Stem cell therapy for type 2 diabetes in india vs,java.settings.cfg download,type 1 diabetes treatment diet nutrition,diabetes type 1 glucagon use - Downloads 2016

These figures may be used freely for academic, noncommercial purposes with the imbedded credit line intact. Contact us if you have any questions. Science, Technology and Medicine open access publisher.Publish, read and share novel research. Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory and Bioethical ConsiderationsAntonio Liras1, 2, Cristina Segovia1 and Aline S.
The Symptoms of juvenile diabetes or type 1 diabetes are basically very similar to adult diabetes or type 2 diabetes. Slowly when the blood sugar level rises these early symptoms of diabetes will grow into more serious conditions and symptomsLike we already said the symptoms of type 1 diabetes are basically very similar to the type 2 diabetes symptoms. The American Diabetes Association has devised a very small and fast online tool with which you can determine if you run a higher risk of getting diabetes. For those who are already checking their blood sugar levels, there are no clear cut criteria but the next are considered as general guidelines.
After these signs and symptoms we're going to take a look at what causes diabetes to see if your circumstances fit the description.
Stem cell treatment of cardiac diseasesAt Euromedic healthcare we are combining technology with healthcare to revolutionize the concept of death forever. The heart is the most important organ of the body as it receives deoxygenated blood and pumps out oxygenated blood. Stem cells are tiny cells obtained from the human body that have the potential to grow and repair just about any tissue in the body. Latest research conducted by scientists at various medical centers treated 17 heart attack patients with infusion of stem cells taken from their own heart. Stem cell transplant cannot only be opted for the treatment of cancers such as leukemia and lymphoma but also for cardiac diseases. Generation of human induced pluripotent stem cells for use in cell therapy, in vitro human pathology modelling and in drug discovery. Induced pluripotent stem cells application to the treatment of haemophilia and diabetes mellitus.
Gaban1, 3[1] Department of Physiology, School of Biology, Complutense University of Madrid, and Cell Therapy and Regenerative Medicine Unit, La Paz University Hospital Health Research Institute-IdiPAZ. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Strategies and new developments in the generation of patient-specific pluripotent stem cells. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.
Here we'll focus on the early symptoms of diabetes mellitus type 1 and 2 and gestational diabetes, because they are the most common typesBe alertIn the early stages there are just a few diabetes symptoms, or they look like symptoms of other health conditions. The difference is that the development of type 2 diabetes symptoms is normally slow and can take many yearsBut symptoms of type 1 diabetes progress fast over weeks or months.
With blood tests he will be able to tell you if you have DiabetesOnly in 40% of the diabetes patients these symptoms of diabetes are observed.
One way to test it is by a fasting glucose test, where you're not allowed to eat and drink 8 hours before the test. Comparison of murine versus human ESC differentiation protocols to insulin-producing cells. Manilay2[1] Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, Merced, CA, , USA[2] Department of Molecular and Cell Biology and Quantitative and Systems Biology Graduate Group, School of Natural Sciences, University of California, Merced, Merced, CA, , USA1.
Stages of normal wound healing with predominant cell types involved at each stage of process.
Scanning electron microscope of co-culture of mesenchymal stem cells and early endothelial progenitor cells in a type 1 bovine collagen scaffold.Table 3.
IntroductionThe prevalence of diabetes mellitus is increasing to epidemic proportions worldwide. The science of medicine is advancing at great speed to conquer death by curing one of the most fatal medical conditions, the heart attack. This oxygenated blood is supplied to the entire body for optimal functioning.  If the heart is diseased, the portion of the heart that is affected stops receiving vital nutrients and oxygenated blood and eventually, that part of the heart stops functioning completely.
It is a big challenge and we at Euromedic Healthcare are already championing this technology with our team of skilled and highly experienced physicians and surgeons. Autologous transplantation of healthy differentiated cells, obtained from iPSCs, into an animal model with haemophilia or diabetes mellitus type 1, normalizes the corresponding altered function by in vivo production of the deficient protein or hormone.5.
This may turn your attention in a different direction but always be aware of this possibilityYou also have to be very aware that in the beginning a lot of people with type 2 diabetes have no type 2 diabetes symptoms yet.
Sometimes it can go so fast that a child will get medical treatment only after an emergency situation has occurred like a coma. The development of pancreatic endocrine cells starts with an early endocrine progenitor and ends with mature hormone-producing islet cell types.
The schematic shows the differences in culture methods to produce IPCs from murine ESC (top) and human ESC (bottom).
IntroductionDiabetes mellitus is a chronic metabolic disease that results in high levels of glucose in the blood. Diabetic foot ulceration can affect up to 25 percent of people with diabetes mellitus throughout their lives. Skilled doctors at our medical centers are providing revolutionary stem cell therapies to patients who may benefit from this boon to mankind. This is extremely detrimental for the body and may even prove fatal.  Thus, if a person gets a heart attack he is put on a lifetime of medications for maintenance therapy to prevent another heart attack and treatment to prevent further damage to the heart. They are unspecialized, rudimentary, life-giving cells that are the seeds of growth of all other cells and tissues of the human body.
The scientists found 50% shrinkage in the damaged cardiac tissue after a year of the procedure. You may now opt for a stem cell treatment and literally get back the lost portion of your precious heart. Cell surface markers (noted above the cell types) and gene expression (in italics) in distinct pancreatic cell lineages are shown. Normally, glucose is transported into cells from the blood for energy, and this transport is initiated in response to the hormone, insulin. The most significant complication of foot ulceration is lower limb amputation, which arises from pre-existing ulcers in the majority of cases. They do not have a specific and set function like other programmed cells of our bodies and can give rise to specialized cell types which is what makes them extremely useful in the treatment of various diseases. This is great news for all those who have suffered from a heart attack before or are on medications to prevent it. With a wholly functioning heart, beating at its best, you can live your life carefree, to the fullest!
In diabetic patients, cellular glucose uptake is defective, in part due to the inability of cells to respond to insulin, or the inability of the body to produce the insulin itself.
Despite current clinical care protocols for ulcer treatment, there exists a high amputation rate. A stem cell has the potential to grow into a variety of heart cells and thus it can be used to repair and regenerate the damaged heart tissue. IntroductionThe potential use of stem cells in advanced therapies such as tissue engineering, regenerative medicine, cell therapy and gene therapy by virtue of their significant therapeutic potential and clinical applications has aroused keen interest among scientists [1,2]. This presents a major burden for individual patients’ health and well-being in addition to significant financial cost for health care systems.
Cell therapy is based on the transplantation of living cells into an organism with a view to repairing tissue or restoring a lost or deficient function. It is an especially challenging health problem, as treatments to both control hyperglycemia as well as the debilitating side effects of diabetes, such as injury to the blood vessels and nerves, must be addressed.
Stem cells are the most frequently used cells for such purposes given their ability to differentiate into other more specialized cells [3]. Amongst the three types of diabetes (Type I - autoimmune, gestational and Type II – adult onset), Type II diabetes is the most prevalent, in which hyperglycemia is uncontrolled due to the body’s inability to produce enough insulin or due to the body’s inability to respond appropriately to lower the blood glucose level. Cell-based therapies offer a novel treatment strategy to augment diabetic wound healing, increase ulcer healing rate and prevent amputation. The chief defining feature of stem cells is their capacity for self-renewal and their ability to differentiate into cells of various lineages. In contrast, in patients with Type I diabetes (TID), insulin-producing beta (?) cells are destroyed by the immune system.
The field of tissue engineering has developed commercially available skin substitutes for diabetic cutaneous wound repair.
Studies of TID by many groups have provided extensive insight to the fields of immunological tolerance and pancreatic developmental biology. They can be found in virtually any kind of tissue including bone marrow, trabecular bone, periosteum, synovium, muscle, adipose tissue, breast gland, gastrointestinal tract, central nervous system, lung, peripheral blood, dermis, hair follicle, corneal limbus, etc. In this chapter, we will briefly describe TID, provide a synopsis of pancreatic ? cell development in mice as compared to humans, review of some of the medical treatments currently available for TID, and discuss current studies that have explored the use of stem cells as alternative possible therapies for TID.
However, having been available for the last decade, the majority have demonstrated only moderate clinical benefit in small clinical trials. In comparison, stem and progenitor cell therapy offer the potential for accelerated wound repair in addition to structural skin regeneration with functional recovery. The clinical application of this type of cell is associated with potentially better prospects than that of embryonic stem cells since use of adult stem cells does not raise any ethical conflicts nor does it involve immune rejection problems in the event of autologous implantation. Stem cells have the ability to self-renew and differentiate into other cell types and are classified into adult stem and progenitor cells, embryonic stem cells and induced pluripotent stem cells. The possibility to generate induced pluripotent stem cells (iPSCs) by reprogramming somatic stem cells through the introduction of certain transcription factors [7-12] is radically transforming received scientific wisdom. TID in humansA fully comprehensive comparison of TID in humans and mice has recently been published, so we will only highlight certain areas (van Belle, Coppieters et al. The mechanisms of action of stem and progenitor cells are not fully elucidated but include 1) differentiation to specialised cells e.g. The pluripotency of these cells, which enables them to differentiate into cells of all three germ layers (endoderm, mesoderm, and ectoderm), makes them an extremely valuable tool for the potential design of cell therapy protocols.
TID is a multigenic disease, and several candidates have been discovered that implicate disruptions in the normal process of negative selection of autoreactive T cells during thymic development, genetic association with specific major histocompatibility complex (MHC) genes in the human leukocyte antigen (HLA) locus, as well as dysregulation of mature T lymphocyte responses. Furthermore, unlike embryonic stem cells, iPSCs are not associated with bioethical problems and are considered a "consensus" alternative that does not require use of human oocytes or embryos and is therefore not subject to any specific regulations. For example, patients with mutations in the FoxP3 or Aire genes, which are important for the development and function of regulatory T cells (Tregs) and expression of “self-antigens” by medullary thymic epithelial cells, often present with autoimmune diabetes, as well as other autoimmune disorders (Michels and Gottlieb 2010). Much research endeavour is determining the benefit of stem cell treatment on diabetic cutaneous wound healing with encouraging results in animal models. On the other end of the spectrum, the inability to downregulate or prevent mature T cell responses due to mutations in cytokine receptor expression (Garg, Tyler et al. Regenerative medicine and tissue engineering specialties are rapidly elucidating the mechanisms of action of stem cells and translating the results of in-vitro and in-vivo experiments to human clinical trials. Researchers have also looked into the application of iPSCs to toxigological and pharmacological screening for the presence of toxic and teratogenic substances [20].Stem cell therapy is emerging as a new concept of medical application in pharmacology. The requirements for success will be patient safety, clinical efficacy and convenience of use.The focus of this chapter is to review the area of topical stem and progenitor cell therapy as a treatment for non-healing diabetic foot ulcers.
For all practical purposes, human embryonic stem cells are used in 13% of treatments, whereas fetal stem cells are used in 2%, umbilical cord stem cells in 10%, and adult stem cells in 75% of cases.
2011) can also result in sustained T cell cytotoxicity or lack of Treg response towards diabetes-related antigens. It will focus on adult stem cells as these are nearer to use in human trials and do not pose the ethical constraints associated with the use of embryonic stem cells. The most significant treatment indications for gene and cell therapy have so far been cardiovascular and ischemic diseases, diabetes, hematopoietic diseases, liver diseases and, more recently, orthopaedics [21].
Topical treatment with endothelial progenitor cell (EPC) and mesenchymal stem cell (MSC) therapy is presented in this review, and more specifically the delivery of these cells using biomaterial scaffolds.
For example, over 25,000 transplants of hematopoietic stem cells are performed every year for treatment of lymphoma, leukemia, immunodeficiency disorders, congenital metabolic defects, hemoglobinopathies, and myelodysplastic and myeloproliferative syndromes [22].Each type of stem cell has its own advantages and disadvantages, which vary depending on the different treatment protocols and the requirements of each clinical condition.
TID in miceThe identification of the mechanisms underlying the autoimmunity of TID in humans has been facilitated by the use of rodent models of the disease (Van Belle, Taylor et al. Thus, embryonic stem cells have the advantages of being pluripotent, easy to isolate and highly productive in culture, in addition to showing a high capacity to integrate into fetal tissue during development. The case for adopting stem and progenitor cell therapy in research and treatment of diabetic foot ulcers will be discussed. By contrast, their disadvantages include immune rejection and the possibility that they may spontaneously and uncontrollably differentiate into inadequate cell types or even induce tumors.
Despite the fact that TID is a multigenic disease with heterogeneous etiologies, there is overall consensus that TID is caused by the destruction of pancreatic ? cells by sets of immune cells that recognize many potential autoantigens (such as insulin, glutamate decarboxylase (GAD), islet antigens, and heat shock proteins-60 and -70) and that the disease can be transferred by the transplantation of autoreactive T lymphocytes that are specific for these antigens (Mallone, Brezar et al.
The benefits of biomaterials and functionalised scaffolds for mediating cell therapy to a wound will be described.
Adult stem cells have a high differentiation potential, are less likely to induce an undesirable immune response and may be stimulated by drugs. For both endothelial progenitor cells and mesenchymal stem cells, the potential mechanisms of action will be discussed with reference to key pre-clinical and clinical studies.
Their disadvantages include that they are scarce and difficult to harvest, grown slowly, differentiate poorly in culture and are difficult to handle and produce in adequate amounts for transplantation.
Several spontaneous, induced and transgenic mouse models currently exist, which have provided important insights into the genetics, cellular mechanisms and immunological aspects of TID. The chapter will also describe strategies to enhance the therapeutic potential of stem and progenitor cells for wound healing. In addition, they behave differently depending on the source tissue, show telomere shortening, and may carry the genetic abnormalities inherited or acquired by the donor.
However, the non-obese diabetic (NOD) mouse strain remains the primary animal strain for studies of TID. The utility of the NOD mouse and comparison of the TID in the NOD mouse with human TID have been extensively reviewed and debated, and we direct the reader to these excellent reviews for further reference (Anderson and Bluestone 2005; Roep and Peakman 2011). The first one is stimulation of endogenous stem cells by growth factors, cytokines, and second messengers, which must be able to induce tissue self-repair. The NOD mouse strain, when housed in specific-pathogen free conditions, displays the onset of diabetes after 12 weeks of age in both sexes. A section of the chapter will focus on translational of these advanced biological medicines to clinical trials.
The second alternative is direct administration of the cells so that they differentiate at the damaged or non-functional tissue sites. Insulitis is observed as mononuclear infiltration in the islets before full blown diabetes is evident, and this cellular infiltrate is comprised of primary T lymphocytes, but other lymphoid and myeloid cells are also present.
This includes issues regarding pre-clinical animal models, optimal cell source, safety and regulatory approval. The third possibility is transplantation of cells, tissues, or organs taken from cultures of stem cell-derived differentiated cells. The insulitis results in the destruction of the pancreatic islets and the insulin-producing ? cells. The US Food and Drug Administration defines somatic cell therapy as the administration to humans of autologous, allogeneic or xenogeneic living non-germline cells, other than transfusion blood products, which have been manipulated, processed, propagated or expanded ex vivo, or are drug-treated. The NOD mouse also displays defects in functional macrophages and natural killer cells, NKT cells and regulatory T cells and complement. The most significant applications of cell therapy as a whole are expected to be related to the treatment of organ-specific conditions such as diabetes —a typically metabolic disease—, liver and cardiovascular conditions, immunological disorders and hereditary monogenic diseases such as haemophilia. The NOD mouse, therefore, has allowed for the investigation of numerous immune mechanisms that contribute to the development of TID. As one of the key advanced therapies —together with gene therapy and tissue engineering— cell therapy will require a new legal framework that affords generalized patient accessibility to these products and that allows governments to discharge their regulatory and control duties. The peripheral neuropathy that accompanies TID in humans is also evident in the NOD mouse, which has allowed for analysis and management of the side effects of TID (Anderson and Bluestone 2005; Roep and Peakman 2011). The biology of cutaneous woundsThe repair of cutaneous wounds is a highly complex biological process.
In this respect, the main advantage of iPSCs lies in the fact that their use does not raise bioethical questions, which means that regulatory provisions governing their use need not be overly stringent.
Similar to human diabetes, the MHC of the NOD mouse contains several loci that are linked to increased susceptibility to TID. The goal of adult wound healing is to repair a skin defect, to ensure the restoration of a barrier and to regain tensile strength. Induced pluripotent stem cells technology and general clinical applicationsiPSCs are obtained through the reprogramming of an individual's somatic stem cells by the introduction of certain transcription factors.
There is involvement of several cell types, cytokines and extra-cellular matrix components. Their chief value is based on their pluripotency to differentiate into cells of all three germ layers, which makes them an useful tool for the discovery of new drugs and the establishment of cell therapy programs. The physiological overlapping pathways that are required for optimal wound healing include haemostasis (which occurs immediately on wounding), inflammation with cell migration and proliferation (neutrophils initially and subsequently macrophages).
Despite the similarities between TID in humans and in the NOD mouse, clear differences have been noted. Moreover, unlike embryonic stem cells, they are not associated with any ethical controversies and therefore regulatory conditions governing their use are much less stringent.
TID in the NOD mouse appears to be a more aggressive disease compared than that in humans, so the NOD mouse may be more beneficial for studies of therapies for long-term TID than for discovery of pre-susceptibility markers of TID. Induced pluripotent stem cells were generated for the first time by Shinya Yamanaka's team [8] from murine and human fibroblasts by transfecting certain transcription factors (Oct4, Sox2, c-Myc, and Klf4) by means of retroviral vectors. ? cells in the NOD mouse may also be more proliferative and could possibly regenerate better than other strains (Sherry, Kushner et al. Angiogenesis (growth of new blood vessels from pre-existing blood vessels) and re-epithelialisation are central processes in wound healing. 2006), and evidence that pancreatic development is altered in the NOD mouse also exists (Homo-Delarche 2001).
This is a superficial description of wound healing and conveys the complexity of the process, but highlights the potential for disruption in a difficult to heal wound.
Therefore, in addition to its clear autoimmune etiology, TID may also result from defects in pancreatic development.
Pancreatic development in the mouse and humanThe adult pancreas organ is comprised of many cell types, including exocrine and endocrine cells, and is highly vascularized. Diabetic wound healingDelayed wound healing as occurs in diabetes mellitus results from dysregulation of the normal healings pathways. Nonetheless, it was later reported that only two transcription factors (Oct4 and Klf4) are needed for generating the iPSCs from neural stem cells that endogenously express high Sox2 concentrations [24].All of these strategies require transfection through retroviral vectors and integration for in vitro and in vivo modeling, which precludes their clinical use because of the potential risks involved. Although TID is a disease that occurs in the fully formed pancreas, strategies to treat or cure TID via ? cell regeneration or ? cell differentiation from embryonic stem cells (ESCs) or induced pluripotent cells (iPSCs) have relied on the current knowledge of pancreatic development during the fetal period. The diabetic wound is complex with contribution from infection, neuropathy and impaired vascular supply.

This is the reason why several research teams have looked into the reprogramming of cells using plasmid vector rather than viral vector transfection [10-12].
Here, we briefly review the development of pancreas in the mouse and human, and then move to a description of development of ? cells only.
Although reprogramming efficacy with plasmid vectors is lower ?as is also the case with non viral gene therapy? this method significantly increases the safety of the procedure, which makes it clinically applicable and also constitutes a source of valuable cell material that can be used for research into reprogramming and pluripotency.Another promising strategy consists in the direct release of reprogramming proteins through modified versions of reprogramming factors in some of their molecular domains.
These include decreased or impaired growth factor production, angiogenic response, macrophage function, collagen accumulation, epidermal barrier function, quantity of granulation tissue, keratinocyte, fibroblast migration and proliferation and bone healing. These protein-induced pluripotent stem cells (piPSCs) bind to the membrane of cells reaching their nucleus [25].
There is an imbalance between the accumulation of extra-cellular matrix components and their re-modeling by matrix metallo-proteinases.(Brem et al. In the first phase, pancreatic formation from the endodermal gut tube begins at embryonic day (e) 8.5. Patterning of the gut tube in the pancreatic region is reliant on retinoic acid (RA) signaling from the mesoderm. On the one hand, this reduces the risk of immune rejection in autologous transplantations by virtue of gene identity. The dorsal and ventral pancreas buds form from the pancreatic epithelium in the primitive gut tube.
On the other, it provides treatment that is customized to the specific characteristics of each patient and takes into account the etiology and severity of the condition.
Recently, the critical role of the Wnt signaling in the pancreatic mesenchyme in the differentiation of pancreatic epithelial cells was shown in vivo (Landsman, Nijagal et al. Angiogenesis and wound HealingThe impaired vascular supply associated with diabetes leads to poor blood flow at the wound site impeding the optimal endogenous reparative response (Jeffcoate & Harding 2003) Impaired angiogenesis is a feature of diabetic wounds. Moreover, induction of pluripotency has been developed for a great variety of tissue types [9,24,27] as it is a relatively straightforward procedure and —as mentioned above— subject to fewer regulatory constraints [28].Important as these advantages are, there are still a few uncertainties that need to be resolved. In addition neovascularisation, or the de novo formation of new blood vessels is critical for granulation tissue formation and tissue regeneration in wound healing. One of the most pressing ones is related to determining the likelihood that these iPSCs may undergo genetic aberrations further to the reprogramming process [29]. Eventually, the ventral pancreas rotates toward the dorsal pancreas, and they fuse into one organ by e12.5. In order for the clinical application of these cells to become a reality both for diagnostic purposes and for the design of cell therapy protocols, a few methodological hurdles must still be resolved in connection, as is often the case with pharmacological products, with their safety profile [30]. This means basically that efforts must be directed at removing the genome in the integrating viral vectors, eliminating the risk of tumor formation and establishing more efficient reprogramming and differentiation protocols. In the second phase (e13.5), the cells within the immature pancreas begin to differentiate into exocrine cells that are required for the production of digestive enzymes, and endocrine cells that secrete hormones into the blood, such as glucagon, insulin, somatostatin, pancreatic polypeptide and ghrelin. In the non-diabetic situation, hypoxia leads to activation of the transcription factor complex HIF-1? (Hypoxia inducible factor-1?), which leads to transcription of multiple genes required for successful wound healing. Clearly our knowledge on the reprogramming mechanisms leading to pluripotency are still insufficient to understand and more importantly control the adverse events that could potentially occur. Each of these hormones is produced by a distinct endocrine cell type (with insulin produced by the ? cells) (Figure 1). Therefore the most important goal for research in this field will be to study genetic modifications in animal models by means of large-scale genome sequencing programs.
The third phase of pancreatic development occurs after birth, and is associated with the ability of the pancreas to respond to dietary intake and maintain glucose homeostasis.Islet development in humans occurs at a slower rate compared to what is observed in mice, but the reasons for this are not understood. This task will require sharing cell lines with other researchers, with appropriate confidentiality protections and, eventually, patenting scientific discoveries and developing commercial tests and therapies. The size of the human islet is comparable to that of the mouse, but in the mouse, the mean area ratio of ? cells is higher than observed in the human (Kim, Miller et al.
It will also be necessary to fully ascertain and confirm that pluripotency confers iPSCs with functions similar to those of embryonic stem cells regardless of the initial source of somatic cells used [14,15].Undoubtedly, the most attractive application of this type of strategy is the production of patient-specific or healthy individual-specific iPSCs for replacement of damaged non-functional tissue. Thus for example skin fibroblast-derived iPSCs have been shown to possess a high potential to differentiate into islet-like clusters and to release insulin, which is highly relevant for diabetes [16].
Fetal antigen-1 is a protein expressed exclusively in human endodermal tissue and pancreatic epithelial cells before islet formation (Tornehave, Jansen et al. Skin regeneration is the regeneration of wounds with restoration of the normal function and anatomy of skin. In biology, foetal wound repair is a regenerative process, and some vertebrate species demonstrate successful tissue regeneration where the initial phase of wound repair is followed by perfect structural and functional regeneration of the organ.
In fact, since they were discovered in 2008, almost one-hundred-and-fifty iPSCs have been established from nearly thirty fibroblast cell lines related to over a dozen conditions, including some complex diseases such as schizophrenia and autism and other genetic or acquired disorders such as cardiovascular or infectious diseases. Islets can be located in the human fetus as early as 12-13 weeks post coitus (Piper, Brickwood et al. 2004), although functional ? cells that are insulin-positive can be detected as early as 8 weeks. The challenge for scientists is to produce tissue engineered products that exhibit extra-cellular matrix re-modeling characteristics seen in embryonic wound repair to produce functional and durable skin.
However, it is still necessary to optimize iPSC protocols, particularly with respect to the possible modifications to their genome, and to increase the efficacy of the transfection process leading to iPSC reprogramming [36,37].
Advanced therapies for monogenic and metabolic diseasesThe progression of the different areas of biology, biotechnology and medicine leads to the development of highly innovative new treatments and pharmacological products. The existence of the self-renewing pancreatic stem cell is still controversial.An overview of pancreatic endocrine cell development is shown in Figure 1. Definitive endodermal progenitor cells can differentiate into pancreatic progenitor cells, which have been identified by the expression of two critical transcriptional factors: Pdx1 and neurogenin 3 (Ngn3). The different schools of thought that advocate the emerging concept of advanced therapies agree that the latter must be used for the treatment of diseases (both hereditary and non-transmissible) caused by the anomalous behavior, or complete lack of function, of a single gene (also called monogenic hereditary diseases) or by an anomaly in several genes (polygenic diseases). Experiments with conditional Pdx1 transgenic reporter mice (inducible Cre-ERTM-LoxP system to mark the progeny of cells that express Pdx1) demonstrated that Pdx1+ cells give rise to all three types of pancreatic tissue: exocrine, endocrine and duct (Gu, Dubauskaite et al. Metabolic diseases, or congenital metabolic errors, are conditions highly amenable to be treated by the new advanced therapies as such treatments have been shown to restore mutation-induced alterations of gene products. Proteins are the most commonly affected gene products, although messenger RNA is also a usual victim. In line with this, Pdx1 deficiency in mouse and humans leads to a complete pancreas agenesis at birth (Ahlgren, Jonsson et al. Pdx1 is also a critical transcriptional activator of insulin and somatostatin in adult islet cells (Ohlsson, Thor et al. Classification of diabetic ulcersDiabetic foot ulcers can be classified as ischaemic, neuropathic or neuro-ischaemic. Of particular interest are the proteins that participate in homeostasis and exert their functions outside the cells that synthesize them.
This is the case of coagulation factors VIII and IX (FVIII and FIX), whose deficiency results in the development of haemophilia A or B, respectively. The typical angiographic pattern of ischaemic diabetic vasculopathy is occluded distal blood vessels.
Another member of this class of proteins is antitrypsin, also of hepatic origin and secreted into the bloodstream, whose function is to prevent the digestion of pulmonary alveoli by proteolytic enzymes. Similar to Pdx1, Ngn3+ cells give rise to all islet lineage cells during embryogenesis (Gradwohl, Dierich et al. Lastly, mention should be made of proteins with such diverse functions as transcription factors, oncogenes, tumor-suppressing genes and even some hormones and their receptors, the latter being specifically related with diabetes mellitus, a typically metabolic disease.The nature of the monogenic or metabolic disease is the main factor that determines whether a treatment that can eradicate or at least mitigate its clinical consequences is possible or not. Although some have reported that Ngn3 is undetectable in adult pancreas (Gradwohl, Dierich et al.
Ulceration develops at sites of excessive pressure predominantly under the first metatarsalphalangeal joint, in the majority due to unperceived trauma. In this context, induced pluripotent stem cells can play a very significant role and hold an enormous therapeutic potential in the fields of cell therapy and tissue engineering.4. 2007), Ngn3+ cells in duct-ligated adult pancreas were detected and observed to differentiate into islet cells (Gu, Dubauskaite et al. Advanced therapies and induced pluripotent stem cells in the treatment of haemophiliaHaemophilia is a recessive X-linked hereditary disorder caused by a deficiency of coagulation factor VIII (haemophilia A) or IX (haemophilia B). The disease is considered to be severe when factor levels are below 1% of normal values, moderate when they are between 1 and 5% and mild when levels range between 5% and 40%. Current treatment strategiesThe management of the diabetic foot is complex requiring a multidisciplinary approach. Haemophilia A is four times more common than haemophilia B and, in terms of severity for both types, 35% of patients have the severe form, 15% the moderate form and 55% have mild haemophilia. Beta cells in the human, like in the mouse, are derived from a heterogeneous population of pancreatic progenitor cells (Scharfmann, Xiao et al. The clinical characteristics of both types of haemophilia are very similar: spontaneous or traumatic hemorrhages, muscle hematomas, haemophilic arthropathy resulting from the articular damage caused by repetitive bleeding episodes in the target joints, or hemorrhages in the central nervous system. In the absence of appropriate replacement treatment with exogenous coagulation factors, these manifestations of the disease can have disabling or even fatal consequences thus negatively impacting patients' quality of life and reducing their life expectancy [39].At present, patients with haemophilia benefit from optimized treatment schedules based on the intravenous systemic delivery of exogenous coagulation factors, either prophylactically or on demand. Pax4 (paired box 4) is required for the commitment to the pancreatic lineages (Wang, Elghazi et al. The current policy in developed countries is in general to administer a prophylactic treatment (2 or 3 times a week) from early childhood into adulthood [39]. 2004), as well as for the regulation of ? cell mass size, proliferation and survival in mice (Brun and Gauthier 2008), and mutations in the Pax4 gene have been linked to susceptibility to both Type I and Type II diabetes in humans (Yokoi, Kanamori et al. Such prophylactic protocols result in a clear improvement in patients' quality of life on account of the prevention of haemophilic arthropaty and other fatal manifestations of the disease as well as a reduction in the long-term costs of treatment because of a decrease in the need of surgical procedures such as arthrodesis, arthroplasty or synovectomy [40].Conventional treatment of haemophilia [41,42] is currently based on the use of plasma-derived or recombinant high-purity coagulation factor concentrates.
Benefit of a cell-based therapy for non-healing diabetic ulcersIt is evident that there is a critical clinical need to develop novel therapies for treatment of non-healing diabetic ulcers in order to prevent amputation and reduce the significant financial drain on healthcare budgets and burden on individuals health. The former are duly treated with heat and detergent to inactivate lipid-coated viruses [43], and the latter are a recently developed product that does not contain proteins of human or animal origin [44,45].
The understanding of the patho-physiology of diabetic wound healing is important in the development of advanced wound healing treatments. Both kinds of factor boast high efficacy and safety profiles, at least for the inactivation-susceptible pathogens known to date. In addition, both human and mice insulinoma cells overexpress Pax4 in comparison to mature ? cells, suggesting that Pax4 may also play a role in ? cell proliferation and resistance to apoptosis.
The choice of one product over the other is usually based on the clinical characteristics of the patient and on cost and availability considerations [46,47].Now that infections by pathogenic viruses (HIV, HCV) that were common a few decades ago have been eradicated, the most distressing adverse effect observed when using either product is the development of antibodies (inhibitors) against the perfused exogenous factors [48,49]. Cell therapy may reverse the biological defects in diabetic wounds by acting as reservoirs for cell and growth factor production.
The appearance of inhibitors renders current treatment with factor concentrates inefficient, increasing morbidity and mortality, leading to the early onset of haemophilic arthropathy and disability and to a consequent reduction in patients' quality of life. Lastly, inhibitors result in higher costs as treatment must be provided both for bleeding episodes and inhibitor eradication (immune tolerance induction).
Limitations with current cell-based therapyTo date clinical trials of topical cell based therapy for non-healing diabetic foot ulcers have yielded limited results.
They are directed at certain regions in the factor molecule that interact with other components of the coagulation cascade and, depending on their titre level and on whether they are transient or persistent, will bring about greater or lesser alterations in the said cascade. Nkx6.1 functions exclusively in ? cells and is required for ? cell development and specification, control of insulin secretion after glucose challenge, and can also induce human and rat ? cell proliferation (Schisler, Fueger et al. One reason is methodological flaws in the clinical trials which have raised concerns over the validity of the results.
Systematic reviews on skin replacement therapy have reported statistical benefit in wound healing endpoints.
However, in vivo overexpression of Nkx6.1 does not have any effect on ? cells in the mouse (Schaffer, Yang et al.
However there was a lack of information reported on safety, method of recruitment, randomization methods and blinding strategy for outcome assessments.
There is a lack of power size calculations in some of the trials and little mention of dropouts in trial.
Current therapies for TIDA fully mature, functional ? cell produces and appropriately secretes the mature form of insulin (i.e.
Whether the factor concentrate used is plasma-derived or recombinant does not have a significant influence on the inhibitor incidence rate [51].Short and medium-term perspectives for the treatment of haemophilia strongly rely on the current research efforts directed at increasing the safety levels of (especially) plasma-derived factors.
Such research focuses on the detection and subsequent inactivation of emerging blood-borne pathogens in donors such as the prions causing variant Creutzfeldt-Jakob disease, or other potential emerging agents [52-54].
The current treatment for TID is long-term insulin replacement therapy that is delivered via injection, insulin pens or pumps, and has been quite successful for control of hyperglycemia.
The current somatic cell therapies do not address the underlying pathology in the diabetic wound i.e. It is also important to increase the efficiency of recombinant factors increasing their half-life (by PEGylating the factor molecule or using fusion proteins [55-58] and attenuating their immunogenic capacity to produce inhibitors, by chemically modifying them [59] or by developing recombinant factors of human origin [60].In the long term, efforts must be directed at the development of advanced therapies, particularly strategies in the field of gene therapy (using of adeno-associated viral vectors) and cell therapy (using of adult stem cells or induced pluripotent stem cells).
However, insulin replacement therapy is not considered a cure for TID, as patients receiving insulin long-term still manifest abnormalities in metabolism, as measured by above-normal levels of glycated hemoglobin (HbA1c). The chief goal of these new strategies will be to address some of the shortcomings associated with current treatment options such as the short in vivo half-life of administered factors, the impending risk of a pathogen-induced infection and the development of inhibitors. Furthermore, for children, the daily requirement for insulin replacement can limit their day-to-day activities, and TID-related complications, such as vascular disease, retinopathy and neuropathy, still persist even with insulin therapy.
An efficient blood supply is central to normal wound healing, and delayed or inefficient angiogenesis will prolong ulceration and increase the probability of amputation. Another goal of the advanced therapies (cell therapy) will be palliative treatment of the articular consequences derived from haemophilic arthropathy [40].Haemophilia is optimally suited for advanced therapies as it is a monogenic condition and does not require very high expression levels of a coagulation factor to reach moderate disease status (Figure 2). Pancreatic transplantation has been performed for TID patients with success, but the main limitations of this strategy are the low number of suitable donors required for transplant and the decision if the risks of surgery and transplantation outweigh the benefits (Jahansouz, Kumer et al.
The current cell treatments do not target angiogenesis (blood vessel formation from pre-existing blood vessels) or neo-vasculogenesis (de novo blood vessel formation). For this reason, significant progress has been possible with respect to these kinds of therapies: cell therapy has broken new ground with the use of several types of target cells and gene therapy has shown particular promise with the use of viral and non-viral vectors.
Furthermore, even with insulin replacement therapy or pancreatic transplantation, autoreactive immune cells that can attack and destroy any residual ? cells in the patient can still remain. For this reason, therapies that modulate the immune response to ? cells and ? cell antigens also exist, and their safety and efficacy have been or are currently being tested in clinical trials (Bluestone, Herold et al.
The employment of these cell treatments result in wound healing by repair and not by regeneration. The studies published so far have, in the most part, not reported any severe adverse effect resulting from the application of such strategies in the clinical trials performed.Specifically, gene therapy trials in haemophilic patients have shown adeno-associated vectors to represent the most promising treatment option given their excellent safety profile, even if on occasion they may create immune response problems. Efforts are currently centered on minimizing the incidence of immune rejection and increasing efficacy and expression time. GAD65 specific-antigen-based immunotherapy with alum adjuvant demonstrated preservation of C-peptide fasting levels in younger new-onset patients four years after treatment (Ludvigsson, Hjorth et al.
Potential superiority of treatment with stem and progenitor cellsEndothelial progenitor cells are a newly described cell type involved in angiogenesis.
In this connection, several studies have been published with a view to optimizing the use of this type of viral vectors. Among them, in a landmark study on patients with severe haemophilia B (<1% FIX), Nathwani et al. Humanized anti-CD3 antibody (in which human Fc immunoglobulin domains are engineered to mouse CD3-binding portions of the antibody) therapy given in a single dose reduced the decline of insulin production and the amount of exogenous insulin required in the patients (Herold, Gitelman et al. Adult mesenchymal stem cell treatment holds promise as this cell type addresses the key wound impairments seen in non-healing diabetic ulcers. They are immuno-modulatory and may create a more favourable inflammatory environment of the diabetic wound.
Significant as they may seem, these results must be considered with caution as the expression levels achieved rather than normalize the patient's phenotype convert it to a mild-to-moderate form. Interestingly, the anti-CD3 antibodies reduced the numbers of circulating T lymphocytes, but whether or not autoreactive T cells are specifically depleted is unclear.
Also, concomitant treatment with glucocorticoids is needed to prevent immune rejection and elevation of liver transaminase levels. There is evidence that the anti-CD3 antibody acts as a weak TCR agonist and stimulates the production of regulatory T cells (Tregs) in humans. Adult mesenchymal stem cells in diabetic wounds may in addition to beneficial paracrine activity, differentiate into other cell types e.g. Due account must also be taken of the fact that the adeno-associated vector has the potential to induce hepatotoxicity. B lymphocytes in TID have been postulated to act as antigen-presenting cells and also to produce autoantibodies that can result in ? cell destruction (Bluestone, Herold et al.
Autologous transplantation of healthy differentiated cells, obtained from iPSCs, into an animal model with haemophilia or diabetes mellitus type 1, normalizes the corresponding altered function by in vivo production of the deficient protein or hormone.Non-viral strategies also have a role to play in the treatment of haemophilia as they could in the long term provide a safer alternative than viral vectors which, as we have seen, are fraught with significant biosafety and efficacy-related problems, which have so far limited their clinical application.
Anti-CD20 antibodies target and deplete B lymphocytes, and lead to a preservation of ? cell function in patients, as measured by C-peptide production and reduction of HbA1c levels compared to controls.
BackgroundThe discovery of putative EPCs by Ashara et al in 1997 (Asahara, et al., 1997) has illuminated the fields of vascular biology and diabetes related vascular dysfunction.
However, the level of autoantibodies in these TID patients was not determined, so the mechanism by which anti-CD20 therapy works in TID patients is still uncertain. For the first time, vasculogenesis or de novo blood vessel formation was determined to occur post-natally, as previously it was assumed to occur only during embryogenesis. As we will explain further below, these immune modulation therapies may also be important for the future success of ? cell regeneration and transplantation. The delivery of EPCs to ischaemic sites in the body offers the possibility of successful treatment of diabetic vascular disease.
This non-viral strategy has made it possible to obtain stable factor VIII secretion in vitro. Worldwide, research groups are testing the hypothesis that EPC therapy may treat peripheral vascular disease and prevent the progression of non-healing diabetic foot ulcers to amputation.
Xenoimplantation of these protein-secreting cell lines into immunocompetent haemophilic mice corrects the severe form of the disease. Experimental alternative treatments for TID: Stem cellsPancreas transplantation has succeeded as a replacement for insulin-secreting ? cells for TID patients, but this relies on a limited source of cadaveric pancreatic tissue (Danovitch, Cohen et al. These cells are suitable for autologous therapy without immunological rejection but this approach may be hindered due to disease associated cell dysfunction.EPC research is complicated by several issues. Such implantation could prove extremely useful as a bioimplant in the context of monogenic diseases such as haemophilia.
Our laboratory has advanced the use of nucleofection as a non-viral transfection method to obtain factor IX expression and secretion in adult adipose tissue-derived mesenchymal stem cells [68].
Early studies demonstrated that isolated human islets did not proliferate in suspension culture, and that adherent islet cells showed limited replication of ? cells (Nielsen, Brunstedt et al.
The reports in the literature describe different identities, sources of isolation, culture methodologies and function. Although it is certainly true that expression efficacy with these types of protocols is lower than when viral vectors are used, it must be underscored that these protocols do offer much higher safety levels, with the additional advantage that increasing factor activity to above 5% of normal values already places the patient in the mild phenotype group.The use of cell therapy in the treatment of haemophilia has to date consisted mainly in the transplantation of healthy cells in an attempt to repair or replace a coagulation factor deficiency.
These procedures have been conducted mainly with adult stem cells and, more recently, with progenitor cells partially differentiated from iPSCs, albeit in most cases the mechanisms by which transplanted cells (to a greater or lesser extent) engraft and go on to proliferate and function remain unknown.Aronovich et al. Therefore, alternative sources of IPCs are needed, and stem cells have been suggested as this source.

These results would seem to indicate that transplantation of a fetal spleen (obtained from a developmental stage prior to the appearance of T-cells) may potentially be used to treat some genetic disorders. Stem cells are undifferentiated cells that are capable of self-renewal and differentiation into any cell type.
The first cell type is named colony forming unit-Hill cells which arise from peripheral blood mononuclear cells which are non-adherent and give rise to a colony after 5 days in culture. Embryonic stem cells (ESCs) are derived from the inner cell mass of implanted embryos (Evans and Kaufman 1981), induced pluripotent stem cells (iPSCs) are adult or fetal cells that have been “reprogrammed” to an ESC-like state (Takahashi and Yamanaka 2006), adult stem cells are isolated from adult tissues (Becker, Mc et al.
The second cell type is a heterogenous collection of cells termed circulating angiogenic cells or early EPCs.
This resulted in a restoration of factor VIII plasma levels and in the correction of the bleeding phenotype.
1963) and germline-derived stem cells are generated from embryonic or adult gonads (Shamblott, Axelman et al.
These arise from mononuclear cells which are adherent to fibronectin or other matrix adhesion proteins after 4-7 days. These iPSC-derived cells express specific membrane markers for these cells such as CD31, CD34 and Flk1, as well as factor VIII.
Tissue-specific pancreatic stem cellsThe existence of pancreatic stem cells was proposed in the last decade (Ramiya, Maraist et al.
These cells are derived from mononuclear cells that adhere to fibronectin and appear after 6-21 days. Following transplantation of these cells into mice with haemophilia A, the latter survived the tail-clip bleeding assay by over 3 months and their factor VIII plasma levels increased to 8%-12%.
These transplanted cells were injected into the liver parenchyma where they integrated functionally and made correction of the haemophilic phenotype. The different cell types may also be characterized by flow cytometry for surface immunophenotype. High levels of FVIII mRNA were detected in the spleen, heart, and kidney tissues of injected animals with no indication of tumor formation or any other adverse events in the long-term. For example, pancreas from pregnant rats and mice displayed an uptake of bromodeoxyuridine (BrdU) during pregnancy, suggesting the proliferation of islets (Parsons, Brelje et al. Induced pluripotent stem cells in the treatment of diabetes mellitusDiseases caused by the destruction or loss of function of a limited number of cells are good candidates for cell therapy. 1999; Rieck and Kaestner) that reduced expression of multiple endocrine neoplasia type 1 (MEN1), a tumor suppressor (Karnik, Chen et al. Diabetes mellitus (DM) is classified into two broad categories: type 1 DM, which is a genetic disease, and type 2 DM, a more generalized variety related with insulin resistance. 2007), and enhanced expression of tryptophan hydroxylase 1 (Tph1), which allows for incorporation of BrdU in the cell (Kim, Toyofuku et al. DM, especially the type 1 form, is associated with microvascular complications, such as retinopathy, neuropathy or nephropathy, as well as cardiovascular problems. Type 1 DM is a T-cell mediated autoimmune disease specifically aimed against pancreatic beta cells, which results in insulin deficiency [75,76]. In addition, expression of Ki-67 (a marker associated with proliferation) and ?-cell mass increase has been described in the pancreas of obese mice, perhaps as a response to insulin resistance (Butler, Janson et al. Symptoms of DM include episodes of lethargy and fatigue, polyuria, enuresis, nocturia, polydipsia, polyphagia, weight loss and abdominal pain. The disorder has a strong genetic component related with the susceptibility to inherit and develop the disease through the HLA complex (HLA-DR and HLA-DQ genotypes) and other loci involved in immunologic recognition and cell-to-cell signaling in the immune system (graft compatibility) [77,78].Abnormal T-cell activation in susceptible individuals results in both an inflammatory response within the Langerhans islets and a humoral immune response involving the production of antibodies against insulin-specific beta cell antigens, decarboxylase glutamic acid or the protein tyrosine phosphatase [79]. The presence of one or more types of antibodies may precede the appearance of type 1 diabetes and its subsequent development [80,81]. Other authors have shown that mature ? cells are capable of self-replication (Figure 2), and that this is the mechanism by which new beta cells in the adult are generated. In any case, the final result is the destruction of beta cells and progressive impairment of the blood glucose metabolism [82].
Young diabetic rats are able to regenerate ? cells after streptozotocin (STZ)-induced ? cell destruction, but this capacity for regeneration declines rapidly during the first days of life (Wang, Bouwens et al.
The mechanism is reported as via paracrine effect, direct incorporation in blood vessels and differentiation into endothelial cells. Some patients with type 1 diabetes may show a higher susceptibility to other conditions such as thyroiditis, Graves disease, Adisson disease, celiac disease, myasthenia gravis or to degenerative skin conditions such as vitiligo [83-85].The greatest incidence of type 1 DM occurs during childhood and in the early years of adulthood with significant variations across different geographies. The field of topical EPC therapy is in the early stages with benefit demonstrated in these studies.
Diagnosis is usually made before the age of 20 (between 16 and 18 in 50-60% of cases) [75].
The factors involved in the development of type 1 DM include the so-called familial predisposing factors, gestational status, age and other iatrogenic causes.
Type 2 DM is characterized by a functional deficiency of insulin per se or by a resistance to the hormone resulting from an alteration of the function or structure of the insulin receptor at the level of the membrane or of any of the molecules involved in the intracytoplasmic signal transduction cascade [86]. In addition the standardisation of cell dose, definition of cell type and animal model is required. The metaboilic effects of insulin vary depending on the action of the molecules that participate in signaling pathways to regulate gene expression in striated muscle cells, adipocytes, hepatocytes and in pancreatic beta cells [87-90]. More recently, human islet precursor cells were identified from IPCs that underwent an epithelial-to-mesenchymal transition, demonstrating that, in vitro, human IPCs from the islets of cadaveric pancreas donors could spontaneously produce highly proliferative “de-differentiated” fibroblast-like cells that could then possibly be expanded and later re-induced into insulin-producing, ?-like cells (Gershengorn, Hardikar et al. The use of human cells in immunocompromised animals are required to further elucidate therapeutic efficacy 4.3. Thus, for example, insulin resistance caused by the impairment of glucose transporter GLUT4 initially results in a metabolic syndrome, type 2 diabetes, lipodystrophy, hypertension, polycystic ovary syndrome or atherosclerosis.
In general, the morbidity and mortality of DM is related with the different long-term cardiovascular complications associated with the disease, also taking into account other proactivating factors such as smoking, obesity, a sedentary lifestyle, hypertension, early onset and prolonged duration of type 1 DM, genetic predisposition and hyperglycemia.Nephropathy, retinopathy and diabetic neuropathy are the most common microvascular complications of DM. Paracrine effectEarly EPCs and Late EPCs may contribute to post-natal neovascularisation by secretion of angiogenic cytokines and growth factors. As regards diabetic neuropathy, this can be a focal complication associated with diabetic amyotrophy or with cranial nerve III oculomotor palsy, or a more generalized occurrence that can take the form of a sensorimotor polyneuropathy affecting the autonomic nervous system, gastric motility and cardiac function.
The secretome of EPCs contains cytokines and growth factors which stimulate wound healing by increasing proliferation, migration and cell survival of the different cell types required for wound healing i.e.
Peripheral neuropathy together with peripheral vascular disease may lead to a diabetic foot syndrome, characterized by ulcerations and poor healing in the lower limbs [91]. As mentioned previously, Pdx1 and Ngn3 are two transcription factors which are essential for endocrine cell differentiation during pancreas development, and have been targets in the search of pancreatic stem cells. As a macrovascular complication, cardiovascular disease accounts for 70% of mortality in individuals with type 2 DM, with the incidence of coronary artery disease being higher in women than in men suffering from type 1 DM [92].
However, it has not been demonstrated if Pdx1+ cells can self-renew (Jiang and Morahan 2011), a requirement for stem cell definition. Atherosclerotic processes are in turn more common in patients with type 1 DM [93].Although treatment and diagnosis of diabetes is well-established, there is a constant quest for new drugs that may be more effective at lowering blood glucose levels, controlling their therapeutical management —especially in younger patients—, and preserving patients' long-term quality of life by reducing the incidence of complications resulting from the disease. Ngn3+ cells from duct-ligated adult pancreas can develop into islet cells (Xu, D'Hoker et al. Current research is centered on unveiling the structure and function of glucose transporters, which may offer significant therapeutic advantages [86], as well as on the development of new fast-acting insulin analogs and more accurate subcutaneous pumps [94-98].
Direct incorporation in blood vesselsThe second mechanism of action is the direct incorporation of EPCs into the growing blood vessel wall or the differentiation of these cells into mature endothelial cells. Commendable as these initiatives are, it is difficult to anticipate and control factors that exert a variable influence upon glucose levels such as nutrition, physical activity or stress. However, Ngn3 expression in the embryo inhibits proliferation by inducing cyclin-dependent kinase inhibitor 1a (Cdkn1a) (Miyatsuka, Kosaka et al. This mechanism is associated with late EPCs This mechanism has been shown in animal models and may not be as significant as the paracrine effect of cell therapy. These factors alter the glycemic environment and consequently the amount of insulin required at each point in time, which reinforces the need to establish sophisticated artificial pumping systems that may simulate the natural endocrine pancreas.
The continuous advancement of our understanding of the mechanisms that govern the physiopathology of diabetes and gene susceptibility together with the multiple possibilities currently offered by biotechnology have fuelled the researchers' interest in the development of all three types of advanced therapies: gene therapy, cell therapy and tissue engineering.
The transplantation of conditioned media or identified therapeutic factors would allow for protein-based therapy. In this regard, although we are still at a very incipient stage [99,100], procedures based on transplantation of insulin-secreting cells or islets obtained from stem cell differentiation may hold valuable hope for the future. One study compared conditioned media from EPCs to EPC treatment alone in an animal model of cutaneous wound healing. The need to justify the human and financial investment made in the development of new advanced therapies is as strong in diabetes as it is in haemophilia.
However, in the case of the former justification is even more compelling taking into account that an optimal and efficient treatment is already available for the disease.
The discovery of insulin as a therapeutic tool for DM constituted an important milestone in the history of medicine even if administration of this hormone does not fully compensate for the function lost. Impaired angiogenesis in diabetes due to EPC dysfunctionIt is known that EPCs are decreased in number and dysfunctional in people suffering from diabetes mellitus.
Moreover, both coagulation factor and insulin treatment are only palliative, never curative, which is the basic idea underlying treatment of DM and haemophilia. Moreover, it is also important to take into account the potential adverse effects of these therapies, and particularly the complications associated with DM, which derive from the fact that it is a long-term disease. This is due to decreased mobilisation from the bone marrow and decreased homing to cutaneous wounds. In addition, advances in terms of the clinical transplantation of Langerhans islets have not met with the expected success as a result of the inadequate number of donors available and the incidence of immune rejection of the newly transplanted beta cells [101]. Built upon the knowledge gained from studies on embryonic cells about the differentiation process, the first studies on iPSCs, whereby human cells were reprogrammed to become in vitro differentiated insulin-producing cells, showed great promise [105,106]. However, as only partial cell differentiation was achieved, those studies failed in their attempt to enrich insulin-producing cell lines or assess their function.
Topical delivery In normal healing EPCs are released into the circulation from the bone marrow in response to ischaemia and travel to sites of tissue injury and participate in angiogenesis. These authors established a quantitative cytometric method to evaluate the efficacy of cell differentiation.
In addition, they increased the level of precision in the assessment of the competence and function of the iPSCs from a rhesus monkey by means of transplantation into immunodeficient mice. These cells were induced to form endodermal structures, pancreatic and endocrine progenitors and insulin-producing cells.
In mice with cutaneous wounds and 4 weeks of streptozocin induced hyperglycaemia, the levels of circulating EPCs were unchanged but the levels of bone marrow derived EPCs within the wound granulation tissue were decreased as compared to non-diabetic controls. By means of a TGF-? inhibitor, generation of endocrine precursor cells capable of generating insulin-producing cells that respond to glucose stimulation in vitro was undertaken. The bone marrow derived EPCs from diabetic mice showed increased apoptosis and decreased proliferation in diabetic wound tissue as compared to non-diabetic controls. Transplantation of these cells into a type 1 DM murine model decreased blood glucose levels in 50% of the mice.
These results show the high efficacy that can be achieved by obtaining iPSCs from a superior animal model as well as the capacity of iPSCs to be transformed into insulin-producing cells, which opens up the possibility for carrying out autologous transplantations in the future.Along the same lines, Jeon et al. This ex-vivo manipulation may restore the EPC functional defect and succeed in restoring diabetic wound healing to the non-diabetic phenotype. Systemic delivery of stem cell results in cells being taken from the circulation in the lungs, spleen and liver and not reaching the wound. The insulin-producing cells obtained in this way express different pancreatic ? cell markers and secrete insulin in response to glucose stimulation.
Transplantation of these cells into non-obese diabetic mice (a model of autoimmune type 1 DM very similar to the human form) results in a kidney graft with a functional response to glucose stimulation and a consequent normalization of blood glucose levels (Figure 2).Until recently, iPSC generation from patients with type 2 DM had not been reported in the literature.
The topical delivery of cells allows for concentrated doses of cells to be delivered to a skin wound and not become trapped in other sites in the body. Matricellular proteins: Osteopontin Osteopontin (OPN) is a matricellular protein and is involved in tissue repair and angiogenesis. These cells were reprogrammed by lentiviral transduction with human transcription factors OCT4, SOX2, KLF4 and cMYC, telomere elongation, and down-regulation of senescence and apoptosis-related genes, and were subsequently differentiated into insulin-producing islet-like cells.
These proteins modulate cell function by interacting with cell-surface receptors, proteases, hormones, and other bioeffector molecules, as well as with structural matrix proteins such as collagens (Bornstein, 2009) Decreased OPN is found in EPCs in diabetes mellitus. Reprogramming of keratinocytes from elderly type 2 DM patients produces efficient iPSCs with a "privileged" senescence status that allows them to transform into insulin-producing islet-like cells, which may lead to the development of a versatile strategy for modeling the disease as well as an advanced therapy for treating it.Generally speaking, several problems must yet be resolved before iPSCs can be applied clinically, specifically to the treatment of haemophilia or diabetes.
In the first place, it is essential to optimize the reprogramming process so that it provides maximum safety assurances against the potential risks derived from undesirable genetic changes in iPSCs [111]. Recent studies have revealed significant chromosomal changes that take place during the long-term culture of iPSCs as well as variations in the number of copies of certain genes and point mutations, which could clearly be related with the reprogramming of somatic cells and result in damage to the DNA [112-115].The second hurdle that must be overcome is the high variability that exists between the different cell lines in the context of differentiation into pancreatic lineages [16]. The epigenetic and functional trials that should be performed in this respect are complicated by the fact that iPSCs have a high epigenetic content [116]. Osteopontin knockout mice have decreased myocardial angiogenesis in response to ischaemia and delayed recovery after hindlimb ischaemia.
The third obstacle has to do with the purification of iPSC-derived ? cells to prevent the transplantation of undifferentiated cells, which could result in the formation of teratomas. Moreover, it is necessary to develop new reagents to make direct differentiation of pancreatic progenitors into functional ? cells more efficient and to design highly specific surface markers for these cells so that a more precise fluorescence analysis can be performed in order to isolate homogeneous populations of this kind of cell so that their function can be rigorously controlled.
Wound healing studies in osteopontin knockout mice show more residual debris and less matrix organisation than wildtype mice.
General regulatory and bioethical issuesCell therapy, as one of the bedrocks of the advanced therapies —together with gene therapy and tissue engineering—, requires a new legislative framework in order to guarantee that patients can avail themselves of the products they need and provide governments with a robust protection, control and regulation mechanism. Delay in diabetic wound healing may arise in part because of the low expression of OPN early in the wound bed after wounding, resulting in the reduced migration of immune cells to the site of injury leading to the accumulation of cell debris, decreased recruitment of endothelial cells, delayed angiogenesis and poor matrix organization. The existing framework regulating advanced therapies will have to be adapted fast in order to keep pace with the proliferation of new knowledge in this rapidly developing field.
However, desirable that this may be, the pace of legislative reform is unfortunately slow and inevitably lags behind the development of new science. For any product based on cells or on tissue, it should be made compulsory to verify that the desired physiological functions are preserved after the preparation process, both in isolation and in combination with other non-biological components, as many of these products will be used with a metabolic purpose [119,120]. Current delivery options include injection of cells, delivery in extra-cellular matrix, delivery on a scaffold and delivery as part of a tissue engineering skin equivalents.
Nevertheless, many things remain to be learned about the procedures that should be followed to guarantee the safety and efficacy of cell therapy products, especially with respect to the biology of stem cells, their self-renewal and differentiation potential and, above all, the evaluation and prediction of potential risks.Most cell therapy products are not controversial from a bioethical point of view.
The exception to this is therapy with human embryonic stem cells, which raises moral and bioethical problems [121,122]. Such consideration refer to the donor's informed consent and to problems associated with the harvesting of oocytes and the destruction of human embryos. In this regard, the guidelines used by the different countries range from total prohibition to regulated authorization. The vascular progenitor cells exit the biomaterial over time and repopulate damaged tissue and participate in the vascular network. In general, there is an international consensus that the results obtained in stem cell research should be applied to humans without prior bioethical scrutiny, with the understanding that scientific research and the use of scientific knowledge must respect human rights and the dignity of the individual in accordance with the Universal Declaration of Human Rights and the Universal Declaration of the Human Genome [123].The main advantage of induced pluripotent stem cells is that their use, unlike that of embryonic stem cells, does not raise moral or bioethical issues as the scientific community, as well as society at large, consider it a valid alternative for the generation of pluripotent stem cells without the need to use human oocytes or embryos.
Furthermore, these cells have shown themselves to be functionally and molecularly similar to embryonic cells, but without their bioethical problems, which means that their use in humans will not require an overly stringent regulatory framework.
The importance of this cannot be overstated as, in many instances, and in some countries more than in others, legislation can hinder the development of science and, consequently, the application of new knowledge and new therapeutic strategies.
Co-culture, gene therapy and hyperoxia It is hypothesised that endothelial progenitor cells act as angiogenic support cells by their paracrine activity. Co-administration of EPCs with smooth muscle progenitor cells increased vessel density in a mouse model of hind-limb ischaemia to a greater degree than administration of either cell alone.
Concluding remarksiPSCs offer an unprecedented alternative for basic, clinical and applied biomedical research. The most significant applications of these cells to the field of cell therapy are related to the treatment of such organ-specific conditions as diabetes ?a typically metabolic disease?, hepatic and cardiovascular diseases, immunological disorders and monogenic hereditary conditions in general such as haemophilia.However, many aspects remain to be unveiled about the safety of iPSCs and about their reprogramming mechanisms, although no-one denies that this technology offers new, until-recently-unimaginable possibilities for correcting alterations in a large number of conditions, particularly in monogenic and metabolic diseases [124]. Also, some technical problems will also have to be resolved such as finding a way to produce these cells using risk-free viral vector transfection as well as safer alternative methods such as viral vector-mediated reprogramming.
Using an ex vivo gene transfer strategy, EPC cell cultures can serve as gene carriers and function as a temporal local production unit of de novo synthesized growth factors within the wound or skin replacement. In the longer term, once the challenges mentioned above have been overcome, both cell and gene therapy will become plausible alternatives.
As far as haemophilia is concerned, the first article discussing the benefits of gene therapy for the treatment of the disease was published a decade ago. At that time, experts in the field anticipated that a cure for haemophilia would be found by the first decade of the 21st century [125], a prediction that did not come true because of multiple problems related to biosafety.
Although many steps have been taken in the right direction with respect to gene therapy, cellular reprogramming of iPSCs and the safety of transfer vectors, efforts must continue in order to resolve problems related to immune response, insertional mutagenesis, efficacy and expression time, the collateral (particularly hepatotoxic) damage caused by viral vectors and the risk of teratoma and neoplasia derived from the application of certain cell types. Increase number of EPCsIncreasing EPC number for topical treatment increases the wound healing benefit of EPCs. Sight should not be lost of the difficulties inherent in recruiting patients for clinical trials and in the large-scale production of vectors and cell lines, needed to facilitate optimal and efficient implementation in the clinical setting. One of the first things that must be addressed when doing research into advanced therapies is whether the expected benefits of such therapies will be able to offset the investment needed.
In-vitro animal studies reveal that proliferation of EPCs derived from the bone marrow can be accelerated by GM-CSF. In the case of haemophilia, the answer is clearly in the affirmative as it is a chronic disease that requires high-frequency life-long treatment, very costly in patients on prophylaxis, and which poses a potential risk of infection by emerging pathogens.
It has been used in human clinical trials for investigation of autologous therapy in critical limb ischaemia. In this regard, application of strategies that are less demanding in terms of efficacy, i.e. As regards diabetes as a typically metabolic disease, advances in the understanding of its physio- and etiopathology, together with the greater biotechnological possibilities available, have made new alternatives possible as a result of the development of advanced therapies to treat it. Transplantation of insulin-secreting cells or of islets obtained a from differentiation of stem cells could hold some hope in the long term.As in haemophilia, in diabetes it is also necessary to justify the investment of human and financial resources required for the development of new advanced therapeutical strategies, taking account of the fact that patients with this condition also benefit from an optimal and efficient treatment at present.
The justification for the said investment is that diabetes gives rise to vascular and neurological complications in the long term and that transplantation of Langerhans islets has not achieved the success that scientists hoped for because of the dearth of donors and the high rate of immune rejection that characterizes diabetic patients. In a nutshell, iPSCs technology has the potential to produce an about-face in the way we conceive cell behavior as iPSCs can be induced to form hormone-producing differentiated cells. In this regard, several authors have reported on the generation of insulin-producing pancreatic cells from iPSCs from rhesus monkey and murine models which, after transplantation, are capable of producing insulin in vivo in response to glucose stimulation. Nonetheless, some general issues affecting iPSCs remain to be resolved before these cells can be used clinically in the treatment of diabetes. Prominent among these are optimizing the reprogramming process as well as their genetic safety, controlling the high differentiation variability of the different pancreatic lines by means of epigenetic trials and enhancing the purification, isolation and characterization of homogeneous populations of iPSC-derived insulin-producing ? cells.NaN.

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