Reversing Type 1 Diabetes: The Magnifying Potential of Stem Cell Encapsulation

Type 1 diabetes (T1D) is one of the most common chronic autoimmune diseases characterized by islet autoimmunity. This is followed by immune destruction of the β cells as T cells attack and destroy insulin-secreting pancreatic β cells, leading to insulin deficiency. Currently, life-long insulin therapy is the primary treatment option for the condition with research being centered around islet transplantation to restore glycemic stability. However, this procedure is limited by risks and supply shortages, highlighting the need for a safer, more effective therapy for the approximately 9 million people across the world with type 1 diabetes. This literature review assesses stem cells and their potential as a vast β cell supply towards the treatment of type 1 diabetes. Based on current findings, stem cells may differentiate to become a self-renewing β cell line that may reverse type 1 diabetes; however, further studies expanding on ncapsulation techniques, methods whereby living cells are entrapped in semi-permeable membranes for the purpose of disease treatment, are required. With this new horizon of possibilities, targeted efforts towards stem cell manipulation in expressing β cell phenotype can pave the way for a high efficiency treatment for type 1 diabetes.


Introduction
Type 1 diabetes typically has a sudden onset during childhood or early adolescence, often diagnosed when the child visits the hospital for one of the condition's symptoms. These symptoms include polyuria, increased thirst, blurred vision, and weight loss¹. If not diagnosed within the rst few weeks, diabetic ketoacidosis (DKA), a condition in which excess blood acids are produced, may develop². This condition is often fatal if left untreated and is marked by abdominal pain, confusion, and nausea. Alternatively, hypoglycemia, a condition in which the patient's blood glucose levels are low, is caused by excess insulin, low levels of eating, or excessive gaps of time between meals³. Hypoglycemia and diabetic ketoacidosis are often caused by missing insulin shots, especially if T1D is still undiagnosed. T1D, diabetic ketoacidosis, and hypoglycemia may all be diagnosed through a urinalysis or blood test that reveals abnormally high levels of glucose and blood acids.
The onset of T1D is typically sudden, making early diagnosis and treatment integral. Stage 1 displays no symptoms, even when a patient's hemoglobin A1C, a blood test for prediabetes and type 2 diabetes, is tested⁴. However, antibodies have already begun to destroy insulin-producing cells. Left unnoticed, stage 2 characterizes itself with increased β cell loss and hence abnormal blood glucose levels as a result. Though symptoms may still not appear, the antibodies' attacks have, at this stage, led to pancreatic damage⁴.
It is at this point, stage 3, where symptoms begin due to the great loss of β cells. Though the direct causes of these stages are still unknown, certain patients appear pre-disposed to the later stages.⁴ While direct causes of T1D are still unknown, some risks have been identi ed. Environmental risks, such as cesarean section births or the time in life when cows' milk is introduced into a child's diet, have been associated with the development of type 1 diabetes⁵. A popular theory linking the environment to T1D links early-life viral infections to the condition's development⁵. Genetics have also been proven to partially cause type 1 diabetes, as the risk of developing the condition ranges between 1%-70% depending on one's genetic proximity to a family member with type 1 diabetes⁵. Additionally, certain drugs and medications can damage β cells, reducing the production of insulin and causing a condition very similar to type 1 diabetes⁵. In some particular cases, such as a rodenticide introduced in America in 1976 by the name of Pyrinuron, the medication can induce the destruction of pancreatic β cells, hence leading to type 1 diabetes⁶.
Pyrinuron was withdrawn from the American markets after just 3 years; however, other drugs may still lead to pancreatic in ammation. These risks for and causes of T1D are in need of further research because, as of now, there is no cure for type 1 diabetes. Previous studies have shown that stem-cell therapy may be developed into an e cient form of cell therapy for Type 1 Diabetes⁷. The goal of this article is to comprehensively review and outline the most recent data surrounding the bene ts, risks, and details of stem-cell therapy as it relates to Type 1 Diabetes treatments. Even though treatments have advanced and the quality of life of T1D patients has improved, T1D prevails as a widespread issue.
Currently, the most prevalent treatments include insulin injections, continuous glucose monitoring devices, utilization of glucose tablets as needed, and a general healthy lifestyle¹. As widespread as these options are, they hold great drawbacks. Insulin injections are a daily maintenance where patients inject insulin subcutaneously in the area between the skin and muscle, typically a location with enough adipose. If injected deeper by mistake, low blood glucose levels combined with increased pain typically occurs. Furthermore, injection sites must be rotated to avoid lipodystrophy, a condition where adipose causes indentations that interfere with insulin absorption⁸. Continuous glucose monitors, in contrast, involve inserting a minuscule sensor under the skin that measures interstitial glucose levels every few minutes⁹. The data is sent to a monitor, which is sometimes a part of the insulin pump, for the patient to examine when needed. While this option minimizes technically di cult self-administered shots, it is limited by a high cost that makes the device inaccessible to a large portion of the Type 1 population. Healthy lifestyles and glucose tablets may be used in combination with traditional blood pricks and glucose monitoring.
However, these are not cures, nor do they reverse the e ects of T1D. Due to the prevalence of this disease, more e cient treatments and therapies are demanded. Stem-cell transplantation is an encouraging procedure in both cost and safety. Stem cells are marked for their capacity to di erentiate into di erent types of cells, creating a large supply of any of the 200+ types of human cells¹⁰, including bone marrow cells, red blood cells, nerve cells, and most importantly for our purposes, islet cells. Hematopoietic stem cells, the primary adult stem cells used in medical settings, are found in bone marrow and are utilized to form blood cells¹¹. This allows for stem cells to be used in integral procedures like bone marrow transplants and holds promise for stem cells to be developed into other cells for other procedures. These unspecialized adult stem cells (AsCs) can develop into islet cells for islet cell procedures¹². Our goal has been to examine this potential procedure that may be the key to a cure of type 1 diabetes.
Pancreatic cell transplantation is able to stabilize blood glucose levels on its own, however, there is a chronic, nationwide shortage of donors and immunosuppression therapy¹³. Islet cell transplantation is an alternative that is less invasive than pancreas transplantation and also proves to be e ective in reversing complications from T1D but has similar limitations to pancreas transplantation, still su ering a lack of donors and hindering immunosuppression therapy. AsCs and embryonic stem cells (ESCs) can be di erentiated into pancreatic islet-like cells to produce insulin in response to change in blood glucose levels¹⁴. This alternative is cost e ective and involves no donor shortages due to the self-producing cell line. However, transplantation is a potential issue. Sourcing of human stem cells requires exploring as new research heavily focuses on umbilical cord stem cells rather than AsCs. Additionally, immunosuppression when stem cells are implanted is still unclear, leading to the potential bene ts of encapsulation devices. Current strategies for this involve implanted cells shielded from the immune system by a physical barrier. Encapsulated stem cell-derived islets may shield β cells from the immune system, ensuring an almost limited supply for islet cells in procedures to cure type 1 diabetes¹⁵. However, encapsulation device strategies need to be improved in order to minimize foreign body response, possibly by targeting di erent sites. Moreover, transplantation of stem cells through encapsulation in minimally invasive areas is still being investigated. Nevertheless, this research emphasizes the possible bene ts within stem-cell therapy based T1D treatment and highlights its need for future research.

Current Cell Therapies
Current cell therapies in use hold signi cant value as they can guide scientists to potentially more e cient and cost-e ective alternatives. Stem cell therapies today include regenerative medicine that causes repair response. A major di culty within these is that organ donors have to t restrictive criteria. The di erent cell types, such as totipotent, pluripotent, and embryonic stem cells, allow for a variety of treatments suited for appropriate diseases¹⁶. It is of utmost importance to carefully select the type of stem cells that are suitable for clinical application.
Speci cally pertaining to type 1 diabetes, islet cell transplantations, where islets are taken from the pancreas of an organ donor, contain beta cells that produce insulin and have been used as a treatment for years now. Naturally, with this treatment, di culties such as limited supply of human islets and poor immunosuppression arise¹⁷. Other potential treatments to reverse T1D involve Mesenchymal stem cells (MSC), whose self-renewal potential and ability to di erentiation into functional cell types can cure diabetes¹⁸.
Potent strategies combatting these transplantation di culties have been explored: alternative transplantation sites, novel immune protective agents, and encapsulation techniques. A clinical trial currently run by the University of Alberta beginning 2021 evaluates pitfalls of islets but also solutions to islet cell transplantation¹⁹. This highly bene cial search for higher e ciency and safety as it pertains to current cell therapies is a major factor as to why islet cells are a great therapeutic treatment method but also why stem cells may be a better option.
Furthering the MSC treatment plan, one stem cell-based clinical trial for Diabetes Mellitus is the intraportal allogeneic cadaveric islet transplantation.
Due to the lack of coverage for transplant costs along with the limitations of cadaveric islets, alternatives have been sought, speci cally regarding Mesenchymal stem cells. As a source for newly generated beta cells, such cells have been proven to be e ective on type 2 diabetes. However, this is not the case for type 1 diabetic patients, as Mesenchymal stem cells cannot di erentiate into beta cells as e ectively in vitro. In vivo, this di erentiation does not occur at all. Instead, human embryonic stem cells are a plausible treatment method that are being studied as surrogates in replace of cadaveric islets. Immune rejection however is a speci c issue to this current cell treatment that can be addressed. One potential research route, which will be explained in more depth later on in this article, is the ability to couple hESC derived organoids that produce insulin with microencapsulation technologies. This optimizes the need for vascularization and can create a more bene cial route in reversing type 1 diabetes²⁰.
This research continues to advance in order to ensure safe therapy, as well as The gure below details stem cell di erentiation, the process by which stem cells form more specialized functions through signaling mechanisms like DNA methylation. The signaling mechanisms are transmitted through nerve cells which generate electrical and chemical signals of action potentials and neurotransmitters to send information. Blood cells are one type of specialized cells that can be derived from stem cells, whose specialized function include its self-renewal potential. In a study published in 2015, the generation of sex cells through stem cell di erentiation is explained, providing new procedures for the e cient generation of such cells from embryonic stem cells, a speci c stem cell discussed later. In the study, mouse embryonic stem cells are signaled to di erentiate into Epiblast-like cells and nally to PGC like cells, or primordial germ cells, a precursor to all germline cells. The versatility of stem cells in di erentiating into many key specialized cells make them optimal for transplantation as it pertains to diabetes, as the self-renewal potential and personalization of the treatment can prove to be more e ective and bene cial²¹. Limited donors comprise a portion of the di culties with stem cell treatments, but economic cost also plays a large role. For example, even costs to create beta cells from stem cells are similar to the cadaveric islet method.
Both mechanisms, though useful and still developing, require more money, and still immunosuppression issues and autoimmune rejection remain a major factor in their ine ciencies. So far, only a handful of trials have used human embryonic stem cells in order to regenerate beta cells. While current cell therapies include wearable insulin delivery devices made possible by modern therapy's increasing normoglycemic ranges-a signi cant improvement over regular insulin pumps-these are not stem cell therapies and are no closer to reversing the e ects of T1D. Rather, the ability to use stem cells to enhance treatment by coupling it with other alternatives like macro or micro encapsulation can prove to be much more bene cial in the long run.

Combined Stem Cell Alternatives
Current stem cell therapies have been combined with other studied therapies and/or biomaterials to explore the ability to heal the e ects associated with T1D or provide reversal treatment altogether. One combined stem cell therapy that has been explored to heal diabetic wounds is through treatment with human umbilical cord-derived mesenchymal stem cell-derived exosomes (hUCMSC-exos) and Pluronic F-127 (PF-127), which is a medicating hydrogel. PF-127's unique thermal properties and porous structure allow for the release of therapeutic proteins, hypothesizing that PF-127 can continuously release hUCMSC-exos directly onto T1D-a ected tissues, thus attracting broblasts and endothelial cells to initiate wound repair22. This treatment was explored through topical application as this delivery was easy, convenient, and non-invasive, with high-e ciency and low toxicity.
The treatment was tested by applying the exosome-hydrogel combination on diabetic rat models, and the researchers observed angiogenesis, cell proliferation, and granulation tissue formation to understand the capabilities of this wound repair mechanism. The diabetic rats were tested in three di erent groups; one group was treated with hUCSMSC-exos only, one with PF-127 hydrogel only, and the last with the combination treatment of hUCMSC-exos/PF-127. It was found that after 7 days the wound area was signi cantly smaller in the combination groups versus the others, and by day 14 the wounds were completely healed for this group²².

I. Macroencapsulation
Macroencapsulation devices can assimilate islets into semi-permeable membranes that elude typical immune responses while simultaneously allowing for transplanted cells to transport insulin. Type 1 diabetes occurs due to an autoimmune response which attacks insulin producing beta cells, When considering macroencapsulation as a potential therapeutic for reversing T1D, it is also crucial to consider implantation sites and shape optimization to maximize the volume of the device within a space. A recent study tested the posterior rectus sheath plane (PRSP) as a potential implant site to host the macroencapsulation device. This plane is in between the muscle belly and the fascia of the rectus abdominis muscle and this site is being explored as implantation and retrieval can be performed without invading the peritoneal space. PRSP has a large blood supply, allowing for greater di usion of nutrients, and the encapsulated cells therefore receive adequate amounts of oxygen. In order to maximize the space in the PRSP, the best shape for a macroencapsulation device here is presented to be a polygonal-shaped device. These polygonal shapes are ideal as they deliver signi cantly more cells as compared to device shapes such as circles or rectangles. Although a polygon-shaped device presents the most ideal solution, the sharp angles pose challenges with manufacturing and patient comfort. The polygonal shaped devices have favorable interactions with the surrounding environment, and the implantation within diabetic pigs has shown to be a minimally invasive procedure, but the long-term performance of these devices remains untested²⁶.
Aside from optimizing the site-speci c sites, a prominent problem with macroencapsulation is being able to supply the encapsulated cells with enough oxygen. In a clinical trial published in 2018 studying the encapsulation sites for optimal delivery of insulin, the βAir device was developed to overcome this obstacle²⁷. The device contained allogeneic human pancreatic islets and was implanted into 4 diabetic patients. Two key sites that ensure easy access to minimal surgical intervention are the pre-peritoneal cavity and under the skin. The signi cance of these sites and the overall use of the βAir device is to ensure retrievability and immunoprotection. The results of the trial provided evidence that such a device that utilized macroencapsulation was indeed safe and capable of preventing rejection of implanted cells. However, metabolic control was impacted with the transplanted cells' limited function. Potential claims for the ine ciency of the transplanted cells include hypoxia and hyperoxia which could contribute to a lesser device volume, which is undesirable when needing to deliver insulin and nutrients at a productive rate.
Macroencapsulation devices must support viability of the transplanted cell at all stages through the maturation process. These devices are bene cial as they allow for immunoprotection of transplantation islet-like stem cells and can also be retrieved with ease in any circumstance. One limitation present with macroencapsulation devices is accessing a space for implantation. The mechanics of the device are acted upon by di erent external forces depending on where implantation occurs, which can in turn limit the functionality and lifetime of the device. Although recent studies have begun exploring new sites and have been able to successfully implant islet-like stem cells in microencapsulated devices in a minimally invasive fashion-the PRSP is a great example of this type of site. Another major limitation is that islet cell survival heavily depends on the supply of oxygen; this is a ected by the devices membrane permeability of oxygen, the rate of oxygen consumption of the encapsulated islet cells, and other factors as well²⁸. One strategy that has been considered to overcome any oxygen de ciency that SC-islet cells may encounter is oxygen delivery to encapsulated cells through oxygen generating materials. In situ oxygen supplementation with the use of an oxysite disk being placed within the center of the macroencapsulation device has been shown to provide adequate oxygen supplementation and improve the survival of cells²⁹.
Unlike macroencapsulated devices, microencapsulated devices avert vessel ingrowth, limiting the supply of nutrients to solely di use through the selectively permeable membrane. This may also result in a hypoxic environment and therefore requires the need for oxygen delivering technology within encapsulation devices.    injected with gastrin and continue the process, as well as take anti-rejection medication, which is crucial because foreign body response as talked about before can hinder the e ects of the study.