Introduction of iPS Cells and ES Cells
Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. Induced pluripotent cells (iPS) are somatic cells that have been reprogrammed artificially and turn on the expression of specific pluripotency genes. This reprogramming can be achieved using a number of techniques with varying efficiencies.
The iPSC technology was pioneered by Shinya Yamanaka’s lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells.
Pluripotent stem cells hold promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic, and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease.
The most well-known type of pluripotent stem cell is the embryonic stem cell. Embryonic stem cells (ES cells or ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo. Embryonic stem (ES) cells can form tissues from all three primary germ layers (ectoderm, endoderm, and mesoderm) of the embryo. However, since the generation of embryonic stem cells involves destruction (or at least manipulation) of the pre-implantation stage embryo, there has been much controversy surrounding their use. Further, because embryonic stem cells can only be derived from embryos, it has so far not been feasible to create patient-matched embryonic stem cell lines.
Tissue Sources, Isolation of iPS Cells
A number of methods have been used to induce the reprogramming of non-embryonic stem cells. The initial techniques described used retro or lentiviral transfections to induce expression of oncogenes into the candidate cell following viral incorporation into the host cell’s genome. Later work demonstrated that adenoviral vectors could be used that would avoid viral incorporation into the host genome. However, the gene expression induced by adenoviral vectors is not maintained long term in transduced cells, limiting their pluripotent longevity. Expression plasmids alone have been used to induce pluripotency genes in target cells, but generation of iPS colonies is extremely inefficient using this method. Recently, a significant advance was described using the introduction of modified mRNA to reprogram adult cells. The modified RNA reprogramming method may prove to be the safest and most efficient strategy to reprogram adult cells and promote subsequent differentiation into the desired cell product for eventual therapeutic use. The field of cellular reprogramming is changing rapidly, and adaptation of techniques developed for other species will likely prove useful to enhance the induction of equine cells to a pluripotent state in the near future. For example, a recent report describes surgical implantation of chemically-induced putative ES-like cells in an equine model of superficial digital flexor tendonitis, leading to improved histological repair in the ES-like cell treated versus control lesions.
Tissue Sources and Isolation of ES Cells
ES cells are derived from the inner cell mass of the blastocyst stage embryo. In humans, this stage occurs at 5–6 days post fertilization, and the mouse at 3–4 days.
Two methods have been used to isolate the inner cell mass of the blastocyst for ES cells isolation. Across species, the most frequently utilized method is microsurgery. This procedure involves mechanical dissection under microscopic guidance and manual separation of the inner cell mass from trophoblastic lineage cells. The second method involves immunodissection using an antibody that targets trophoblast lineage cells of the blastocyst. Complement is added to the antibody-labeled blastocyst, leading to destruction of trophoblastic lineage cells while the inner cell mass remains unharmed.
Advantages of ES Cells and iPS Cells
Pluripotent stem cells and embryonic stem cells hold promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic, and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease. The following are some advantages of these two kinds of stem cells.
Firstly, embryonic stem cells have the potential to replicate indefinitely under defined conditions without differentiation, making an “off the shelf” preparation possible. Next, ES cells are able to form many committed cell types for regenerative tissue repair when differentiated prior to implantation. Another significant advantage is that there is minimal genetic manipulation of ES cells and there is a consequently decreased risk of aberrant tumor formation compared to iPS cell lines. ES cells have potential value in the treatment of genetic diseases through therapeutic cloning applications. ES cells have been used extensively in the production of transgenic mice, demonstrating proof of principle of their potential value with genetic manipulation. However, iPS cells have both advantages and disadvantages, they should be less prone to immunorejection since they can be patient-derived or MHC class I-matched for compatibility. Production of iPS cell lines also avoids the ethical controversy of embryo destruction associated with ES cell generation. In the horse, abundant donor tissues (e.g. dermal fibroblasts isolated from skin biopsies) are available to provide ample initial adult cells for reprogramming.
Disadvantages of ES Cells and iPS Cells
As for iPS, the use of viral vectors (especially retro or lenti viruses that randomly incorporate into the host genome) for iPS colony generation increases the risk of tumor formation and leads to concern for transplantation of reprogrammed cells in clinical trials. iPS cells can also show dramatic variability in the completeness of reprogramming and require extensive screening to select the most ES-like cells. Similarly to cultured ES cells, cultured iPS cells need to be frequently monitored for genomic abnormalities to ensure clinical safety.
Despite the tremendous potential ES cells hold for clinical benefit, a number of disadvantages need to be addressed. Routinely utilized methods for inner cell mass isolation necessitate destruction of an embryo, leading to ethical concerns across species, but especially in human ES cell research. Another concern for clinical application of ES cells is the potential for allogenic immunogenicity. Since ES cells are somewhat immunoprivileged, this may be less of a concern in therapeutic uses. One important disadvantage is the need for specific, complicated culture conditions to propagate and maintain ES cell lines in an undifferentiated state and the requirement for frequent monitoring of cultured cells for changes in genomic state to assure phenotypic stability. Additionally, there is a risk for tumor formation if ES cells are not fully directed into a differentiated cell type prior to surgical implantation. For therapeutic applications in people, another major concern is the use of non-human materials such as fetal bovine serum and mouse feeder cells to derive ES cells. An added problem to address is that the genetic background of the blastocyst plays an important role in the efficiency of deriving ES cell lines, as has been clearly demonstrated in the mouse. Horses have much more genetic variability than typical inbred mouse strains, therefore equine ES cell line generation may be complicated by their inconsistent genetic background. Horses have the additional disadvantages of low embryo numbers to harvest during blastocyst(s) collection and lack of optimal culture conditions to promote equine ES cell expansion without differentiation.