MY, Han MJ, Cho YS, Chung HM, Kim KS, Cha HJ. Inhibition of pluripotent stem cell-derived teratoma formation by small molecules. Proc Natl Acad Sci U S A. 2013; 110:E3281–3290. DOI: 10.1073/pnas.1303669110. PMID: 23918355. PMCID: 3761568.

60. Ben-David U, Nudel N, Benvenisty N. Immunologic and chemical targeting of the tight-junction protein Claudin-6 eliminates tumorigenic human pluripotent stem cells. Nat Commun. 2013; 4:1992. DOI: 10.1038/ncomms2992. PMID: 23778593.

61. Ben-David U, Gan QF, Golan-Lev T, Arora P, Yanuka O, Oren YS, Leikin-Frenkel A, Graf M, Garippa R, Boehringer M, Gromo G, Benvenisty N. Selective elimination of human pluripotent stem cells by an oleate synthesis inhibitor discovered in a high-throughput screen. Cell Stem Cell. 2013; 12:167–179. DOI: 10.1016/j.stem.2012.11.015. PMID: 23318055.

62. Cho SJ, Kim SY, Jeong HC, Cheong H, Kim D, Park SJ, Choi JJ, Kim H, Chung HM, Moon SH, Cha HJ. Repair of ischemic injury by pluripotent stem cell based cell therapy without teratoma through selective photosensitivity. Stem Cell Reports. 2015; 5:1067–1080. DOI: 10.1016/j.stemcr.2015.10.004. PMID: 26584542. PMCID: 4682089.

63. Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S. Generation of mouse induced pluripotent stem cells without viral vectors. Science. 2008; 322:949–953. DOI: 10.1126/science.1164270. PMID: 18845712.

64. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009; 324:797–801. DOI: 10.1126/science.1172482. PMID: 19325077. PMCID: 2758053.

65. Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, Kim KS. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 2009; 4:472–476. DOI: 10.1016/j.stem.2009.05.005. PMID: 19481515. PMCID: 2705327.

66. 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; 7:618–630. DOI: 10.1016/j.stem.2010.08.012. PMID: 20888316. PMCID: 3656821.

67. Zhao Q, Gregory CA, Lee RH, Reger RL, Qin L, Hai B, Park MS, Yoon N, Clough B, McNeill E, Prockop DJ, Liu F. MSCs derived from iPSCs with a modified protocol are tumor-tropic but have much less potential to promote tumors than bone marrow MSCs. Proc Natl Acad Sci U S A. 2015; 112:530–535. DOI: 10.1073/pnas.1423008112. PMCID: 4299223.

68. Nakagawa M, Takizawa N, Narita M, Ichisaka T, Yamanaka S. Promotion of direct reprogramming by transformation-deficient Myc. Proc Natl Acad Sci U S A. 2010; 107:14152–14157. DOI: 10.1073/pnas.1009374107. PMID: 20660764. PMCID: 2922531.

69. Villa-Diaz LG, Ross AM, Lahann J, Krebsbach PH. Concise review: the evolution of human pluripotent stem cell culture: from feeder cells to synthetic coatings. Stem Cells. 2013; 31:1–7. DOI: 10.1002/stem.1260. PMCID: 3537180.

70. Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, Clark AT, Plath K. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci U S A. 2008; 105:2883–2888. DOI: 10.1073/pnas.0711983105. PMID: 18287077. PMCID: 2268554.

71. Chan EM, Ratanasirintrawoot S, Park IH, Manos PD, Loh YH, Huo H, Miller JD, Hartung O, Rho J, Ince TA, Daley GQ, Schlaeger TM. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nature Biotechnol. 2009; 27:1033–1037. DOI: 10.1038/nbt.1580.

72. Wu DT, Seita Y, Zhang X, Lu CW, Roth MJ. Antibody-directed lentiviral gene transduction for live-cell monitoring and selection of human iPS and hES cells. PLoS One. 2012; 7:e34778. DOI: 10.1371/journal.pone.0034778. PMID: 22536330. PMCID: 3334894.