Celprogen Inc., has designed a cell culture medium for growing stem cells of all types in the laboratory that does not contain any animal products. Such a medium is called “Xeno-free.”
This new xeno-free medium, XFS2 can successfully culture induced pluripotent stem cells, primary human cells, stem cells and progenitor cells. XFS2 can also be used to grow cancer cell for research purposes.
Growing stem cells in cell culture requires a unique mixture of growth factors that stimulate cell proliferation and cell survival. Because stem cells can grow in XFS2 without differentiating, it can be used to grow cells for clinical trials s well.
Celprogen scientists have used XFS2 to grow human embryonic stem cells, fat-derived stem cells, and stem cells isolated from specific human organs. A chief advantage of XFS2 is that it does not contain any human serum, which is essential for clinical applications, since patients could have serious immunological reactions against serum that does not come from their own blood.
When stem cells grown in XFS2 were compared with stem cells grown in other cell culture media, the XFS2-grown cells did not display any detectable differences from their serum-grown counterparts. Furthermore, according to Celprogen, stem and progenitor cells grew robustly in XFS2. We will certainly need to see the reactions of laboratories who chose to use XFS2 to grow their stem cells before we can confirm or deny this.
Celprogen hopes that their safe and xeno-free XFS2 medium will facilitate the potential application of stem cell transplantation for the treatment of various diseases, and Celprogen is equally hopeful that their new medium will prove itself to be safe, give reproducible results, and a high-quality medium for the propagation of stem and progenitor cells in the laboratory.
Has anyone used XFS2 to grow their stem or progenitor cells in the laboratory yet? If so, let me know how it works.
When mouse embryonic stem cells were first derived in 1981 independently by Gail Martin at UCSF and Evans and Kaufman at Cambridge University, the inner cell mass cells from the blastocyst-stage mouse embryos were cultured on a layer of mouse skin cells that had been treated with a drug that prevented them from dividing or with radiation that did the same. These single layers of mouse skin fibroblasts secreted growth factors that prevented the embryonic stem cells from differentiating and drive them to divide. These layers of cells were known as “feeder” cells, because the secretions of the cells fed the growing embryonic stem cells.
When James Thomson at the University of Wisconsin derived the first human embryonic stem cell lines in 1998, he also used mouse feeder cells to keep the cells growing and undifferentiated. Once the embryonic stem cells were taken from this culture system, they began to differentiate.
However, it became equally clear that using mouse feeder cells represented a problem if human embryonic stem cells were going to be used for clinical purposes because animal cells can harbor occult viruses and other infectious agents that can infect human cells. Also, animal cells possess unusual sugars that are transferred to human cells when they are together in culture. Such foreign sugars can elicit robust immune responses against the cells if they are used for clinical purposes See Martin et al., Nature Medicine 2005; 11:228-232; and Stacey et al., Journal of Biotechnology 2006;125:583-588). Therefore, it became clear that finding ways to grow embryonic stem cells in the absence of feeder lines was an important goal if these cells were going to be used for clinical purposes.
Several laboratories successfully derived so-called “Xeno-free” embryonic stem cells by using protein substrata to grow the cells. These protein substrata included matrigel (animal), human laminin, E-cadherin, and vitronectin (see Xu C,, et al (2001) Nat Biotechnol 19:971–974; Miyazaki T,, et al. (2008) Biochem Biophys Res Commun 375:27–32; Nagaoka M,, et al. (2010) BMC Dev Biol 10:60; Chen G,, et al. (2011) Nat Methods 8:424–429). When Yamanaka and his colleagues discovered procedures for making human induced pluripotent stem cells, once again, feeder lines were initially used, but feeder-free protocols were also developed for deriving xeno-free induced pluripotent stem cells (iPSCs; see Chen G,, et al. (2011) Nat Methods 8:424–429; Nakagawa M,, et al. (2014) Sci Rep 4:3594).
A new report from Luis Gerardo Villa-Diaz, Jin Koo Kim, Joerg Lahann, and Paul H. Krebsbach from the University of Michigan, Ann Arbor, Michigan, has described a way to derive and grow human iPSCs on a completely synthetic substratum. This substratum, poly2-(methacryloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide, or PMEDSAH, forms a hydrogel that is completely synthetic. Therefore, the cells do not touch anything made from genetically manipulated cells or animal products.
Krebsbach and his group used fibroblasts from human gum tissue as their cell source. These cells were reprogrammed into iPSCs by means of infection with recombinant Sendai viruses. These viruses cause expression of the four genes required for reprogram cells (Oct4, Klf4, Sox2, and c-Myc), but they do not insert their viral genomes into the chromosomes of the host cell. Therefore, these viruses only express the reprogramming factors transiently, and afterwards, no trace of them can be found in the iPSC line, provided you properly screen for the absence of the virus. The reprogrammed cells were grown on the PMEDSAH and the cells not only were reprogrammed on this substratum, but also grew on it rather well.
The gum-based fibroblasts were nicely reprogrammed and made iPSCs that expressed all the right genes and produced tumors called teratomas when implanted into nude mice. The teratoma-production assay is an important test for pluripotency, because teratomas are tumors that consist of the mishmash of different tissue types. The fact that implanted cells produce these tumors with a mixed cell population of such wildly different cell types is an important indication of their pluripotency.
Even more importantly perhaps is the genetic integrity of these cells. Karyotypes of these iPSC lines (karyotypes lay our the chromosomes of the cell to see if there are a normal number of chromosomes and if the chromosomes appear normal) revealed that they were beautifully normal. However, hCGH (array-based comparative genomic hybridization) analysis, which uses specific chromosome-specific probes to finding missing or duplicated bits of chromosomes that are too small to see in a karyotype revealed a few missing and added bits to the genomes of all three derived iPSC lines. None of these were in known cancer loci. As shown in the figure below, these lines had only a few mutations. The karyotypes used cells from nine months after their derivation. Thus these cells proved to be rather stable.
This paper demonstrates that it is possible to generate transgene-free, stable iPSCs on a synthetic substrate. This type of platform has the potential to meet the good manufacturing practices that must be used to make products for clinical use.
A new study, published today in the journal Applied Materials & Interfaces reports the discovery of a new method for growing human embryonic stem cells that does not depend on feeder cells from human or animal cells.
Traditionally, embryonic stem cells are cultivated with the help of feeder cells derived from animals. Feeder cells secrete a host of growth factors and other signaling molecules that prevent the embryonic stem cells from differentiating and maintain their pluripotency. However, the use of animal products in the production of human cells lines rules out their use in the treatment of humans, since they can become contaminated with animal proteins that will cause rejection by the immune system or animal viruses that can infect the patient and cause significant disease.
The team of scientists led by the University of Surrey and in collaboration with Professor Peter Donovan at the University of California have developed a scaffold of carbon nanotubes upon which human stem cells can be grown into a variety of tissues. These nanotube networks mimic the surface of the body’s natural support cells and act as scaffolding for stem cells to grow on. Even cultured cells that have previously relied on feeder cells can now be grown safely in the laboratory, which paves the way for revolutionary steps in replacing tissue after injury or disease.
Dr Alan Dalton, senior lecturer from the Department of Physics at the University of Surrey said: “While carbon nanotubes have been used in the field of biomedicine for some time, their use in human stem cell research has not previously been explored successfully.”
“Synthetic stem cell scaffolding has the potential to change the lives of thousands of people, suffering from diseases such as Parkinson’s, diabetes and heart disease, as well as vision and hearing loss. It could lead to cheaper transplant treatments and could potentially one day allow us to produce whole human organs without the need for donors.”