Chicken Induced Plurpotent Stem Cells Made With Minicircles

The safety of induced pluripotent stem cells (iPSCs) haws been debated in several studies and publications.  Original studies of the genetic differences between the cellular sources of iPSCs and the iPSCs derived from them tended to show a whole gaggle of new mutations that seemed to not appear in the original cells.  Therefore, several commentators warned about the “dark side of pluripotency.”. However, other studies that utilized higher-resolution techniques showers that many of these mutations that occurred in iPSCs did exist in the original cells before their reprogramming, but that these mutations occurred at low frequencies, but became amplified during the culturing of reprogrammed cells.

One feature that has received less attention in these discussions of the safety of iPSC derivation is that the method by which iPSCs are made has distinct consequences for the stem cells that are made.  Typically, methods that utilize gene vectors that do not integrate into the genomes of the host cells are inherently safer than those vectors that do integrate.  PiggyBac transposon vectors integrate, but self-excise soon after their integration, and, therefore, do not leave a trace or their previous integration.  Minicircles also do not integrate and tend to produce safer iPSCs.  For this reason, this present paper is of interest to us.

Franklin West and his colleagues at the University of Georgia have made chicken iPSCs using minicircles to reprogram adult cells.  West was interested in using iPSCs to make recombinant chickens, since chickens are a rather primary food source and major component of economic development in several countries.  Making transgenic or recombinant chickens by means of stem cell technology makes it possible to make animals with improved meat and egg production or disease resistance.

To this end, West and his group made chicken (c) iPSCs from skin fibroblast cells by means of a nonviral minicircle reprogramming method.  This resulted in ciPSCs that showed excellent stem cell appearance and expressed key stem cell marker genes (alkaline phosphatase, POU5F1, SOX2, NANOG, and SSEA-1).  These cells also showed very rapid growth in culture and expressed high levels of the enzyme telomerase, which is an enzyme that is vital for the maintenance of chromosomes.

When West and his research group transplanted late-passage ciPSCs into stage X chicken embryos, the cIPSCs successfully integrated into the growing embryo and contributed to tissues derived from all three primary germ layers (ectoderm, mesoderm, and endoderm).  These ciPSCs also contributed to the gonads, which means that the ciPSCs could make gametes that could contribute to the production of a new generation of chicken.

These ciPSCs provide an exciting new tool to create transgenic chickens and has broad and exciting implications for agricultural and transgenic animal fields at large.  However, it also demonstrates that iPSCs can be safely produced and used for agricultural purposes.  This means that if non-integration-based or non-viral-based techniques are used to make iPSCs it should be possible to make them safely for therapeutic purposes also.

Making Better Induced Pluripotent Stem Cells

On July 2nd of this year, a paper appeared in the journal Nature that performed complete genomic analyses of embryonic stem cells derived from embryos or cloned embryos, and induced pluripotent stem cells (iPSCs), which are made from reprogrammed adult cells.  They found that both embryonic stem cells made from cloned embryos and iPSCs derived from the same types of adult cells contained comparable numbers of newly introduced mutations.  However, when it came to the epigenetic modification of the genome (the small chemical tags attached to specific bases of DNA that gives the cell hints as to which genes to turn off), the epigenetic pattern of the embryonic stem cells made from cloned embryos more closely resembled that from embryonic stem cells.  The iPSCs still had some similarities with the adult cells from which they were derived whereas the embryonic stem cells made from cloned embryos were more completely reprogrammed.  From this the authors claimed that making embryonic stem cells by means of cloning is ideal for cell replacement therapies.

There is a big problem with this conclusion:  This was tried in animals and it did not work because of immunological rejection of the products from the stem cells.  For more information on this, see my book, The Stem Cell Epistles, chapter 18.

Despite this “bad news” for iPSCs, two recent papers have actually provided some good news for stem cells that can heal without destroying embryos.  The first paper comes from Timothy Nelson’s laboratory at the Mayo Clinic in Rochester, Minnesota.  Differentiation of iPSCs is, in some cases, rather efficient and the isolation procedures fail to effectively isolate the differentiated cells from potentially tumor-causing cells.  However, in other cases, the differentiation is inefficient and the isolation procedures are also rather poor, which leaves a large enough population of undifferentiated tumor-causing cells.

Nelson’ group has discovered that treating iPSCs and their derivatives with anti-cancer drugs like etoposide (a topoisomerase II inhibitor for those who are interested) increases engraftment efficiency and decreases the incidence of tumors.  My only problem with Nelson’s paper is that he and his colleagues used lentiviral vectors to make their iPSCs.  These vectors tend to produce iPSCs that are rather good at causing tumors.  I would have rather that he tried making iPSCs with other methods that do not leave permanent transgenes in the cells.  Nelson and his group transplanted their iPSC-derived cells into the hearts of mice where they could use high-resolution imaging to determine the number of cells that integrated into the heart and the presence of cell masses that were indicative of tumors.  None of the ectoposide-treated cell transplants caused tumors whereas 4 of the 5 transplants not treated with ectoposide caused tumors.  This paper appeared in Stem Cells and Development.

The second “good news” paper for iPSCs comes from Junji Takeda at the University of Osaka and Ken Igawa from the Tokyo Medical and Dental University, Japan.   In their paper from Stem Cells Translational Medicine, the Japanese groups collaborated to make iPSCs from skin based fibroblasts and then differentiate them into skin cells (keratinocytes).  However, they made the iPSCs in two different ways.  The first protocol utilized the piggyBac transposon system to make iPSCs.  The piggyBac system comes from moths, but it is highly active in mammalian cells.  It can deliver the genes to the cells, but the segment of DNA is then easily excised from the host cells without causing any mutations.  This system, therefore, will generate iPSCs that do not have any transgenes in them.  The second protocol used a system based on cytomegalovirus that leaves the transgenes in the cells but gradually inactivates their expression.

When these two types of iPSCs were compared, they seems to be essentially identical when grown in culture.  Thus in the pluripotent state, the cells were equivalent for the most part.  But once the iPSC lines were differentiated into skin cells, the transgene-free iPSCs formed skin cells that looked, behaved and had the same gene expression profile as normal human skin cells.  The transgene-containing iPSCs differentiated into skin cells, but they did not look quite like skin cells, did not have the same gene expression profile as normal human skin cells, and did not behave like normal human skin cells.

The moral of this story is that not all iPSC lines are created equally and the way you derive them is as important as the cell type from which they were derived.  Also, even incomplete differentiation does not need to be an obstacle for iPSCs, since the cancer-causing cells can be removed by means of specific drugs.  Finally, not all that glitters is gold.  Cloned embryos may give you stem cells that look more like embryonic stem cells, but so what.  These might still suffer from many of the same set backs.  Add to that the ethical problems with getting women to give up their eggs for research and cures (see Jennifer Lahl’s movie Eggsploitation for more disturbing information about that), and you have a losing combination.