Stem cells used to treat diabetes symptoms,best chinese medicine for diabetes,type 2 diabetes target range yelp,shoes for diabetic feet look - PDF Review

The main symptoms of the diabetes mellitus type 2 are excessive thirst, excessive urination, weakness, fatigue and dry mucous membranes. One of the most common complications of diabetes mellitus are cardiovascular diseases, renal disease and retinal eye disease (retinopathy). For the treatment of diabetes mellitus type 2 the medicines, capable to reduce the level of blood sugar and insulin, are applied. Therefore, immunobiological methods of cell and regenerative therapies are increasingly used in the treatment of diabetes mellitus and its complications in the leading medical centers around the world. The Institute of Cell Therapy developed and clinically tested the method of treatment of diabetes mellitus, the 1st and 2nd type, by means of hematopoietic stem cells transplantation. Treatment of diabetes mellitus type 2 with the use of stem cells does not only increase the effectiveness of treatment by traditional means and methods, but sometimes enables to refuse completely from them.
Do not waste your time and contact us, because the sooner you apply for this method, the more effective the treatment will be.
There has been considerable discussion about the whether the media hyped the recent Harvard Diabetes stem cell paper (see top 10 takeaways of that paper) .
This Harvard publication reported production of insulin-secreting cells from human embryonic stem cells (hESC). Newspapers around the world widely exaggerated the potential impact of this paper.
I have tremendous respect for the Harvard researchers on this team and this paper is very important, but this situation got totally out of control.
I’m not a Diabetes expert, but my understanding is that in fact there may be no such thing as a cure for Diabetes.
This entry was posted in Uncategorized and tagged Diabetes Cure, Diabetes Hype, Diabetes stem cells, Diabetes Treatment, Harvard stem cells, Medical research hype, stem cell hype, Stem Cells by admin. Unfortunately hyping is an effective technique for getting some superficial researches spreading out as solid discoveries and, more sadly, a successful approach for attracting money into some eventually useless research areas.
The material on this blog is intended for educational purposes only and absolutely should not be considered medical or financial advice. This website does not do sponsored posts or receive payment for posting about products, companies, etc.
Doug Melton's laboratory is interested in the genes and stem cells that give rise to the pancreas and insulin-producing beta cells, with possible therapeutic implications for diabetes. Type 1 or juvenile diabetes is a genetically complex disease caused by an autoimmune destruction of insulin-producing beta (?) cells.
We analyze the genes and cells that form the pancreas and use that information to direct differentiation of multipotent stem cells toward the ?-cell fate.
We are extending our work now, with collaborators, to explore ways to protect stem cell-derived beta cells from immune destruction folllowing transplantation.
We use a wide variety of techniques, including functional genomics, chemical screening, tissue explants and grafting for analyzing inductive signals, and developmental genetics for direct assays of gene function. Should we be successful in directing the differentiation of human cells into functional ? cells, or find signals that cause ? cell replication and regeneration in vivo, we will extend our findings to clinical applications for the treatment of diabetes.
Some of these projects were also supported by grants from the National Institutes of Health, the Beta Cell Biology Consortium, the Juvenile Diabetes Research Foundation, and the Harvard Stem Cell Institute. Free resources for science teachers and students, including animations, short films, and apps. HHMI’s science magazine explores biomedical research through in-depth features, news, and perspectives. HHMI’s innovative research center where scientists pursue challenging problems in a collaborative setting.
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Transplantation of pancreatic islets offers a direct treatment for type 1 diabetes and in some cases, insulin-dependent type 2 diabetes.
Diabetes mellitus is a chronic metabolic disorder manifested by hyperglycemia due to a deficiency of insulin production by pancreatic ?-cells.
One explanation for the poor long-term outcome is recurrent immune destruction of the transplanted islets despite immunosuppression. Stem cells are cells that are able to proliferate while maintaining an undifferentiated status (self-renewal) and retaining a capacity to differentiate into specialized cell types under appropriate conditions.
During early embryogenesis, the epiblast and primitive endoderm arise from the inner cell mass (ICM) of the blastocyst, from which pluripotent embryonic stem (ES) cells are derived. In mice, progression from foregut endoderm to insulin-producing cells is rapid (between E9.5 and E10). Insulin is the most abundant hormone detected during the first trimester in human pancreas [22]. In mice, embryonic pancreatic epithelial cells express nuclear Pdx1 and cytoplasmic cytokeratin-19 (CK19) [22].
Pancreatic endocrine cell fate specification is also ensured by a lateral inhibition process mediated by Notch signaling pathways.
Islet1 expression in pancreatic endodermal cells is required for the formation of dorsal mesenchyme and generation of all endocrine islet cells.
Insulin mRNA is translated as a single chain precursor called preproinsulin, and the removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin. Several studies have revealed the possibility that endocrine precursors lie within pancreatic ducts.
Guided by the expression of ngn3, ?-cell precursors were identified in an injury model of adult pancreas [58]. The endocrine cells of the adult rat pancreatic islets of Langerhans, including ?-cells, turn over every 40-50 days by a process of apoptosis, and are replaced by neogenesis from progenitor epithelial cells located in the pancreatic ducts [55, 57]. In another study, rat and human pancreatic islets were isolated post-mortem and cultivated in medium supplemented by growth factors. However, controversy exists regarding the significance of nestin-expressing cells in islet neogenesis.
Human cells derived ex vivo from pancreatic exocrine tissue isolated from healthy donors de-differentiated into a ductal phenotype (CK19- and CK7-positive) after adherence to culture surfaces [63]. The ability of adult stem cells to produce differentiated cells from embryologically unrelated tissues is an example of metaplasia and shows that embryological commitments can be changed or reversed under certain circumstances [66]. Although no significant in vivo differentiation of bone marrow into ?-cells was observed in a study of adult mice [70], transdifferentiation from liver to pancreas has been seen.
Islet-like cell clusters (ICC) cultured from human fetal pancreatic tissue (18 to 24 weeks gestation) were able to mature functionally and morphologically when grafted to kidney or pancreas in normoglycemic mice [73]. In another study, nicotinamide treatment induced maturation of human fetal pancreatic islet cells, but not adult ?-cells, and resulted in otherwise unresponsive glucose-stimulated insulin release [75]. Apart from the derivation of ?-cells from various pancreatic stem cells in adult tissues and pancreas, pluripotent ES cells are another source to search for renewable ?-cells.
Assuming that techniques were available for the efficient production of ?-cells from HESC, there are still other factors restricting its widespread use for application in therapy.
In the first successful attempt to induce pancreatic differentiation from MESC, Soria et al.
Several genes, such as nkx6.1, bind to the insulin promoter and regulate insulin transcription in ?-cells. Pancreas organogenesis shares similarities with the development of the nervous system, despite pancreas and CNS originating from different germ layers [16, 87, 88].
With minor modifications in Lumelsky’s differentiation protocols, populations of nestin-expressing MESC derivatives differentiated into insulin-producing cells showing increased levels of ?-cell transcripts and insulin production. The discrepancy in insulin1 mRNA and insulin protein expression, and the lack of C-peptide release in these two studies leads us towards the hypothesis that cells differentiated with the nestin protocol take up exogenous insulin from the media. Constitutive expression of transcription factors with a role in pancreatic development such as pdx1 or pax4 has also been used to enhance differentiation of insulin-producing ?-cells from MESC [101].
Inducible expression of Pdx1 in MESC also resulted in increased expression of pancreatic endocrine transcription factors during EB differentiation [102]. Expression of genes characteristically involved in the establishment of the mouse embryonic pancreas, including NGN3 and PDX1 transcription factors as well as INSULIN, GCK and GLUT2 were detected by RT-PCR analysis in HESC cultures during EB and monolayer culture differentiation [103]. Three years after the first report with MESC was published, it was shown that HESC could be induced to form insulin-producing islet-like clusters similar to immature pancreatic ?-cells [104].
Given the inefficiency and controversies for deriving ?-cells from nestin-expressing neuroendocrine progenitors, a more plausible approach is to direct ES cells through a process that mimics normal pancreas development. Studies with MESC revealed that by restricting serum in the presence of activin A in media induced the formation of Bracyhury-expressing mesendoderm cells in EB, from which endoderm cells may develop later [15]. These DE cells can further differentiate into pancreatic endocrine cells in the presence of Hedgehog- and Notch-signaling inhibitors [107].
It is reasoned that in vivo maturation might be necessary for the functional maturation of HESC-derived pancreatic endocrine or ?-cells. Our own laboratory has described an efficient method to enhance ?-cell differentiation from HESC [111]. Generally, the formation of a ?-cell from a pluripotent stem cell represents a part of the complex choreography of embryonic development. Several findings have underscored the necessity for deriving more HESC for research purposes. The derivation of iPSC opens new possibilities to reprogram pluripotent stem cell lines from individual patients to avoid immunological rejections in cell therapy.
An attractive alternative to eliminate the age concern and to reduce the likelihood of graft rejection in stem cell therapy is through the use of human leukocyte antigen (HLA)-haplotype cell banking, from which a best match could be selected.
However, whether cell transplantation will ultimately be the approach of choice for regenerative medicine is a moot point. The authors are grateful to Juvenile Diabetes Research Foundation (JDRF) for funding some of the work presented in this article.
Peltoniemi P, Yki-Jarvinen H, Oikonen V, Oksanen A, Takala TO, Ronnemaa T, Erkinjuntti M, Knuuti MJ, Nuutila P. Shapiro AM, Ricordi C, Hering BJ, Auchincloss H, Lindblad R, Robertson RP, Secchi A, Brendel MD, Berney T, Brennan DC, et al. Researchers have used injections of patients' own stem cells to reverse the course of type 1 diabetes, reports a research team from the University of SA?o Paulo in Brazil and Northwestern University in Chicago.The findings, published in the current issue of the Journal of the American Medical Association, exemplify the remarkable gains made by diabetes researchers, who are battling a continuously spreading disease that now affects nearly 8% of adults and children.
A follow-up to their previous work, Voltarellia€™s team reported the same success with eight more patients, confirming that the majority of the stem cell transplants led to a considerable repopulation of insulin-producing beta cells, which are found in the pancreas.
The blood levels of C-peptide, which is a protein produced by beta cells, were traced for confirmation that the patienta€™s remaining beta cells were able to begin growing again, repopulation the pancreas and producing insulin. More researchers are studying the advantages of adult stem cells, including stem cells grown from skin, which would eliminate the need to harvest cells from bone marrow. Advanced Oral and Maxillofacial Surgery (AOMS) in Elmhurst, IL Partners with Provia Labs to make Store-A-Tootha„? Dental Stem Cell Banking Available to their Patients.
Born with a windpipe less than a tenth of an inch wide, he was the first child in the world to get a transplant made from a donor organ and his own stem cells. Eventually scientists hope to get to the point where any replacement body part or organ you need would simply be manufactured in a lab, man-made, just for you..
How transplanted stem cells help the braina€™s repair mechanism following traumatic brain injury. Patients with diabetes mellitus type 2 make about 90% of the total number of cases of diabetes mellitus. According to various sources, there are from 120 to 180 millions people, suffering from diabetes mellitus in the world, accounting for 2-3% of the world’s population.
The incidence of diabetes has a family character, and your risk is 40%, if your close relatives suffered from this disease.
In type 2 diabetes, the progressive increase of the blood glucose level and reduced ability to capture glucose by tissues occur, whereby the body utilizes free fatty acids and amino acids as the sources of energy, but the latter are necessary for the body for other purposes. Approximately 65% of deaths from diabetes mellitus are due to the myocardial infarction or stroke. Due to the multiple life-threatening complications of the disease, diabetes mellitus is an extremely important medical and social problem. For this purpose the cultures of xenogeneic cells from Langerhans islets were investigated, as well as bone marrow and cord blood stem cells.
The main purpose of such treatment is the prevention of complications of diabetes mellitus, normalization of the level of glycosylated hemoglobin in blood, reduction of the dose of insulin in diabetes type 1, and the normalization of blood glucose level in diabetes type 2.
As an example, the data on the treatment of 25 patients with diabetes mellitus type 2, who participated in the clinical trials on the hematopoietic stem cell transplantation, performed by the Coordination Centre on Organs, Tissues and Cells Transplantation of the Ministry of Health of Ukraine on the basis of the Institute of Cell Therapy. Stem cells restore sensitivity of cells and tissues to the action of insulin, significantly improve pancreatic function, which leads to the normalization of blood sugar level.
The principle of treatment is based on the fact that stem cells are able to transform into ?-cells of the pancreas and promote the formation of stem cells of the patient. Clinical tests confirm that after stem cell treatment the positive tendency towards the increase of the level of insulin and decrease of the blood sugar. If people won?t need insulin anymore after this trial, I think they would be cured and this would be a great step for stem cell research.
Those submitted to this blog will be posted at the sole discretion of the editor and may be edited for content. The opinions here are only those of the authors and do not reflect those of the University of California, Davis. Thus, the challenge finding new treatments or a cure can be divided into two problems: blocking or reversing the autoimmune attack and providing new ? cells. Genetic marking in mice has allowed us to map the lineage of progenitor cells that give rise to the exocrine, endocrine, and ductal components of the pancreas. The tool works online so you will not have to install anything onyour system to configure your custom config.
This can be a direct consequence of autoimmune destruction of ?-cells, as seen in type 1 diabetes (T1D) [1-3].
Indeed, late graft loss is commonly observed although graft function measured by C-peptide immunoreacticity was retained. The sources of such stem cells for the purpose of generating ?-cells were firstly identified through several studies on adult and fetal pancreas.
The primitive endoderm gives rise to extraembryonic tissue, whereas the epiblast differentiates to form the three primary germ layers during gastrulation. Nonetheless, in humans, although pancreatic differentiation from foregut endoderm is initiated at an equivalent time in human (26 days post conception, dpc), significant insulin expression is delayed.
Pdx1 plays an important role in the transactivation of the insulin gene and so is required to maintain normal ?-cell homeostasis [21]. Genetic studies that involved ectopic expression of neurogenin3 (ngn3) and intracellular Notch in early pancreas progenitors collectively confirm the function of ngn3 in controlling endocrine cell fate. A number of genes control the differentiation of specific pancreatic endocrine cell subsets.
Pdx1 regulates the expression of ?-cell-specific genes such as INSULIN, IAPP (islet amyloid polypeptide), ?-cell-specific glucose transporters glucokinase (GCK) and GLUT2. A functional ?-cell exhibits an acute three-fold stimulatory insulin release in response to glucose. Proinsulin consists of three domains: an amino-terminal B chain (30 amino acids), a carboxy-terminal A chain (21 amino acids) and a connecting peptide in the middle known as the C-peptide. In the adult pancreas, endocrine cell formation from ductal epithelial cells has been observed both in experimental models of pancreas injury [52] and in various clinical pathologies [53].
Following partial duct ligation, cells located in the ductal lining were found to reactivate ngn3. However, the administration of glucose or GLP-1 to rats for 48 hours resulted in a doubling of islet cell mass, suggesting that islet progenitor cells were able to proliferate and that the precursors may reside within the islets themselves [59]. Focal regions of nestin-positive cells were identified in large, small, and centrolobular ducts of the rat pancreas. Selander and Edlund did not find nestin expression in the pancreatic ductal epithelium [60], where the potential progenitors reside. After 2 days in culture, these cells reactivated PDX1 expression, suggesting their pancreatic precursor potential; PDX1 re-expression in ductal cells has been considered to be a prerequisite for their differentiation.
For example, both rodent and human bone-marrow stromal cells have been demonstrated to differentiate into other mesodermally derived tissues such as cardiac muscle cells [67], liver or neurons [68, 69], which are ectodermal in origin. Additionally, exendin-4, used to treat those human fetal pancreas-derived ICC in vitro, upregulated PDX1 expression [74]. Although fetal ?-cells failed to proceed through the differentiation process to achieve a terminal phenotype in culture, this finding suggests that they are able to go through the maturation process in vivo. Mouse and human ES cells are derived from the inner cell mass of pre-implantation blastocysts [76, 77], but they require different signaling pathways to maintain pluripotency. Besides ethical concerns, because of their derivation from human embryos, difficulties remain due to tissue rejection following transplantation.
It was reasoned that insulin-producing cells could be enriched if cells that activated its upstream promoters were isolated and expanded.
Pancreatic endocrine cells also share several characteristics with neurons, for instance, islet cells are electrically excitable [38, 87].
By adding fluorescein-labeled insulin in the media, cells were found to absorb these insulin molecules, hence were immunoreactive to anti-insulin antibody.
During in vitro differentiation, important changes in expression levels of pancreatic genes were detected in Pax4-positive cells, and to a lesser extent, in Pdx1-positive cells. A MESC line, in which exogenous Pdx1 expression was regulated by a tet-off system integrated into the ROSA26 locus, was employed to examine the effect of Pdx1 expression during in vitro differentiation using a protocol similar to Lumelsky's protocol. Immunohistochemistry analysis revealed that 60-70% EB contained insulin-expressing cells, although only up to 2% of cells in an EB stained for insulin. The differentiation strategy in this study was a modification of the 'nestin-selection' method.


B: RT-PCR analysis of NESTIN expression in H1, H7 and H14 HESC lines during in vitro differentiation.
HESC grown to confluency gave rise to spontaneously differentiated cells that yielded FOXA2 and PDX1 co-expressing definitive endoderm (DE) cells at the periphery of the colonies. In brief, KAAD-cyclopamine and FGF10 were used to promote endoderm patterning and proliferation from DE cells. Pancreatic endocrine cells, owing to their precursor status, may be more potent than ?-cells in undergoing maturation should they respond to in vivo milieu. We first overexpressed Pax4 in HESC from a constitutively active CAG (human cytomegalovirus major immediate early enhancer- chicken ?-actin- rabbit ?-globin) hybrid promoter and induced differentiation in EB model. If the formation of a ?-cell in vivo is to be strictly followed, expression of key regulatory genes for this sequential commitment should be regulated in a time-dependent manner.
As HESC lines are derived from a genetically heterogenous population, their biological variations, heterogeneity, genetic and epigenetic differences contribute to the variations in developmental potential. However, time and costs necessary for derivation, differentiation and safety testing of good manufacturing practice (GMP)- or clinical-grade iPSC from each individual may exceed practical and economical limits. The results of pancreas transplantation will also be improved by minimizing HLA mismatches [115]. Many technical challenges remain to be overcome, and it is possible that a better understanding of the basic biology of the pancreas, from the point of view of both its development and subsequent homeostasis, may offer insight that will provide alternative approaches to curing diabetes and related diseases. Chee Liew is a recipient of California Institute of Regenerative Medicine (CIRM) Postdoctoral Fellowship. Regulation of glycogen synthase and phosphorylase activities by glucose, insulin and basal enzyme activity in human skeletal muscle. Resistance to exercise-induced increase in glucose uptake during hyperinsulinemia in insulin-resistant skeletal muscle of patients with type 1 diabetes. Growth and development of the islets of Langerhans: implications for the treatment of diabetes mellitus.
Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.
Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats.
Enriched human pancreatic ductal cultures obtained from selective death of acinar cells express pancreatic and duodenal homeobox gene-1 age-dependently. Stimulated endocrine cell proliferation and differentiation in transplanted human pancreatic islets: effects of the ob gene and compensatory growth of the implantation organ.
Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.
There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development. A quantitative immunofluorescent study of the endocrine cell populations in the developing human pancreas. Novel SOX9 expression during human pancreas development correlates to abnormalities in Campomelic dysplasia.
Morphology and function of cultured human fetal pancreatic cells transplanted into athymic mice: a longitudinal study. Richard Burt says, a€?I wouldna€™t use the word cure, but it appears we changed the natural history of the disease.
In type I diabetes, a patienta€™s immune system attacks the beta cells that are responsible for the production of insulin, which is hormone that breaks down the glucose in food. They began by carefully extracted stem cells from the bone marrow of the diabetes patients. The likelihood of the development of diabetes mellitus is 50%, if the father had diabetes, and 35%, if the mother was sick. Chronic hyperglycemia leads to the increase of the osmotic pressure of blood and damage of the vessel wall (angiopathy).
However, the currently known methods of correction of metabolic disturbances in diabetes mellitus do not provide the desired effect and do not prevent disability. American scientists have shown a positive effect of the umbilical cord blood infusions in patients with diabetes mellitus, enabling to reduce the dose of insulin in such patients. The principle of treatment is based on the fact that the hematopoietic cells are able to transform into ?-cells in the pancreas as well as to stimulate the formation of these cells from the patient’s own stem cells (such cells are present in pancreatic ducts).
The diagrams clearly show that within 3 months after transplantation the level of glucose and glucosylated hemoglobin reduced almost to normal values and remain stable for 1 year. If you suffer from diabetes mellitus type 2, we invite you to our clinic for a high quality and effective treatment of this disease. This can result not only the restoration of the normal level of sugar in blood but also anables to prevent the complications of diabetes mellitus, such as the damage of blood vessels of the retina, kidney, heart and legs. This therapy also causes the healing of the ulcers and tissue defects of the foot, improves microcirculation and reduces the incidence of diabetic angiopathy. There is a fine line between maintaining appropriate energy and enthusiasm with the language that one uses and going too far. Comments cannot include personal attacks, promotional material for stem cell clinics, criminal allegations (e.g.
We have focused on the latter, pursuing three complementary approaches aimed at making new ? cells for diabetics. In parallel, using biological and chemical screens, we investigate the regulatory genes that specify pancreatic cell fates.
Many extant studies have focused on deriving ?-cell progenitors from pancreas and pluripotent stem cells. Other types of diabetes, collectively known as type 2 diabetes (T2D), occur because of a combination of reduced insulin sensitivity (non-insulin dependent) and impaired ?-cells function [4]. For successful islet transplantation a sufficient amount of ?-cells needs to be included in the transplant to control the blood glucose level without repeated insulin injections. This phenomenon of ‘islet graft exhaustion’ could be attributed to the toxicity of immunosupression, autorejection or recurrence of autoimmunity [8].
It is generally assumed that islet neogenesis or replication occurs throughout life with a gradual decrease in capacity in elder humans. Cells from the epiblast are recruited into the primitive streak and migrate out to form the mesoderm and definitive endoderm (DE), from which the pancreas develops. This observation supports the notion that embryological stages in the mouse and the human are not as closely equivalent as previously assumed [17]. Another more recently described pancreas-related transcription factor, Sox9, is predominantly expressed throughout the early developing pancreas (prior to 14 weeks of gestation). Mice lacking ngn3 function fail to generate pancreatic endocrine cells and die postnatally from diabetes [25]. Pax4 is required for the initial commitment of early endocrine precursors to become ?- and ?-cells, while pax6 is required for the early differentiation of ?-cells [28-33]. Hence, the expression of Pdx1, Gck and GLUT2 can be used as a key indicator of ?-cell functionality [38]. Zinc is required for packaging insulin, an integral part of insulin crystals for 2-Zn-insulin hexamer, as well as free ionized zinc in the extragranular space that acts as a reservoir for granular zinc pools [43-46]. Within the endoplasmic reticulum, proinsulin is exposed to several specific endopeptidases that excise the C-peptide, thereby generating the mature form of insulin. Islet neogenesis and replication may provide the source of pancreatic stem cells from which normal renewal of islets occurs throughout life [54].
The pancreatic stem cells generated insulin-producing stem cells (IPSC) in a monolayer, from which islet progenitor cells (IPC) budded. The cells were grown in serum-free media with glucose, insulin, transferrin and selenium (ITS) supplements, nicotinamide and keratinocyte growth factor (KGF).
GLP-1 is capable of restoring normal glucose tolerance in aging mice and increasing islet mass in adult animals previously subjected to subtotal pancreatectomy [9]. These Ngn3-positive cells express ductal cytokeratins, but did not stain for insulin, suggesting they are of islet progenitor phenotype. Instead, nestin was detected in mesenchymal cells, called pancreatic stellate cells [61, 62] and was not detected during pancreas development [21]. This phenomenon of 'transdifferentiation' is questionable, since these in vitro studies are subject to ductal cell contamination [64].
Nine genes that exhibit ?-cell phenotypes when mutated were selected for initial reprogramming experiments. Although insulin content did not increase in culture, ICC transplanted under the kidney capsule were found to give rise to grafts that had significantly higher levels of insulin compared to adult islets and completely reversed diabetes in STZ-treated nude rats. Taken together, these tissue culture models have provided groundwork for further studies to dissect the molecular mechanisms of islet cell differentiation from developmentally earlier multipotent or pluripotent stem cells such as ES cells. Recently, cells from epiblast of post-implantation mouse and rat embryos have also been shown to give rise to pluripotent stem cells.
Much effort has been spent in the development of alternative methods of generating patient-specific stem cells.
The cells were transfected with a plasmid containing a neomycin resistance gene under the control of the insulin promoter, and hygromycin selection cassette driven by a constitutively active phosphoglycerate kinase (PGK) promoter.
Additionally, insulin-producing cells have also been observed in the invertebrate nervous system [89-91] and in primary cultures of mammalian fetal brain [92]. An explanation for this active insulin uptake is the prevalence of apoptosis and necrosis due to suboptimal culture condition.
In this study, the use of ITSF (insulin, transferrin, selenium and fibronectin) and bFGF were avoided after replating EB on monolayer, so that selection or enrichment of specific cell types before induction of pancreatic differentiation would not occur. IAPP, glut2 and insulin transcripts were upregulated in Pax4-positive cells at a later stage of differentiation, although ngn3 expression remained constant [101].
Not surprisingly, with such low numbers of insulin-positive cells, further characterizations were impossible. Similarly, HESC were differentiated in EB and replated on monolayer to allow the growth of nestin-positive neuroendocrine progenitors.
These early pancreatic progenitors were manually picked and co-transplanted with E11.5 mouse embryonic dorsal pancreas under the kidney capsule of SCID mouse. 80% of cells in activin A-treated cultures were immunoreactive to FOXA2 and SOX17, suggesting the enrichment of DE cells.
The outcome of this stage was the generation of PDX1-expressing gut-tube endodermal like cells. Harvested pancreatic endocrine cells went on to form aggregates on gelatin foam sponges and matrigel overlays. We reasoned that a proportion of DE cells spontaneously differentiated from EB, substantiated by the detection of FOXA2 and NEUROD1 transcripts.
Cells were re-aggregated in suspension cultures to yield islet-like clusters consisted of cells expressing pancreatic endocrine hormones. A major challenge is that ES cells support the early stages of endoderm and pancreatic endocrine cell formation in vitro but do not robustly generate glucose-responsive ?-cells, making diabetes treatment using stem cell therapy untenable. Various studies have reported that HESC lines exhibited marked differences in differentiation propensity into specific lineages [112, 113]. There were transient expressions of NGN3, PAX4 and NKX6.1 transcription factors at early stages of H14 EB differentiation.
Another concern is the age of the patient; iPSC obtained from young adults are healthier than those derived from mature adults.
Studies of ES cells and other stem cells may well play a substantial role in acquiring the necessary knowledge, and the development of patient-specific iPSC certainly offers a new approach to advancing disease models as well as tools for screening new drugs that could play a role in novel treatments in the future.
The groupa€™s initial accomplishment was reported in 2007, with as many as fifteen patients with Type 1 Diabetes receiving injections of their own stem cells.
Sooner or later, the immune cells will essentially eliminate all of the beta cells in the body and glucose levels will begin to increase.
Then each patient was given radiation treatments, similar to the treatment given to cancer patients, to destroy the immune system. In diabetic micro-and macroangiopathy the vascular permeability is disrupted, vessel fragility is increased and the tendency to thrombosis and atherosclerosis occurs. Also it was shown that the use of endothelial progenitor cells, capable to form new blood vessels, promotes neangiogenesis in patients with obliterative vascular disease of the lower extremities on the background of diabetes mellitus type 2.
Furthermore, hematopoietic cells restore the endothelial cells of blood vessels, that are damaged by glycosylated hemoglobin in diabetes mellitus.
In addition, in these patients the indices of fat metabolism also were normalized, the atherogenic index decreased, the biochemical parameters of liver function improved. Also the recovery of “red blood” occurs, namely the increase of the level of hemoglobin and number of red blood cells. These studies have identified a set of transcription factors and intercellular signaling molecules (growth factors) that are responsible for the stepwise differentiation of normal pancreatic development.
Efforts to generate ?-cells in vitro will help elucidate the mechanisms of ?-cell formation and thus provide a versatile in vivo system to evaluate the therapeutic potential of these cells to treat diabetes. Diabetes can be inherited, such as that seen in maturity onset diabetes of the young adult (MODY), or caused by mutations in an autosomal dominant gene resulting in the disruption of insulin production. In 2000, Shapiro and colleagues described the successful cure of T1D in a small number of patients using a procedure known as the 'Edmonton Protocol' [6]. Long term failure of the early transplantation therapies has prompted the search for more defined sources of ?-cells.
It is also assumed that the generation of ?-cells can take place by two pathways: firstly via replication of already existing ?-cells and secondly via neogenesis (differentiation) from pancreatic endocrine or ductal progenitors [9, 10]. Nodal and activin, members of transforming growth factor beta (TGF-?) superfamily, are essential for the initial endoderm specification, where they regulate the activation of many key regulatory genes, such as foxa2, sox17 and mixl1 in a strict temporal sequence.
In humans, by 35 dpc, the ventral pancreatic bud begins to migrate backwards and comes into contact and eventually fuses with the dorsal pancreatic bud during 6 weeks post conception (wpc) [18].
At this stage, islets are comprehensively vascularized and contain cells independently immunoreactive for insulin, glucagon, somatostatin and pancreatic polypeptide (PP). In contrast to Pdx1, the expression of Sox9 is down-regulated once endocrine cells are developed and is later restricted to ductal cells. Similarly, neuroD1 knock-out mice fail to develop islets and develop severe diabetic ketoacidosis and perinatal death [26]. Fully differentiated ?-cells first appear around E13 at the start of a massive wave of ?-cell differentiation, which is known as “secondary transition” [34]. The dual action of Pdx1, as a pancreas commitment factor during embryogenesis and as a regulator of islet cell physiology in mature islet cells, underscores the unique role of PDX1 in maintaining the function of human pancreatic endocrine cells [39]. The ability to regulate glucose uptake by the islet-specific glucose transporter GLUT2 is the first step necessary for the activation of the regulatory region of the Insulin gene to glucose [47]. Insulin and free C-peptide are packaged in the Golgi into secretory granules, which accumulate in the cytoplasm.
The pancreatic islet mass significantly increased by growing IPC in culture; many of the cells expressed both glucagon and insulin, a phenotype reported for immature islet cells on their path to end-stage differentiation [56]. The epithelial cells formed three-dimensional cystic structures, characteristic of cultivated human islet buds. Additionally, endogenously pdx1-positive rat (ARIP) and stably pdx1-transfected human (PANC-1) cell lines attained a ?-cell phenotype upon GLP-1 treatment.
However, they developed into cells that subsequently proliferated, both in situ and in vitro, and were found to also express Pdx1.
Following tamoxifen induction, a human placental alkaline phosphatase (HPAP) reporter gene was expressed ('pulse').
When these cells were grown in medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), they were able to proliferate. Nonetheless, many groups have tested nestin selection protocols to derive ?-like cells from mouse embryonic stem cells (MESC) later. However, a recent report has demonstrated that pancreatic exocrine cells can rapidly adopt a ?-cell phenotype when the genome of these cells was altered and reprogrammed [65].
Of these, the combination of Ngn3, Pdx1 and MafA transcription factors could efficiently reprogram 20% of differentiated pancreatic exocrine cells into insulin-positive ?-cells in vivo. A follow-up study indicated that insulin- as well as glucagon-producing cells were found mainly located in the proximity of central veins [72]. These epiblast stem cells share many features with human embryonic stem cells (HESC), and differ from mouse embryonic stem cells (MESC), suggesting that the differences between mouse and human ES cells are due to their relationship to different developmental stages, rather than distinct species differences in the control of pluripotency [78]. One way to circumvent the problem is to reprogram adult cells back to pluripotent ES-like cells. The hygromycin-resistant clones were cultured in suspension to induce embryoid body (EB) differentiation. The hygromycin-resistant clones were first differentiated as EB in the presence of several exogenous agents.
Hence, many protocols for deriving ?-cells from MESC were designed first to produce or select for neural progenitors defined by nestin expression [93] and then to direct pancreatic islet differentiation in subsequent steps.
The outcome of their protocol was the formation of cell clusters connected by a web of neurons, with 10-30% insulin-expressing cells localized inside these clusters. LY294002 has been shown to increase total endocrine cell number and insulin content from the human fetal pancreas [96], as well as to prevent neurite outgrowth from neuroendocrine cells [97]. Although these cells release insulin in response to glucose, C-peptide release was never detected, suggesting that de novo insulin synthesis was not a common outcome of cells differentiated from nestin-expressing neuroendocrine progenitors [98, 99]. Functional ?-like cells were generated following pancreatic differentiation by serum-free medium containing nicotinamide and laminin. There was also a significant increase in the number of nestin-expressing cells in Pax4-positive cells, and this increase was corroborated by an increased level of intracellular insulin. The results showed that Pdx1 induction enhanced the expression of the insulin, somatostatin, Kir6.2, glucokinase, ngn3, p48 and pax6 genes.


This strategy was later refined by placing the ?-geo gene under the control of the promoter of Nkx6.1 (ii). There was no significant difference in the levels of NESTIN expression when the cells were grown in ITSF serum-free medium, indicating that ITSF medium did not increase the expression of NESTIN in HESC. Eight weeks after transplantation, clusters consisted mainly of proinsulin-expressing ?-like cells and a few glucagon and amylase-secreting cells were detected in the grafted section. These cells were subsequently cultured in retinoic acid (RA), ?-secretase inhibitor and exendin-4 to promote the transition towards hormone-expressing pancreatic endocrine cells. Inefficiencies in deriving a scalable source of ?-cells from HESC in vitro likely reflect the inability of undifferentiated stem cells to mimic these normally intricate in vivo gene expression patterns. Notably, genes that play the key role in maintaining pluripotency, such as OCT4, NANOG and CRIPTO showed little variation; however, marker expression of multiple tissue-specific lineages became variable after the cells began to differentiate [114].
Renal disease in diabetes (diabetic nephropathy) is one of the factors of the development of chronic renal failure. It is noteworthy, that the transplantation of the large doses of hematopoietic stem cells eliminates the autoimmune component of the disease in patients with diabetes mellitus type 1 and 2. This genetic and cellular knowledge of pancreatic developmental biology guides our approach to the directed differentiation of stem cells.
Various successful experiments using ?-cells in animal models have generated extensive interest in using human embryonic stem cells to restore normoglycemia in diabetic patients. Gestational diabetes is another less known form of diabetes, but it imposes an increasingly prevalent risk factor for the development of T2D after pregnancy [5]. The authors achieved independence of insulin injections in seven T1D patients by transplanting a large number of islet cells (obtained from two donor pancreases) combined with the use of glucocorticoid-free immunosuppressive regimen.
Intensive research is being conducted to look for alternative sources of ?-cells because of the shortage of cadaver donors.
During fetal life, the majority of new ?-cells develop from precursors, but newly developed ?-cells also proliferate.
The primitive gut tube then arises from the DE, which later forms foregut, midgut and hindgut [15].
Both insulin and glucagon expression could only be detected at 8 wpc [19, 20], two weeks later than expected compared to the mouse [21]. A study using sox9 heterozygous mouse mutants suggests that the role of sox9 is as a determinant of multipotent pancreatic endocrine cells in the pancreas [24]. It has been shown that maturity onset diabetes of the young type 6 (MODY-6) in humans is also associated with mutations in NEUROD1 and that the abnormality of islet morphogenesis is due in part to inadequate expression of the INSULIN gene [27]. Nkx6.1 expression is required by this second phase of ?-cell neogenesis in the developing pancreas [35, 36]. Apart from PDX1, mutations of which have been linked to MODY-4 [40, 41], several other genes have also been shown to be required in humans and in mice to assure ?-cell functionality. In the absence of GLUT2, the endocrine pancreas shows the loss of first phase glucose-stimulated insulin secretion and an inverse ?- to ?-cell ratio [48].
In pancreatic ?-cells, C-peptide and mature insulin are present in equimolar ratios and co-localize in the secretory granules [51]. These IPSC-derived islets secreted insulin in response to glucose challenge, and upon addition of nicotinamide they further matured and differentiated into fully functional islets.
These cultivated human islet buds consisted of both CK19-positive ductal cells and hormone-positive islet cells. This study provides a significant basis for determining the minimum biological requirements for a non-?-cell to become a ?-cell, whilst supporting the previous finding that pancreatic ductal cell lines are capable of giving rise to pancreatic endocrine cells. Ngn3-positive cells were then isolated and injected into ex vivo embryonic mouse pancreas, and were found to autonomously (not by fusion) increase glucose-responsive ?-cell mass in the explants.
Although emergent extra-islet insulin-positive cells did not appear to self-cluster as islets, angiogenesis was remarkably increased.
This indicates that pdx1 plays an important instructive role in pancreas differentiation, not only from primitive gut endoderm but also to reprogram extra-pancreatic tissue towards a ?-cell phenotype [72].
Neomycin antibiotic pressure was applied later to select for cells expressing factors that activate the insulin promoter.
As described in the previous section, nestin has been detected in a small population of pancreatic cells that have been proposed as possible islet precursors [59, 87]. Although the differentiated cells were immunoreactive to insulin, RT-PCR revealed that insulin2, glut2 and pdx1 mRNA levels were low and ?-cell-specific insulin1 mRNA was never detected. This modification yielded insulin-producing cell clusters (IPCC) with morphological similarities to pancreatic islets. However, ITSF were still added in the later stage of the differentiation protocol to compensate for serum-free culture conditions.
However, by reducing the glucose concentration in later stages of differentiation and by reaggregating these clusters in suspension, insulin expression and secretion was increased. Intriguingly, transplanting DE cells alone did not result in the appearance of ?-like cells.
This protocol generated 7-12% of insulin-positive cells, and similar to fetal ?-cells, they released C-peptide in response to depolarizing reagents such as potassium chloride (KCl) and tolbutamide. Although no significant level of human C-peptide was detected within the first month, long term engraftment data revealed that glucose-stimulated serum levels of human C-peptide increased rapidly during the next sixty days [110]. These cells also expressed markers of voltage-gated Ca2+-channels (VGCC) at comparable levels to human fetal pancreas. Furthermore, published studies so far revealed that HESC-derived ?-cells were not glucose-responsive, but acquired further maturation once transplanted into mice.
Some lines exhibited a marked potential to differentiate into one lineage, often with more than 100-fold differences in lineage-specific gene expression. Up-regulation of NEUROD1 and ISLET1 transcripts was detected in the course of EB differentiation.
They proposed that about 150 HESC lines would be needed for most of the UK population, and that as few as ten might be sufficient if one were to prospectively identify cell lines that could serve a larger number of patients, such as lines homozygous for common HLA types [116, 117]. Therefore, one possible treatment for the disease may be get rid of entire existing immune system, replacing it with a new one that comes from stem cells that do not have this destructive trait. Using both embryonic and induced pluripotent stem cells (ES and iPS, respectively, from mice and humans), we aim to create functional ? cells, using a stepwise differentiation protocol wherein specific signals are used to tell the cells which fate to adopt.
While new techniques are continually unveiled, the success of ?-cell generation rests upon successful manipulation of culture conditions and the induction of key regulatory genes implicated in pancreas development. In the adult pancreas, the rate of both ?-cell neogenesis and replication is more limited than in newborn, but it is thought to take place [11-13]. Signals from the notochord and mesenchyme specify the pancreatic domain at the endodermal region. The expression of these markers implies that fetal ?-cells may be capable of processing and secreting insulin. A recently described transcription factor, MafA is induced at the final stage of ?-cell differentiation and functions as a potent activator of insulin gene transcription [37]. After transplantation, the mice were able to regulate the levels of glucose in their blood within a week, and survive without further need for insulin.
There was a significant increase in DNA and insulin content over 3-4 weeks of culture, suggesting that these human islet buds were immature and still in the process of differentiation. Surprisingly, all islets analyzed in adult pancreas in the ‘pulse and chase’ experiments contained numerous HPAP-immunoreactive ?-cells. These findings provide evidence that pancreatic islets themselves, apart from the ducts contain multipotential progenitor cells that are able to differentiate to ?-cells.
Importantly, exocrine cells that adopted a ?-cell phenotype improved hyperglycemia in STZ-induced diabetic mice. It was also sufficient to direct the production and secretion of mature, biologically active insulin from a restricted population of cells in liver in vivo [71]. In one clone, the cells released insulin in response to glucose, although the insulin content was low. An almost pure population of insulin-positive cells was produced, of which 20% were Pdx1 co-expressing cells from several clones.
Immunocytochemistry staining showed that Pdx1, GLUT2 and glucokinase were co-expressed by insulin-positive cells. Although it is not clear whether the addition of ITSF to the medium would select for nestin-positive cells, they concluded that ?-cell precursors transiently expressed nestin during differentiation in vitro.
Pax4-positive cells were then cultivated in Spinner rotation cultures to promote their histotypic maturation into spheroid islet-like clusters grown in suspension. Insulin-producing clusters produced by this method resemble immature pancreatic embryonic cells. This finding suggests that the stimulatory cues from co-grafted pancreas and an in vivo milieu were necessary to coax HESC to differentiate into ?-cells. Nonetheless, because purification steps were not used, teratomas containing various other tissues of ectodermal and mesodermal were formed in most of the animals.
In addition, the increase in VGCC genes correlated with an enhanced responsiveness of EB to muscarinic receptor agonists such as KCl.
DE cells responded to hedgehog and Notch inhibitors, giving rise to cells that express pancreas endocrine hormones. These findings add support to the contention that these cells mimic ?-cells in the human fetal pancreas. When gene expression levels of the same HESC lines with different passage numbers were compared, consistent patterns within the same line but significant variations between lines remained.
Differentiated EB expressed INSULIN, GLUT2 and GLUCOKINASE ?-cell markers at a later stage of EB differentiation, suggesting that H7 and H14 cells were able to produce ?-cells in the course of spontaneous differentiation.
In this review, we compare successfully conducted protocols, highlight essential steps and identify some of the remarkable shortfalls common to these methods.
However, despite short term success, long term insulin independence is usually not sustainable. This article reviews the current knowledge on the role of stem cells in pancreatic development and as a source for ?-cell transplantation.
The pancreas develops from the posterior foregut and the subsequent formation of the pancreatic anlagen relies on retinoid signaling and on inhibition of Indian and Sonic hedgehog (Shh) signaling. However, the human fetal pancreas is not glucose-responsive, despite being able to secrete insulin upon stimulation with ?-cell secretagogues [23]. The synthesis of the glucose-phosphorylating enzyme glucokinase is a late event in ?-cell maturation [50].
Taken together, those studies suggest that fully differentiated or even somatic cells could serve as an important source of tissue for generating functional insulin-producing cells in diabetic patients. Insulin secreted from the liver of pdx1-transfected mice ameliorated streptozotocin (STZ)-induced diabetes. They later improved the protocol by eliminating C-MYC due to its function as an oncogene [81]. These insulin-positive cells normalized hyperglycemia when transplanted into the spleen of STZ-induced diabetic mice. Subsequent transplantation of these cells into STZ-induced diabetic mice normalized their glycemia. Not surprisingly, with such small amounts of insulin, the cells failed to correct hyperglycemia when grafted into STZ-treated diabetic mice. However, the cells were also distinct from islets in numerous ways; while 95-97% of the cells were insulin-positive, and 2-3% were glucagon-positive, no staining for somatostatin and PP was detected. Electron microscopy detected insulin-labeled secretory granules in Pax4-derived cells, and notably, these cells were able to normalize blood glucose levels in STZ-treated diabetic mice.
A substantial number of cluster cells were co-stained for insulin and glucagon or somatostatin.
Thus, future improvement will require purification of pancreatic cell types and depletion of inappropriate cell types prior to transplantation.
In order to recapitulate pancreas organogenesis in vitro, an improved three-dimensional EB differentiation model and manipulation of extrinsic signals may be employed to support the formation of pancreatic mesenchyme, acini, islets and ?-cells in a spatially and temporally controlled pattern.
This observation argues against the possibility that variability of culture technique or senescence of HESC contributed to marked differences in differentiation propensity in different lines. Violation of the microvasculature and secondary immunodeficiency cause the infections of the soft tissues of the foot, nonhealing wounds, trophic ulcers. In addition, we discuss recent advancements in the derivation of patient-specific pluripotent stem cells that may facilitate the use of autologous ?-cells in stem cell therapy.
Their international clinical trial showed that only 13% of the patients (5 out of 36 subjects with T1D) maintained insulin independence at 2 years, and 28% had complete graft loss 1 year after the final transplantation [7]. In addition, it discusses recent advancements in the derivation of patient-specific pluripotent stem cells that may facilitate the use of autologous ?-cells in stem cell therapy. The developing pancreas is composed of pancreatic and duodenal homeobox 1 (Pdx1)-expressing epithelial precursor cells that give rise to the three differentiated compartments of endocrine, exocrine and ductal cells found in the adult pancreas. These studies notwithstanding, the mechanisms that control endocrine cell formation from human neonatal pancreas are poorly understood because of the apparent scarcity of material and difficulties in obtaining human fetuses. Notably, partial pancreatectomy resulted in the same observation but with increased bromodeoxyuridine (BrdU) incorporation, indicating that these ?-cells retained their full proliferative capacity following injury. Zhou’s study was largely based on earlier studies that have provided the evidence that these lineage-committed cells can further be reprogrammed back to an ES cell fate, a characteristic termed induced pluripotency, or more commonly referred to as reprogramming. This potentially allows the generation of iPSC from patients with genetic disorders, providing a platform in which human diseases including diabetes mellitus may be studied in vitro [82], apart from the prospect of patient-specific cells that would obviate immune rejection in transplantation regimens. In these mice, blood glucose levels remained normal and insulin-positive cells were present in the liver and in the spleen even after 42 weeks of transplantation [84].
The same group later demonstrated that substitution of insulin for pancreas-conditioned medium did not result in differentiated ?-cells, suggesting that the differentiation effect was mostly due to soluble factors released by the pancreatic rudiment [86]. Nonetheless, these IPCC increased circulating insulin levels, reduced weight loss, improved glycemic control and completely rescued the survival of diabetic mice.
RT-PCR detects the expression of immature islet genes such as NGN3 but low expression of ISLET1, PDX1 and GCK. These cells could be isolated by fluorescence activated cell sorting (FACS) using Newport Green dye based on the high zinc content in insulin-containing cells. B: ?-like cells were enriched from Pax4-expressing EB and replated on monolayer with nicotinamide and low glucose. We have also observed variations of pancreatic lineage differentiation potential from different HESC lines (Figure 4). Thus, the production of HLA-mapped clinically-safe iPSC would greatly minimize the ethical concern and enable the creation of more efficient HLA-haplotype banks. These patients develop necrotic processes of the ulcerated foot, lesions of bones and joints.
The endocrine cell mass aggregates in interstitial clusters adjacent to the ductal epithelial cells, to form the islets of Langerhans [16].
Thus, their observation indicates that terminally differentiated ?-cells are capable of self-renewal and differentiation into new ?-cells.
The grafted mice were able to maintain their body weight and survived for longer periods of time than hyperglycemic sham-grafted controls. Intriguingly, when LY294002 was not used, transplanted cells formed tumors that resembled teratomas, suggesting that undifferentiated cells were still present.
Although they showed a response to the different ?-cell agonists and antagonists, their responsiveness to glucose was minimal. Similarly, these aggregates produced cells secreting various pancreatic endocrine hormones, and notably, released C-peptide in response to glucose. Purified ?-like cells were enriched in PDX1 and INSULIN transcripts but depleted in OCT4 expression, suggesting the absence of non-pancreatic cell types and residual undifferentiated cells. Thus, it would be advantageous to work with HESC that are inclined toward pancreatic differentiation.
The diabetic foot often leads to the necessity of the limb amputation in diabetic patients.
To date, this study remains controversial because it does not prove the absence of stem cells during neonatal life or after pancreas injury, and the possible transcriptional activity of insulin promoter in such overlooked stem cells. Although the nature of insulin-staining cells derived by this method remains controversial, other groups have successfully used variations on this procedure to generate similar cell types. According to our experience, nestin is expressed by undifferentiated HESC and its expression remained constant during differentiation (Figure 2).
The use of xeno-free HESC will be prerequisite for utilizing these cells in stem cell therapies [108, 109]. Isolated cells also synthesized proinsulin, expressed C-peptide and responded to tolbutamide.
These cells went on to form C-peptide-positive cells that responded to ?-cell secretagogues.
Thus, the protocol to differentiate ?-cells using nestin selection should be reevaluated considerably. Whereas this study shows the possibility to increase the differentiation of ?-cells from HESC, further work is required to characterize in vivo maturation and function of these cells.
It remains to be determined whether these isolated ?-cells rescue hyperglycemia in a mouse model.



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