MIC115 2024 Induced Pluripotent Stem Cells Lecture Notes PDF

MIC115 Recombinant DNA Cloning 2024FQ Lecture 16 –Induced pluripotent stem cells and cancer Breaking news! A groundbreaking study in regenerative medicine has demonstrated the successful use of induced pluripotent st...

MIC115 Recombinant DNA Cloning 2024FQ Lecture 16 –Induced pluripotent stem cells and cancer Breaking news! A groundbreaking study in regenerative medicine has demonstrated the successful use of induced pluripotent stem cells (iPSCs) to treat severe corneal damage caused by limbal stem-cell deficiency (LSCD), a condition that leads to vision loss due to the depletion of stem cells in the cornea's limbal region according to a report published in the Lancet. Researchers in Japan reprogrammed donor blood cells into iPSCs and developed corneal epithelial cell sheets, which were transplanted into the eyes of four patients with LSCD. Over a two-year observation period, the treatment showed no serious adverse effects, such as tumor formation or immune rejection, even in patients who did not receive immunosuppressants, and most patients experienced significant improvements in vision and corneal health. These results mark a significant advance in regenerative medicine, paving the way for larger clinical trials to confirm the efficacy and broader application of this innovative therapy. Learning goals - Understand early evidence of somatic cell reprogramming to embryonic state using somatic cell nuclear transfer (SCNT) - Understand how Dr. Yamanaka determined the defined factors for iPSC reprogramming - Understand the differences between ES cells and iPS cells - Understand how iPSC can be utilized for regenerative medicine and other fields (in combination with genetic engineering) I. An overview of stem cells Stem cells are functionally defined by their dual capacity for self-renewal and differentiation: Self-renewal refers to the ability to go through numerous cycles of cell division while maintaining the undifferentiated state. Differentiation refers to the ability to differentiate into specialized cell types under certain conditions. Stem cells are categorized into different classes according to their capacity for differentiation, i.e. “potency”. A. Stem cell potency Totipotent: these cells can produce all embryonic cell types as well as extraembryonic structures, such as the placenta. As such, they can construct an entire, viable organism. During the development of a fertilized egg, up until the eight-cell stage, each cell within the embryo is totipotent to give rise to a complete animal when implanted into the uterus of a pseudo-pregnant animal. Pluripotent: these cells can produce all embryonic cell types but not the extraembryonic structures, e.g. the placenta. The inner mass cells of the blastocyst are pluripotent stem cells. They are incapable of generating a viable organism in its entirety. Embryonic stem cells (ESCs) are the classic example of pluripotent cells. Multipotent: these cells can give rise to multiple different cell types present in one specific organ or tissue. Most adult stem cells are multipotent stem cells, including hematopoietic, mesenchymal, and neural stem cells. B. Medical applications of human stem cells 1 MIC115 Recombinant DNA Cloning 2024FQ ES cells could contribute to regenerative medicine: similar to mouse ES cells, the human ES cells can produce various specialized human cells in a large quantity, e.g. neural cells, hepatic cells, and cardiac cells. Then, we should be able to treat patients suffering from various disease and injuries, such as Parkinson disease, spinal cord injury, hepatic failure, or diabetes, by transplanting those specialized cells. ES cells are also immensely useful for drug development: patient-derived stem cells can be differentiated into diseased specialized cells for disease modeling and various aspects of drug discovery, including target identification, metabolic profiling, and toxicity evaluation. C. Technical and ethical challenges facing human stem cell research Although multipotent stem cells can be found widely distributed throughout the body in different physiological systems, they tend to proliferate more slowly than pluripotent stem cells, and have a limited capacity for expansion, particularly in vitro. Generating new embryonic stem cell lines involves destruction of human embryos, thus poses ethical and political problems. Additionally, the use of stem cells for transplantation introduces the challenge of immune rejection, especially with allogeneic therapies, where the donor cells come from a genetically different individual. This can lead to the recipient’s immune system identifying the transplanted cells as foreign and attacking them, necessitating the use of immunosuppressive drugs, which carry their own risks. In contrast, autologous stem cell therapies, which use the patient’s own cells, eliminate the risk of immune rejection but are often more time-consuming and costly, as the cells need to be harvested, reprogrammed, and expanded specifically for each individual. Can we bypass these problems by inducing pluripotency from fully differentiated somatic cells? D. Somatic cell nuclear transfer (SCNT)-based cloning demonstrates that fully differentiated somatic cells can still be reprogrammed into an embryonic state In 1958, John Gurdon transferred the genetic material of an intestinal epithelium cell of a Xenopus tadpole into an enucleated frog oocyte. The modified egg developed into a normal tadpole. These tadpoles were exactly genetically identical to the Xenopus larva from which the original somatic cell nucleus was taken. Subsequent SCNT experiments have generated cloned mammals, including “Dolly” the sheep. These experiments proved that the mature cells still contained the genetic information needed to form all types of cells and can be reprogrammed into an embryonic state. E. Converting somatic cells to pluripotent stem cells by expression of define factors One of the most important tasks in the generation of induced pluripotent stem cells (iPSCs) was the identification of the key pluripotency factors. To establish the list of candidate genes, Yamanaka et al. first used a subtraction approach to identify cDNAs that are specifically enriched in mouse embryonic stem cells (ES cells) in comparison to differentiated somatic cells. Subsequently, 24 genes were selected as candidate factors to test their ability to revert the fully differentiated somatic cells into the ES state. Full length cDNAs of the 24 genes were obtained and cloned into retroviral expression vectors. An assay system in which the induction of the pluripotent state could be detected as resistance to G418 is constructed: a beta-geo cassette (a fusion of the beta-galactosidase and neomycin resistance genes) is cloned downstream of the Fbx15 gene in mouse ES cells by homologous 2 MIC115 Recombinant DNA Cloning 2024FQ recombination. Although specifically expressed in mouse ES cells and early embryos, Fbx15 is dispensable for the maintenance of pluripotency and mouse development. ES cells homozygous for the beta-geo knock-in construct were resistant to extremely high concentrations of G418 (up to 12 mg/ml), whereas somatic cells derived from Fbx15 beta-geo/beta-geo mice were sensitive to a low concentration of G418 (0.3 mg/ml). Activation of the Fbx15 locus (i.e. induction of the ES state) would result in resistance to G418. Using this convenient assay system, 4 of the 24 genes-KLF4, Sox2, Oct3/4, and c-myc-were found in 2006 to be necessary and sufficient to transform fully differentiated mouse somatic cells into pluripotent stem cells. In 2007, the same 4 factors were demonstrated to transform fully differentiated human somatic cells into pluripotent stem cells. II. Applications with iPS cell technology A. Macular degeneration Macular degeneration is caused by progressive degeneration of the retinal pigment epithelial (RPE) cells, which is the leading cause of vision loss in elderly people in developed countries. iPS cell technology can be used to replace the degenerated RPE with a healthy layer of RPE, which can be derived from patients or donor’s cells. So far, in Japan, 7 patients received iPS cell-derived RPE cells to repair eye disorders, and three participants had markedly improved vision one year after their operations (reported in 2020). B. Ischemic cardiomyopathy There was a case report on transplantation of iPS cell-derived cardiomyocyte transplantation for ischemic cardiomyopathy. Ischemic cardiomyopathy is a heart disease of losing cardiomyocytes due to restricted blood flow (ischemia). There was a 51yr old male patient, who was repeatedly admitted to hospitals due to cardiomyopathy. Blood cells from a donor were reprogrammed to iPS cells and differentiated into cardiomyocyte patches. The clinical symptoms improved 6 mohts after surgery without any major adverse events. Further clinical evaluations are needed. C. Stem cell therapy for diseases caused by certain genetic mutations Example – cystic fibrosis (CF), caused by mutations in CFTR gene (cystic fibrosis transmembrane conductance regulator) iPS cell reprogramming from patient’s somatic cells  Genetic modification (e.g., CRISPR)  Differentiation of iPS cells into lung epithelial cells  Transplant D. Disease modeling and drug discovery Example – ALS (Amyotrophic lateral sclerosis) ALS is caused by the death of neurons controlling voluntary muscles (motor neuron). The exact cause of this disease is largely unknown, but it is believed to involve both genetic and environmental factors. Because of this reason, there is no good animal model for this disease. In addition, it is not feasible to obtain enough cells from ALS patients because motor neurons do not multiply. From patients, we can obtain any somatic cells (e.g., skin cells) and 3 MIC115 Recombinant DNA Cloning 2024FQ reprogram into iPS cells and differentiate into specific neuronal cells to study (e.g., drug screen, genetic modifications). 4