FAQ About Feeder Cells for Stem Cells –Part One

The cost of preparing feeder cells for induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) is mainly due to 1. serum and media, 2. labor for growing and treating cells, and 3. expenses for freezing media and vials. Ready-to-use feeder cells saves one important labor-intensive step of iPSC generation, it should be an important help for iPSC and stem cell researchers. We know that most of our colleagues are tired of preparing fresh early passages of MEFs and treating them with expensive mitomycin C or finding an irradiator to pre-treat the MEFs. A lot of iPSC researchers lost iPS stem cells due to the lack of patience in handling MEF feeders. The offering of Allele’s feeder cell product line is really an easy solution and convenience to iPSC researchers.

Question 1: There are companies offering drug-resistant feeder cells such as MEF cells expressing neo-, puro-, or hygromycin-resistance genes. Is it important to have such drug-resistance genes when choosing feeder cells?

Adding drug resistant markers to these cells should not be necessary because iPSCs grown on feeder cells are usually not cultured in antibiotics-containing medium. The feeder cells will not be selected by drug resistance nor will they contaminate iPS cells since they can not propagate after irradiation. However, for those who do need to use drug selection for any reason, we will provide drug-resistant feeder cells upon request.

Question 2: There are publications showing the use of cells lines as feeder cells instead of primary fibroblasts, e.g. SL10, MRC-5, STO. Are there any advantages of using these cell lines?

Not really. Handling primary cells requires certain amount of experience and may be tedious; using cell lines, on the other hand, would be easier for preparing feeder cells. We provide feeder cells from immortalized early passage human foreskin fibroblasts at prices often lower than those from cell lines.

Question 3: Should I choose fluorescent protein expressing feeder cells for easy separation from iPSCs?

You do not need to include fluorescent protein in feeder cells, as feeder cells are quite different in morphology from iPS cells or ES cells. In fact, many labs use iPS factors that are co-expressed with fluorescent markers, in which cases feeder cell expressed fluorescent proteins will confuse the readout.

Question 4: What are the main advantages of using bFGF-expressing feeder cells?

Our bFGF-feeder cells not only eliminate the needs for added recombinant bFGF to stem cell cultures, but also form very nice cell lawn to serve iPSC colony formation because of their strictly controlled passage and growth conditions. We have used these cells without coating dishes with gelatin and obtained nice iPSC colonies.

Preview: Next Part of FAQ on Feeder Cells: choosing mouse or human fibroblasts, selecting iPSC colonies…

Announcement: An audience-orientated User Forum will be added to Allele Biotech webpages so that people can freely discuss or review products and technologies. A distilled version of discussions will be presented in a related but separate FAQ section, which will also include all Allele eNewsletters sent to our contacts about every quarter. Look for the links on www.allelebiotech.com in coming weeks.

Tags: bFGF, biology FAQ, biology forum, biotech enewsletter, ES cells, ESC, ESCs, feeder cells, human fibroblasts, iPS cells, iPSC, iPSCs, irradiation, MEF, MEFs, mitomycin C, stem cell culture

Retroviral Vectors with Integrated oriP/EBNA1 for IPSC

The new product of the week of Sep 21-27 is the retrovirus plasmid sets that contain a built-in episomal expression system. As we have discussed previously, OriP/EBNA1 system originated from Epstein-Bar virus, which allows the establishment of stable episomes at 5-20 copies per cell, and duplication once per cell division.

By using the oriP/EBNA1 episomal system, reprogramming cDNAs can be expressed at prolonged time period in reference to plasmid transfection, without integration into chromosomal DNA. A paper published in PLoS One on Sep 18, 2009 by Marchetto et al. showed that by using such a system (on different plasmids) the authors were able to create induced pluripotent stems cells (iPS cells,) effectively from human embryo neural precursor cells.

The Allele pCHAC-EBNA system has dual functions: it can be ready-to-use plasmids for episomal expression of Oct4, Sox2, c-Myc, Klf4, or Nanog and Lin28 by a simple transfection into target cells; it can also be packaged into retroviruses by transfecting into the Allele Phoenix Retrovirus packaging Eco or Ampho cells. This product group is officially launched today. It should become a highly convenient and unique tool for iPSC-related studies.

Tags: Ampho cells, EBNA, Eco cells, episomal expression, iPS cells, iPSC, iPSCs, New Product of the week, oriP, Phoenix Retroviral System, reprogramming, retroviral vector, retrovirus