Since the isolation of embryonic stem cells in 1998, stem cell research has been considered the most promising research platform for developmental studies, disease treatment, tissue repair engineering, and regenerative medicine. However, embryonic stem cell research has been widely regulated and restricted due to the ethical issues surrounding research using embryonic tissue. Induced pluripotent stem cells (iPS cells) are stems cells that are derived through the genetic reprogramming of a somatic cell. iPS cells are nearly identical to embryonic stem cells, possessing the potential to give rise to every cell type in an organism, with the exception of extraembryonic tissues. Consequently, induced pluripotent stem cells promise the same research and clinical benefits as embryonic stem cells, without the ethical concerns. This presentation explores the process of generating induced pluripotent stem cells and investigates potential applications of induced pluripotent stem cells in both a research and clinical setting.
19. Chromatin immunoprecipitation assay
Microarray analysis of iPS cells revealed differences in methylation and
revealed slight differences in acetylation patterns of the promoter
gene expression profiles of iPS regions of pluripotency and
and embryonic stem cells. development associated genes between
iPS and ES cells.
20. Tissues from all three germ layers present in teratomas
derived from iPS cells.
21. Contribution of iPS cells to mouse embryonic development.
iPS cells were
microinjected into
blastocysts. Embryos
were analyzed with a
fluorescence
microscope
The chimeric embryo
was sectioned and
stained with anti-GFP
antibody (brown). Cells
were counterstained
with eosin (blue).
Chimeric embryos
contained cell from all
three germ layers.
22. Cardiac
Cells
Drug
Hepatocytes
screening Biopsy
and disease
modeling Neuron
Differentiation
of iPS Cells
Cardiac Reprogramming
Cells
Hepatocytes
Neuron Differentiation Genetic Repair
of iPS Cells of iPS Cells
32. Parkinson’s Disease
Symptoms
Cognitive: loss of
executive functions.
Physical: tremors,
stiffness, and slowed
movement
Inside the Brain
Dopaminergic neurons
within the substantia
nigra degenerate.
Dopaminergic Neurons
33. Isolation of Fibroblasts
Fibroblast Culture
Retroviral • Oct3/4
Rat Model of transduction of • Sox2
Parkinson’s Disease pluripotency factors • C-Myc
• Klf4
Transplantation of iPS Cells
Dopaminergic Neurons
Neuronal • Sonic Hedgehog
Differentiation • Fibroblast Growth
Factor 8
Dopaminergic
Neurons
iPS cell treated rats display improved
behavior and motor function
Stem cells are unspecialized cells with two unique properties. First, stem cells have the ability to self-renew through mitosis while maintaining their undifferentiated state.
Secondly, stem cells are pluripotent, meaning that they possess the ability to differentiate into all of the specialized cell types of an organism.
There are two broad categories of stem cells: embryonic stem cells and adult stem cells. Embryonic stem cells, as their name suggests, are derived from embryos.
Specifically, they are extracted from the inner cell mass of the blastocyst, an early embryo structure that forms five days after fertilization.
The blastocyst consists of two layers: the inner cell mass and the trophoblast. The inner cell mass contains cells that are destined to form the embryo, while the trophoblast contains cells that will form extraembryonic tissues such as the placenta.
Embryonic stem cells posses the potential to differentiate into every cell of the organism, with the exception of extraembryonic tissues.
Adult stem cells are undifferentiated somatic cells, found among differentiated cells in a tissue or organ. Adult stem cells can self-renew and differentiated into some or all of the specialized cells within an organ or tissue. Therefore, the major function of adult stem cells is to repair and maintain the tissue or organ in which they are found. While embryonic and adult stem cells promise advancements in both research and clinical medicine, both types of stem cells have obstacles that interfere with stem cell research.
Embryonic stem cells have been widely contested due to ethical concerns of performing research on embryonic tissues. These ethical issues have led to legislative restrictions on the creation and use of embryos in stem cell research, which pose a significant barrier to clinical breakthroughs in stem cell research.
Adult stem cells, while largely free of ethical concerns or legislative restrictions, possess a functional restriction. They are difficult to isolate from the body and are fewer in number than embryonic stem cells. Additionally, in the clinical setting, adult stem cells posses many of the same challenges that exist in organ donation. Like whole organs and tissues used for donation, adult stem cells possess the same unique genetic markers that are specific for each individual. Consequently, recipients of adult stem cells would have to be genetically matched to the stem cell donor in order to prevent rejection.
In response to the ethical and functional challenges of embryonic and adult stem cells, during the early 2000’s, stem cell research shifted its focus towards inducing pluripotency in somatic cells. In other words, researchers attempted to reprogram somatic cells to an undifferentiated state, with the hope that they could then be differentiated into specific, specialized cell types that could be used for cellular therapies and tissue regeneration.
In 2006, Shinya Yamanaka and his research team at Kyoto University successfully created induced pluripotent stem cells using adult mouse fibroblasts. In 2007, Yamanaka’s group successfully generated induced pluripotent stem cells using adult human fibroblasts, signifying a major breakthrough in the field of molecular biology, for which he has been awarded the Nobel Prize in Physiology or Medicine.
Yamanka’s group based their research on previous studies, which demonstrated that somatic cells could be reprogrammed to a pluripotent state by transferring their nuclear contents into oocytes or by fusing them with embryonic stem cells. This process, called somatic cell nuclear transfer, demonstrates that unfertilized eggs and embryonic stem cells contain factors that can confer pluripotency to somatic cells. Yamanaka’s group then proceeded to identify these factors, which maintain the pluripotency of embryonic stem cells and could induce pluripotency in somatic cells.
They began with 24 candidate genes, and narrowed down the key factors to just four genes which were capable of inducing pluripotency in somatic cells: Oct3/4, Sox2, c-Myc, and Klf4. Yamanaka’s group found that the addition of Oct3/4, Sox2, c-Myc and Klf4 to mouse fibroblasts, cultures under embryonic stem cell conditions, was capable of inducing pluripotency in the fibroblasts.
The four key pluripotency factors were added to the fibroblast cells using a technique called retroviral transduction. Retroviral transduction begins with the use of a retroviral vector, or a retrovirus that has been genetically modified so that it does not affect the physiology of target cells upon infection. The viral replication genes of the retrovirus have been replaced with the genes that the researchers wish to incorporate into the target cells. In Yamanaka’s study, the viral genes were replaced with genes coding for Oct3/4, Sox2, c-Myc, and Klf4.
The retroviral vector, containing the four key genes, was then allowed to infect the fibroblast culture, inserting the four key genes into the genome of the fibroblast cell.
It is this incorporation of the four key factors into the fibroblast genome that induces pluripotency in the fibroblast cells. After the generation of induced pluripotent stem cells, Yamanaka’s team carried out an extensive characterization process that compared the iPS cells with embryonic stem cells. The iPS cells were evaluated with regards to their morphology, global gene profile, epigenetic characteristics, pluripotency in vitro and teratoma formation in vivo.
iPS cells are morphologically indistinguishable from embryonic stem cells.
Microarray analysis of iPS cells revealed slight differences in gene expression profiles of iPS and embryonic stem cells.Chromatin immunoprecipitation assay demonstrated slight differences in methylation and acetylation patterns of the promoter regions of pluripotency and development associated genes between iPS and ES cells.
Pluripotency of the iPS cells was evaluated by teratoma formation. A teratoma is a tumor that contains tissues from all three germ layers; therefore, a cell’s ability to form teratomas demonstrates pluripotency, as it shows that the cell is capable of differentiating into any cell type. Yamanaka’s iPS cells were demonstrated to form teratomas, confirming their pluripotency.
Additionally, another important hallmark of ES cells as pluripotent stem cells is their ability to form chimeric mice. In this process, iPS cells are injected into a mouse blastocyst, which is then implanted into a surrogate mother for gestation. The iPS cells give rise to the embryo, demonstrating that iPS cells are pluripotent and capable of giving rise to tissues from all three germ layers. These characterization procedures demonstrate that iPS cells possess the same phenotypic qualities of embryonic stem cells, particularly self-renewal and pluripotency. However, it is important to note the gene expression and epigenetic differences between iPS cells and ES cells.
Much like embryonic stem cells and adult stem cells, induced pluripotent stem cells promise countless clinical applications, particularly in the form of cellular therapies and regenerative medicine.However, while iPS cells are free from ethical concerns, they possess their own unique barriers to clinical applications.
A primary concern of iPS cells is their intrinsic predisposition to teratoma formation. In the context of characterization, teratoma formation is a positive indicator of pluripotency. However, in a clinical setting, teratoma formation in patients poses obvious concerns. While tumorigenic activity of iPS cells is lost following differentiation of iPS cells, it is difficult to guarantee that all iPS cells undergo full differentiation. Consequently, improved purification techniques must be established to ensure that a pure sample of differentiated cells are used in therapeutic applications in order to avoid the risk of tumor formation in patients.
A second major concern regarding the use of iPS cells in a clinical setting is the use of viral vectors to induce pluripotency. While retroviral vectors successfully integrate key pluripotency genes into the target cell genome, retroviral insertion can cause gene disruption at the site of integration, leading to potential upregulation of adjacent genes. This is particularly worrisome, as the upregulation of many genes, specifically genes related to cell proliferation, can cause the formation of tumors. Furthermore, the four key pluripotency genes (Oct3/4, Sox2, c-Myc, and Klf4) have all been demonstrated as common mutations in a variety of cancers. Consequently, it is vital that the virally transduced genes be permanently switched off following successful differentiation, in order to eliminate the threat of tumorigenesis.
Recent advancements in iPS cell research have been made regarding the delivery method of pluripotency factors into somatic cells.The most promising technique delivers Oct3/4, Sox2, c-Myc, and Klf4 proteins directly into the cell. Unlike retroviral transduction, this method does not insert the genetic code for these proteins into the somatic cell genome, reducing the risk of tumorigenesis.
The induction of pluripotency via the direct delivery of proteins is mediated by cell penetrating peptides (CPPs), which are peptide sequences containing a high proportion of basic amino acids (predominantly lysine and arginine). Unlike typical proteins, cell penetrating peptides can easily cross the plasma membrane. CPPs thus act as a shuttle to carry cargo proteins across the plasma membrane and into the cell.
The mechanism by which CPPs translocate across the plasma membrane is not well understood, although several hypotheses have been suggested, including endocytosis and the formation of inverted micelles. In 2009, induced pluripotent stem cells were successfully generated using CPP mediated direct delivery of reprogramming factors. While this method provides improved safety, the efficiency of generating iPS cells is greatly reduced.
iPS cells have the potential to be invaluable tools for biomedical research.Currently, iPS cells are used to evaluate the safety and effectiveness of drugs.iPS cells provide a platform to address unanswered questions about the normal developmental pathway and process of cell differentiation.iPS cells are an ideal tool for the development of personalized models of disease
The potential therapeutic applications of iPS cells are by far the most promising and exciting.iPS cells can be directed to differentiate into any cell of an organism and thus, can be used to generate patient-specific treatments for countless genetic, metabolic, degenerative and autoimmune diseases, including sickle cell anemia, liver disease, Parkinson’s disease, and diabetes mellitus.Easily accessible cells (such as fibroblasts) can be biopsied from a patient, cultured, and induced to a pluripotent state. The iPS cells can then be directed to differentiate into a specific cell type that is free of the patient’s disease pathology. These healthy cells can then be transplanted into the patient.
The use of iPS cells in cell-based therapies will significantly reduce immune rejection of transplanted tissues, as the patient’s own cells will serve as the source of the new, healthy tissue.
Parkinson’s Disease (PD) is a common, chronic neurodegenerative disorder characterized by the selective degeneration of dopaminergic (DA) neurons in the substantia nigra of the midbrain. The cause of PD is not well understood, although research indicates that ubiquitin-proteasome system dysfunction, inflammation, and increased apoptosis may contribute to the progression of the disease.
Symptoms of Parkinson’s Disease include the gradual loss of cognitive functions, as well physical symptoms such as tremor and slow movement. Currently, there are no effective treatments for PD. Therefore, iPS cells provide a promising source of cell-based therapy for the treatment of PD.
iPS cell-derived dopaminergic neurons have demonstrated successful synaptic integration and innervation in a rat model of PD. Analysis of the iPS cell-treated rats demonstrated marked improvements in behavior and motor function.
Further studies have demonstrated that dermal fibroblasts of PD patients can be efficiently reprogrammed and then differentiated into dopaminergic neurons. Additionally, the integration technique used in this study allowed for the successful excision of pluripotency factors after reprogramming and before differentiation, thereby eliminating risks of tumor formation. Therefore, with further study, this method may be a successful treatment for Parkinson’s Disease.
Induced pluripotent stem cells provide a new platform for the successful application of stem cell derived therapies. While iPS cell-derived therapies have yet to be studied in human clinical trials, their limitless potential in both research and clinical settings promise increased treatment options and improved quality of life for countless patients.