NutriStem® hPSC XF Medium

Product Overview

NutriStem® hPSC XF Medium is a widely published, defined, xeno-free, serum-free cell culture medium designed to support the growth and expansion of human induced pluripotent stem (hiPS) and human embryonic stem (hES) cells. Protocols have been established around the world for applications ranging from derivation to differentiation. NutriStem® hPSC XF Medium offers the ability to culture cells in a completely xeno-free medium without the need for high levels of basic FGF and other stimulatory growth factors and cytokines. In addition, superior cell attachment and proliferation is observed with NutriStem® hPSC XF Medium aid high-throughput screening applications. NutriStem® hPSC XF Medium exhibits a consistent media performance and predictable cellular behavior derived from a defined xeno-free formulation as well as increased reproducibility shown in long-term growth of over 50 passages.

Low-protein formulation that contains stable L-alanyl-L-glutamine and HSA.

Features

• Defined, serum-free, and xeno-free 
• Flexible and compatible with multiple matrices
• Amenable to weekend-free culture
• FDA Drug Master File (DMF) available, produced under cGMP
• Enables efficient expansion and growth of hES and hiPS cells in feeder-free culture systems
• Extensively tested and widely used on multiple hES and iPS cell lines, including H1, H9.2, I6, I3.2, and CL1

Figure: 1 Human Embryonic Stem Cells (H1, passage 6) were seeded in 96-well plates (Matrigel coated) in BI's NutriStem® hPSC XF and competitor's media. Stem cell media were changed every 24 hours. Number of cells was determined using CyQuant™ cell proliferation assay kit.

Cell morphology

Figure: Normal Colony Morphology. H1 hES cells (top panel) and ACS-1014 hiPS cells (bottom panel) cultured in NutriStem® hPSC XF Medium on Matrigel-coated plates display colony morphologies typical of normal feeder-free hES and hiPS cell cultures, including a uniform colony of tightly compacted cells and distinct colony edges.

Immunostaining

Figure: H1 cell morphology and immunofluorescence analysis of hESC markers red SSEA-4, green OCT4 and blue DAPI. H1 cells stained positive for the expression of pluripotency markers.

Embryoid body formation

Figure: Embryoid bodies (EBs) were generated from H9.2 hES cells cultured for 16 passages in NutriStem® hPSC XF Medium on Matrigel matrix as an evaluation of pluripotency. The pluripotent H9.2 cells were suspended in serum-supplemented medium, where they spontaneously formed EBs containing cells of embryonic germ layers. The following cell types were identified by examination of the histological sections of 14-day-old EBs stained with H&E: (A) neural rosette (ectoderm), (B) neural rosette stained with Tubulin, (C) primitive blood vessels (mesoderm), and (D) megakaryocytes (mesoderm).

Taratoma formation

Figure: H9.2 hES cells were cultured for 11 passages in NutriStem® hPSC XF Medium using a human foreskin fibroblast (HFF) feeder layer. The hES cells were subsequently injected into the hind leg muscle of SCID-beige mice for in vitro evaluation of pluripotency. The following tissues from all three germ layers were identified in H&E-stained histological sections of the teratoma 12 weeks post-injection: (A) cartilage (mesoderm), (B) epithelium (endoderm), and (C) neural rosette (ectoderm).

NutriStem® and Cardiomyocytes

1. R. Ophir et al. Inflammation And Contractility Are Altered By Obstructive Sleep Apnea Children's Serum, In Human Embryonic Stem Cell Derived Cardiomyocytes. American Journal of Respiratory and Critical Care Medicine 2017
2. J. KRISTENSSON, Optimization of Growth Conditions for Expansion of Cardiac Stem Cells Resident in the Adult Human Heart. Master’s thesis in Biotechnology, Department of Physics, Division of Biological Physics, Chalmers University of Technology, Gothenburg, Sweden 2016
3. K. Tryggvason et al. Differentiation of pluripotent stem cells and cardiac progenitor cells into striated cardiomyocyte fibers using laminins LN-511, LN-521 and LN-221. US Patent Application 20160122717, 2016
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4. P. Menasché et al. Human embryonic stem cell-derived cardiac progenitors for severe heart failure treatment:first clinical case report. European heart journal (2015): ehv189.
5. L. Jacquet et al. Three Huntington’s Disease Specific Mutation-Carrying Human Embryonic Stem Cell Lines HaveStable Number of CAG Repeats upon In Vitro Differentiation into Cardiomyocytes. PloS one 10.5, 2015
6. S. Rajasingh et al. Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a NovelMethod of Measuring Contractility. PloS one 10.8, 2015: e0134093
7. W. Siqin et al. Spider silk for xeno-free long-term self-renewal and differentiation of human pluripotent stem cells. Biomaterials 35.30 (2014): 8496-8502.
8. E. Di Pasquale et al. Generation of human cardiomyocytes: a differentiation protocol from feeder-free humaninduced pluripotent stem cells. JoVE (Journal of Visualized Experiments) 76 (2013): e50429-e50429
9. G. Földes and M. Mioulane. High-content imaging and analysis of pluripotent stem cell-derived cardiomyocytes.Imaging and Tracking Stem Cells. Humana Press, 2013.
10. P.W. Burridge and E.T Zambidis. Highly efficient directed differentiation of human induced pluripotent stem cellsinto cardiomyocytes. Pluripotent Stem Cells: Methods and Protocols. Methods in Molecular Biology, volume 997, pp 149-161, Humana Press, 2013.
11. R. S. Song et al. Generation, Expansion, and Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs)Derived From the Umbilical Cords of Newborns. Current protocols in stem cell biology (2013): UNIT 1C.16.
12. Warren, L., et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7: 618-630, 2010




Cancer Stem Cells

13. Y Qin, et al. Laminins and cancer stem cells: partners in crime? Seminars in Cancer Biology, 2016




Differentiation of Pluripotent Stem Cells

14. R. De-Santis et. al. FUS Mutant Human Motoneurons Display Altered Transcriptome and microRNA Pathways with Implications for ALS Pathogenesis. Stem Cell Reports (2017), https://doi.org/10.1016/j.stemcr.2017.09.004
15. R.A. Hazim et al. Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization. Stem Cell Research & Therapy 2017
16. S. Petrus-Reurer et al. Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy. Retinal Cell Biology, February 2017
17. Lenzi, J., et al. Differentiation of control and ALS mutant human iPSCs into functional skeletal muscle cells, a tool for the study of neuromuscolar diseases. Stem Cell Research: Volume 17, Issue 1, Pages 140–147, 2016.
18. D. VOULGARIS, Evaluation of Small Molecules for Neuroectoderm differentiation & patterning using Factorial Experimental Design. Master Thesis in Applied Physics, Department of Physics, Division of Biological Physics, Chalmers University of Technology, Göteborg, Sweden 2016
19. X. Yuan et al. A hypomorphic PIGA gene mutation causes severe defects in neuron development and susceptibility to complement-mediated toxicity in a human iPSC model, PLOS ONE, 2017
20. P. Bergström et al. Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation, Scientific Reports, 2016
21. Sweeney, CL et al.Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction,Molecular Therapy, 2017
22. Tieng, V. ae al. Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase, Molecular Therapy — Methods & Clinical Development 6, Article number: 16069, 2016
23. Brykczynska, U, et al. CGG Repeat-Induced FMR1 Silencing Depends on the Expansion Size in Human iPSCs and Neurons Carrying Unmethylated Full Mutations Stem Cell Reports, 2016
24. Sellgren ,C.M. et al. Patient-specific models of microglia-mediated engulfment of synapses and neural progenitors Molecular Psychiatry, 2016
25. Bailly, J., et al. Method for differentiation of pluripotent stem cells into multi-competent renal precursors. US Patent 20,160,145,578, 2016
26. K. Tryggvason et al. Differentiation of pluripotent stem cells and cardiac progenitor cells into striated cardiomyocyte fibers using laminins LN-511, LN-521 and LN-221. US Patent Application 20160122717, 2016
27. A. Reyes, et al. Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model Plaza. Stem Cell Reports: Volume 6, Issue 1, p9–17, 2015
28. M.V. Krivega et al. Cyclin E1 plays a key role in balancing between totipotency and differentiation in human embryonic cells. Mol. Hum. Reprod, 2015
29. S. Rajasingh et al. Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a NovelMethod of Measuring Contractility. PloS one 10.8, 2015: e0134093




Growing Methods of hESC and iPSC (Derivation, Expansion, Scaling up, and Suspensions)

30. Maroof M Adil, David V Schaffer. Expansion of human pluripotent stem cells. Current Opinion in Chemical Engineering 2017, 15:24–35
31. Tateno, H. et al. Development of a practical sandwich assay to detect human pluripotent stem cells using cell culture media Regenerative Therapy, Volume 6, June 2017, Pages 1–8
32. Baker, D. et al. Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells Stem Cell Reports, 2016
33. Vega-Crespo, A., et al. Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells. Molecular Therapy — Methods & Clinical Development 3, Article number: 16050 (2016)
34. W. Zhang, et al. Distinct MicroRNA Expression Signatures of Porcine Induced Pluripotent Stem Cells under Mouse and Human ESC Culture Conditions. PLOS ONE, 2016
35. M. Mikkola et. al. A Method for Generating Induced Pluripotent Stem Cells. US Patent Application 20160068818, 2016
36. K. Alessandri et. al. A 3D printed microfluidic device for production of functionalized hydrogel microcapsules forculture and differentiation of human Neuronal Stem Cells (hNSC). Lab on a Chip: 16(9), 2016
37. Y.Y. Lipsitz, P.W. Zandstra, Human pluripotent stem cell process parameter optimization in a small scale suspensionbioreactor. BMC Proceedings, 9(Suppl 9), O10, 2015
38. S. Eminli-Meissner et al. A novel four transfection protocol for deriving iPS cell lines from human blood- derivedendothelial progenitor cells (EPCs) and adult human dermal fibroblasts using a cocktail of non-modified reprogrammingand immune evasion mRNAs. Sientific Poster, REPROCELL, 2015
39. S. Wu et al. Efficient passage of human pluripotent stem cells on spider silk matrices under xeno-free conditions. Cellular and Molecular Life Sciences: 73(7):1479-88, 2015
40. S. Gregory et al. Autophagic response to cell culture stress in pluripotent stem cells. Biochemical and Biophysical Research Communications, doi:10.1016/j.bbrc.2015.09.080, 2015
41. H Tateno et al. Undifferentiated Cell Detection Method And Complex Carbohydrate Detection Method. US Patent Application 20150204870, 2015
42. S. Herz, Optimization of RNA-based transgene expression by targeting Protein Kinase R. Dissertation for the degree “Doctor rerum naturalium”, 2015
43. J. Lenzi et al., ALS mutant FUS proteins are recruited into stress granules in induced Pluripotent Stem Cells (iPSCs)derived motoneurons. Disease Models & Mechanisms: 8, 755-766, 2015
44. N. Desai, P Rambhia and A. Gishto, Human embryonic stem cell cultivation: historical perspective and evolutionof xeno-free culture systems. Reproductive Biology and Endocrinology 13.1 (2015): 9.
45. T. Yokobori et al., Intestinal epithelial culture under an air-liquid interface: a tool for studying human and mouse esophagi. Diseases of the Esophagus, doi: 10.1111/dote.12346. 2015
46. T. Cerbini et al., Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targetedwith a GFP Gene into the AAVS1 Safe Harbor, JoVE (Journal of Visualized Experiments), 2015
47. L. Healy, L Ruban, Derivation of Induced Pluripotent Stem Cells, Atlas of Human Pluripotent Stem Cells in Culture, pp 149-165. Springer US 2015
48. N.Y. Thakar et al., TRAF2 recruitment via T61 in CD30 drives NFkB activation and enhances hESC survival andproliferation, Molecular Biology of the Cell: 26(5):993-1006 2015
49. V. Bellamy et al., Long-term functional benefits of human embryonic stem cell-derived cardiac progenitors embedded into a fibrin scaffold, The Journal of Heart and Lung Transplantation, 2014, in press
50. A. J. Schwab, A.D. Ebert, Sensory Neurons Do Not Induce Motor Neuron Loss in a Human Stem Cell Model of SpinalMuscular Atrophy. PLoS One. 2014; 9(7): e103112
51. G. Finesilver, M. Kahana, E. Mitrani. Kidney-Specific Micro-Scaffolds and Kidney Derived Serum FreeConditioned Media support in vitro Expansion, Differentiation, and Organization of Human Embryonic Stem Cells. Tissue Engineering Part C: Methods. -Not available-, ahead of print. doi:10.1089/ten.TEC.2013.0574.
52. M. Amit, J. Itskovitz-eldor. Novel Methods and Culture Media for Culturing Pluripotent Stem Cells. US Patent Application 20130236961, 2013
53. M. Amit, J. Itskovitz-Eldor. Atlas of Human Pluripotent Stem Cells: Derivation and Culturing. Stem Cell Biology and Regenerative Medicine, 2012
54. R. Bergström, Xeno-free culture of human pluripotent stem cells, Methods Mol Biol. 2011;767:125-36
55. S. Sugii et al., Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells. PNAS February 23, 2010 vol. 107 no. 8 3558-3563
56. J.Collins et al,. Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells withSynthetic Modified mRNA. Cell Stem Cell 7 (5): 618-630 (2010).

Induction of Pluripotency of hESC and iPSC

58. S. Eminli-Meissner et al. A novel four transfection protocol for deriving iPS cell lines from human blood- derivedendothelial progenitor cells (EPCs) and adult human dermal fibroblasts using a cocktail of non-modified reprogrammingand immune evasion mRNAs. Sientific Poster, REPROCELL, 2015
59. Sahin, U., et al. Diagnosis and therapy of cancer involving cancer stem cells. US Patent Application 20150314018, 2015
60. M. Brouwer et al. Choices for Induction of Pluripotency: Recent Developments in Human Induced Pluripotent StemCell Reprogramming Strategies. Stem Cell Reviews and Reports: Volume 12, Issue 1, pp 54–72, 2015
61. H.X. Nguyen et al., Induction of early neural precursors and derivation of tripotent neural stem cells from humanpluripotent stem cells under xeno-free conditions. Journal of Comparative Neurology: Volume 522, Issue 12, pp 2767–2783, 2014




iPSC Derivation Patents

62. U. Sahin et al. Method for Cellular RNA Expression. US Patent Application 20150314018, 2015
63. M.F Yanik, M. Angel. Methods for Transfecting Cells With Nucleic Acids. US Patent Application 20140073053, 2014
64. C.E Buensuceso et al., Induced Pluripotent Stem Cells Prepared from Human Kidney-Derived Cells. US Patent Application 20140073049, 2014
65. C. Buensuceso et al., Induced Pluripotent Stem Cells from Human Umbilical Cord Tissue-Derived Cells. US Patent Application 20130157365, 2013
66. A. De Fougerolles, S.M. Elbashir, J.P. Schrum. Modified Polynucleotided for the Production of Factor IX. US Patent Application 20130245103, 2013




Different Basement Matrices

67. Cosset, E. et al. Human tissue engineering allows the identification of active miRNA regulators of glioblastoma aggressiveness Biomaterials, 2016
68. Wilkinson, D.C, et al. Development of a Three-Dimensional Bioengineering Technology to Generate Lung Tissue for Personalized Disease Modeling Stem Cells Translational Medicine, 2016
69. O. Simonson. Use of Genes and Cells in Regenerative Medicine. Karolinska Institutet, 2015
70. Nacalai USA Inc. Vitronectin-398™ (Xeno-free). Nacalai USA website
71. S. Rodin et al., Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions. Nature Protocols 9, 2354–2368 (2014) doi:10.1038/ nprot.2014.159
72. Rodin S, et al. Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Nat Commun. 5:3195. doi: 10.1038/ncomms4195, 2014
73. StemAdhere™ Defined Matrix for hPSC. Primorigen Biosciences website.
74. I. lenz et al., Automated 3D Culture to Undifferentiated hESC. (Scientific Poster)
75. J.L. Weber et al,. The Corning® Synthemax™ Surface: A Synthetic, Xeno-Free Surface for Long-Term Self-Renewal of Human Embryonic Stem Cells in Defined Media. presented in 2010 world stem cell summit




Proteins and Antibodies Expression and Isolation

76. J. Mata-fink et al. Methods and Compositions For Immunomodulation. US Patent Application 20170020926, 2017
77. Abcam, Immunocytochemistry / Immunofluorescence abreview for Anti-Oct4 antibody - ChIP Grade. Abcam website




Clinical Applications- Derivation and Expansion of hESC and IPSC

78. K. Jacobs et al. Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and GenomeInstability. Stem Cell Reports: 6(3), pp 330–341, 2016
79. D. Keefe et al. A Method for a Single Cell Analysis of Telomere Length. US Patent Application 20160032360, 2016
80. M. Di Salvio et al. 2015. Pur-alpha functionally interacts with FUS carrying ALS-associated mutations. Cell Death & Disease.
81. L. de Oñate et al. 2015. Research on Skeletal Muscle Diseases Using Pluripotent Stem Cells. DOI: 10.5772/60902
82. P. Menasché et al., Towards a Clinical Use of Human Embryonic Stem Cell-Derived Cardiac Progenitors:A Translational Experience. European Heart Journal: Volume 36, Issue 12, pp 743-50, 2015
83. T. Seki, K. Fukuda, Methods of induced pluripotent stem cells for clinical application, World Journal of Stem Cells: Volume 7, Issue 1, pp 116–125, 2015
84. S. Abbasalizadeh, H. Baharvand. Technologies progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies. Biotechnology Advances: Volume 31, Issue 8, pp 1600-23, 2013.
85. Y. Luo et al.,Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator-Like Effector Nucleases, STEM CELLS Translational Medicine: Volume 3, Issue 7, pp 821-35, 2014
86. J. Durruthy-Durruthy et al. 2014. Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions. PlOS one: http://dx.doi.org/10.1371/journal.pone.0094231
87. H. Tateno et al. 2014. A medium hyperglycosylated podocalyxin enables noninvasive and quantitative detection of tumorigenic human pluripotent stem cells. Scientific Reports 4, Article number: 4069.
88. J. P. Awe et al. Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status. Stem Cell Research & Therapy, 2013, 4:87
89. Hovatta, Outi. Infectious problems associated with transplantation of cells differentiated from pluripotent stem cells. Seminars in Immunopathology: Volume 33, Issue 6, pp 627-30, April 2011
90. Susanne Ström. Optimisation of human embryonic stem cell derivation and culture – towards clinical quality. Karolinska Institutet, Stockholm, Sweden, 2010.




Patents

91. Dietrich M. EGLI, Methods for making and using modified oocytes . US Patent Application 20140308257, 2016
92. O. Hovatta, K. Tryggvason, Methods of Producing RPE Cells. US Patent Application 20150299653, 2015
93. A. De Fougerolles, S.M. Elbashir, Delivery and Formulation of Engineered Nucleic Acids, US Patent Application 20150017211, 2015
94. E. Zambidis, P Burridge. Compositions and Methods of Generating a Differentiated Mesodermal Cell. US Patent Application 20130136721, 2013.
95. A. Kurtz, A. Bosio, and S. Knoebel. Highly efficient differentiation of hPSC into hepatocyte-like cellsby selection of CXCR4 (CD184) definitive endoderm (DE) cells




Drug Screening

96. Z. Ye et al., Differential sensitivity to JAK inhibitory drugs by isogenic human erythroblasts and hematopoieticprogenitors

Material Safety Data Sheet

•  NutriStem® hPSC XF Medium (MSDS)

Manuals and Protocols

•  NutriStem® hPSC XF Medium Instructions for Use
•  NutriStem® hPSC XF Medium Technical Manual
•  Weekend-free culture of human pluripotent stem cells on LN-521 (BioLamina)

Webinars & Videos

• On-Demand Webinar: Human Pluripotent Stem Cells (hPSC) – An Overview of Morphology and Culture Methods (BI-USA)
• On-Demand Webinar: Reprogramming and iPSC culture considerations for translational research (RegMedNet)
• Protocol video: Passaging Human Embryonic Stem Cells and Induced Pluripotent Stem Cells in NutriStem® XF/FF Culture Medium (Stemgent)

Product Literature

• NutriStem® hPSC XF Medium Product Sheet
• Case Study: Xeno-Free hiPS Cell Culture using NutriStem® hPSC XF Medium on a Human Vitronectin Substrate without the need for ROCK Inhibitors
• Application Note: Corning® PureCoat™ rLaminin-521 Cultureware: A Ready-to-use, Animal-free Vessel Platform for the Culture and Single Cell Passaging of Human Pluripotent Stem Cells (Corning)
• Application Note: NutriStem® hPSC XF Medium Supports Longterm Culture of Human Pluripotent Stem Cells (Corning)

Frequently Asked Questions

• FAQ's: NutriStem® hPSC XF Medium and culturing Human Pluripotent Stem Cells