these blog posts are written by Dr. alfredo G. Tomasselli, a Retired bio-Chemist and Bio-physicist who spent his life in Academia and The Pharmaceutical Industry researching cures and treatments for diseases such as HIV/Aids, Diabetes, and Rheumatoid arthritis. His daughter sara inspires this blog and helps to write posts. sara was diagnosed with type 1 diabetes on april 4th, 2014 at the age of 30. 

2015 Type 1 Diabetes: Research Advances to develop a cure

This document is only for information and cannot be taken to make medical or other personal decisions

In our blog we have striven to stay updated with progress in areas concerning disease etiology, prevention with life style and vaccines, islets/β-cells transplant/encapsulation, stem cell research, re-education of the faulty immune system, improvements in glucose monitoring and insulin delivery, and amelioration of disease management to eliminate or delay disease caused complications.

Here's a summary of what's being posted

(1) The main feature of Type 1 Diabetes (T1D) is the inability of the body to produce insulin, a protein hormone that regulates glucose levels. Insulin is produced by the β-cells housed within the islets of Langheran (footnote i) of the pancreas. Without insulin the body’s glucose raises to life-threatening levels. Daily injections of insulin are the standard treatment, but even when tightly matched to meals’ carbohydrate content, injected insulin is not the same as its natural counterpart and complications may arise in the long run; hence a need to find a cure for this disease. (2) Type 1 Diabetes is an autoimmune (footnote ii) disease whereby certain white blood cells (footnote iii) of the immune system attack β-cells. It is triggered by environmental factors in genetic predisposed people; though the triggering factors remain largely unknown (a leading hypothesis is a viral infection), more is known about genetic factors as a number of them have been identified and are related to mutations in genes related to functions of the immune system. Years before the symptoms occur, it can be predicted that people who carry specific antibodies will develop the disease within a certain time frame. Indeed, clinical trials intended to prevent T1D are carried out in people without disease symptoms, but who have T1D predictive antibodies and those with a family history of the disease. (3) For people who cannot control blood sugar and experience dangerous glucose level swings (especially glucose lows), a total pancreas transplant (a very demanding surgery), or islets transplant (a relatively mild procedure) become options, but these clinical procedures have their own set of shortcomings, mostly related to (a) a lack of pancreas donors, (b) a high rate of failure of the transplanted islets, (c) a need for chronic use of immunosuppressive medications because of both autoimmunity and alloimmunity (footnote iii), and (d) a relatively short duration of the transplant. So far, the islets have been transplanted in the liver, but this organ has drawbacks and alternative options are explored. Recently, the first successful islets transplant in the omentum of a female patient has been carried out and several more will follow. If this technique works, it might offer a new islets transplant paradigm and solve some of the transplants’ drawbacks. Moreover, it has been observed that supplementation of the islets with anti-ageing glycoprotein improves their survival and insulin secretion in animal models. (4) Scientists have successfully performed β-cells transplants in animal models; it would be interesting to see how β-cells work when transplanted directly in humans (presently we are transplanting them indirectly as a part of the islets). However, shortage of both human islets and β-cells is a problem, and many groups are at work to produce them in large quantities by using stem cell technologies. Two types of stem cells are found in the human body, embryonic stem cells (ESCs) and adult stem cells (ASCs). Human ESCs are considered “the master cells” as they have the remarkable properties of: (a) being able to proliferate (self-renew) indefinitely, and (b) they can become any type of cell; ASCs, are more limited than ESCs in which type of cells they can become, but some specific ASCs have been employed in thousands of applications, including T1D. More recently, scientists have found techniques to reprogram cells of the body (e.g., adult skin cells) to generate cells (referred to as induced pluripotent stem cells, or iPS cells, or iPSCs) that closely resemble embryonic stem cells. Like true ESCs, iPSCs can be instructed to become any cells in the body, e.g., β-cells. Interestingly, it is already possible to take patient’s donated skin cells, reprogram them to become iPSCs, and transform them into β-cells; it is hoped that in a near future, we will be able to return the transformed β-cells to the patient’s body for therapy; this will be a big step to avoid alloimmunity! (5)  A combination of bone marrow and umbilical cord stem cells transplants has produced positive results with respect to patients amelioration of  insulin secretion, fasting blood glucose, HbA1c, C-peptide, hypoglycemic events, and reduction in daily insulin requirements. (6) Two companies have started transplants of encapsulated islets in several patients. Islets are encapsulated into semipermeable membranes that separate them from physical contacts with the cells of the immune system, while allowing the free flow of glucose and insulin. This technology may offer an important means of islets protection against autoimmune and alloimmune attacks. (7) Progress has been made regarding the reeducation of the disfunctional immune system of T1D patients by replacing the faulty or lost Treg cells with new functional ones (footnote iii).

Footnotes

i- The islets of the Langheran are small (about 0.2 mm diameter) clusters of cells disseminated across the pancreas constituting 1-2% of its mass. They contain glycemic regulatory hormones producing cell species such as: (I) β-cells that produce insulin and amylin (each islet contains approximately 1,000 β-cells, i.e., they constitute the majority of the cells of the islet); amylin, co-secreted with insulin, is involved in preventing spikes in blood glucose after meals by slowing down gastric emptying and fostering satiety; and (II) α-cells that produce glucagon that raises the glucose concentration in the blood, thus opposing the insulin glucose-lowering activity.

ii- Autoimmunity is an immune response whereby a person’s immune system attacks the body’s own cells causing their destruction, e.g., in T1D the β-cells are destoyed; Alloimmunity is an immune response against a foreign entity (non-self), e.g., a transplant from one person to another of an organ like the pancreas, or of pancreas’ islets.

iii-Evidences show that CD8+ T cells (also called cytotoxic T cells) attack and kill the insulin-producing β-cells with the help of CD4+ T cells. Both CD8+ T and CD4+ T cells are Lymphocytes (a subtype of white blood cell in the vertebrate immune system). The actions of the above T cells are kept in check by another subtype of T cells called regulatory T cells (or Treg). It has been shown that in autoimmune diseases, such as T1D, Treg are defective.

Part 1

Background information.

Type 1 Diabetes is an autoimmune disease, triggered by environmental factors in genetic predisposed people. In Type 1 Diabetes, the insulin-producing β-cells, housed within the islets of Langheran are mistakenly recognized as foreign entities and killed by misguided immune cells (footnote iii).  Insulin is a protein hormone that helps certain body cells, such as the muscles and adipose tissue, to take up blood glucose which is then used to produce energy to sustain life; without insulin the level of glucose raises to dangerous levels and failure to bring it back within a normal range causes health complications and can be fatal. Since its discovery in 1921 by Banting, Best, McLeod, and Collip, at the University of Toronto, insulin has become the mainstay of T1D. Various chemical/biochemical modifications to insulin and continuous improvements in glucose monitoring technology (e.g., see information within our blog), over the span of nearly 95 years, have transformed a once deadly disease into a manageable one. Indeed, insulin therapy has raised the bar so high that any other approach studied thus far to treat Type 1 Diabetes has fallen short of becoming a therapy with the exception of pancreas and islets transplants that have brought benefits to a relatively small percentage of patients, but suffer drawbacks. Yet, exogenous insulin therapy, even when tightly matched to the carbohydrate content and sizes of the meals, cannot restore the precise physiological glucose control offered by the insulin produced naturally by β-cells. Managing the disease properly to avoid, or at least delay long-term health complications, is a demanding task requiring a combination of daily insulin injections, good diet, exercise, and a careful monitoring of glucose levels; and the majority of people, for various reasons, do not strictly comply with the required strict life-style. Hence, a cure for the already affected people (estimated between 1.5 to 3.0 million in the USA alone) is needed. It is also expected that restoration of the proper β-cells’ number and functions in type 1 diabetic people will also benefit Type 2 Diabetics (estimated to be about 30 million in the USA alone) since a large number of them have an impaired insulin production. We will be considering T1D only in this blog. World-wide research efforts to understand this complex disease and find a cure for it are underway both in academic institutions and in the pharmaceutical industry, with a robust financial help by research advocacy groups [primarily, the Juvenile Diabetics Research Foundation (JDRF)], governments’ agencies, and private donations.

Part 2

Etiology: genetic predisposed people and environmental factors are needed for T1D to develop

 Though it is widely accepted that T1D is triggered by both genetic and environmental factors, a convincing mechanism through which they cooperate leading to pancreatic β-cells destruction and manifest hyperglycemia has yet to be demonstrated. In regard to the genetic risk for T1D it has been found that if one monozygotic (identical) twin has type 1 diabetes, the other one has ~50% probabilities to also get it (compared to 8% of a dizygotic twin), while a sibling has ~6% probability; the risk of contracting the disease by the general population is ~0.4%.  World-wide studies to find genetic factors that are responsible for T1D have identified numerous genes associated with predisposition to the disease. The majority of the identified genes have important roles in the regulation of the immune system.  Indeed the most predictive factors are mutated forms of the Human Leukocyte Antigen (hla) system which contains more than 200 genes coding for proteins that reside on the surface of most of our cells bodies (except for red blood cells) and are critical for these cells to interact with the cells of the immune system. These proteins allow a properly functioning immune system to distinguish between what is self and what is foreign; in other words, it spares the self and destroys anything perceived as foreign or potentially dangerous cells, e.g., cancer cells. So, it also makes intuitive sense that mutations in proteins like those of the HLA system, so critical to distinguish self from foreign, may trick the immune system to mistake the self for the foreign. Numerous other proteins, some involved in the immune system, and some not involved, have also been associated to T1D, but their contribution is estimated to be more modest than that afforded by the HLA. T1D is autoimmune because, not only does it involve malfunctioning of immune regulatory proteins such as HLA, but also because various auto-antibodies (or antibodies against pancreatic proteins) are present in the blood of patients with T1D.  With regard to factors related to environmental factors, various theories have been put forward, though no one is definitive; and it could well be that multiple factors are at work and might be not be constant across the population of  affected individuals. In fact, several papers have appeared over the years relating certain viruses to the onset and progression of T1D, with the coxsackie virus B (CVB) being the prominent suspected co-factor since it has also been found in the pancreas of patients with T1D. The potential viral pathogenic mechanism in human type 1 diabetes has been reviewed, e.g., see paper by A. Schneider & M. G. von Herrath at the link.

Indeed, a critical step for disease prevention and, eventually, for a cure would be an unambiguous discovery of causes and processes leading to the destruction of the pancreas β-cells. Though such a discovery has not yet been made, it is believed that the disease progresses asymptomatically for years until the number of functional β-cells has become so low that they are no longer able to supply sufficient insulin to maintain normo-glycemia. The Juvenile Diabetes Research Foundation and the Disease Interception Accelerator (DIA) group of Janssen Pharmaceuticals are now partnering “in a novel scientific approach, called disease interception, to identify the root causes of disease and enable the development of interventions that stop its onset and/or its progression” (Link to press release )

Biomarkers (footnote iv) to predict who will get Type 1 Diabetes

 As pointed out by Dr. Ezio Bonifacio in a recent review (Bonifacio et al., 2015) there are now various biomarkers that can predict the risk of getting T1D to the point of including children and adults without clinical diabetes in prevention trials conducted through networks such as Trial Net. Typical biomarkers to predict diabetes are serum antibodies against β-cell antigens. These antibodies include insulin (IA), Glutamic Acid Decarboxylase (GAD), Insulinoma-Associated-2 (IA-2A), and zinc transporter 8. The Bonifacio’s review points out that, even in the absence of hyperglycemia, “the presence of two or more of those four auto-antibodies can be considered asymptomatic disease, and usually progresses to hyperglycemia. As an example, in a cohort of 1,000 three-year old children with multiple islet auto-antibodies, hyperglycemia is expected to develop in 50% of the children within 6 years and in 80% within 12 years and that diabetes would develop in the last individuals in the cohort by 60 years of age”. We would like to point out that, when hyperglycemia develops, the above auto-antibodies are also critical to diagnose whether a person has Type 1 or Type 2 diabetes.

References

Bonifacio, E. (2015) “Predicting Type 1 Diabetes Using Biomarkers” Diabetes Care; 38:989–996.

Footnotes

iv -A biomarker is “any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease” as defined by Strimbu K, Tavel JA. What are biomarkers? Curr Opin HIV AIDS 2010;5:463–466.

 

Part 3

Islets Transplants

Among the large population of  Type 1 Diabetes patients, estimated between 1.5 and 3.0 million people in the USA alone, there are thousands of  them that, in spite of a disciplined life-style, experience sudden swings of glucose levels; particularly dangerous is frequent unaware hypoglycemia (low glucose) that can lead to coma or even death. These patients become eligible for pancreas or islets transplants; while the former procedure is a very invasive and demanding surgery, the latter is much simpler and much less risky.

Pioneering studies by Paul Lacy and co-workers at Washington University in St. Louis in the early ‘70s led to reversal of diabetes in mice. These studies together with the development of an automated method for isolation of human pancreatic islets (the Ricordi Method3) and by a new immunosuppressive drug, tacrolimus, paved the way to the first successful human islets transplant in the liver at the University of Pittsburg in 1990 by Camillo Ricordi and co-workers4. Yet, in the first 10 years of its application only a modest number of patients were able to reach normo-glycemia and they did so for a brief period of time. The advent of a procedure known as the Edmonton Protocol, pioneered by James Shapiro and coworkers5, has been a breakthrough in the field. The Edmonton Protocol is a procedure to purify islets from the cadaveric pancreas by using enzymes. Upon purification, the islets are delivered to the liver through the portal vein and are protected from rejection by the host immune system by the chronic use of two immunosuppressive drugs, sirolimus and tacrolimus; and by a monoclonal antibody called daclizumab that is administered immediately after transplantation and then discontinued. In the original work with the Edmonton Protocol, Shapiro and co-workers5 treated seven patients; all of them were able to reach and sustain normoglycemia for one year without insulin, but only 15% remained insulin free, five years after receiving the procedure. Continuous improvement of the technology resulted in prolonged normoglycemia without insulin injections over a five year time-span as reported by the Collaborative Islet Transplant Registry (CITR) 6, which supplies the largest registry of islet transplant data. In their 2010 report, the CITR showed that 60% to 70% of the 481 patients treated between 1999 and 2009 with an improved Edmonton protocol reached 5-year insulin independence. The protocol improvement consisted in the addition of a tumor necrosis factor (TNF) antagonist to reduce inflammation and a T-cell depletion step prior islets transplant (certain T-cell types attack the insulin-producing β-cells, see footnote iii). Beside the 5-year insulin independence result, there was a good improvement of glycated hemoglobin (A1C) levels and a drastic reduction of hypoglycemic unawareness. The islets transplant is an approved procedure in many European countries and Canada, but is still experimental in the US. The islets transplant still suffers from several drawbacks, including (a) the need for 800,000 to 1,000,000 islets per patient (requiring normally two or more patient-matched pancreas donors), (b) the very limited number of pancreas donors; (c) the requirement for toxic chronic immunosuppression against both auto- and allo-immunity, the former caused by the autoimmune process that caused the disease in the first place, and the latter by the introduction of foreign islets into the host. Ironically, the chronic use of immunosuppressive drugs is both toxic to the β-cells and diabetogenic; consequently, it leads to the graft gradual loss of efficiency and, in many cases, to islets inability to produce quantity of insulin adequate to support normoglycemia. When this happens, another islets transplant is needed. It is also important to mention that a weak immune system caused by chronic use of the immunosuppressive drugs promotes the risk of opportunistic infections and cancer; and (d) before they are isolated from the pancreas, the islets are properly connected to blood vessels from which they get the blood supply rich in oxygen and nutrients. On the other hand, when the islets are transplanted into the liver many of them die, while a relatively small percentage are able to establish their own connection to the blood supply to survive and function. In fact, the islets are implanted in the portal vein of the liver and travel to the portal vein branches. Unfortunately, there is an immediate contact with blood during the transplant that exposes the islets to deleterious reactive oxygen species (ROS) and inflammatory processes. In addition, the liver is a rather hostile environment for the transplanted islets compared to their natural pancreatic habitat as it constantly processes body waste yielding substances toxic to the islets. In conclusion, the liver is not the best place for the islets to survive and thrive! Yet, until recently, in spite of numerous efforts and many years of research to find a site alternative to the liver, this organ has been the only site that has led to insulin independence upon islets transplant, but a recent application has potential to offer a better alternative.

 In fact, a recent application of a technology referred to as the BioHub, developed by Dr. Ricordi and co-workers at the Diabetes Research Institute (DRI), a Center of Excellence at the University of Miami Miller School of Medicine, is based on the transplant of islets in the omentum, the apron-like membranous double layer of highly vascularized fatty tissue that covers the abdominal organs. This technology has the potential to offer a better site for islets transplants than that offered by the liver; and, if successful, may start a new chapter for the islets transplant therapy. One of the DRI BioHub platforms is referred to as the Biodegradable Scaffold and consists of mixing the donor islets with the patient plasma and to deliver the mixture to a portion of the omentum. The clinical grade enzyme thrombin is then added to the mixture to form a gel that sticks to the omentum and keeps the islets in place. The portion of  the omentum containing the islet is folded and stitched to protect the islets. Over time the gel is absorbed by the body and the islets are left intact in the omentum while new blood vessels are formed and connect to the islets to support their survival and function. The procedure is minimally invasive as it is done by laparoscopy. The description of the Biodegradable Scaffold Platform presented here is based on the very interesting video from the Diabetes Research Institute that also shows a second platform referred to as Bioengineered Scaffold. Watch the BioHub video down below:

On August 18, 2015, a team led by Dr. Ricordi applied the DRI BioHub Biodegradable Scaffold platform to successfully transplant donor islets in the omentum of a 43-year old woman who had Type 1 Diabetes since she was 17 years old and had a difficult time to control her glucose levels, especially hypoglycemic events. The transplant into omentum therapy included the immunosuppressive regimen currently used for islets transplant in the liver (see above). On September 09, 2015 the DRI announced that the patient was insulin free. Dr. Ricordi commented that he had never seen before this level of functioning of transplanted islets in a patient. Apparently, some of the negative aspects described for the transplants of  islets into the liver were removed or, at least, mitigated by this new approach. Specifically: (a) by avoiding a direct contact of the transplanted islets with blood they have removed the biggest source of inflammation which is a killer of many islets; (b) the omentum is highly vascularized allowing the provision of oxygen and nutrients quickly; (c) overtime, blood vessels grow around islet cells permitting the latter to survive and function properly. Importantly, for this approach a single donor is needed versus two or more pancreas donors required for islet transplant into the liver. The clinical result accomplished by the scientists at the DRI looks like advancement, but the magnitude of this advancement needs to be evaluated when more patients (which is underway) and several years of consistent and, hopefully, positive results will be available. However, it is not a cure, because a cure would require the same features described for transplant in the liver, i.e., a life-long reestablishment of normoglycemia in the absence of injected insulin. Moreover, it also requires normalization of the malfunctioning immune system without use of immunosuppressive drugs. As a first step, the scientists at the DRI aim at inhibiting islets rejection and autoimmune killing of β-cells by limiting immunosuppression to the place where the islets are transplanted (omentum); yet, the ultimate goal is to re-educate the immune system. IT IS NOT A CURE, but, it reiterates AN IMPORTANT PROOF OF CONCEPT, i.e., “INTRODUCTION OF FUNCTIONAL β-CELLS IN A TYPE 1 DIABETIC PATIENT RESTORES, at least temporarily, ENDOGENOUS INSULIN PRODUCTION AND NORMOGLYCEMIA” (both, total pancreas transplants, and islets transplants in the liver have also demonstrated, many times over, this important proof of concept).  

A new discovery might improve both the efficacy of islet transplant and islet survival.  A recent paper by Dr. Shapiro’s team (published online before print November 18, 2015, link to the abstract; see also post from Science News shows that exposure of both mouse and human islets to the rather toxic immunosuppressant drug tacrolimus, used in the Edmonton protocol, inhibits the release of insulin from the islets. However, restoration of insulin release from the islets was attained, both in vitro and in vivo, upon supplementation of the islets with anti-aging glycopeptide (AAGP), a synthetic analogous of the antifreeze proteins (AFP) found in the Arctic fish; the AFP is recognized for its cytoprotective capabilities. Besides a robust increase in insulin secretion, AAGP treatment improved both the efficacy of islet transplant and islet survival by a mechanism involving the decrease of expression of pro-inflammatory proteins (cytokines). It would be interesting to see sometime soon an application of this new discovery in the transplant of islets in humans with T1D.

Even in the best scenario of both successful transplant and good immune suppression limited to the omentum, and even in the eventuality of future improvements brought about by the AAGP treatment, a wide application of the implant into the omentum technology would be prevented by the number of pancreas donors. Here, enters the stem cell technology, which will provide an unlimited number of β-cells or at least, enough β-cells to cover all patients. Moreover, the stem cell technology aims at developing a personalized therapy whereby the patient will receive β-cells generated from his/her own body cells which have been instructed to become β-cells (described below).  There is also a need to reeducate the broken immune system to behave normally, and numerous efforts are underway to accomplish this difficult task; we have already described one of them in relation to the umbilical cord blood cell therapy (see this blog) and other research will be also reported.

References

(3)  Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. (1988) Automated method for isolation of human pancreatic islets. Diabetes. 37:413

(4) Tzakis AG, Ricordi C, Alejandro R, et al. (1990) Pancreatic islet transplantation after upper abdominal exenteration and liver replacement. Lancet 336:402–405

(5) Shapiro AJ, Lakey JT, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343(4):230–238

(6) CITR comprises U.S. and Canadian medical institutions and two European centers.

 

Part 4

STEM CELL RESEARCH

Medical science is addressing three critical outstanding issues towards a cure for type 1 diabetes: (1) the preparation of large quantities of β-cells that can be safely transplanted; (2) the re-education of the autoimmune process that has led to T1D by β-cells killing; and (3) the bypass of the allo-immune process caused by the introduction of foreign islets into the host.

To us, a true cure for Type 1 Diabetes can be simply defined by a comparison with a successful cure of an infection with antibiotics: a few weeks treatment gets rid of the infection with no or minimal short-lasting side effects. On the other hand, we see a functional cure as a treatment whose results are the same, or better, than those produced by insulin therapy, but does not have drawbacks comparable to those of continuous body pricking by insulin injections and glucose monitoring.

Human Stem Cells

Modern medicine is devoting significant resources to stem cell research as stem cells hold the potential to replace lost or damaged cells in many diseases (e.g., the pancreatic β-cells lost by type 1 diabetics, cardiocytes in heart attacks, and neurons in stroke).  Human stem cells are considered “the master cells” of the human body as they have the remarkable properties of: (1) being able to proliferate (self-renew) indefinitely, and (2) they can become any type of cell in the body (there’s around 200 different cell types in the body). There are two types of stem cells found in the body, embryonic stem cells and adult stem cells. Moreover, scientists have found techniques to reprogram cells (e.g., adult skin cells) to generate other cells closely resembling embryonic stem cells that, like stem cells can be instructed to become any cells in the body. The reprogrammed cells are called induced pluripotent stem cells (or iPS cells or iPSCs).

Embryonic stem cells (hESC).  Three to five days after an egg fertilization, a ball of 100-150 cells is formed called a blastocyst. The inner cells of the blastocyst are pluripotent stem cells because they will eventually generate nearly all cells of an organism. Scientists typically use the inner cells (Figure 1) of the blastocysts which are obtained through in vitro fertilization (a process taking place outside of the human body). Scientists are able to collect these cells in a Petri dish that contains nutrients to make them divide and proliferate. Think of it as a little “stem cell factory.” This factory is making only one type of cell (a replica of the original embryonic cell). Once they have obtained the desired quantities of undifferentiated embryonic cells, they add specific factors to make them differentiating into the desired cells, e.g., liver, muscle, heart, adipocytes, neuron, pancreatic beta cells, and others. However, embryonic stem cells suffer from several drawbacks which include: (1) they are not patient-matched and their transplant into patients has the same graft-versus host immune rejection issues encountered when an organ is transplanted, or when the pancreatic islets are transplanted; (2) they induce teratomas (tumors) in the host; (3) they raise ethical concerns because blastocysts need to be destroyed in order to obtain them.

Adult Stem Cells. Adult stem cells are a unique type of cells that are not yet developed into specialized cells; they have been discovered residing among the already differentiated cells in tissues such as the liver, brain, skin, bone marrow, skeletal muscles, blood, and blood vessels and have the ability to replace dying cells and regenerate tissues that have been damaged. Present understanding, is that adult stem cells are more limited than embryonic stem cells in their ability to differentiate into different cell types: they typically differentiate into the cell types of the tissue of origin and are therefore, regarded as multi-potent stem cells. Moreover, because of their limited amount, it is still difficult to separate them from tissues and it is much more difficult to make them proliferate than embryonic stem cells. Adult stem cells separated from a patient’s own tissue are induced to proliferate in the laboratory and when re-introduced in the same patient body these cells are accepted by the body without immune rejection; moreover, they do not have ethical issues. Bone marrow, containing adult stem cells, has been used for many years to treat diseases such as leukemia and cancer.

Adult stem cells can be easily extracted from the umbilical cord; the umbilical cord stem (UCS) cells are biologically younger than other adult stem cells and are more flexible than the other adult stem cells in their ability to be differentiated in more varieties of cells; moreover, though they are typically allogenic (i.e. not extracted from the person under treatment) they have a much weaker graft versus host response (immune rejection) than allogenic embryonic stem cells.

Induced pluripotent stem cells (or iPS cells or iPSCs, see Figure 1). Japanese scientists led by Dr. Shinya Yamanaka were the first to develop technology whereby, after painstaking research, they were able to identify four specific genes encoding transcription factors (proteins) that, when introduced into adult skin cells isolated from mice (Takahashi, K., and Yamanaka, S. 2006) or human (Takahashi, K., et al., 2007) were able to reprogram them into cells with properties equivalent to embryonic stem cells. Induced iPS cells have the properties of self-renewal, and differentiation into all body cells observed for the embryonic stem cells (ESCs) and, in principle, could offer advantages over the latter cells in medical applications. In fact, in addition to their ability of self-renewal and differentiation into many cell types typical of ESCs, they would offer the advantages of been derived from adult tissues (no embryos involvement, hence no ethical issues) and their employment in the replacement of damaged or diseased cells of the patient would avoid the graft versus host problem (immune rejection) of the ESCs as the patient is also the cells’ donor.

Since the first reports by the Yamanaka group, various approaches to make stem cells have been reported in the scientific literature each one with its virtues and drawbacks, and this area of research is moving quickly with frequent discoveries being made; notwithstanding, iPSCs have not reached the safety requirements to be advanced to therapeutic transplants in humans. Yet, iPSCs are increasingly important in drug discovery and in studying patient-specific disease.

  The past year has been very prolific with regard to both the development of technology for in vitro preparation of human pancreatic β-cells and the starting of clinical trials. Hopefully a cure, or, at least a practical cure, is nearing, though the timeline for it really happening is still unclear.

References
Takahashi, K., and Yamanaka, S.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors (2006) Cell, 126, 663 – 676.


Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. (2007) Cell, 131:861-872

In vitro preparation of Beta cells

Exciting developments concerning the production of human β-cells in vitro were reported by two groups: the Kieffer/Beta Logic group (Rezania A et al. 2014) in Canada and a team led by Douglas Melton (Pagliuca et al., 2014) at Harvard. The two groups were able to differentiate both human embryonic stem cell (hESC) lines and human induced pluripotent stem cell (iPSC) lines, into insulin-producing β-cells. They achieved these results by adding soluble factors, such as growth factors, cytokines and small molecules, to the growth media rather than following the earlier strategy to use DNA encoding transcription factors inside the iPSC. They obtained cells that looked like, and exhibited features characteristic of beta cells and, when implanted in a mouse model of T1D, responded to glucose variations like normal beta cells would do. β-cells have proven very difficult to obtain from both hESC and iPS; and a single patient would require in the range of 350 million to 750 million β-cells per transplant (roughly 1,000 more β-cells than islets). Interestingly, Melton et al. report that their procedure can yield about 300 million β-cells per 500 ml flask; this is impressive and, if scalable, it would mean having the supply to cover all patients’ in the world. Moreover, as the authors point out, the technology would provide sufficient β-cells for drug screening in order to identify drug that are safe and effective in augmenting the insulin production; this could be of benefit to those patients with type 2 diabetes that have still β-cells, but do not produce sufficient insulin. Yet, the β-cells produced by both groups still contained ~15% cells expressing other hormones (polyhormonal cells); polyhormonal cells are undesirable because of drawbacks, including unresponsiveness to increase of blood glucose. More recently, Russ et al (2015) were able to optimize the β-cells differentiation process which brought down the polyhormonal cells level to ~ 3%.  All this is important, but there is still some way to go until this technology will become useful to patients as there are still unanswered questions such as: (1) How close are these β-cells made outside the organism (ex-vivo) to the native ones? (2) Can these ex-vivo made β-cells be implanted safely in humans without triggering induction of cancer or other undesired effects (once done the transplant cannot be reversed); (3) transplantation of β-cells derived from embryonic stem cell is an allogeneic (cells come from another person) transplant and the cells will be attacked by the host immune system as foreign entities (the so-called graft-versus-host disease or GVHD); (4) In principle the allogenic β-cells could be by-passed by producing autogenic β-cells (from patient own cells), but how safe are the latter? Moreover, the newly implanted autogenic β-cells will also be attacked by the same mechanism that has caused the disease in the first place that would require the patients still receive chronic anti-suppressive drugs for the rest of their lives unless re-education of the faulty immune system is accomplished (this is the ultimate goal!) unless other means of protection are used (e.g., β-cell encapsulation); and (5) How long is the transplant going to last?

References

Rezania A, Bruin JE, Arora P, Rubin A, Batushansky I, et al. (2014) Reversal of diabetes with insulin producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol; 32:1121-33.

Pagliuca FW, Millman JR, Gurtler M, Segel M, Van Dervort A, et al. (2014) Generation of Functional Human Pancreatic beta Cells In Vitro. Cell 159: 428–439

Russ et al (2015) Controlled induction of human pancreatic progenitors produces functional beta-like cell in vitro. EMBO J. 2;23(13):1759-7

Part 5

Applications of Stem cells in medicine

The company Viacord reports that cord blood cells are used in the treatment of nearly 80 diseases, including a wide range of cancers, genetic diseases, and blood disorders; these cells can be frozen after isolation, a process that prevents their ageing, damage, and viral infection.  

Combinations of bone marrow and umbilical cord stem cells have been transplanted in patients

Interestingly, a paper has just been published concerning the treatment of Type 1 Diabetes patients with combinations of bone marrow and umbilical cord stem cells: Cai et al (2015). The major objective of this trial was “To determine the safety and effects on insulin secretion of umbilical cord (UC) mesenchymal stromal cells (MSCs) plus autologous bone marrow mononuclear cell (aBM-MNC) stem cell transplantation (SCT) without immunotherapy in established type 1 diabetes (T1D)”. In this open-label trial, 42 type 1 diabetes patients, age 18 to 40 years, were randomly divided in two groups of  21 patients/group. One group, received SCT (1.1million/kg UC-MSC, 106.8 million/kg aBM-MNC through supraselective pancreatic artery cannulation), while the other group received standard care (control). The participants were diagnosed with T1D for more than 2 years, but less than 16 years, had a daily insulin requirements of  less than 100 units, and their HbA1c concentrations ranged between 7.5% and 10.5%. They were followed for 1 year at 3-month intervals. Encouraging results were observed in the patients who received cotransplantation of allogeneic UC-MSC and autogenic aBM-MNC; specifically: (1) improved insulin secretion, as indicated by significantly improved of both fasting C-peptide levels, and C-peptide during an oral glucose tolerance test at 1 year; (2) decreased in HbA1C and fasting blood glucose; (3) reductions in daily insulin requirements; (4) fewer severe hypoglycemic events (5) patients quality of life improved; and (6) the treatment was well tolerated. They concluded: “Transplantation of UC-MSC and aBM-MNC was safe and associated with moderate improvement of metabolic measures in patients with established T1D.” This paper also cites the positive results obtained by other groups that had treated both T1D animal models and/or T1D patients with umbilical cord and bone marrow stem cells. Collectively, all these promising results underscore the potential utility of UC-MSCs and aBM-MNC in the treatment of T1D.

 Part 6

 Islets encapsulation to avoid auto- and allo-immunity  

Encapsulation. Two biotechnology companies, Viacyte in San Diego (USA), and Beta O2 in Israel have received approval from the Food and Drug Administration (FDA) to proceed with Phase I/II clinical trials that will test their islets encapsulation technology. The technology consists of preparing and incorporating islet cells inside semi-permeable devices to protect them from being killed off by the immune system. Islets encapsulation to treat T1D is a several decades old concept that has already been tested multiple times both in human, yielding inconsistent results and, more successfully, in various animal models (e.g., see a recent review by Dr Meirigeng Qi here.

Concerning the Viacyte’s clinical trial, the first patient of the approximately 40, scheduled at multiple sites, was implanted with the clinical candidate VC-01™ in San Diego (USA) towards the end of October 2014, while the start of implanting patients at a second site in Edmonton (Canada) has been announced at the end of July 2015. The main purposes of this Phase I/II trial are to evaluate safety and effectiveness of the VC-01™ product in replacing the lost β-cells. The clinical candidate VC-01™, consists of pancreatic progenitor cells (PEC-01TM), that can be obtained in unlimited amounts with Viacyte proprietary techniques, and of a device (Encaptra®) surrounded by a semi-permeable membrane inside which the progenitor cells are housed. This device has the size of a credit card and is implanted under the patient’s skin. The semi-permeable membrane’s function is to allow glucose, nutrients, and oxygen to go inside the membrane and insulin and some toxic waste products to flow out of it; moreover, the semi-permeable membrane separates and protects the enclosed progenitor cells from a direct deadly contact with cells of the immune system. As time goes, the progenitor cells complete their maturation into insulin-producing islets, while blood capillaries form around the device and promote the delivery of key nutrients and oxygen for islets survival. Other glucose’s regulatory hormones, such as glucagon, somatostatin, and amylin are produced along with insulin.  Years of experimentation in various animal models have validated the efficacy of the Viacyte system raising hopes that an effective therapy for T1D is nearing. However, it is still a hope as there is a long list of drug candidates that have worked well in animal models, but have not done comparably well in humans. Some of the potential issues that the trial will hopefully clarify are: (1) how efficiently blood capillaries will be able to form around the device to provide nutrients and oxygen for the islets to survive and function; (2) the semi-permeable membrane allows insulin crossing, but how efficiently will prevent the entrance of proteins such as antibodies and pro-inflammatory cytokines that, once inside the device, could promote islets deaths; (3) how well are the encapsulated islets able to mimic their natural counterparts in responding to raising glucose levels by releasing the correct physiological amounts of insulin; (4) how often the implant needs to be replaced as deposition of both fibrotic material around the device and waste inside it will occur overtime; (5) is the immune system going to attack the device since it is a foreign object?

Beta-O2 approach is to encapsulate islets obtained from human pancreas of deceased donors. Their device is approximately the size of an ice-hockey puck (about 2.5-inch diameter) and is composed of  two Teflon membranes.  By impregnating the membranes with high mannuronic acid alginate, Beta-O2’s scientists have rendered them impermeable to islets’ potentially harmful macromolecules such as antibodies and pro-inflammatory cytokines produced by the host, while maintaining the free passage of glucose and insulin. The device is equipped with an external system capable to periodically send oxygen inside the membrane to promote survival of the oxygen-hungry islets. An application of the technology was carried out in Germany in a 63-year old patient that was followed for 10 months post implant. Over the course of the implant, it was observed that the islets preserved their morphology and were functional, as attested by the increase of C-peptide (though it did not reach physiological levels), the lowering in hemoglobin A1c, and a reduction in insulin requirement (total insulin requirement pre-transplantation 52+/- 5.8 IU/d versus post-transplantation 43 +/- 4.9 IU/d). All this was accomplished without any immunosuppressive therapy. Analysis of the site of implant showed that the device was well vascularized and there was no sign of inflammation. Upon disassembly of the device, the islets were found well preserved and able to respond to glucose.  As the authors point out, only moderate improvements were observed, though they indicated that they used only 2,100 islet equivalents (IEQ)/kg body weight versus a minimal mass of 10,000 IEQ/kg body weight required to achieve insulin independence (based on their analysis of larger cohorts of patients that had islet transplanted in the liver).  Major issues are the shortage of viable islets donors (hopefully will be taken care by stem cell technology), the daily manual injections of oxygen and the replacement of the reservoir of oxygen every three months. Other concerns are the duration of the implant, the efficiency of insulin release and the protection of the membranes against entrance of islet’s toxic substances. 

Beta-O2 announced that a study with 8 participants has started in Sweden with the first patient being implanted with the βAir Bio-artificial Pancreas from Beta-O2 in October 2014, and 3 others have followed since. 

Part 7

Attempts to re-educate the immune system

A recent paper by Bluestone and Coworkers (Bluestone 2015)  regards efforts to re-educate a faulty immune system in T1D patients by replacing the faulty or lost Treg cells with new functional ones (7). In their Phase I clinical trial these investigators enrolled 14 patients who were 18 to 45 years of age and diagnosed with T1D within 3 to 24 months of screening for clinical trial participation.  Upon collection of about 400 ml fresh blood per patient, they employed a technology referred to as fluorescence-activated cell sorting (FACS) to isolate highly purified (average purity 98.4%) Treg  cells. FACS is able to sort out Treg  cells from all the other cells in the blood based on specific protein markers that Treg  cells carry on their surfaces. They obtained between 4.2 million and 11.8 million purified Tregs, which they were able to expand ex vivo (out of the patient body) many folds: specifically, from 29.8-fold to 1366.8-fold. Patients received the expanded Tregs ranging from ~5 million to ~2.6 billion cells in a single infusion via a peripheral intravenous line over 10 to 30 min (each patient received his/her own Tregs).  The main objective of this Phase I study was to prove safety, and this objective was achieved by the results they obtained. Moreover, in several individuals, C-peptide levels persisted when checked 2+ years after cell transfer, but the Tregs infusion did not result in lower requirement for exogenous insulin. They concluded that “the results support the development of a phase 2 trial to test efficacy of the Treg therapy”.

 More impressive, in our opinion, are studies performed in children by Marek-Trzonkowska and coworkers (8,9) by employing a procedure similar to that used by Bluestone and coworkers. In their more recent paper, Marek-Trzonkowska and coworkers reported results of one year follow-up of 12 T1D children that received either one or two infusions autologous Tregs that were expanded ex vivo (up to the total dose of 30 million autologous Tregs /kg). Major findings of these studies were: (1) Tregs number in peripheral blood increased as a result of Tregs infusion, (2) C-peptide levels increased in 8/12 patients after the first infusion and in 4/6 after the second infusion; (3) Tregs therapy lowered exogenous insulin amounts in 8/12 treated patients as compared to 2/10 untreated controls in remission; remarkably, two children were completely insulin independent at one year; and (4) Treatments with Tregs is safe, can prolong survival of β-cells in DM1, and does not impair post-immunization antibody responses.

Another trial intended to educate the immune system to behave properly and not attack β-cells

Results of the Pre-POINT Phase I/II clinical study were published (Bonifacio et al 2015). The aim of the overall study is to elicit an immune response that would protect against T1D. This preliminary study was conducted between 2009 and 2013 in Germany, Austria, the United States, and the United Kingdom. The participants in this study included 25 children aged 2 to 7 years with a family history of T1D and harboring the human leukocyte antigen class II genotypes making them susceptible to the disease. 15 children split into 5 groups (3/group) were treated orally (not injection) with different daily doses (up to 67.5mg/day) of insulin, while 10 children received a placebo. The purpose of the study was to find out the immune responses and side effects associated with orally administered insulin in these children. The results showed that daily oral administration of insulin resulted in a desired immune response without hypoglycemia (which you would expect if injected); yet, it was observed that the insulin-treated group had nearly twice as many side effects than the placebo group.  However, the positive immune response that occurred in the insulin-treated group gives hope that ingested insulin could re-educate the immune system to recognize the β-cells as self. A phase III study involving many more children is required to assess if the orally administered insulin can prevent islet autoimmunity and T1D in such patients.

References

J. A. Bluestone, J. H. Buckner, M. Fitch, S. E. Gitelman, S. Gupta, M. K. Hellerstein, K. C. Herold, A. Lares, M. R. Lee, K. Li, W. Liu, S. A. Long, L. M. Masiello, V. Nguyen, A. L. Putnam, M. Rieck, P. H. Sayre, Q. Tang. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Science Translational Medicine, 2015; 7 (315) ra189

Marek-Trzonkowska N, Myśliwiec M, Dobyszuk A, et al. Administration of CD4+CD25highCD127− Regulatory T Cells Preserves β-Cell Function in Type 1 Diabetes in Children. Diabetes Care. 2012;35(9):1817-1820.

Marek-Trzonkowska N, Myśliwiec M, Dobyszuk A, Grabowska M, Derkowska I, Juścińska J, Owczuk R, Szadkowska A, Witkowski P, Młynarski W, et al. Clin Immunol. 2014 Jul; 153(1):23-30.

Bonifacio, E. et al. (2015) “Effects of High-Dose Oral Insulin on Immune Responses in Children at High Risk for Type 1 Diabetes The Pre-POINT Randomized Clinical Trial”. JAMA; 313(15):1541-1549.

 

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