Product FAQ

At what density should I plate my stem cells before transfecting them using Lipofectamine Stem Transfection Reagent?

Answer

Cell confluency can impact the amount of Lipofectamine Stem Transfection Reagent that is needed for efficient transfection. Stem cells that are near confluency do not transfect well. Plating density should be such that cells are not overgrown 24 hours after transfection. To obtain the highest percent of stem cells transfected, we highly recommend that you dissociate both colony type and monolayer cultures into small clumps of 3-5 cells. If you are growing human pluripotent stem cells in a 24-well plate in Essential 8 Medium on Vitronectin, plate the cells to achieve 30-60% confluency on the day of transfection. For additional details, please refer to the Lipofectamine Stem Transfection Reagent protocol pertinent to your cell type and media on the product page (https://www.thermofisher.com/order/catalog/product/STEM00008?ICID=search-product), under Documents, Product Literature.

Answer Id: E15214

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Product FAQ

Can Gibco Essential 8 Medium and Gibco Vitronectin (VTN-N) support long-term growth of pluripotent stem cells (PSCs)?

Answer

Gibco Essential 8 Medium and vitronectin have been shown to support PSC growth for >50 passages without any signs of karyotypic abnormalities, and maintain the ability of PSCs to differentiate into all three germ line lineages. As published by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) in the laboratory of James Thomson, the VTN-N variant of vitronectin supports human pluripotent stem cell attachment and survival better than wild-type vitronectin when used in conjunction with Gibco Essential 8 Medium.

Answer Id: E12425

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Product FAQ

Can pluripotent stem cells (PSCs) previously cultured in other media and on other substrates be cultured in Gibco Essential 8 Medium and on Gibco Vitronectin?

Answer

Cells cultured in other feeder-free media systems, such as mTeSR Medium with Matrigel Basement Membrane Matrix, or StemPro hESC SFM with Geltrex Matrix, can be successfully cultured in Gibco Essential 8 Medium and VTN-N. In addition, PSCs grown on feeders with KnockOut SR have also been shown to be successfully cultured in Gibco Essential 8 Medium on VTN-N. However, when changing media systems, cells must be passaged either manually, or with EDTA prior to culturing in Gibco Essential 8 Medium on VTN-N.

Answer Id: E12427

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Product FAQ

Do you offer human neural stem cells (NSCs)?

Answer

We offer two types of human NSCs:

- Gibco StemPro Neural Stem Cells (Cat. No. A15654 or A15655): Isolated from human fetal cortex brain and manufactured under good manufacturing practice (GMP). Each lot is generated from the same master bank (same donor) so that the lot-to-lot variability is low. Cell doubling time is ~ 100 hours.

- Gibco Human Neural Stem Cells (H9-derived) (Cat. No. N7800): Induced from H9 human ESC using a proprietary method. Cell doubling time is ~ 40-50 hours and increases with increasing passage number.

Both cells are tested for their ability to retain their proliferation and differentiation potential for at least 3 passsages after thawing. Both are able to differentiate into neurons, astrocytes, and oligodendrocytes. However, StemPro Neural Stem Cells are recommended to grow in suspension culture as adherent culture would trigger differentiation, whereas Human Neural Stem Cells (H9-derived) can grow under both suspension and adherent conditions.

Answer Id: E12542

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Product FAQ

With Lipofectamine Stem Transfection Reagent, how can I improve the transfection efficiency in my stem cells?

Answer

To obtain the highest percent of stem cells transfected with Lipofectamine Stem Transfection Reagent, we highly recommend that you follow cell culture conditions that promote formation of a monolayer versus clumps of cells. If the density of your stem cells is greater than 50% or they are being grown as colonies in other media, more Lipofectamine Stem Transfection Reagent may improve transfection efficiency. For additional details, please refer to the Lipofectamine Stem Transfection Reagent protocol pertinent to your cell type and media on the product page (https://www.thermofisher.com/order/catalog/product/STEM00008?ICID=search-product), under Documents, Product Literature.

Answer Id: E15216

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Product FAQ

Can Lipofectamine Stem Transfection Reagent be used to transfect stem cells in suspension?

Answer

Yes. High transfection efficiency can be achieved in stem cells in suspension with reverse transfection using Lipofectamine Stem Transfection Reagent. Stem cells can easily be transfected at the time of passaging and during re-plating, when they are in suspension. For simple instructions on using reverse transfection with Lipofectamine Stem Transfection Reagent, please take a look at the transfection protocols for pluripotent stem cells, listed under Documents, Product Literature on the product page (https://www.thermofisher.com/order/catalog/product/STEM00008?ICID=search-product).

Answer Id: E15203

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Product FAQ

What are neural stem cells?

Answer

Neural stem cells (NSCs) are self-renewing multipotent cells of the nervous system capable of differentiating into neurons, oligodendrocytes, and astrocytes. NSC can be generated by induced differentiation from embryonic stem (ES) cells, or isolated from various regions of the brain including the cortex, the subventricular zone (SVZ), and the ventricular zone, or generated from bone marrow-derived mesenchymal stem cells (MSCs) (J Cell Biochem 114:764 (2013)). NSCs are valuable tools for the study of neurogenesis and neurotransmitter and receptor function. NSCs were used in the investigation of different CNS disorders such as PD and Huntington's disease in various animal models (J Cell Biochem 114:764 (2013)).

Answer Id: E12189

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Product FAQ

What are induced pluripotent stem cells?

Answer

Induced pluripotent stem cells (iPS or iPSCs) are pluripotent stem cells directly generated by introducing combination of genes coding for “reprogramming factors” into adult cells. These reprogramming factors include Oct4, Sox2, c-Myc, KLF4, NANOG, and LIN28. Yu, et al, generated iPS from a human mesenchymal cell line using lentiviral vectors carrying Oct4, Sox2, NANOG, and LIN28 genes (Science 318:1917 (2007)). Using a similar approach, Takahashi et al, generated iPS from human primary fibroblast cells by introducing genes coding for Oct3, Sox2, KLF4, and c-Myc into these cells (Cell 131:861 (2007)). iPS generated by reprogramming are similar to human ES cells in morphology, the capacity for unlimited proliferation, surface-antigen expression, gene expression, the ability to differentiate into cell types representing the three germ layers in vitro, and the ability to form teratomas after injection into SCID mice.

Answer Id: E12180

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Product FAQ

Can I use Lipofectamine Stem Transfection Reagent to transfect neural stem cells (NSCs)?

Answer

Yes. Lipofectamine Stem Transfection Reagent can be used to transfect a wide range of stem cell types, while supporting continued proliferation without inducing differentiation. It is our recommended solution for transfecting neural stem cells.

Answer Id: E15213

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Product FAQ

What growth conditions do you recommend when using the AlgiMatrix 3D Culture System for umbilical cord stem cells?

Answer

For umbilical cord stem cells, you might want to try a relatively high inoculation density of 100,000-300,000 cells per sponge with daily or every-other-day re-feeding of cultures, as the medium turns “yellow”.

Answer Id: E11793

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Product FAQ

What are mesenchymal stem cells?

Answer

Mesenchymal stem cells (MSCs) are multipotent cells isolated primarily from bone marrow or fat tissues that exhibit the ability to differentiate into bone, cartilage, and fat cells. Under normal cell culture conditions, MSCs isolated from bone marrow are spindle shaped with the unique ability to adhere to uncoated plastic culture dishes (Arthritis Res Ther 9:204 (2007)).

Answer Id: E12184

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Product FAQ

Can Dopaminergic Neuron Maturation medium be used to differentiate neural stem cells?

Answer

We have tested neural stem cells (NSCs) isolated from fetal tissue or derived from pluripotent stem cells (PSCs), and have seen that both populations can benefit from maturation medium (DMEM/F12 + Dopaminergic Neuron Maturation Supplement) to have nicely spread homogenous neurons with reduced progenitor population. Matured neurons can be further maintained in neurobasal medium supplemented with Dopaminergic Neuron Maturation Supplement.

Answer Id: E13084

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Product FAQ

Which transfection reagent should I use for delivering my gene of interest into stem cells?

Answer

We recommend using Lipofectamine Stem Transfection Reagent for delivery of DNA, mRNA and co-transfection (siRNA and plasmid DNA) in a wide variety of stem cells. Lipofectamine Stem Transfection Reagent also delivers Cas9-gRNA ribonucleoproteins for gene editing applications. For more information on the transfection efficiency and versatility of the reagent, please visit: https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-stem-transfection-reagent.html

Answer Id: E9010

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Product FAQ

Which kits do you offer for stem cell reprogramming?

Answer

We offer the Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518) which is a non-integrating system that uses Sendai virus vectors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit contains three Invitrogen CytoTune 2.0 reprogramming vectors that are used for delivering and expressing key genetic factors necessary for reprogramming somatic cells into iPSCs. Only one application of the vectors is required for successful reprogramming.

Additionally, we offer the Episomal iPSC Reprogramming Vectors (Cat. No A14703) or Epi5 Episomal iPSC Reprogramming Kit (Cat. No A15960). This is a non-integrating system that reprograms somatic cells into induced pluripotent stem cells (iPSCs). These two products contain a mixture of six or five vectors designed to provide the optimal system for generating transgene-free and virus-free iPSCs in a feeder-free environment. Originally developed by Junying Yu and James Thomson (http://www.ncbi.nlm.nih.gov/pubmed/19325077) and further optimized by Cellular Dynamics International, these Episomal iPSC Reprogramming Vectors have proven successful in reprogramming a number of different somatic cell types.

Answer Id: E12483

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Product FAQ

How should I prepare my stem cells when I use Lipofectamine Stem Transfection Reagent?

Answer

Proliferating stem cells in culture need room to expand. We recommended plating cells so they will be 30-60% confluent at the time of transfection. For additional details, please refer to the Lipofectamine Stem Transfection Reagent protocol pertinent to your cell type and media on the product page (https://www.thermofisher.com/order/catalog/product/STEM00008?ICID=search-product), under Documents, Product Literature.

Answer Id: E15208

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