Safer Culture Conditions for Stem Cells


Jeanne Loring from the Scripps Institute is the senior author of a very important study that examined the culture conditions for pluripotent stem cells.

Several scientists have discovered that induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) can accumulate cancer-causing mutations when grown in culture for extended periods of time (for example, see Uri Weissbein, Nissim Benvenisty, and Uri Ben-David, J Cell Biol. 2014 Jan 20; 204(2): 153–163). However, some laboratories have managed to keep ESCs in culture for extended periods without observing instabilities.

To try to tease apart why this might be the case, Loring and her group examined various culture methods and determined that some stem cell culture methods are associated with increased incidence of mutations in the DNA of stem cells.

“This is about quality control; we’re making sure these cells are safe and effective,” said Loring, who is a professor of developmental neurobiology at Scripps Research Institute (SRI) in San Diego, CA.

All cells run the risk of accumulating mutations when they divide, but previous research from Loring and her colleagues showed that particular culture conditions could potentially select for faster growth and mutations that accelerate growth. Such growth-enhancing mutations are sometimes associated with tumors.

“Most changes will not compromise the safety of the cells for therapy, but we need to monitor the cultures so that we know what sorts of changes take place,” said Ibon Garitaonandia, who is a postdoctoral research fellow in Loring’s laboratory at SRI.

New research from Loring’s group has shown how particular culture conditions can reduce the likelihood of mutations. Loring and her colleagues tested several different types of surfaces upon which the cells were grown. They also used different ways of propagating or “passaging” the cultures. When cells are grown in culture, the culture dishes must be scraped to get the cells off them and then the cells must be transferred to a fresh culture dish. How you do this matters: do you use enzymes to detach the cells, or do you mechanically scrape them off? Other culture techniques use layers of “feeder cells” that do not divide, but are still able to secrete growth factors that improve the health of the growing stem cells.

Loring and her crew tested various combinations of surfaces, passaging methods and feeder cell populations and grew the cells for three years with over 100 passages. Over the course of this experiment, the cells were sampled and analyzed for the presence of new mutations in their genomes.

It turns out that stem cells grown on feeder cells that are passaged by hand (manually) show the fewest growth-enhancing mutations after being cultured for three years.

Loring’s study also demonstrated the importance of monitoring cell lines over time. In particular, deletion of the TP53 gene, a tumor suppressor gene, in whose absence cancer develops, should be closely watched.

“If you want to preserve the integrity of the genome, then grow your cells under those conditions with feeder cells and manual passaging,” said Loring. “Also, analyze your cells. It’s really easy, she added.

When Thomson made the first human ESC lines, he used feeder cells derived from mouse skin cells.  However, the use of animal materials to make ESCs might pollute them with animal viruses and specific sugars from the surfaces of the animal cells might also contaminate the surfaces of the ESCs, making them unsuitable for regenerative medicine (see Stem Cells 2006; 24:221-229).  To address this problem, several laboratories have made “Xeno-free” ESC lines that were made without touching any animal products.  Some of these Xeno-free lines were made without feeder cells (see C. Ellerström, et al., Stem Cells. 2006 Oct;24(10):2170-6)., but others were made with human feeder cell lines (see K Rajala, et al., Hum Reprod. 2007 May;22(5):1231-8). Therefore, it appears, that the use of human feeder cell lines are preferable to feeder-free systems, given Loring’s findings.  However, it is also possible that such culture systems are also preferable for iPSCs, which do not have the problem of immunological rejection for patients, and do not require the killing of the youngest members of humanity.  Therefore, Loring’s work could very well benefit iPSC cultures as well.

Derivation and Culture of Induced Pluripotent Stem Cells on an Artificial Substrate


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.

Human and mouse embryonic stem cells. (A) Colony of Human Embryonic Stem Cells (Cat# GSC-1103) growing on mitotically arrested feeder layers (Cat Nr GSC-6001M); colony morphology is characteristic of undifferentiated human ES cells. (B) Mouse Embryonic Stem Cells (Cat# GSC-5002) in culture.
Human and mouse embryonic stem cells. (A) Colony of Human Embryonic Stem Cells (Cat# GSC-1103) growing on mitotically arrested feeder layers (Cat Nr GSC-6001M); colony morphology is characteristic of undifferentiated human ES cells. (B) Mouse Embryonic Stem Cells (Cat# GSC-5002) in culture.

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.

Evaluation of pluripotency of human induced pluripotent stem cells (iPSCs) derived and cultured on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The pluripotency of the three human iPSCs derived and cultured on PMEDSAH was tested by embryoid body (EB) formation, directed in vitro cell lineage differentiation, and teratoma induction. (A): Representative micrograph of EBs from human foreskin fibroblast induced pluripotent stem cells 9 months after derivation and continuous in vitro culture. (B): Graph showing relative RNA transcription levels of genes expressed in cells after directed in vitro differentiation of human iPSCs on PMEDSAH-GPs. (C–E): Representative micrographs of directed in vitro cell lineage differentiation on PMEDSAH-GPs of human iPSCs 9 months after derivation and continuous in vitro culture. Neural differentiation (ectoderm) was achieved after treatment with Noggin (B, C). Definitive endoderm/pancreatic differentiation was induced by activin A treatment (B, D). Mesoderm lineage was obtained after treatment with activin A and BMP4 to induce cardiac muscle differentiation (B, E). Teratoma formation was performed 6 months after derivation and continuous in vitro culture of human iPSCs. (F–H): Representative micrographs of neurons (F), gut glandular epithelium (G), and cartilage (H) identified in teratomas. Scale bars = 200 μm (A), 100 μm (C–E), and 50 μm (F–H). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.
Evaluation of pluripotency of human induced pluripotent stem cells (iPSCs) derived and cultured on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The pluripotency of the three human iPSCs derived and cultured on PMEDSAH was tested by embryoid body (EB) formation, directed in vitro cell lineage differentiation, and teratoma induction. (A): Representative micrograph of EBs from human foreskin fibroblast induced pluripotent stem cells 9 months after derivation and continuous in vitro culture. (B): Graph showing relative RNA transcription levels of genes expressed in cells after directed in vitro differentiation of human iPSCs on PMEDSAH-GPs. (C–E): Representative micrographs of directed in vitro cell lineage differentiation on PMEDSAH-GPs of human iPSCs 9 months after derivation and continuous in vitro culture. Neural differentiation (ectoderm) was achieved after treatment with Noggin (B, C). Definitive endoderm/pancreatic differentiation was induced by activin A treatment (B, D). Mesoderm lineage was obtained after treatment with activin A and BMP4 to induce cardiac muscle differentiation (B, E). Teratoma formation was performed 6 months after derivation and continuous in vitro culture of human iPSCs. (F–H): Representative micrographs of neurons (F), gut glandular epithelium (G), and cartilage (H) identified in teratomas. Scale bars = 200 μm (A), 100 μm (C–E), and 50 μm (F–H). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

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.

Genetic stability of the human iPSCs derived on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The genomic stability of the three human iPSC lines derived on PMEDSAH-GPs was tested 9 months after derivation and continuous in vitro culture. (A): Representative standard G-banding metaphase karyotyping of one of the three human iPSCs derived on PMEDSAH-GPs showing normal male karyotype. (B): Ideogram summarizing chromosome losses and gains (left and right, respectively) of the three human iPSCs as detected by high-resolution array-based comparative genomic hybridization. No mutations are localized in chromosome loci where genes related to stem cells, cancer, or culture adaptation are localized. Abbreviations: chr, chromosome; CNG, copy number gain; CNL, copy number loss; hFF, human foreskin fibroblast; hGF, human gingival fibroblast; iPSC, induced pluripotent stem cell.
Genetic stability of the human iPSCs derived on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The genomic stability of the three human iPSC lines derived on PMEDSAH-GPs was tested 9 months after derivation and continuous in vitro culture. (A): Representative standard G-banding metaphase karyotyping of one of the three human iPSCs derived on PMEDSAH-GPs showing normal male karyotype. (B): Ideogram summarizing chromosome losses and gains (left and right, respectively) of the three human iPSCs as detected by high-resolution array-based comparative genomic hybridization. No mutations are localized in chromosome loci where genes related to stem cells, cancer, or culture adaptation are localized. Abbreviations: chr, chromosome; CNG, copy number gain; CNL, copy number loss; hFF, human foreskin fibroblast; hGF, human gingival fibroblast; iPSC, induced pluripotent stem cell.

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.

Ending the Reliance on Feeder Cells for Stem Cell Growth


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.”