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CAN STEM CELLS THERAPY OFFER HOPE TO PEOPLE WITH KIDNEY FAILURE? By NATHAN LEUNG Grade awarded: Pass with Merit RESEARCH PAPER BASED ON PATHOLOGY LECTURES AT MEDLINK and VET-­‐MEDLINK 2014 ABSTRACT This paper will provide an overview of the use of stem cells in medicine including ethical considerations and other challenges in the use of stem cells. In particular, I have placed emphasis on the application of stem cells as regenerative therapy in renal disease. Kidney disease is currently a global public health problem, with an incidence that has reached epidemic proportions. The kidneys perform essential homeostatic function of fluid balance and waste metabolism. When the kidneys fail, people have to either start dialysis or have a kidney transplant. Stem cell therapy to regenerate function may one day be an option of therapy along side the traditional replacement therapy of dialysis or transplantation. INTRODUCTION WHAT IS A STEM CELL? A stem cell is defined as a cell that is capable of asymmetric division, giving rise to a daughter cell that retains “stemness” and to another daughter cell that is a progenitor for a particular cell lineage. Thus stem cells are characterized by their capacity for self-­‐renewal and ability to differentiate into specialized cell types (Figure 1)[1]. Levels of competence form the basis of their classification as totipotent (giving rise to all three embryonic germ layers, as well as extra-­‐
embryonic tissues), pluripotent (able to contribute to all three germ layers of the embryo, i.e., they can make any cell of the body) or multipotent (only make cells within a given germ layer) and unipotent (make cells of a single type). 2
Figure 1 Classically, the potency of cells relates to the time of embryonic development of the organism. That is, cells that arise from the first few cell divisions following fertilization of the egg are generally the only cells that have totipotency. Pluripotent cells are cells derived from either the inner cell mass of the blastocyst (a pre-­‐implantation stage of development occurring approximately 7 to 10 days after fertilization in the human) or nascent germ cells in the embryo. Cells cultured and cell lines established from these structures are called embryonic stem (ES) cells and embryonic germ cells respectively. SOURCES OF STEM CELLS Stem cells can be derived from human embryos, somatic tissues in the adult or they can be created by inducing greater potency in an already differentiated somatic cell, so called induced pluripotent stem cells (iPSCs). Embryonic Stem (ES) Cells 3
ES cells are typically derived from a pre-­‐implantation blastocyst (7 to 10 days post fertilization). Pluripotent ES cells are the most primitive cell type likely to find application in cell therapy. Their potential to generate any given cell type of the embryo (pluripotency) makes them in some ways the most attractive stem cell for cell therapy. Induced Pluripotent Stem Cells (iPSCs) Induced pluripotent stem cells (iPSCs) are made by reprogramming adult, specialised cells of the body to act like embryonic stem cells. They have the ability to develop into any cell or tissue in the body. Shinya Yamanaka and his colleagues took genes that were expressed in pluripotent ES cells, but not generally in mature cells and introduced them into mature cells[2]. The genes would now be “ectopically” expressed in a cell type where the gene is normally not expressed and a number of the mature cells reverted back to a highly immature cell state that resembled an ES cell. This process is called reprogramming, induced a pluripotent state in a previously differentiated cell type (Figure 2)[1]. Figure 2 Adult Stem Cells 4
Adult stem cells are thought to be present in most, but not all, tissues and to persist throughout life. Their role is thought to be tissue maintenance, particularly true for tissues where there is high cell turnover, such as the blood, skin and intestine. A number of organs are thought to be largely non-­‐regenerative and non-­‐proliferative such as the heart, kidneys and brain. Therefore in settings of extensive injury, a tissue that depends upon residual mature cells to replace the injured cells may be more limited in its regenerative capacity than tissues that can rely on resident stem cells. This is one hypothesis for the failure of repair of pancreatic islet damage in type I diabetes mellitus, whereas extensive cell damage to the blood with cancer chemotherapy can result in complete restoration of tissue function. Thus the use of stem cells can potentially provide healing through regeneration of tissues. THE STEM CELL JOURNEY Yamanaka, for his discovery of iPSCs[2] shared the 2012 Nobel Prize in Physiology or Medicine with John Gurdon who had shown decades before that adult somatic cell nuclei transfer (SCNT) transplanted into enucleated frog oocytes could be reprogrammed and give rise to new frogs[3]. In 1997, Wilmut et al[4] showed that fundamentally the same procedure SCNT could be used to “clone” Dolly the sheep. Factor (or factors) in oocyte cytoplasm was capable of reprogramming the genome present within the transplanted somatic cell nucleus back to a state of pluripotency. Thus, autologous (patients’) cells could potentially be generated for cell therapy of patients with degenerative disease, including kidney injury. Using somatic cells from people with diseases may avoid the risk of rejection often seen in the sphere of cell transfer or transplantation. This application of SCNT was termed “therapeutic cloning,” to distinguish it from a procedure in which the blastocyst created via SCNT would be implanted in the uterus of a surrogate mother to give birth to a new organism. STEM CELL IN MEDICINE The paradigm for stem cells as a means of replacing injured or diseased tissue was first explored in response to the threat of nuclear warfare after World War II. In 1963, researchers in Toronto demonstrated that a bone marrow-­‐derived cell could replace all the blood elements and rescue an otherwise lethally-­‐irradiated animal by simple infusion of donor cells into the blood[5]. There are 3 main uses of stem cells in medicine: stem cells as targets of drug therapy, stem cells to generate differentiated tissue for in vitro study of disease models for drug development and stem cells as therapy (either to replace cell lines that have been lost or destroyed, or to modify the behavior of other cells). The rest of the paper will focus on stem cell therapy and in particular its application in kidney disease. STEM CELL THERAPY 5
The therapeutic potential for ES cell-­‐derived somatic cells has been demonstrated in animal models of retinal blindness, Parkinson's disease, Huntington disease, spinal cord injury, myocardial infarction and type I diabetes mellitus. In human, stem cell therapy has been used in a number of medical conditions with clinical benefits, including diabetes mellitus, Parkinson’s disease, multiple sclerosis, organ transplants, retinal disease and in immune-­‐inflammatory conditions. The approach of using induced human pluripotent cells (iPCs) has been used in Fanconi anemia. A normal version of the gene mutated in Fanconi anemia was introduced into fibroblasts from patients, and the gene-­‐corrected cells were reprogrammed to generate patient-­‐specific iPCs. The iPCs carrying the normal gene were able to generate blood cell types in a manner similar to cells from normal volunteers. IPCs have also been used in Huntington disease, a neurodegenerative disease. Fibroblasts from patients were reprogrammed to generate patient-­‐specific iPCs, which were then differentiated into gamma-­‐aminobutyric acid-­‐producing neurons. Using human cells as opposed to ES cells also circumvent the ethical dilemma of using embryos as source of stem cells. Before advocating the use of ES cells or iPCs as panacea for diseases, caution has to be exercised. In one case, a child was given cultured fetal brain cells intrathecally and subsequently developed multiple brain tumours of donor origin. As iPCs are pluripotent, they have the greatest potential for inducing tumours. Moreover the use of retrovirus to reprogram cells runs the risk of mutagenesis by virtue of viral integration into the host genome. THE KIDNEY Kidney diseases are currently a global public health problem, with an incidence that has reached epidemic proportions. These trends correlate with the global rise in the aged population and the increasing prevalence of conditions that cause renal complications, namely cardiovascular disease, hypertension and diabetes. The kidney performs essential roles including metabolic waste excretion, fluid and acid base balance, vital homeostatic and endocrine functions. It is a complex organ made up of heterogeneous cells (Figure 3)[6]. Nephrons have three major parts: (1) a glomerulus that filters the blood, (2) a tubule that modifies the filtrate to reabsorb and secrete solutes as the fluid passes through proximal, intermediate, and distal segments, and (3) a duct that carries the urine into a centralized collecting system. 6
Figure 3 ACUTE KIDNEY INJURY (AKI) Acute kidney injury (AKI) involves a rapid loss of kidney function from sudden renal cell damage, which can be caused by pre-­‐renal, renal and post-­‐renal causes (Figure 4). AKI leads to necrosis of renal proximal tubule cells as a result of ischemic or toxic insult. AKI affects more than 15% of all hospital admissions and is associated with increased rates of death and morbidity. The mortality rate can range from 15% in patients with isolated renal failure to 50-­‐80% in severe cases, in which dialysis is needed. The recovery of renal function following AKI depends on the ability of renal tubules to regenerate and to replace necrotic tubular cells with functional tubular epithelium. The absence or reduction of epithelial regeneration may predispose to tubulo-­‐interstitial scarring and chronic kidney disease. 7
Figure 4 CHRONIC KIDNEY DISEASE Chronic kidney disease (CKD) is typified by the progressive loss of kidney function over time due to fibrosis and the erosion of healthy tissue. Approximately 6% of the UK adult population has chronic kidney disease (CKD) stages 3-­‐5 (NHS Kidney Care, 2010). RENAL REPLACEMENT THERAPY When end stage renal failure (ESRF) is reached, people will need to have either a kidney transplant or dialysis. The current cost of treating people with ESRF has been estimated at 1-­‐2% of the NHS budget at a cost of 1-­‐2 billions, yet they comprise only 0.05% of the population. Kidney transplants offer a hope for cure, but thousands of patients die each year due to a shortage of donor organs. In 2006/7 over 3,000 patients in the UK received an organ transplant (Figure 5), but another 1,000 died whilst waiting or after being removed from the waiting list because they had become too ill. Even patients who are lucky enough to receive transplants run the risk of their immune systems 8
rejecting the donor kidney and they have to take immunosuppressive drugs with serious side effects of cancer development and infections. Thus, there is considerable drive to develop new therapies for renal failure with the capacity to replace a wider range of the kidney's functions, reducing morbidity, mortality, development of ESRF and need for dialysis, as well as the overall economic impact associated with this condition. Figure 5 KIDNEY DISEASE AND STEM CELL THERAPY There is emerging evidence that human kidneys possess innate regenerative abilities. Diabetic patients with CKD exhibited reversion of fibrotic lesions in their kidneys ten years after receiving a pancreas transplant. Bussolati et al (2005)[7] since discovered cells in the renal papilla with characteristics suggesting that they may represent the elusive adult kidney stem cell. 9
Bone-­‐Marrow Derived Cells (BMDC) BMDCs appear in the kidney in response to renal injury. BMDCs can transdifferentiate into renal tubular epithelial cells, mesangial cells, glomerular endothelial cells, and podocytes[8]. There is evidence that BMDCs significantly contribute to the regeneration of the renal tubular epithelium, differentiate into renal tubules, or promote proliferation of both endothelial and epithelial cells after injury. Stem cell factor and granulocyte colony-­‐stimulating factor (G-­‐CSF) induced BMDCs home to the injured kidney, leading to the significant enhancement of the functional recovery of the kidney. BMDCs might contribute to the regenerative process by producing protective and regenerative factors, rather than by differentiating to directly replace damaged cells. These data suggest that the delivery of BMDCs to the kidney hold potential for the treatment of acute kidney injury. However boosting of peripheral stem cell numbers was also associated with worsening renal failure and mortality[9]. The contradictory results in the localization of BMDCs and the degree of the BMDC contribution to kidney regeneration after injury may be due to methodological limitations in tracking BMDCs and differences in the protocols used in these studies. Mesenchymal Stem Cells (MSC) The other possible BMDC responsible for ameliorating renal damage is the MSC. In a phase-­‐II clinical trial[10], autologous MSC decreased the incidence of acute rejection in patients undergoing kidney transplantation, although their effects on graft survival and long-­‐term outcomes require further studies. In a rat sepsis model, MSC protected major organs from damage[11], including the kidney by reducing inflammatory cell infiltration and kidney cell apoptosis. Preliminary results in a phase-­‐1 clinical trial[12] using aortic injection of allogeneic bone-­‐marrow-­‐
derived MSC after cardiac surgery showed that post-­‐operative AKI was reduced (20%) as were the length of stay and readmission (40%). Importantly, this study showed that suprarenal, postoperative administration of allogeneic MSC did not led to adverse events. Thus, the use of MSC to protect the kidney or to prevent AKI in high-­‐risk patients seems to be feasible and safe. Further research is needed to establish whether the use of MSCs could lead to a treatment for patients. Adipose tissue-­‐derived Stem Cells Adipose tissue-­‐derived stem cells are an alternative source of stem cells with regenerative properties similar to those of BMDCs. Adipose tissue-­‐derived stem cell therapy minimised kidney 10
damage or improved renal dysfunction in ischemic injury[13], a mouse progressive renal fibrosis model[14] and in AKI[15]. Induced Pluripotent Cell (iPCs) Recently researchers have been able to use iPCs to produce kidney cells in a very early stage of development. These very early kidney cells resemble cells found in the embryo that will turn into the cells that eventually make up the kidney in foetal development. These cells could have the potential to make the glomerulus and tubules, the building blocks of the nephron. Recently, unique methods for stimulating the differentiation of human iPS cells into kidney lineages[16] or three-­‐dimensional structures of the kidney[17] have been developed. IPCs from normal human mesangial cells[18], renal tubular cells present in urine[19] and fibroblasts of patients with autosomal dominant polycystic kidney disease[20] have been established. Human Amniotic Fluid Stem Cells Human amniotic fluid stem cells are multipotent cells with characteristics of both embryonic and adult stem cells. They are another promising candidates for stem cell therapy. Beneficial therapeutic effects of amniotic fluid stem cells have been shown in kidney injury models including AKI injury induced by glycerol, a mouse model of Alport’s syndrome and a mouse unilateral ureteral obstruction model[21]. TRANSDIFFERENTIATION The main attraction of the transdifferentiation approach is that it offers the possibility of avoiding complications from immunogenicity of introduced cells by obtaining the more easily accessible stem cells of another tissue type from the patient undergoing treatment, expanding them in vitro, and reintroducing them as a therapeutic agent. This is based on findings that showed adult stem cells, long thought to be highly restricted in their differentiative potential, may possess a considerable degree of plasticity. However a lack of an emerging consensus has served to dampen the initial optimism over transdifferentiation of BMSCs. Further work is required to settle the controversy. SEEDLING An alternative and simpler way of potentially restoring the kidney function is seeding through the vasculature or through the ureter. Bonandrini et al (2014)[22] seeded mouse ESCs through the renal artery and reported an even distribution of cells with over 97% cell attachment. Similarly, Caralt et al (2015)[23] reported that renal arterial perfusion seeding of human renal cortical tubular epithelial cells at led to about 50% coverage of the renal area. 11
TISSUE AND BIOENGINEERING APPROACHES The tissue and bioengineering approaches are, in general, based on in vitro manipulation of the cells of interest and their association with biomaterials to produce a device for implantation or incorporation into an extracorporeal circuit. At the modest end of the scale are strategies such as the implantation of a single differentiated cell type to replace a metabolic or catabolic function, whereas at the far (as yet theoretical) end of the spectrum sits the goal of “growing” a functioning organ for transplantation. ORGAN SCAFFOLDS An alternative approach to organ replacement is the use of organ scaffolds to produce whole, transplantable organs. An effective alternative to traditional organ transplantation is needed in order to increase the number of organs available for transplantation, decrease patient wait-­‐list times, and improve long-­‐term outcomes. Organ scaffolds are organs from which all the cells have been removed. What remains is the extracellular matrix – the part of the organ that supports its shape. This matrix can be seeded with a patient’s own cells, which can be carefully nurtured to grow and multiply to re-­‐cover the scaffold. By using the patient’s own cells, the complications of immune rejection that can occur with organ transplantations are drastically reduced. Decellularization has been successfully adapted for the generation of a whole-­‐heart scaffold, intact lungs, kidneys, pancreas from rodents, pigs, primates and humans have been decellularized using similar approaches[24]. The next challenge is effective recellularization. For complete organ regeneration, the parenchyma, vasculature and support components must be reestablished prior to implantation; requiring appropriate cell sources, an optimal seedling method and a physiologically relevant culture method. A common theme in the organ bioengineering literature is the use of fetal cells derived from the organ of interest. Rat lung scaffolds seeded with neonatal or fetal rat lung cells participated in gas exchange after implantation and similar success in liver recellularisation with restoration of urea and albumin production. Neonatal renal cells showed similar success when seeded rat kidneys produce urine in vivo[18,25]. Fetal cells provide proof-­‐of-­‐concept but, nevertheless, are not viable cell types for clinically relevant organ engineering. The use of iPSCs by reprogramming somatic cells from patients could one day provide a cell source for the construction of patient-­‐specific organs. Encouragingly recent investigations have harnessed iPSCs for lung recellularization[26]. WHOLE ORGAN ENGINEERING: IMMUNOGENICITY 12
One of the major drawbacks of traditional organ transplantation is the requirement for life-­‐long immunosuppression to reduce risk of rejection of organ due to immune response to major histocompatibility complex (MHC) antigens. Decellularization of rat lungs was shown to significantly reduce the presence of MHC class I and II molecules and DNA[27]. ETHICAL DILEMMA The use of stem cells is fueled by ethical controversy. ES cells are obtained mainly from surplus embryos from in vitro fertilization (IVF), which entails their destruction. Today, with the advances of biology, the debate of the moral status of the human embryo has turned into a philosophical one. Back in the early 1990s, the definition of the human embryo was based on a purely biological fact: the mixture of the male and female gametes, that is, what science has traditionally called fertilization. The entity arising as a result of human fertilization was considered an embryo. The scenario, however, changed dramatically in February 1997, when cloning resulted in Dolly the sheep[4]. The traditional definition of human embryo suddenly became obsolete, as it was possible to create human beings without fertilization. However, the use of stem cells from umbilical cord blood or the use of iPSCs from humans are not subject to ideological restrictions of embryos research. STEM CELL TOURISM Unfortunately, due to their high and very promising potential, stem cells are sometimes used in unwanted and non-­‐ethical ways. Cell therapy-­‐based medical tourism is a phenomenon when people suffering from fatal diseases search for help. Often patients’ expectations are unrealistically raised, or told that cells won't be rejected, or that the risk of other disease is avoided as using one own cells or that it is more ‘ethical’. In recognition of the growth of unproven stem cell treatments, the International Society for Stem Cell Research (ISSCR) in 2007 developed guidelines for the clinical translation of stem cell research addressing these concerns of “potential physical, psychological, and financial harm to patients”[28]. CONCLUSION CAN STEM CELLS BE USED TO TREAT KIDNEY DISEASE TODAY? Stem cell treatments for kidney disease have been tested in vitro and in animal models of kidney disease. There is evidence of benefits in animal models of AKI[8]. Intra-­‐arterial seedling of iPSCS could prove to be applicable in acute kidney injury or chronic renal failure to accelerate recovery or to restore function. Using human iPSC cells as opposed to ES cells circumvent the ethical dilemma of using embryos as source of ES cells. Reprogrammed kidney iPSCs may also aid in the study of genetic kidney diseases and lead to the development of novel therapies. 13
The kidney is a very complex organ consisting of a large number of different types of cells. To make a new kidney in the laboratory, all these different cells would need to be produced in a different way and mixed together in the hope that they would eventually recreate a functional kidney. What's more, there are many causes of kidney disease with diverse etiologies with different parts of the kidney affected. Thus treatments aiming to replace damaged cells within a patient's kidney would need to supply different types of cells for different patients. Organ scaffolds with decellularization and recellularizaton, plus a cell based regenerative strategy requires considerable study still. Although regeneration of a kidney is envisioned, this is likely to be a long-­‐term goal still years away. REFERENCES 1. UpToDate: physician-­‐authored clinical decision support resource http://www.uptodate.com/home 2. Takahashi, K.(2006).Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures bt defined factors. Cel,126(4),663-­‐676. 3. Gurdon, J.(1962).Developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J.Embryol.Exp.Morph,10,622-­‐640. 4. Wilmut, I.(1997).Viable offspring derived from fetal and adult mammalian cell. Nature,385,810-­‐3. 5. Becker, A.(1963).Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature,197,452. 6. Regenerative medicine for the kidney: http://www.clintransmed.com/content/2/1/11 stem cell prospects & challenges 7. Bussolati, B.(2005).Isolation of renal progenitor cels from adult human kidney. Am.J.Pathol,166,545-­‐555. 8. Bussolati, B.(2008).Contribution of stem cells to kidny repair. Am.J.Nephrol,28,813-­‐
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