The Stagnant Adaptation of Defined and Xeno-Free Culture of iPSCs in Academia
- 1. InvivoSciences, Inc., USA
Pluripotent Stem Cells were originally derived and cultured using a feeder layer of cells. Movements have been undertaken to transition from this method to one more defined, high-throughput, and without xenogenic factors. Tremendous research has been done in this area and many products have been developed, however, based on our analysis of recent publications in stem cell related journals many in academia are still using older methods like a feeder layer. In this short communication, we discuss the feasibility of transitioning to defined, xeno-free methods, how a standardized method could improve the field and industry, and that a study bringing together multiple institutions comparing culture methods could be done to evaluate the efficacy of these new methods.
Vecchi JT, Wakatsuki T (2016) The Stagnant Adaptation of Defined and Xeno-Free Culture of iPSCs in Academia. Arch Stem Cell Res 3(1): 1016.
• Stem cells
iPSC: Induced Pluripotent Stem Cells; MEF: Mouse Embryonic Fibroblasts; ESC: Embryonic Stem Cells; FBS: Fetal Bovine Serum; BSA: Bovine Serum Albumin
Several revolutionary technologies [1,2] have been developed recently to improve the consistency and clinical potential of iPSC research, however, many academic labs have yet to transition to these culture systems. This brief review will discuss some of the issues that pertain to this topic, in particular the history, the current state of the field, the costs associated with the culture techniques, and what this means for the goals of the field as a whole.
Pluripotent stem cells were first derived and cultured using a MEF feeder layer [3,4]. This was never meant to be their permanent culture method and there was a movement to eliminate the feeder layer , xenogenic sources [6,7], and define all needed components of the culture early on [8,9]. The goal is to identify a defined, xeno-free culture system including the media, substrate, and dissociation reagent. Defined culture systems use chemically defined media whose every component and concentration is known while xeno-free culture systems for human (i.e., clinical) applications use by definition the biological components derived from human or are produced in culture recombinantly using human genetic sequences. Defined and xenofree culture systems would minimize the inherent variability in biological components and standardize the experimental system in the research community and clinical products by providing the more stringent quality control necessary. While there are tremendous amounts of research and products available now for defined and xeno-free culture, this movement of applying these techniques has fallen flat so far. In 2016 (Jan – June), well over half of the articles published on iPSCs or ESCs in two highly regarded stem cell journals (Cell Stem Cell [10-27] and Stem Cells [28-55]) still use a feeder layer (27 of 46) [10-19,28-44], while no study utilized truly defined and xeno-free conditions. The remaining (20 of 46) did not use a feeder layer, but utilized undefined or xenogenic conditions in one way or another (e.g., Matrigel®, FBS, or BSA) [14,20-27,45-55]. One study utilized both in comparison so it was included in both lists . This phenomenon of defined and xeno-free cultures not being published on is also seen when you look at all articles on PubMed. The share of “Stem Cell” articles with the phrases “defined culture” and “free” is not increasing over the last 20 years, see Figure (1). If these techniques were being implemented, it would be expected that these shares would be increasing. Scale-up and development of clinical grade products using human pluripotent cells need defined and xenofree cultures, so why is the research community lagging behind in the adoption of these methods when many of the projects are translational research rather than basic science?
These methods may seem to be more expensive or prohibitive by the cost of materials or new reagents needed for the culture, as seen by the cost of materials (Table 1). However, when you look at the cost of implementation, defined and xeno-free methods are competitive in price and very close to that of the feeder system and undefined systems, see Figure (2). This may still be prohibitive for some labs with inherited feeder systems or cheaper ways to make media, but for the labs that can afford this minor increase what else needs to be done to promote the adoption of these changes? The companies supplying these alternatives have provided methodology for transitioning to defined, xeno-free systems and have shown validation of the products [1,2,57-60]. While the end goal of some of these group’s studies may not be a therapy or large scale production, it will undoubtedly help the field if researchers are united in utilizing the same, consistent experimental system instead of those with tremendous inherent variability  or those that garner large patient by patient inconsistency . So what is the next step for those in this industry, does the field need to validate the technologies more or on a larger scale? There are some claims of lower efficiency of the new methods [63,64], so perhaps it is that research groups are attempting to adopt the new techniques and are obtaining poor results?
Defined, xeno-free cultures of pluripotent cells are imperative for their industrial scale production and clinical application. In 2010 the International Stem Cell Forum funded a project comparing the performance of different media for culturing human ESCs . Emerging stem cell products  including those in clinical trials  will benefit from a similar organized tandardization project for evaluating the advanced culture technologies. Our cost analysis results shown in Figure (2) and Table (1) indicate that the difference in cost between undefined/ xenogenic and defined/xeno-free culture systems is becoming negligible. Therefore, it is an ideal time to validate and adopt those technologies by the laboratories not only in industry but also in academia. Benefits and limitations of the new technologies should be evaluated objectively to establish standards. These standardizing efforts streamline the materialization of revolutionary technology discovered in academic and start-ups laboratories.
1. TeSRΤΜ-E8ΤΜ. Stemcell Technologies.
2. Vitronectin (VTN-N) Recombinant Human Protein, Truncated. Thermo Fisher Scientific.
3. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic Stem Cell Lines Derived from Human Blastocysts. Science. 1998; 282: 1145-1147.
4. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell. 2007; 131: 861-872.
5. Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA. Feeder-independent culture of human embryonic stem cells. Nat Methods. 2006; 3: 637-646.
6. Chase LG, Firpo MT. Development of serum-free culture systems for human embryonic stem cells. Curr Opin Chem Biol. 2007; 11: 367-372.
7. Eremeev AV, Svetlakov AV, Polstianoy AM, Bogomazova AN, Philonenko ES, Sheina YI, et al. Derivation of a novel human embryonic stem cell line under serum-free and feeder-free conditions. Dokl Biol Sci. 2009; 426: 293-295.
8. Ludwig T, A Thomson J. Defined, feeder-independent medium for human embryonic stem cell culture. Curr Protoc Stem Cell Biol. 2007.
9. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, et al. Chemically Defined Conditions for Human IPSC Derivation and Culture. Nature Methods Nat Meth. 2011; 8: 424-429.
10. Lamm N, Ben-David U, Golan-Lev T, Storchová Z, Benvenisty N, Kerem B, et al. Genomic Instability in Human Pluripotent Stem Cells Arises from Replicative Stress and Chromosome Condensation Defects. Cell Stem Cell. 2016; 18: 253-261.
11. Ji X, Dadon DB, Powell BE, Fan ZP, Borges-Rivera D, Shachar S, et al. 3D Chromosome Regulatory Landscape of Human Pluripotent Cells. Cell Stem Cell. 2016; 18: 262-275.
12. Pastor WA, Chen D, Liu W, Kim R, Sahakyan A, Lukianchikov A, et al. Naive Human Pluripotent Cells Feature a Methylation Landscape Devoid of Blastocyst or Germline Memory. Cell Stem Cell. 2016; 18: 323-329.
13. Zhang H, Gayen S, Xiong J, Zhou B, Shanmugam AK, Sun Y, et al. MLL1 Inhibition Reprograms Epiblast Stem Cells to Naive Pluripotency. Cell Stem Cell. 2016; 18: 481-494.
14. Otani T, Marchetto MC, Gage FH, Simons BD, Livesey FJ. 2D and 3D Stem Cell Models of Primate Cortical Development Identify SpeciesSpecific Differences in Progenitor Behavior Contributing to Brain Size. Cell Stem Cell. 2016; 18: 467-480.
15. Krijger PH, Di Stefano B, de Wit E, Limone F, van Oevelen C, de Laat W, et al. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming. Cell Stem Cell. 2016; 18: 597-610.
16. Beagan JA, Gilgenast TG, Kim J, Plona Z, Norton HK, Hu G, et al. Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State during Somatic Cell Reprogramming. Cell Stem Cell. 2016; 18: 611-624.
17. Kang E, Wang X, Tippner-Hedges R, Ma H, Folmes CD, Gutierrez NM, et al. Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human IPSCs. Cell Stem Cell. 2016; 18: 625-636.
18. Zhu Z, Li QV, Lee K, Rosen PB, González F, Soh LC, et al. Genome Editing of Lineage Determinants in Human Pluripotent Stem Cells Reveals Mechanisms of Pancreatic Development and Diabetes. Cell Stem Cell. 2016; 18: 755-768.
19. Chen Y, Xiong M, Dong Y, Haberman A, Cao J, Liu H, et al. Chemical Control of Grafted Human PSC-Derived Neurons in a Mouse Model of Parkinson’s Disease. Cell Stem Cell. 2016; 18: 817-26.
20. Mascetti VL, Pedersen RA. Human-Mouse Chimerism Validates Human Stem Cell Pluripotency. Cell Stem Cell. 2016; 18: 67-72.
21. Krishna kumar R, Chen AF, Pantovich MG, Danial M, Parchem RJ, Labosky PA, et al. FOXD3 Regulates Pluripotent Stem Cell Potential by Simultaneously Initiating and Repressing Enhancer Activity. Cell Stem Cell. 2016; 18: 104-117.
22. Respuela P, Nikoli? M, Tan M, Frommolt P, Zhao Y, Wysocka J, et al. Foxd3 Promotes Exit from Naive Pluripotency through Enhancer Decommissioning and Inhibits Germline Specification. Cell Stem Cell. 2016; 18: 118-133.
23. Zhou Q, Wang M, Yuan Y, Wang X, Fu R, Wan H, et al. Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro. Cell Stem Cell. 2016; 18: 330-340.
24. Rao J, Pfeiffer MJ, Frank S, Adachi K, Piccini I, Quaranta R, et al. Stepwise Clearance of Repressive Roadblocks Drives Cardiac Induction in Human ESCs. Cell Stem Cell. 2016; 18: 341-353.
25. Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, et al. CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human IPSCs. Cell Stem Cell. 2016; 18: 541-553.
26. Luo S, Lu JY, Liu L, Yin Y, Chen C, Han X, et al. Divergent lncRNAs Regulate Gene Expression and Lineage Differentiation in Pluripotent Cells. Cell Stem Cell. 2016; 18: 637-652.
27. Parfitt DA, Lane A, Ramsden CM, Carr AJ, Munro PM, Jovanovic K, et al. Identification and Correction of Mechanisms Underlying Inherited Blindness in Human IPSC-Derived Optic Cups. Cell Stem Cell. 2016; 18: 769-781.
28. Hermanson DL, Bendzick L, Pribyl L, McCullar V, Vogel RI, Miller JS, et al. Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment of Ovarian Cancer. Stem Cells. 2016; 34: 93-101.
29. Wu CY, Persaud SD, Wei LN. Retinoic Acid Induces UbiquitinationResistant RIP140/LSD1 Complex to Fine-Tune Pax6 Gene in Neuronal Differentiation. Stem Cells. 2016; 34: 114-123.
30. Barta T, Peskova L, Collin J, Montaner D, Neganova I, Armstrong L, et al. Brief Report: Inhibition of MiR-145 Enhances Reprogramming of Human Dermal Fibroblasts to Induced Pluripotent Stem Cells. Stem Cells. 2016; 34: 246-251.
31. Collin J, Mellough CB, Dorgau B, Przyborski S, Moreno-Gimeno I, Lako M. Using Zinc Finger Nuclease Technology to Generate CRX-Reporter Human Embryonic Stem Cells as a Tool to Identify and Study the Emergence of Photoreceptors Precursors During Pluripotent Stem Cell Differentiation. Stem Cells; 2016; 34: 311-321.
32. Iseki H, Nakachi Y, Hishida T, Sugahara YY, Hirasaki M, Ueda A, et al. Combined Overexpression of JARID2, PRDM14, ESRRB, and SALL4A Dramatically Improves Efficiency and Kinetics of Reprogramming to Induced Pluripotent Stem Cells. Stem Cells. 2015; 34: 322-333.
33. Banerjee P, Dutta S, Pal R. Dysregulation of Wnt-Signaling and a Candidate Set of miRNAs Underlie the Effect of Metformin on Neural Crest Cell Development. Stem Cells. 2016; 34: 334-345.
34. Todorova D, Kim J, Hamzeinejad S, He J, Xu Y. Brief Report: Immune Microenvironment Determines the Immunogenicity of Induced Pluripotent Stem Cell Derivatives. Stem Cells. 2016; 34: 510-515.
35. Muñoz-López Á, van Roon EH, Romero-Moya D, López-Millan B, Stam RW, Colomer D, et al. Cellular Ontogeny and Hierarchy Influence the Reprogramming Efficiency of Human B Cells into Induced Pluripotent Stem Cells. Stem Cells. 2016; 34: 581-587.
36. Qian X, Kim JK, Tong W, Villa-Diaz LG, Krebsbach PH. DPPA5 Supports Pluripotency and Reprogramming by Regulating NANOG Turnover. Stem Cells. 2016; 34: 588-600.
37. Findlay SD, Postovit LM. Brief Report: Common Genetic Variation in Chromosome 10 Q22.1 Shows a Strong Sex Bias in Human Embryonic Stem Cell Lines and Directly Controls the Novel Alternative Splicing of Human NODAL Which Is Associated with XIST Expression in Female Cell Lines. Stem Cells. 2016; 34: 791-796.
38. Knappe N, Novak D, Weina K, Bernhardt M, Reith M, Larribere L, et al. Directed Dedifferentiation Using Partial Reprogramming Induces Invasive Phenotype in Melanoma Cells. Stem Cells. 2016; 34: 832-846.
39. Carter MG, Smagghe BJ, Stewart AK, Rapley JA, Lynch E, Bernier KJ, et al. A Primitive Growth Factor, NME7AB, Is Sufficient to Induce Stable Naïve State Human Pluripotency; Reprogramming in This Novel Growth Factor Confers Superior Differentiation. Stem Cells. 2016; 34: 847-859.
40. Chin CJ, Cooper AR, Lill GR, Evseenko D, Zhu Y, He CB, et al. Genetic Tagging During Human Mesoderm Differentiation Reveals Tripotent Lateral Plate Mesodermal Progenitors. Stem Cells. 2016; 34: 1239- 1250.
41. Sweeney CL, Teng R, Wang H, Merling RK, Lee J, Choi U, et al. Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis. Stem Cells. 2016; 34: 1513-1526.
42. Tang Y, Hong YZ, Bai HJ, Wu Q, Chen CD, Lang JY, et al. Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation Ofpmaip1. Stem Cells. 2016; 34: 1527-1540.
43. Fujita A, Uchida N, Haro-Mora JJ, Winkler T, Tisdale J. β-GlobinExpressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs. Stem Cells. 2016; 34: 1541-1552.
44. Naujock M, Stanslowsky N, Bufler S, Naumann M, Reinhardt P, Sterneckert J, et al. 4-Aminopyridine Induced Activity Rescues Hypoexcitable Motor Neurons from Amyotrophic Lateral Sclerosis Patient-Derived Induced Pluripotent Stem Cells. Stem Cells. 2016; 34: 1563-1575.
45. Wu Y, Chen K, Liu X, Huang L, Zhao D, Li L, et al. Srebp-1 Interacts with c-Myc to Enhance Somatic Cell Reprogramming. Stem Cells. 2016; 34: 83-92.
46. Bhinge A, Namboori SC, Bithell A, Soldati C, Buckley NJ, Stanton LW. MiR-375 Is Essential for Human Spinal Motor Neuron Development and May Be Involved in Motor Neuron Degeneration. Stem Cells. 2016; 34: 124-134.
47. Ramachandra CJ, Mehta A, Wong P, Shim W. ErbB4 Activated P38? MAPK Isoform Mediates Early Cardiogenesis Through NKx2.5 in Human Pluripotent Stem Cells. Stem Cells. 2016; 34: 288-298.
48. Lee H, Haller C, Manneville C, Doll T, Fruh I, Keller CG, et al. Identification of Small Molecules Which Induce Skeletal Muscle Differentiation in Embryonic Stem Cells via Activation of the Wnt and Inhibition of Smad2/3 and Sonic Hedgehog Pathways. Stem Cells. 2016; 34: 299-310.
49. Fang L, Zhang J, Zhang H, Yang X, Jin X, Zhang L, et al. H3K4 Methyltransferase Set1a Is A Key Oct4 Coactivator Essential for Generation of Oct4 Positive Inner Cell Mass. Stem Cells. 2016; 34: 565- 580.
50. Aksoy I, Marcy G, Chen J, Divakar U, Kumar V, John-Sanchez D, et al. A Role for RE-1-Silencing Transcription Factor in Embryonic Stem Cells Cardiac Lineage Specification. Stem Cells. 2016; 34: 860-872.
51. Livesey MR, Magnani D, Cleary EM, Vasistha NA, James OT, Selvaraj BT, et al. Maturation and Electrophysiological Properties of Human Pluripotent Stem Cell-derived Oligodendrocytes. Stem Cells. 2016; 34: 1040-1053.
52. Neganova I, Shmeleva E, Munkley J, Chichagova V, Anyfantis G, Anderson R, et al. JNK/SAPK Signaling Is Essential for Efficient Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells. Stem Cells. 2016; 34: 1198-1212.
53. Veluscek G, Li Y, Yang SH, Sharrocks AD. Jun-Mediated Changes in Cell Adhesion Contribute to Mouse Embryonic Stem Cell Exit from Ground State Pluripotency. Stem Cells. 2016; 34: 1213-1224.
54. Lambers E, Arnone B, Fatima A, Qin G, Wasserstrom JA, Kume T. Foxc1 Regulates Early Cardiomyogenesis and Functional Properties of Embryonic Stem Cell Derived Cardiomyocytes. Stem Cells. 2016; 34: 1487-1500.
55. Ohlemacher SK, Sridhar A, Xiao Y, Hochstetler AE, Sarfarazi M, Cummins TR, et al. Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration. Stem Cells. 2016; 34: 1553-1562.
56. PubMed. National Center for Biotechnology Information. U.S. National Library of Medicine.
57. Beers J, Gulbranson DR, George N, Siniscalchi LI, Jones J, Thomson JA, et al. Passaging and Colony Expansion of Human Pluripotent Stem Cells by Enzyme-free Dissociation in Chemically Defined Culture Conditions. Nat Protoc. 2012; 7: 2029-2040.
58. Versene Solution. Thermo Fisher Scientific.
59. Braam SR, Zeinstra L, Litjens S, Ward-van Oostwaard D, van den Brink S, van Laake L, et al. Recombinant Vitronectin Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells. 2016; 26: 2257-2265.
60. Goh, Pollyanna A., Sara Caxaria, Catharina Casper, Cecilia Rosales, Thomas T. Warner, Pete J. Coffey, and Amit C. Nathwani. A Systematic Evaluation of Integration Free Reprogramming Methods for Deriving Clinically Relevant Patient Specific Induced Pluripotent Stem (iPS) Cells. PLoS ONE. 2013; 8: e81622.
61. Unger C, Skottman H, Blomberg P, Dilber MS, Hovatta O. Good manufacturing practice and clinical-grade human embryonic stem cell lines. Hum Mol Genet. 2008; 17: R48-53.
62. Kulkeaw K, Horio Y, Mizuochi C, Ogawa M, Sugiyama D. Variation in hematopoietic potential of induced pluripotent stem cell lines. Stem Cell Rev. 2010; 6: 381-389.
63. Ojala M, Rajala K, Mari PM, Miettinen M, Huhtala H, Katriina AS. Culture Conditions Affect Cardiac Differentiation Potential of Human Pluripotent Stem Cells. PLoS ONE. 2012; 7: e48659.
64. Chen KG, Mallon BS, McKay RD, Robey PG. Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell. 2014; 14: 13-26.
65. Trypsin-EDTA (0.25%), phenol red. Thermo Fisher Scientific. 66.Stem Cell Feeder Cells. MTI-Global Stem.
67. Shahjalal HM, Shiraki N, Sakano D, Kikawa K, Ogaki S, Baba H, et al. Generation of insulin-producing β-like cells from human iPS cells in a defined and completely xeno-free culture system. J Mol Cell Biol. 2014; 6: 394-408.
68. mTeSR™-1. Stemcell Technologies.
69. Matrigel® Matrix. Corning®.
70. Accutase® Cell Detachment Solution. Innovative Cell Technologies, Inc.
71. ATCC® Stem Cell Culture Guide. Manassas, VA: ATCC®. 2015. PDF
72. iCell® Products. Cellular Dynamics International.
73. Sargent B. Fifteen Cell and Stem Cell Therapies in Clinical Trials. Cell Culture Dish. Mar.-Apr. 2013.
74. ISC-2. International Stem Cell Forum.
75. Trounson A, McDonald C. Stem Cell Therapies in Clinical Trials: Progress and Challenges. Cell Stem Cell. 2015; 17: 11-22.