Diabetes Type 1: Stem Cell Research
Stem cell therapy involves the direct transplant of islet cells to potential areas in the pancreas that have the ability to store and facilitate the differentiation of beta cells in the body. Such treatment is currently under progressive study in terms of its effectiveness and the possibility of fortifying the islet transplant’s resistance to autoimmune attacks antibodies. We discuss the actual procedures and different alternatives of stem cell therapy for DMT1 patients. The discussion covers the potential problems being confronted by such treatment, such as stem cell scarcity, autoimmune attacks against the islet transplants, etc.
Lastly, discussion also covers the potential alternatives of the treatment, specifically (1) human embryonic stem cells, (2) cultured stem cells and (3) potential xenogeneic resources. In the conclusion, we have found several problems currently being faced by stem cell therapy. These problems include the scarcity of available islet grafts or transplants and the autoimmune risks that can dramatically hinder to the success of the therapy.
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However, various studies are currently being explored in order to obtain potential alternatives, such as xenogeneic stem cell resources, embryonic or progenitor alternatives, etc.
Furthermore, we discover different methodologies in stem cell culturing and preparation techniques that confront the immunity problems most especially in post-transplant phase. These include the usage of different immuno-suppressing drugs, such as gastrin, etc. 2. Introduction 1. 1 . Type 1 Diabetes DMT1 is essentially the absence or severe insufficiency of insulin due to the autoimmune (e. g. CD4 interleukin attacks, cellular necrosis, macrophagial reactions, etc. ), environmental or viral destruction of beta cells (e. g. iral infections from mumps, etc. ) or insulin-secreting cells in the pancreas. Although, autoimmune reasons are the most commonly associated etiology that cause DMT1 condition. Apparently, the body antibodies, specifically interleukins and minor interferons, recognize the antigenicity present on pancreatic islets as foreign substances, which consequently triggers autoimmune responses. The prevalence of DMT1 in United States is approximately 1 in 2500 for the age group of 5 years old, which 1 in 300 for every 20 years of age group.
Although the most considerable nature of DMT1 is its autoimmune nature, prevalence of DMT1 in United States and European nations largely depends on two causations: (1) genetics and (2) lifestyle. According to the EURODIAB collaborative study, a registry involving 44 countries in Europe, states an annually increasing rate of DMT1 with approximately 3 to 4%, with a larger increase in some central and eastern European countries . The prevalence of DMT1 among 191 World Health Organization (WHO) member states and for all age groups worldwide is estimated to be 2. % in 2000 and 4. 4% in 2030 . DMT1’s beta cell destruction does not only consider the negative effects towards insulin production. Deficiency in insulin can directly lead to moderate to severe hyperglycemia that can further trigger problems, especially in (1) neural systems, (2) peripheral and central vascular regions, (3) cardiac and (4) kidney areas. Vascular complications among DMT1 are associated to different cellular dysfunctions, such as Endothelial Progenitor Cells (EPC) that induce metabolic stress and vascular angiogenicity especially when the cells are decreased.
The primary principle that explains the metabolic and cardiovascular dangers of this illness is the increased of tonicity in the blood or also known as fluid hypertonicity. Due to the increased surge of blood glucose levels, fluids, such as blood, lymph, interstitial fluid, etc. , become thicker than its normal viscosity. With this fluid condition, the circulation exerts tremendous vascular and hyperosmolar pressure from major vessels to minor arterioles and veinuoles. Eventually, the prolonged pressure can lead to various complications, such as eye retinopathy, nephronic damage, nerve ending necrosis, etc.
The common treatment being prescribed among DMT1 patients is the continuous administration of insulin injectables in order to fill in the body’s insulin requirements. This is done to temporarily replace or fill in the insulin insufficiency of the body. However, insulin therapy and maintenance are lifetime measures that require continuous commitment, which can greatly interfere in the person’s self-esteem and lifestyle progression. To resolve these potential emotional and psychosocial damages of the temporary insulin therapy, permanent treatments, such as stem cell implants, autoimmune suppressors, etc. are currently being studied with hopes of permanently curing the disease. Stem cell studies have carefully focused in determining the potential strategies in order to induce beta cell differentiation and cellular regeneration, especially among those damaged or destroyed islet cells. Clearly, with cellular differentiation and regeneration s the goal of stem cell treatment, vast numbers of research discussed in the latter part of the studies have intensively focused their explorations in the disease’s autoimmune nature.
Modern studies of beta cells have always been associated to the macrophagial and lymphocytic activities of T-cell mediated antibodies, such as CD4+CD25+, CD+ T-cells, etc. Most studies are determined in configuring the possible strategies of resolving, preventing and/or countering the DMT1’s autoimmune response on both original islets and implant islet grafts. In animal trials, most commonly rodents, autoimmune elements of the disease are somehow resisted when significant dosage of immune-inhibiting drugs (e. g. nfliximab, daclizumab and sirolimus, etc. ) are applied on the islet implants prior to the commencement of stem cell implantation.
Several studies (e. g. Gastrin applicationetc. ) have found promising strategies that can immunize the transplant grafts and possibly the original islets themselves from the autoimmune destruction rendered by the disease; although, modern science has not yet considered the safe applicability and effectiveness among human trials due to the conflicts encountered by the studies, such variations of drug responses or autoimmune actions. On the other hand, the signs and symptoms of DMT1 and DMT2 are both related to the two principal components of diabetes: (1) hyperglycemia and (2) hypoinsulinemia.
DMT1 commonly presents its condition with the classic manifestations of polydipsia, polyphagia and polyuria . Physiologically, the three principal signs of DMT1 are extremely integrated and fostered by the body’s sympathetic natural response. For example, due to the hyperglycemic state of the body, the satiety centers of the brain triggers polydipsia in order for the body to increase its fluid intake aiming to dilute the tonicity or increased blood glucose levels. In the process, the body increases the fluid contents in the blood increasing as well the kidney workload in processing urinary output; therefore, producing polyuria.
Consequently, fluid loss also causes significant electrolyte losses and glucose malabsorption that trigger body weakness. In order to compensate, the body triggers polyphagia that aims to increase food consumption. The three latter manifestations are considered the cardinal or principal manifestations of DMT1 common to all patients. Weight loss, fatigue, blurred vision, pruritis and muscle wastage are the secondary symptoms that follow with the continuous manifestations of DMT1 cardinal signs .
The secondary complications of DMT1 can further aggravate if the physiological hyperglycemia and other associated signs and symptoms are not resolved. Tertiary complications involve severe manifestations that can be fatal in nature, such as diabetic ketoacidosis and possibly diabetic coma. 1. 2. Causes of DMT1 DMT1 has three potential origins that are currently under extensive study, namely (1) chronic autoimmune destruction of beta cells, (2) environmental destruction of beta cells that is commonly viral in nature, and (3) genetic abnormality in beta cells and/or insulin receptors .
The autoimmune etiology of DMT1, as discussed earlier, involves the activity of interleukin-1 protein cytokine that principally triggers the immunologic response of CD4+ T cells against beta cells. The autoimmune nature has proven the relationship between beta cell destruction and islets’ inflammation due to interleukin invasion; however, studies have not yet determined the principal source of this cytokine production . The issues surrounding the autoimmune proposition in the DMT1 condition is the communicating element/s induced by the disease that activates antibodies’ response against the islet cells.
As of the recent studies, no specific communicating agent has been discovered linking both DMT1 condition and its autoimmune reaction towards islet cells; although, there are numerous evidences that reveal the exact autoimmune attacks against pancreatic islet cells, most significantly on the beta cells. Meanwhile, viral causations have also been associated to the occurrence of DMT1. Common viruses, such as mumps, rubella and coxsackie, have been associated to the destruction of beta cells, which eventually triggers the chronic drop of insulin production .
Cytokine-interferon alpha (IFN-alpha) has been associated with the occurrence of DMT1 with hypothetical viral origin. According to clinical reports, IFN-alpha is brought by environmental viruses (enteroviruses) that trigger immune-mediated beta cell destruction. Significantly, therapeutic agents targeting IFN-a may potentially be beneficial in the prevention of type 1 diabetes and autoimmunity . Lastly, genetic abnormalities min beta cell progenitors and cellular differentiations are also becoming part of the controversial cause of DMT1.
The idea of genetic causation of DMT1 involves the reduced activity of embryonic progenitors in pancreatic endothelial, which consequently lessens the cellular differentiation of beta cells. With small beta cell count in the body, insulin production becomes insufficient causing cellular tension for insulin production. Prolonged state of hypoinsulinemia or complete absence of insulin in the blood usually results to DMT1 complications. Islet transplantation or stem cell therapy considers the destroyed islet areas that need replacement.
According to Rother and Harlan, if patients with greater body mass indices and/or with insulin resistance were also considered for an islet transplant, the 3,000 transplantable islet preparations presently achievable would likely be sufficient to restore euglycemia to fewer than 1,000 patients per year, or less than 0. 1% of patients with T1DM, or approximately 0. 005% of those with either form of diabetes. Despite of the technological advancements of stem cells and islet transplants, most parts of DMT1 condition and autoimmune functionalities are still left undetermined.
The scarcity of islet stem cells is not the only problems being faced by islet transplant therapy but also the impending variations of autoimmune activities of the body. Controlled experiments have been conducted on both rodents and primates; however, the results most of the time vary when applied to human samples. Although, such islet therapy have already been applied to human sample and proven to cause independent insulin production; although, medical issues, such as alternative stem cell or islet graft sources, risk of anaphylactic rejections, etc, are still being studies extensively.
Therefore, scarcity and further study of the procedure are necessary to further the application of islet stem cell therapy among DMT1 patients. 1. 3. Therapy for DMT1 Stem cell transplant of islets of langerhans, specifically the ß -cells, is now considered as alternative treatment in treating Diabetes Mellitus Type 1 (DMT1); although, not all DMT1 patients are applicable candidates of stem cell therapy. Antigenicity testing and severity of DMT1 manifestations as well as autoimmune response to the treatment are usually evaluated before considering stem cell transplant.
Through the process of genetic engineering, the autoimmune response of DMT1 towards the islet cells can now be countered by replacing the cellular necrosis of ß-cells. The study explores the different sections of ß–cells stem cell transplant, particularly on (1) the actual procedure, (2) allogeneic and xenogeneic possibilities, (3) the actual condition of DMT1 and (4) the pathophysiological principles involved in the process of disease progress and stem cell therapy.
The case of DMT1 is autoimmune by nature wherein the body acts negatively to the islet cells by recognizing these cells as a form of foreign objects. Theoretically, the body’s macrophages and interleukins are alarmed by the foreign or abnormal structuring of islet antigens, which probably resulted due to the extensive response of the cells thriving within high insulin-needing environment. In response, the body’s immunologic centers trigger macrophagial and anti-body mediators (e. g. GAD65 Ab – Glutamic Acid Decarboxylase Antibodies, Iinsulinoma Antigen 2, etc. attacking and destroying the body’s own pancreatic tissues . During these conditions, islet cells chronically declines in number as macrophagial actions subdue and destroy both progenitor cells in the pancreas and those differentiated islet cells, which include the beta cells. With the destruction of progenitor cells, the rate of cellular differentiations for further beta cells and other islet cell types (e. g. alpha cells, etc. ) decline leaving the body deficient of these endocrine hormones.
Furthermore, as the existing and pre-existing beta cells die due to autoimmune damages, the capacity of the islet cells to regenerate also decline, which eventually decreases the number of existing beta cells within the islets. Theoretically, According to Xu, Wang and Hou (2008), as the body’s insulin requirement heightens and prolonged, the remaining beta cells experience physiological stress in insulin production, which, if not prevented, can lead to a negative feedback mechanism wherein insulin production complete shuts off its production.
DMT1 patients experience decreased and/or absence of insulin production, and usually peaks between early adolescence (10 to 14 years of age) to middle adulthood (30 and above) . Pancreas manifests lymphocytic infiltration and destruction of islets of langerhans, which consequently causes depletion of insulin production. During the past few decades, studies on islet transplantation through mesenchymal stem cells (MSC) have shown to improve the metabolic conditions of DMT1 patients. However, the performances and study results using MSC remains to be questionable.
Trans-differentiation of MSCs into insulin-producing cells (IPCs) is considered the principal concept of the therapy; however, other reports have negated these results claiming that it is too difficult to assume and determine the timing and extent of improvement by only analyzing the effects through trans-differentiation. Cellular differentiation and self renewal can greatly vary depending on various conditions, such as existing drug therapies, immunologic sensitivity, duration of the illness, other existing disorder including complications dealt by DMT1, etc.
Similar to other beta-stimulating treatments, MSC is considered growth factor stimulant of the surrounding beta cells, which aids in the mechanism of self duplication rather than cellular proliferation. According to Xu, Wang and Hou (2008), “MSCs transplantation into diabetic animals may prevent apoptosis of injured pancreatic beta cells and enhance regeneration of endogenous progenitor cells through paracrine actions” (e. g. angiogenic, cytoprotective, anti-inflammatory, mitogenic and anti-apoptotic effects, etc. ). MSC studies are still on the process of development along with animal trials.
MSC therapy alternative is process for treating principally the occurrence of hyperglycemia in DMT1; however, the process remains an assumption and currently being studied. In the study of Ezquer, Ezquer and Parrau (2008), MSC procedure has been detected to also contribute to tissue regeneration (e. g. bones, cartilage, infracted heart, brain and kidney). In the study, a test subject with streptozotocin (STZ)-induced type 1 diabetes (C57BL/6 mice) has shown significant cellular neogenesis on pancreatic and renal function as well structure.
Somehow, MSC has triggered a potential role of becoming a promising alternative as pancreatic progenitor cells that possess the capacity to initiate cellular differentiations. After the subject received a 0. 5 x 10(6) MSCs via ex vivo expansion, the sample has shown significant reduction in blood glucose levels and euglycemic values after a month. With MSC acting as the islet’s alternative progenitor cells, beta cell differentiation can progress to the development of other beta cells, which if continued can trigger cellular regeneration among produced existing beta cells.
According to final conclusions of Ezquer, Ezquer and Parrau (2008), “MSC administration resulted in beta-pancreatic islets regeneration and prevented renal damage in diabetic animals. ” This evidence shows the possibility of using MSC in initiating both cellular differentiation and self-duplication. Altthough, Xu, Wang and Hou (2008) still consider this process as an experimental alternative therapy for DMT1 condition. However, the study sample did not consider the potential effects of human autoimmune responses against these MSC grafts.
Autoimmune responses can risk the success of graft transplant considering the increased antigenicity present among these islet transplants, which is a considerable issue that arises in the results of their study. Meanwhile in the study of Feng, De-quan and Yan-hua (2008), they have focused on MSCs derived from human umbilical cord blood (UCB) in order to facilitate cellular transdifferentiation into beta cell alternatives via in vitro. In the study, UCB samples are obtained, while presenting MSCs are isolated for analysis via flow cytometer.
In the process, islet-cellular differentiation has been induced for 15 days with or without extracellular matrix gel. This extracellular matrix gel provides an enriched environment that nourishes cellular requirements aiding in their differentiation and consequent self-duplication. With the help of chemiluminescent immunoassay system (CIS) in detecting glucose activity and insulin response, the studied found out that insulin positive cells (25. 2±3. 4%; UCB n=42) within ECM gel have produced functional islet proteins after 9 days of pancreatic differentiation.
Considering the feasible environment setup by ECM, the possibility of creating a zone wherein autoimmune reactions are considered nullified has also become one of the propositions that theoretically explained the results of the study. According to the conclusion of their study, MSC can actually differentiate into islet like cells in vitro and ECM gel. Fortunately, with the advent of modern technology and introduction of somatic stem cell transplant, the depletion of ß–cells can now be replaced with new generating ß–cells through stem cell implantation.
In 1990, Scharp et al. has brought reports of success in the process of transplanting islet cells to patients with DMT1 through the process of improved islet isolation techniques (developed by Ricordi, Lacy and Finke et al. 1988) . Isolation techniques aim in discovering alternative progenitor sources of progenitor cells that possess the capacity to differentiate into insulin-producing cells that can serve as essential alternative for beta cells.
Aside from pancreatic progenitor cells, the study has also discovered potential sources in the kidney, liver, bone marrow and other vital organs of the body. Isolation techniques usually require individualized culturing of islet transplants prior to the actual therapy. With the introduction of ß–cells implantation, different forms of islet transplant (e. g. billiary installation of islet cells, xenogeneic sources of islets, etc. ) have been considered throughout the process of stem cell therapy. On the other hand, certain reaction problems produced during the process (e. . anaphylactic response, incompatible cellular transplant, insulin-sensory impairment, etc. ) have also been observed in throughout the process of therapy. Despite of the potential therapeutic permanence of islet transplant therapy against DMT1 condition, most medical specialists (Kabelitz, Geissler and Soria, 2008; Xu, Wang and Hou, 2008) consider this treatment as last resort therapy for severe cases of DMT1. Stem cell therapy is not yet considered as a general treatment applicable for all sorts of DMT1 conditions.
According to Kabelitz, Geissler and Soria (2008), the concepts in the cellular treatment of DMT1 are (1) the replacement of islet cells by islet-like cells derived from embryonic or adult stem cells, and (2) promotion and establishment of immunological tolerance of islet cells towards self-antigens through regulatory T cells and/or tolerance-promoting monocyte-derived cells. Studies have explored possible ways in dealing with the confronting problems of the procedures, such as scarcity, autoimmune sensitivity, etc.
In the preceding sections of the discussion, the two concepts are further explained considering the possibility of merging the two procedures in order to attain maximum efficiency in the DMT1 cellular therapy. 3. Modern Techniques in Treatments of DMT1 1. 1 Islet Cell Transplant The principal concept of stem cell therapy is the harvesting of potential and/or adult health cells that are transferred to failing or degenerating organs. As for DMT1 conditions, islet transplantation, specifically on ß–cells implantation, is the most impressive treatment that shows promising permanent cure for islets’ autoimmune degradation.
According to Hussain and Theise, “stem-cell therapy here implies the replacement of diseased or lost cells from progeny of pluripotent or multipotent cells. ” According to Haller, Viener and Wasserfall et al (2008), UCB-derived MSCs are significant autologous progenitor inducers that can initiate cellular self duplication or regeneration. In their study using 12 autologous UCB infusions, preliminary results show significant slowing of endogenous loss of beta cell degradation among DMT1 children subjects.
Aside from the slowing of hyperglycemic actions induced by DMT1, Keymeulen (2008) has proposed the possibility of actually blocking or preventing the autoimmune destruction of beta cells in DMT1 conditions. In the study, Keymeulen (2008) proposes the short-term humanized anti-T-cell antibody treatment that aim to inhibit the t-cell activities and preserve the residual beta-cells for at least 18 months in order to induce cellular regeneration and stabilize metabolic control of the body over the rising glucose levels.
By applying Anti-Thymocyte-Globulin, tacrolimus and mycophenolate mofetil to a non-uremic C peptide negative DMT1 patient, marked decrease in autoimmune activities has risen to more than 80%. Another principle of stem cell transplant in islet cell therapy is biologic differentiation wherein a pool of undifferentiated precursors (e. g. Human Islet-derived Precursor Cells or hIPCs, etc. in pancreas appears to be a series of stem cell that further differentiate to islet-endocrine cellular population: (1) Glucagon-producing alpha-cells, (2) insulin-producing beta-cells, (3) somatostatin-producer in Delta cell, (4) pancreatic polypeptide secreting cells . Both of these cellular somas act as the cellular surrogate of ß–cells that shall replace the depleted or damaged cellular source in the pancreas . Cellular differentiation holds the key in inducing growth to the depleted beta cells in the islet of langerhans.
According to the study of Abdi, Fiorina and Adra (2008), islet transplantation (ppluripotent stromal cells) provides great potential for diversifying the cellular lineage even with postnatal damaged tissues. The study of of Abdi, Fiorina and Adra (2008) support the idea of cellular renewal and differentiation giving more emphasis on the mesodermal origin. In such case, the study introduces the concept similar to other studies (e. g. immuno suppression of T-cell activity, increasing beta cell antigenitcity resistance, etc. wherein the introduction of MSCs or islet transplant pluripotent cells may induce an immunomodulatory effect, which eventually facilitates cellular regeneration. The study of Seissler and Schott (2008) also supports the idea of cellular differentiation and self-renewal; however, they have questioned the capacity of supporting the cellular capabilities of stem cells derived from adult pancreas or non-pancreas. During cellular differentiation of endocrine tissue, precursor cells secrete multiple hormones prior to final maturation of differentiated cells that secret single classification of hormone.
Most of these hormones are actual growth hormones that enhance cellular differentiations and regeneration. Although these actions are most of the time slow-phased and are very much vulnerable to immunologic attacks, some studies (e. g. Piper, Brickwood and Turnpenny, 2004; Lai, Schneider and Kidszun, 2005) suggest that once islet cells have regained its stable cellular disposition, which can varies depending on the prevailing physiological atmosphere (e. g. decreased immune activity, prolonged hypoinsulinemia, etc. , the cellular proliferation and restorative scheme can pursue more rapidly than its common phasing. In the process of islet transplant, beta cells are produced as part of the general cellular differentiation produced by broad cellular differentiations . According to Rosenburg, Lipsett and Yoon et al (2004), once islet cell quantity have increased to a stable position and the environment requires extensive insulin production, autoimmune response of the body against these cells are seen to decline dramatically.
Once islets have differentiated from progenitor populations, the cells migrate towards the surrounding exocrine tissues. With the help of angiogenesis resulted by vascularization of islet’s arteriolar blood flow, specific cells present in the islet progenitors, beta cell progenitor, increase its differentiation phase, which consequently increases the number of beta cells present in the pancreas . As beta cells increase, the body’s glucose-perception also enhances considering the increased quantity of glucose sensing beta cells.
The differentiated beta cells react against the decreased body insulin levels by producing insulin, which further stimulate beta cell’s massive proliferation in islets of langerhans . Upon stimulation of cellular differentiation under insulin deficient environment, islet transplant may significantly continue with its differentiation and regeneration schemes without the heightened danger of autoimmune attacks. This theoretical physiology can serve as the actual basis for considering the value of restoring stable beta cell count within the body.
However, the conflict that needs resolution is the safety of islet grafts upon its initial stage of transplant. Differentiation of beta cells is the primary target of islet stem cell therapy among DMT1 patients. These cells are highly specialized cell type, phylogenetically developed, and regulators of glucose homeostasis in higher forms of organisms. However, some studies suggest (Montanya, 2004; Vinik, Rosenberg and Pittinger, 2004; Hermann, Margreiter and Hengster, 2007) the inverse relationship present between cellular proliferation and differentiation of islet implanted stem cells.
The most common problem that arises during post-transplant phase is the decreased differentiation of beta cells, which, in some cases, are not enough to fill in the body’s insulin requirements . However, Dor, Brown and Martinez (2004) assert that Beta cells, during post-stem cell therapy, do not base the production of additional beta cells in the rate of differentiation; rather, beta-cells proliferate through the process of self-duplication .
This is considered as an argument in the idea proposed in the latter section wherein it proposes the nullity in achieving cellular stability in both differentiation and regeneration once specific rate of beta cells are reached in the process. Although the proposed theory does not entirely in-distant with the latter, the argument suggests that beta cell proliferation solely derives from the pre-existing beta cells obtained via transplant, which further proliferates via the process of cellular regeneration and not entirely differentiation.
As for the critique, cellular differentiation is regarded as of little importance due to its low contribution in cellular proliferation. According to Dor, Brown and Martinez (2004), “Our analysis shows that pre-existing beta-cells, rather than pluripotent stem cells, are the major source of new beta-cells during adult life and after pancreatectomy in mice… These results suggest that terminally differentiated beta-cells retain a significant proliferative capacity in vivo and cast doubt on the idea that adult stem cells have a significant role in beta-cell replenishment. Xunrong, Hua and Soo (2005) support the argument through their study indicating the process of autoimmune blockage (Transforming Growth Factor-TGF-[beta]1) rather than the concept of cellular differentiation brought by stem-cell therapy . In the study, they have mention the capacity of growth factors, such as TGF, to provide temporary autoimmune suppression that blocks the hazardous effects of this bodily responses.
With increased angiogenesis or vascularization, the newly introduced cells (beta cells) can rapidly and freely proliferate as long as adequate oxygenation from rapid blood supply is present, and autoimmune suppression is being facilitated by the growth factors. According to Xunrong, Hua and Soo (2005), “Syngeneic islet grafts failed by day 17 in all untreated mice, whereas Ad-hTGF- [beta]1 therapy prolonged survival of islet grafts. Our data demonstrate that systemic TGF-[beta]1 gene therapy blocks islet destructive autoimmunity, facilitates islet regeneration, and cures diabetes in diabetic NOD mice”.
TGF-[beta]1 possesses the functions of temporarily blocking the autoimmune response against the transplanted islet graft as well as triggering cellular regeneration channeled through self-duplication. Considering the arguments propose by the two latter studies, this study still concludes the essential contributions of cellular differentiations brought by pre-existing progenitor cells from stem transplant or original sources; since, these component holds the appropriate physiological distribution of islet cell re-categorization and reproduction. 1. 2 Stem Cell Transplantation
Contrary to the concept of cellular differentiation and proliferation, post-stem cell transplant on islet cell is said to induce aggressive self-renewal due to the presence of significant growth components (e. g. TGF-[beta] 1, hemo-erythropoetin,etc. ) that enhance pre-existing beta cell proliferation and protect the cells from autoimmune attacks. Through the use of a DNA analog-based lineage-tracing technique , the study has found that precursor cells do not actually contribute to further differentiation of adult beta cells, and not even during acute beta cell regeneration.
Rather, beta cells are being produced through self-renewal or duplication wherein a programmed cell division occurs through a refractory period preventing excessive or massive beta cell proliferation. Although, as argued by various studies (Lee, Grossman and Chong, 2008; Gershengorn, Anandwardhan and Wei, 2004), theoretically, differentiation rate usually surges during the initial phase of cellular implantation; however, once the cellular count of these differentiated cells stabilize, self-renewal or cellular regeneration of the existing beta or islet differentiated cells follow.
Thus, explaining the inverse relationship between beta-cell proliferation and differentiation. Current studies in both allogeneic and xenogeneic stem cell sources are now being studied with marked emphasis on autoimmunity reversal or even autoimmunity tolerance. According to Lee, Grossman and Chong (2008), “stem cells from hematopoietic sources, such as bone marrow and fetal cord blood, pancreas, intestine, liver, and spleen, promise either new sources of islets or may function as stimulators of islet regeneration”.
Through stem cell introduction of pancreatic cells, specifically islets of langerhans, the adult human beta cells pre-existing in the stem cell transplant exhibit hormonal expression . Contrary to the concept of cellular proliferation, stem cell transplant essentially increases beta-cell resistance to autoimmune destruction of DMT1, which consequently facilitates the proliferation of beta cell in the islets of langerhans.
According to various studies (Linning and Madkuhar, 2004; Strobel, Yuval and Stirman, et al. 006), aggressive beta cell self-duplication is the actual cause of beta cell proliferation whether by implantation of TGF-[beta] 1- induced islet cells or the traditional islet replacement. Implanted islet progenitors, when cultured, expresses 1% of endocrine cell proliferation during the first 48 hours up to 6% after five days . According to Rosenberg, Lipset and Yoon (2004), increasing the mass of beta cells after the event of post-immune destruction induces a 175-amino acid pancreatic acinar cell protein called, Islet Neogenesis-Associated Protein (INGAP) peptide, which acts as a stimulator of beta cell mass stimulator.
INGAP peptide, similar to TGF-Beta growth factor, triggers cellular neogenesis enabling the rapid rate of cellular regeneration after significant results from cellular differentiation. The production of INGAP protein is commonly cited during post-phase of islet transplant. However, according to Lai, Irina and Eugen et al. (2008), gene modification present in cell transplantation process is problem considering the extensive cellular processes involved in the adaptation and transplant reception.
Although, applications of several viral vectors (e. g. adenovirus-associated vectors, etc. have proven to be successful, but hESC is considered a more potent alternative due to its feasibility for genetic manipulation and self-renewal. During the mass replication of beta cells, the small portion of the cells stops in the process of neogenesis, while other beta cells are reserved for continuous replication at a very slow phase. After this scenario, the counter-attack of autoimmunity is usually expected; hence, treatment regimen that suppresses immunologic reaction on islet grafts are usually being instilled to the transplant sample prior to the therapy.
This procedure increases the resistance of the graft cells against the autoimmune reactions triggered by the body. With a disorder such as DMT1, the chances of beta cell recovery become lesser due to the persistent autoimmune destruction of beta cells . The decreased capacity cellular replication in the adult beta cell is very much limited to result in a significant regeneration rate following autoimmune damages . Likewise, chronically increased metabolic requirements, such as increased insulin demand, can cause beta cells’ incapacity to fully cope in the given physiologic environment.
This gives the appropriate rationale for implanting islet cells in the area of depleting beta cell in order for the progenitors to differentiate and proliferate mass beta cells in the area. According to the study of Urban, Kiss and Kovacs et al. (2008), hematopoetin centers of the body, such as bone marrow, may harbor cells that can actually influence the self-duplication of beta cells. Such concept is greatly associated to the principle of angiogenesis implying the value of appropriate oxygenation in the area of developing cellular clusters.
In the study, sex-mismatched bone marrow cells (BMCs) and syngeneic or allogeneic MSCs are administered to a mice sample with streptozotocin induced DMT1, and consequently led to the rapid tissue regeneration after a single injection of a mixture of 10(6) BMCs per 10(5) MSCs. Other agents that can forcefully differentiate beta cells during post-islet transplant are INGAP (Rosenberg, Lipset and Yoon et al. , 2004; Weir, Toschi and Inanda et al. , 2004), GLP-1 and GLP-1 receptor agonist exendin-4 (Li et al. , 2004), betacellulin and activin A (Brubaker and Drucker, 2004), and the combination of EGF and Gastrin (Rooman and Bouwens, 2004) .
These agents can actually force the cellular differentiation providing immediate and ensured processing new beta cells with much more lessened risks of being attacked by immunologic elements. Betacellulin, Activin A and Gastrin are the common immuno-suppressants being applied to most controlled studies on islet transplants today due to its availability and decreased result variations; although, some studies still explore the applicability and effectiveness of these agents in the process of triggering cellular differentiation.
Meanwhile, Melleoul (2006) suggests that cellular differentiation of beta cell during post-islet transplant is controlled by series of genetic activators and transcription factors . Its absence in mice and humans during embryogenic to postnatal development may actually lead to pancreatic agenesis. After such condition, cellular differentiation becomes restricted principally to ß cells wherein cellular regulation of genetic expression in ß cell-specific genes occurs.
Furthermore, such condition facilitates the mediation of the glucose effect on insulin gene transcription, which shows that any exposure of ß cells to high glucose even with short period of time can actually stimulates insulin gene expression. However, chronic exposure to high glucose levels can actually trigger negative effects, such as alteration in ß-cell functions and gene transcription. PDX-1 transcription breaks down upon exposure to chronic hyperglycemia, while stimulation of beta activity is seen during acute hyperglycemia.
Such genetic modifications can actually enhance the survivability of islet transplants within a new host considering the autoimmune function being rendered by continuous DMT1-induced CD4 immunoglobulins. According to Phillips and Tang (2008), using cellular, molecular and gene manipulation strategies, each islet transplant can actually be guarded or attain enhanced resistance even with the hostile environment directing immune rejection, inflammation, hypoxia and apoptosis.
Genetic engineering provides cellular modification for constructing gene sequences. Considering the conflict existing in mass beta cell replication and autoimmune destruction, high quantities of beta cell replication during post-islet transplant has been associated to the reduced impact of autoimmune damage. With the help of CTL antagonists in terms of restricting T-cell activity, the regenerative capacity and neogenesis of ß-cells are expected to progress through forced-differentiation therapies.
Initial activities between autoaggressive Cytotoxic T-lymphocytes (CTL) and beta cells are terminal event leading to cellular agenesis of ß-cell, which consequently affects both progenitor beta cells and those potential self-replicating beta cells from the pool of potential ß-cell replenishment . Progression of CTL is unlikely to be stopped; hence, the only appropriate idea of treating the pathogenesis of DMT1 is the replenishment of beta cells that have been damaged throughout the ongoing autoimmune attacks.
According to Dor (2006), progenitor cells present in the pancreas, specifically on pancreatic ducts, acini, islets of Langerhans, and other parts of the body (e. g. bone marrow, spleen, etc. ) are even more potent source of beta differentiation . However, these progenitor cells provide variable cellular differentiation rate that can compromise the process of stem cell therapy especially if the non-ideal progenitor cell source are used in the procedure.
To compensate, most studies have explored the possibility of using embryonic-obtained stem cells that contain the most feasible progenitor cells aside from the ideal pancreatic progenitors. Although beta cells are differentiated from progenitor cells during embryonic phase of pancreatic development, the progenitors (marked by expression of neurogenin 3 and the paired box protein Pax-4) are seen to disappear upon birth . Such disappearance actually implicates a significant process that are undergoing with beta cells, which actually trigger fundamental change in their mode of maintenance and expansion.
The cellular process begins from the embryonic progenitor-cell-based differentiation and further progress to massive self-regeneration. In the study of Nagaoka, Fukuda and Hashizume (2008), betacellulin (BTC) is analyzed as another potential growth factor that can induce progenitor-cell-based differentiation and cellular self-duplication. BTC possesses ErbB receptor tyrosine kinases that induces differentiation and cellular mitosis, especially among acinar-derived AR42J cells, transforming these cells into insulin-producing or beta functioning cells. As supported by Parnaud, Bosco and Berney et al. 2008), BTC-induced purified beta cells within allogeneic islet transplant graft enhanced by ECM have yielded a population of 91. 4±2. 8%. Nagaoka, Fukuda and Hashizume (2008) mention that BTC “independently and preferentially binds to two type I tyrosine kinase receptors, the EGF receptor (ErbB1) and ErbB4”. Significantly, BTC induced graft transplants are seen to contain mutant protein that promotes the rapid differentiation of pancreatic acinar AR42J cells to insulin-producing cells, which is actually the opposite with AR42J cells that contain wild-type BTC protein.
Rapid differentiation is not entirely beneficial in nature as this can cause hyperplasia. According to Min Cho, Lim and Yoo et al. (2008), BTC, together with Nicotinamide sustained PDX1 expressions, actually induced cellular differentiation C-peptide proteins; although, insulin mRNA is found to be very low. 4. New Advances in Stem Cell Research The theory between stem cell differentiations versus beta cell progenitor self-duplication still coincide the need to restore pre-existing beta cell pool from the ongoing damage made by the autoimmune CTL.
Stem cell is still an important consideration in replenishing these depleted resources. However, due to the extensive problem on stem cell donors and sources, stem cell therapy is not yet considered part of an ideal DMT1 treatment. According to Korsgren, Lundgren and Felldin (2008), new alternatives for stem cell therapy are currently being explored with aims of determining other contributing components that induce cellular graft survivability and reduction of immunoresponse against DMT1 mediated antibodies.
During the process of transplantation, the isolated islets transplant grafts are induced to embolise the liver after its introduction via the hepatic portal vein, which is a procedure that is unique in the area of stem cell implantation. However, such procedure is only an example of low efficacy procedure. A novel view on the engraftment of intraportally transplanted islets is presented that could explain the low efficacy of the procedure. As supported by Rother and Harlan (2004), and Hardikar (2004), only 750 patients have already been treated using allogeneic islet transplants since 1974 despite of the billions of DMT1 cases worldwide.
Various alternatives have been proposed in order to counter such scarcity, specifically: (1) embryogenic blastocyst and post-natal resources, (2) culturing of stem cells, and (3) stem cell grafting using xenogeneic resource (e. g. umbilical cord, etc). The isolation of human embryonic stem (hES) cells has been introduced as a potential prospect for filling in the scarcity of beta cells, specifically through islet transplantation . Embryonic stem cells are harvested from blastocysts, while adult stem cells are from postnatal organisms.
The process involves (1) the culturing and plating of embryoid bodies in insulin-transferrin-selenium-fibronectin medium, (2) supplementation and maintenance using N2, B27, and basic fibroblast growth factor (bFGF), (3) lowering of glucose concentration to reduce the physiological pressure on premature beta cell, (4) bFGF is withdrawn to prevent excessive growth stimulation, and (5) nicotinamide addition . Counteracting transcription-polymerase chain reaction found out an enhanced cellular expression of pancreatic genetic chains within the site of cellular differentiated cells.
Using the Immunofluorescence and in situ hybridization analysis, the findings have revealed a significantly increased percentile range of insulin-expressing cells within the cellular clusters. According to the study of Xia, Ayala and Thiede et al. (2008), hESCs, with the help of drug-inducing transgene expression (in vitro and in vivo) forms >95% purity level, which significantly implies the high possibility of regulating genetic expression of hESCs. After the islet transplantation, genetic expression of the cells remained stable and regulated with the help of an orally administered drug.
Although, according to Chung and Stainer (2008), cellular origins that regulate pancreatic B cell induction and genetic expression is not yet fully understood. Differentiation of embryonic stem (ES) cells to islet phenotype, identification and utilization of pancreatic precursor/stem cell from adult sources, and the cultivation of new islets from adult stem cells obtained from various tissue types or directly form other terminally differentiated cell types are the common areas being covered by islet transplant or stem cell research for DMT1 immunogenetics research .
In such case, cultured embryogenic or adult somatic islet cells are transferred from its original placement to appropriate locations in the body of a DMT1 patient. Human Embryonic Stem Cell (HESCs) or ES possesses the capacity to continuously differentiate to cells that express both endoderm and pancreatic progenitor function, such as Foxa2, Sox17, Pdx1, and some islet endocrine hormones (e. g. beta cells) . According to Kroon, Martinson and Kadoya et al (2008), cellular therapy for DMT1 requires the renewal of human beta cells and not entirely the replacement of the degraded ones.
In order to induce regeneration, pancreatic endoderm must be stimulated through the use cellular mediated glucose-responsive endocrine cells present within hESCs. The hESC-derived insulin-producing islet-like clusters (ILCs) comprises of 2 to 8% of human C-peptide-positive cells, glucagon-positive and somatostatin-positive cells. The study has detected a content of 70 ng of insulin/mug of DNA being produced through these hESC-derived ILCs, which is statistically higher than the innate fetal islets.
In addition, cellular differentiation of hESCs induces the formation of Embryoid Bodies (EBs) that stimulate the gene expressions of POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin as long as the glucose environment is within 25mM levels . In the essence, implantation of hESCs in autoimmune-damaged islets can mobilize the islet cell differentiation through genetically expressed progenitors from the islet transplant medium. Furthermore, continuous genetic expression is expected since the body’s glucose levels also influence the cellular differentiation of beta cells.
Stem cells derived from hESCs places markers of development for endoderm, pancreatic and ß-cell development, glucose sensing, and production of mature insulin . Meanwhile, most studies have also centered in protein-based cellular communication involved during cellular differentiation phase after stem cell implants have been introduced. According to Kroon, Martinson and Kadoya (2008), therapeutic tests using a mice sample with 3000 transplanted human islet cells indicate that hESC derived pancreatic endoderm can actually aid in antibody resistance.
In the study’s conclusion, they have pointed the definitive evidence proving the capacity of hESCs in generating glucose-responsive and insulin secreting implanted cells. Interestingly, in the study of Yu, Vodyanik and Smuga-Otto et al (200), hESCs are found to be programmed by specific four genes, OCT4, SOX2, NANOG, and LIN28, which actually determines the pluripotent capacity of the embryonic stem cells and the characteristic of cellular differentiation. Although, the study concludes that the genetic mapping and processes involved within these newly discovered hESC genes are still in the process of intensive studies.
Implanted stem cells actually integrate their needed functions for initiating the mechanism of glucose responsive regulation present as pre-proinsulin mRNA and expression of insulin C-peptide in vitro (Clark, Yochem and Axelman, 2007). Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. The results of the study suggest that embryonic germ cell derivatives (e. g. ILCs, etc. ) may eventually function as a potent insulin producing cells .
The use of islet-derived or stem cell therapy using embryonic cells remain experimental due to the challenges of cellular differentiation. Currently, the problems being faced by the treatment is the availability of stem cells that can possess the appropriate capacity to induce cellular differentiation and regeneration. According to the mentioned studies, simple cellular implantation is not entirely enough due to the greater risks imposed by the body’s physiological reaction against islet grafts.
Hence, another issue arises in determining the best anti-immunity function or tolerance enhancer of islet graft transplants; although, latter studies have already discovered potential enhancers that can disregard or at least lessen the impact of cellular degradation brought by DMT1 immunity. Lastly, new advances of genetic modification techniques that shall increase cellular differentiation and renewal rates are already in the process of development. 5. Discussion In the research of Froud, Ricordi and Baidal, islet stem cells are cultured under steroid-free immunosuppression and are transplanted to 16 DMT1 samples.
The cultured islet stem cells have undergone a period of in vitro culture-process with heightened necrosis resistance through TNF- a (Tumor Necrosis Factor) blockade that aim to improve islet engraftment and provide alternative to fresh human islet transportation. The results of the study suggest that the implantation of cultured human islet allografts cause a reproducible insulin independence in all subjects under the series immunosuppressant infusions (a. intial Infliximab infusion, b. daclizumab and c. irolimus maintenance), comparable to that of freshly transplanted islets (Edmonton protocol) .
In the absence of supplemental infusions (nfliximab, daclizumab and sirolimus), the results of the study have incurred 11/14 (79%) subjects that produced insulin independence at 1 year, while other 6/14 (43%) samples have gained this capacity after 18 months. Surprisingly, the same test subjects have maintained their insulin independence until 33 ± 6 month span. Furthermore, the findings have observed that patients are able to maintain their graft function while under the immunosuppressing infusions.
According to the results, 8 out of 14 patients have suffered chronic partial graft losses that are likely immunological in nature considering that 5 of these already received supplemental infusions. Currently, 11 out of 14 subjects are in the receiving immunosuppressing infusions, and 8 (73%) of these are already manifesting insulin independence. The study significantly demonstrates the possibility of withholding the immunologic response upon exposure to certain immunosuppressant (e. g. nfliximab, daclizumab and sirolimus, etc. ).
Although, the study has not mentioned the possible side effects and complications that such infusion can provide towards the body as a whole. However, since the stem cells are the only ones infused with these immunosuppressants, the chances of systemic immunosuppression are less likely as long as the dosage infused with the stem cells remain appropriate and feasible to the body’s normal function. In another culture study brought by Pinzon, Lakey and Brand (2005), they have used the combination of epidermal growth factor (EFG) and gastrin in order to induce beta cell neogenesis specifically on pancreatic exocrine duct cells .
These growth factors also carry the risk of triggering extensive cellular neoplasia over-cellular multiplication; although, studies have already found drug induced techniques that can contain the cellular differentiation and regeneration upon introduction within the body system. In the study, human islet cells are placed under four weeks culture study in a serum-free medium with EGF (0. 3 µg/ml) as the control variable and gastrin content of 1. 0 µg/ml.
Beta cells have shown significant increase in cultures with the combined medium of EGF and gastrin (+118%), while +81% for cultures with EGF alone. The EGF-gastrin culture has been observed again for the next four weeks, but without the said combination. Impressive results have shown beta cells progressive increase in quantity for the culture previously infused with both EGF and gastrin (+232%). Comparing these results from the latter discussed studies, EGF and gastrin have actually trigger cellular differentiation and self-duplication due to their growth factor properties.
In the study of Suarez-Pinzon and Rabinovitch (2008), gastrin growth factor combined with epidermal growth factor (EGF) can actually restore pancreatic islet beta-cell mass and even reverse hyperglycemia even in the absence of immunotherapy in mice samples with artificially induced-DMT1. Reversal of hyperglycemia is most likely due to the increase in insulin production that counters the effects of DMS1. With the appropriate amounts of insulin secretion in the blood, the glucose tonicity will consequently be absorbed by the cells granted that the diabetic anomaly does not consider the insulin receptor functionalities within cellular surfaces.
In the study, EGF dose of 10 microg/kg and gastrin dose of 30 microg/kg via intraperitoneally have been administered to 10 sample DMT1 mice. In terms of glucose levels, the samples have shown a marked decline from blood glucose of 23 +/- 2 mmol/L to 12 mmol/L within 36 days of individual EGF administration, while 19 days in individual gastrin administration. When combined, the decline in the samples’ glucose levels is already present within 11 days.
In addition, the cellular islet counts have increased from 13. 0 +/- 0. x 10(5) cells to 29 +/- 2 x 10(5)cells, and considering the marked decrease of surrounding CD45+ leukocytes have also been observed. Therefore, such combination (EGF plus Gastrin) is confirmed to reduce blood glucose levels, prevent autoimmune activity of DMT1 mediated CD4 cells and increase cellular differentiation. Lastly, aside from hESC’s and cultured islet transplants, another potential source of stem cells currently being studied is from animals, known as xenogeneic sources . Pig islets are considered the best option available for xenogeneic transplants.
According to Rother and Harlan (2004), such potential alternative are now being studied for different considered potentials, such as: Pig islets have been considered as potential source of islet stem cells aside from human source (a) The fact that humans had been treated with pig insulin for more than 60 years (b) Favorable husbandry — in that the species has large litters with offspring that attain adult size rapidly and with relatively robust islet numbers (c) The fact that pig islets respond to glucose in the same physiological glucose range as human islets (d) Existence of suitable societal-cultural relationship between the species
Despite of the potential capacity of pig islets in acting as alternative stem cell resource, studies (Hering, Wijkstrom and Graham et al. , 2006; Rood, Buhler and Bottino, 2006) have identified its increased immuno-response towards CTL and autoimmune attacks initiated by DMT1 disease. Autoimmune attacks are the principal conflict considered in the process of islet transplantation wherein even if the graft has been successfully implanted, the risk of failure in the procedure is still considered possible considering the effects of autoimmunity triggered by increased antigenicity in the graft transplant.
In one study, acute rejection caused the death of two macaque samples through cellular rejection mediated by CD4+ and CD*+ T cells and macrophages . In order to increase the effectiveness of xenografts after post-transplant phase, different culture infusions have been studied to prolong the life of pig islets xenografts. CD4 antibodies are usually being activated upon detecting significant system foreign antigens, which are usually introduced by bacteria, virus or any material that enters the body systems.
In this principle, researchers (Kirchhof, Shibata and Wikkstrom et al. 004) have pointed their assumptions in the possible presence of antigens within xenotransplanted islet grafts. In addition, cellular infusions are considered to be at great risk due to the potential intrusion of incompatible antigens that might induce transplant rejection, and eventually autoimmune degradation of transplanted islet cells in the body. This condition is currently under extensive analysis and consideration since even with successful islet transplant, autoimmune response due to heightened cellular antigenicity can still pose the failure of the stem cell therapy.
Due to this genetic dilemma, some studies (Kirchhof, Shibata and Wikkstrom et al. 2004; Komoda, S. Miyagawa and T. Omori et al. , 2004) have focused in determining the potential drug enhancers that can improve transplant antigenicity, especially among xenogeneic sources. First, with the infusion of islets from N-acetylglucosaminyltransferase-III (GnT-III) transgenic pigs, pig islet’s xenoantigenicity have significantly declined prolonging the survival of islets for the next five days of culture study. In another study, pig islets subjected for xenotransplantation are tested with alginate encapsulation.
The transplant to tested in a primate, specifically a monkey-Cynomolgus maccacus . Adult pig islets encapsulated in alginate under optimal conditions (n=7) or not (n=5) are transplanted under the kidney capsule of the non-diabetic primate sample. Meanwhile, additional samples have received empty capsules (n=1) and non-encapsulated pig islets (n=2) as controls . The results of the study show the rapid inviability of non-encapsulated and encapsulated islets with no alginate and not in optimal condition.
Implanted pig islets under optimum alginate encapsulation showing significant prolonged islet survival for as long as six months. However, despite of the experimental success, the study still regards the conflicts encountered by the processes (e. g. variations of graft antigenicity, etc). 6. Conclusion DMT1 is a condition manifested by increased and frequent manifestations of hyperglycemia caused by the insufficient production or depleted insulin levels. The most universally recognized cause of beta cell destruction is the autoimmune etiology caused by CD4 interleukins, and other associate antibodies.
The aims of the therapy are the induction of cellular differentiation while facilitating as well the renewal of the existing and pre-existing beta cells in the islet graft transplant or in the remaining original islets. However, the principal conflict of the procedure is the interference caused by the autoimmune reaction of the body towards the transplanted islet grafts; although, recent studies have continuously explored different possibilities of suppressing autoimmune responses and forcing cellular activities.
Stem cell therapy is a potential prospect for permanently treating the condition of DMT1 considering the main concept involved in its pathogenesis – destruction of beta cell or insulin producing cells. The processes, physiology and pathological considerations in the stem cell therapy of islet transplant involve the criticality of autoimmune response towards the islet transplant.
The controversy of such treatment is the effectiveness of implanting whether the islet cells containing stem cells based on the concept of cellular differentiation or islet cells with pre-existing beta cells based on the concept of cellular self-renewal. Despite of the argument between the two perspectives involve, another main issue arises, specifically the scarcity of stem cell from allogeneic donors. According to the approximated statistics, only 750 cased of DMT1 have successfully obtained the stem cell transplant of islet cells considering the billions of other DMT1 patients existing.
In order to resolve such scarcity, various forms of stem cell resources have been proposed and are currently under extensive studies, specifically (1) human embryonic stem cells, (2) cultured islet stem cells, and (3) xenogeneic sources specifically the pig islet stem cells. According to most studies, autoimmune damage progress if cell count of beta cells is introduced insufficiently to the recipient body; although, stem cell therapy is nearing towards its potential of being a significant cure as beta cell replacement and insulin producer.