1. What are feeder cells?
Mouse embryonic fibroblasts (MEF) are often referred to as feeders or feeder cells and have routinely been used to support the culturing and maintenance of embryonic stem cells (ESCs) in an undifferentiated state without losing their pluripotency. Human Newborn Fibroblasts are also used as feeder cells for supporting healthy undifferentiated human ES and iPS cells.
2. Why are mouse embryonic fibroblast (MEF) cells used when culturing stem cells?
MEF serve as a substrate for ES and iPS cells to grow on. They also secrete a number of essential growth factors that are important to maintain pluripotency.
3. At what density should I plate my MEF feeder cells?
We recommend that you plate your MEFs at the same density used to initially bank your ES/iPS cells. At MTI-GlobalStem we use our MEFs at a density of 30,000-50,000 cells/cm2. However, our MEF can be plated at lower densities (e.g. 20,000-30,000 cells/cm2) and still retain their ability to support undifferentiated ESC.
4. Can I use MTI-GlobalStem MEF for human embryonic stem (ES) cell culture?
Yes, every lot of our MEFs is comprehensively tested for contamination and health on both mouse and human ES stem cells to ensure robust and consistent performance.
5. How many passages can MEF cells be expanded?
Most of our MEFs are mitotically arrested (mitomycin C-treated or irradiated) so that they will not proliferate when used as feeders for ES/iPS cell culture. MEFs die away after ES/iPS cells are split.
6. Can I use gelatin when plating MEF cells?
If using gelatin, we recommend using qualified ready-to-use commercial gelatin such as Thermo Fisher Cat. no. S-006-100. Our MEFs adhere well to many tissue culture plastic plates without coating the plastic with matrix proteins.
7. How soon after plating my MEF can I plate cells?
MEF should be plated 24 hours before being used as a feeder layer.
8. What is the difference between the two mouse strains of MEF feeders, CF-1 and C57B/6 (Black 6)?
The only difference between the CF-1 and the C57BL/6 (also known as Black6) MEF is the mice that they are made from. CF-1 mice are outbred, while C57BL/6 are inbred. All of our MEF are tested for their ability to support human ESC in the undifferentiated state.
9. What are the mouse pathogens that you screen for?
MTI-GlobalStem screens for 22 different pathogens: Sendai virus, Mouse hepatitis virus, Pneumonia virus of mice, Minute virus of mice, Mouse parvovirus (MPV1, MPV2, & MPV3), Theiler's murine encephalomyelitis virus, Murine norovirus, Reovirus 3, Mouse rotavirus, Ectromelia virus, Lymphocytic choriomeningitis virus, Polyoma virus, Lactate dehydrogenase‐elevating virus, Mouse adenovirus (MAD1, MAD2), Mouse cytomegalovirus, K virus, Mouse thymic virus, Hantaan virus, and mycoplasma.
10. What method does MTI-GlobalStem use for screening mouse pathogens?
All of the mouse pathogens listed above (#9) are assayed by PCR.
11. What are NuFF cells?
Human Newborn Fibroblasts (NuFF) are feeder cells for supporting healthy undifferentiated human ES, and iPS cells. They can also be successfully reprogrammed.
12. What medium should I use to grow MTI-GlobalStem Mitomycin-C treated NuFF?
Our NuFFs have been expanded in DMEM containing 2 mM glutamine with the addition of 15% FBS. We recommend that the Mitomycin-C treated NuFFs be thawed and plated into the same medium. If using the NuFFs as a feeder layer, the medium can be switched to stem cell medium when the stem cells are plated on the feeders.
13. How many times can the NuFF be expanded?
MTI-GlobalStem guarantees that our untreated NuFFs (NuFF3-RQ™) can be passaged to P11 and maintain very good pluripotency. While cells can be expanded much further, and most likely be fine, we do not test past passage 11. NuFFs that have been inactivated via Mitomycin C or Irradiation will not proliferate.
14. What type of test do you perform to ensure consistence between different lots?
For each lot of NuFFs we perform all the following quality tests:
- Viability – typically >95%
- Negative for Human Pathogen
- Negative Mycoplasma
- Identity Testing - Cell identity is tested by Short Tandem Repeat (STR) analysis using 16 probes – 15 STR loci plus Amelogenin.
- Supports human pluripotent stem cell culture through multiple passages.- Based on the morphology, growth, and immunocytochemisty of multiple undifferentiation markers
15. Are the cells prepared under GMP conditions?
No. Our cells are not prepared under GMP conditions, but they are produced under a very well controlled laboratory process that includes defined SOPs and controls to ensure quality and consistency lot-to-lot.
16. How long do you have to differentiate the HIP™ NSCs to have mostly MAP-2 positive and β3-Tubulin positive cells?
The neural progenitor cells need to be differentiated 2 - 3 weeks to obtain a majority population of beta tubulin positive neurons by mitogen withdrawal.
17. Can I generate a single neuronal subtype from the HIP™ Neural Stem Cells?
Yes. HIP™ Neural Stem Cells (NSCs) can be directed to differentiate efficiently toward a single neuronal subtype, such as Tyrosine Hydroxylase (TH)-expressing Dopaminergic neurons. There are several published methods for generating these different cell types from similar iPSC-derived NSCs.
18. Is it possible to derive neurons, astrocytes and oligodendrocytes from your HIP NSCs?
Yes. While HIP™ NSCs are predisposed to spontaneously differentiate into neurons they are also capable of generating the other major cell types of the brain: astrocytes and oligodendrocytes. There are several published methods for generating these different cell types from similar iPSC-derived NSC and we can send you some references.
19. Can I generate a master bank from the neural progenitor cells?
Yes, HIP™ NSCs can be propagated and banked for later use. We have expanded our production lots over 5 passages and confirmed robust neuronal differentiation. Note that as the passage number increases there may be a reduced production of neurons relative to other cell fates. HIP™ Neurons cannot be propagated.
20. Do you have a protocol for differentiating the HIP™ NSC into neurons?
Upon withdrawal of the mitogen FGF2, the differentiating progeny of the HIP™ NSCs will be primarily neurons.
21. What percent of neurons can I expect to see with HIP™ Neurons?
HIP™ neurons will consist between 50-90% neurons, depending on the maturity of the culture. Mature differentiated cultures may contain greater than 90% neurons identified by β-III Tubulin.
22. How do I differentiate HIP™ hNSC to Astrocytes?
HIP™ hNSC are biased toward neuronal differentiation. However, they will differentiate toward GFAP-positive astrocytes under certain conditions. Nearly pure astrocytes have been generated from human pluripotent stem cells (via NSC intermediates) in several laboratories:
Emdad L, D'Souza SL, Kothari HP, Qadeer ZA, Germano IM (2011). Efficient Differentiation of Human Embryonic and Induced Pluripotent Stem Cells into Functional Astrocytes. Stem Cells Dev. 2011 Jul 26. [Epub ahead of print].
Krencik R, Weick JP, Liu Y, Zhang ZJ, Zhang SC (2011). Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nature Biotechnology 29(6):528-34.
23. How do I differentiate HIP™ hNSC to Oligodendrocytes?
As noted in the above question, hNSC will differentiate toward oligodendrocytes under certain conditions. Several protocols have been used to successfully generate oligodendrocytes from human pluripotent stem cells:
Hu BY, Du ZW, Zhang SC (2009). Differentiation of human oligodendrocytes from pluripotent stem cells. Nat Protoc. 2009;4(11):1614-22. Epub 2009 Oct 15.
Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, Steward O (2005). Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci. 2005 May 11;25(19):4694-705.
24. How do I differentiate HIP™ hNSC to Neurons?
This will depend on the type of neurons you want to generate Upon withdrawal of bFGF from culture media HIP™ hNSCs spontaneously differentiate primarily into neurons,
Various protocols can be used for directed differentiation to specific neural cell types:
Zeng H, Guo M, Martins-Taylor K, Wang X, Zhang Z, Park JW, Zhan S, Kronenberg MS, Lichtler A, Liu HX, Chen FP, Yue L, Li XJ, Xu RH (2010). Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluripotent stem cells. PLoS One. 2010 Jul 29;5(7):e11853.
25. How do I know if my cells are still undifferentiated?
Antibodies to Nestin and Sox1, or Sox2, are commonly used to assess the undifferentiated state of hNSC. These markers are down-regulated upon differentiation. Early neuronal differentiation of the cells can be detected by antibodies for Beta-III Tubulin or Microtubule-Associated Protein 2 (MAP2).
26. How do I know if my cells are growing quickly enough?
When following the supplied protocol, you should expect to see a doubling time for HIP™ hNSC of 50±6 hours. Many factors can lengthen the doubling time, especially higher passage number or poor quality FGF2. Poor recovery at passage can also affect the measurement of doubling time.
27. Should I add LIF?
If you have difficulty maintaining the hNSC in an undifferentiated state, adding 10 ng/mL of human leukemia inhibitory factor (LIF) to your media may help. Note: Only human LIF will work; mouse LIF will not bind the human receptor.
28. When should I passage the cells?
Cells should be allowed to become 100% confluent prior to subculturing. Gently dissociate the culture to single cells and replate at a density of 30,000-50,000 cells/cm2.
29. Can I passage HIP™ hNSC with Trypsin or TrypLE or scraping?
Yes, but it is not recommended. Accutase® is commonly used and recommended for gentle dissociation of HIP™ hNSC. Be aware that the use of serum to inactivate trypsin may alter the performance of HIP™ hNSC.
30. How many times can I passage the cells?
When following our maintenance protocol, HIP™ hNSC are guaranteed to maintain their differentiation potential for at least 5 passages after thawing.
31. Can I use PluriQ™ G9™ Maintenance Medium with matrices other than vitronectin?
32. Do I need to add anything to the PluriQ™ G9™ Cloning Medium?
No. The PluriQ™ G9™ Cloning Medium comes complete, ready to thaw and use for expansion of plutipotent stem cells starting from a single cell. It can be used post genome-editing or after creating induced pluripotent stem cells to isolate cells no longer harboring the reprogramming vectors.
33. Are 2102Ep EC cells sensitive to media?
Medium is best used within a month of first opening it, or of thawing the serum. If the medium is fresh but the pH is high, equilibrate the medium in a 5% CO2 incubator before use. Using the dipeptide L-alanine-L-glutamine in place of L-glutamine can also extend the life of your medium.
34. What tissue culture plate coating should I use to grow the cells?
We grow 2102Ep on uncoated tissue culture-grade plasticware. No coating of the plastic with polylysine, fibronectin, etc. is needed. As a result of such coating, cells may take longer than the recommended 1- 2 min to trypsinize.
35. How do I tell if cells in my stem cells are differentiating?
Difference in morphology of differentiated cells can depend on the culture system being used. They will down-regulate the usual pluripotency markers so it is more accurate to evaluate for expression of the markers periodically.
36. What effect does my choice of promoter have on evaluation of transfection efficiency?
The basic answer is that different promoters can have different expression levels based on the cell type being used. When one uses a GFP or other reporter, therefore, to monitor transfection efficiency, it is necessary to consider the promoter strength and the mode of detection of expression, in order to get the true answer about transfection efficiency. For example, in many cell types we have looked at there is not a major difference in CMV vs EF1α promoters and both work well. However, in induced pluripotent stem cells and neural stem cell as seen below, we saw a major difference. Using the same exposure times as the other cells tested we see little to no GFP with the CMV promoter.
Cells were transfected side by side using the reagent preferred with plasmid DNA where the GFP gene is under control of either the CMV or EF1α promoter. Cells were analyzed by fluorescent microscopy after 24 hours.
37. How can I enhance detection of weak expression to get more representative results?By increasing the exposure time of the cells transfected with the plasmid with GFP under the CMV promoter, we can see a bit more GFP than above. However, by using an anti-GFP antibody to enhance detection, we can see the transfection was indeed effective, yet the level of GFP expression is low. We used un-transfected cells as a negative control for the antibody detection. Images were taken with same exposure time (1/6 sec).
38. Are there references for how different promoters perform in different cells as a guide?Yes. There are several publications that look at differences in promoter effectiveness in different cell types:
- Qin, J. Y., Zhang, L., Clift, K. L., Hulur, I., Xiang, A. P., Ren, B.-Z., & Lahn, B. T. (2010). Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter. PLoS ONE, 5(5), e10611.
- Pasleau F, Tocci MJ, Leung F, Kopchick JJ (1985) Growth hormone gene expression in eukaryotic cells directed by the Rous sarcoma virus long terminal repeat or cytomegalovirus immediate-early promoter. Gene 38: 227–232.
- Martin-Gallardo A, Montoya-Zavala M, Kelder B, Taylor J, Chen H, et al. (1988) A comparison of bovine growth-hormone gene expression in mouse L cells directed by the Moloney murine-leukemia virus long terminal repeat, simian virus-40 early promoter or cytomegalovirus immediate-early promoter. Gene 70: 51–56.
- Oellig C, Seliger B (1990) Gene transfer into brain tumor cell lines: reporter gene expression using various cellular and viral promoters. J Neurosci Res 26: 390–396.
- Manthorpe M, Cornefert-Jensen F, Hartikka J, Felgner J, Rundell A, et al. (1993) Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice. Hum Gene Ther 4: 419–431.
- Yew NS, Wysokenski DM, Wang KX, Ziegler RJ, Marshall J, et al. (1997) Optimization of plasmid vectors for high-level expression in lung epithelial cells. Hum Gene Ther 8: 575–584.
- Xu ZL, Mizuguchi H, Ishii-Watabe A, Uchida E, Mayumi T, et al. (2001) Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene 272: 149–156.
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