Nutristem hPSC XF Medium

Description

NutriStem hPSC Overview

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.

NutriStem® hPSC XF Medium is a 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. Nutristem hPSC is widely published and numerous protocols have been established for applications that incorporate the complete cell culture process from derivation to differentiation. NutriStem hPSC XF Medium allows for pluripotent stem cells to be cultured in a completely xeno-free system, without the need for high levels of basic FGF and other stimulatory growth factors and cytokines. The superior cell attachment and proliferation NutriStem hPSC XF Medium provides makes it an excellent medium choice to permit high-throughput screening applications. The reliable and consistent cellular morphology and growth of cells cultures in NutriStem hPSC XF Medium increases experimental reproducibility and has been shown in long-term growth of over 50 passages whilst preserving normal karyotypes.

NutriStem hPSC XF Features

• Defined, serum-free, and xeno-free
• Allows for flexibility to derived cells with a variety matrices
• Allows for 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
• Low-protein formulation that contains stable L-alanyl-L-glutamine and HSA
• Extensively tested and widely used on multiple hES and iPS cell lines, including H1, H9.2, I6, I3.2, and CL1

Sample Data

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).

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).

Instructions for Use  

• Upon thawing, the medium may be stored at 2 – 8ºC for 10 to 14 days
• Media should be aliquotted into smaller working volumes to avoid repeated freeze/thaw cycles
• Avoid exposure to light

Note: A common feeder-free basement membrane matrix is Matrigel, which is not xeno-free. Effective xeno-free alternatives to Matrigel is recombinant laminin, such as LaminStem(R) 521 (BI Cat. No. 05-753-1F) which has been validated to successfully culture human ES and iPS cells using NutriStem® hPSC XF medium.

Legal : NutriStem® hPSC XF is registered as an In-vitro diagnostic (IVD) medical device. NutriStem® is a registered trademark of Biological Industries​.

A Drug Master File (DMF) for NutriStem® hPSC XF is available.

Additional information

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

1. O.M. Russell et al. Preferential amplification of a human mitochondrial DNA deletion in vitro and in vivo. Scientific Reports, volume 8, Article number: 1799 (2018)
2. J. Rosati et al. Production and characterization of human induced pluripotent stem cells (iPSCs) from Joubert Syndrome: CSSi001-A (2850). Stem Cell Research, Volume 27, March 2018, Pages 74–77
3. Maroof M Adil, David V Schaffer. Expansion of human pluripotent stem cells. Current Opinion in Chemical Engineering 2017, 15:24–35
4. 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
5. Baker, D. et al. Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells Stem Cell Reports, 2016
6. 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)
7. 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
8. 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
9. 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.
10. 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
11. L. Healy, L Ruban, Derivation of Induced Pluripotent Stem Cells, Atlas of Human Pluripotent Stem Cells in Culture, pp 149-165. Springer US 2015
12. 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.
13. 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.
14. M. Amit, J. Itskovitz-Eldor. Atlas of Human Pluripotent Stem Cells: Derivation and Culturing. Stem Cell Biology and Regenerative Medicine, 2012
15. R. Bergström, Xeno-free culture of human pluripotent stem cells, Methods Mol Biol. 2011;767:125-36
16. 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).
17. 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

 Differentiation of Pluripotent Stem Cells

1. K.M. Gray et al. Self-oligomerization regulates stability of Survival Motor Neuron (SMN) protein isoforms by sequestering an SCFSlmb degron. Molecular Biology of the Cell, 2017 mbc.E17-11-0627
2. D.C. Wilkinson et. al. Development of a Three-Dimensional Bioengineering Technology to Generate Lung Tissue for Personalized Disease Modeling. Stem cells translational medicine, 6(2), 2017
3. E. Welby et al. Isolation and Comparative Transcriptome Analysis of Human Fetal and iPSC-Derived Cone Photoreceptor Cells. Stem Cell Reports (2017), https://doi.org/10.1016/j.stemcr.2017.10.018
4. 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
5. R.A. Hazim et al. Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization. Stem Cell Research & Therapy 2017
6. 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
7. 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
8. 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.
9. 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
10. 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
11. P. Bergström et al. Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation, Scientific Reports, 2016
12. 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
13. 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
14. Sellgren ,C.M. et al. Patient-specific models of microglia-mediated engulfment of synapses and neural progenitors Molecular Psychiatry, 2016
15. Cosset, E. et al. Human tissue engineering allows the identification of active miRNA regulators of glioblastoma aggressiveness Biomaterials, 2016
16. M. Di Salvio et al. Pur-alpha functionally interacts with FUS carrying ALS-associated mutations. Cell Death & Disease, 2015
17. 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
18. Tieng, V. et al.Engineering of Midbrain Organoids Containing Long-Lived Dopaminergic Neurons. Stem Cells and Development. February 2014, 23(13): 1535-1547.
19. 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
20. 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
21. A. Kurtz, A. Bosio, and S. Knoebel. Highly efficient differentiation of hPSC into hepatocyte-like cellsby selection of CXCR4 (CD184) definitive endoderm (DE) cells

Cardiomyocyte differentiation

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. S. Rajasingh et al. Generation of Functional Cardiomyocytes fro Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility. PloS one 10.8, 2015: e0134093
4. L. Jacquet et al. Three Huntington’s Disease Specific Mutation-Carrying Human Embryonic Stem Cell Lines Have Stable Number of CAG Repeats upon In Vitro Differentiation into Cardiomyocytes. PloS one 10.5, 2015
5. 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
6. E. Di Pasquale et al. Generation of human cardiomyocytes: a differentiation protocol for feeder-free human induced pluripotent stem cells. JoVE (Journal of Visualized Experiments) 76 (2013): e50429-e50429
7. P.W. Burridge and E.T Zambidis. Highly efficient directed differentiation of human induced pluripotent stem cells into cardiomyocytes. Pluripotent Stem Cells: Methods and Protocols. Methods in Molecular Biology, volume 997, pp 149-161, Humana Press, 2013.

Gene Editing

1. 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
2. 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
3. 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

Material Safety Data Sheet

• NutriStem® hPSC XF Medium (MSDS)

Manuals and Protocols

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

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)
• Application Note: Single Cell Passaging with NutriStem® hPSC XF on Laminin-521 without the Need for ROCK Inhibitor
• Technical Resources: Passaging Methods for hPSCs under Feeder-Free Conditions

Frequently Asked Questions

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