The origins of low-temperature tissue storage research date back to the late 1800s. Over half a century later, osmotic stress was revealed to be a main contributor to cell death during ...cryopreservation. Consequently, the addition of cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), or propylene glycol (PG), although toxic to cells at high concentrations, was identified as a necessary step to protect against rampant cell death during cryopreservation. In addition to osmotic stress, cooling and thawing rates were also shown to have significant influence on cell survival during low temperature storage. In general, successful low-temperature cell preservation consists of the addition of a CPA (commonly 10% DMSO), alone or in combination with additional permeating or non-permeating agents, cooling rates of approximately 1ºC/min, and storage in either liquid or vapor phase nitrogen. In addition to general considerations, cell-specific recommendations for hepatocytes, pancreatic islets, sperm, oocytes, and stem cells should be observed to maximize yields. For example, rapid cooling is associated with better cryopreservation outcomes for oocytes, pancreatic islets, and embryonic stem cells while slow cooling is recommended for cryopreservation of hepatocytes, hematopoietic stem cells, and mesenchymal stem cells. Yields can be further maximized by implementing additional pre-cryo steps such as: pre-incubation with glucose and anti-oxidants, alginate encapsulation, and selecting cells within an optimal age range and functional ability. Finally, viability and functional assays are critical steps in determining the quality of the cells post-thaw and improving the efficiency of the current cryopreservation methods.
To dissect therapeutic mechanisms of transplanted stem cells and develop exosome-based nanotherapeutics in treating autoimmune and neurodegenerative diseases, we assessed the effect of exosomes ...secreted from human mesenchymal stem cells (MSCs) in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model. We found that intravenous administration of exosomes produced by MSCs stimulated by IFNγ (IFNγ-Exo) (i) reduced the mean clinical score of EAE mice compared to PBS control, (ii) reduced demyelination, (iii) decreased neuroinflammation, and (iv) upregulated the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice. Co-culture of IFNγ-Exo with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive cytokine indoleamine 2,3-dioxygenase. IFNγ-Exo could also induce Tregs in vitro in a murine splenocyte culture, likely mediated by a third-party accessory cell type. Further, IFNγ-Exo characterization by deep RNA sequencing suggested that IFNγ-Exo contains anti-inflammatory RNAs, where their inactivation partially hindered the exosomes potential to induce Tregs. Furthermore, we found that IFNγ-Exo harbors multiple anti-inflammatory and neuroprotective proteins. These results not only shed light on stem cell therapeutic mechanisms but also provide evidence that MSC-derived exosomes can potentially serve as cell-free therapies in creating a tolerogenic immune response to treat autoimmune and central nervous system disorders.
Five-Year Follow-Up After Clinical Islet Transplantation
Edmond A. Ryan 1 ,
Breay W. Paty 1 ,
Peter A. Senior 1 ,
David Bigam 2 ,
Eman Alfadhli 1 ,
Norman M. Kneteman 2 ,
Jonathan R.T. Lakey 2 and
...A.M. James Shapiro 2
1 Department of Medicine, Clinical Islet Transplant Program, University of Alberta and Capital Health, Edmonton, Alberta, Canada
2 Department of Surgery, Clinical Islet Transplant Program, University of Alberta and Capital Health, Edmonton, Alberta, Canada
Address correspondence and reprint requests to Edmond A. Ryan, Clinical Islet Transplant Program, 2000 College Plaza, 8215
112th St., Edmonton, Alberta, Canada T6G 2C8. E-mail: edmond.ryan{at}ualberta.ca
Abstract
Islet transplantation can restore endogenous β-cell function to subjects with type 1 diabetes. Sixty-five patients received
an islet transplant in Edmonton as of 1 November 2004. Their mean age was 42.9 ± 1.2 years, their mean duration of diabetes
was 27.1 ± 1.3 years, and 57% were women. The main indication was problematic hypoglycemia. Forty-four patients completed
the islet transplant as defined by insulin independence, and three further patients received >16,000 islet equivalents (IE)/kg
but remained on insulin and are deemed complete. Those who became insulin independent received a total of 799,912 ± 30,220
IE (11,910 ± 469 IE/kg). Five subjects became insulin independent after one transplant. Fifty-two patients had two transplants,
and 11 subjects had three transplants. In the completed patients, 5-year follow-up reveals that the majority (∼80%) have C-peptide
present post–islet transplant, but only a minority (∼10%) maintain insulin independence. The median duration of insulin independence
was 15 months (interquartile range 6.2–25.5). The HbA 1c (A1C) level was well controlled in those off insulin (6.4% 6.1–6.7) and in those back on insulin but C-peptide positive
(6.7% 5.9–7.5) and higher in those who lost all graft function (9.0% 6.7–9.3) ( P < 0.05). Those who resumed insulin therapy did not appear more insulin resistant compared with those off insulin and required
half their pretransplant daily dose of insulin but had a lower increment of C-peptide to a standard meal challenge (0.44 ±
0.06 vs. 0.76 ± 0.06 nmol/l, P < 0.001). The Hypoglycemic score and lability index both improved significantly posttransplant. In the 128 procedures performed,
bleeding occurred in 15 and branch portal vein thrombosis in 5 subjects. Complications of immunosuppressive therapy included
mouth ulcers, diarrhea, anemia, and ovarian cysts. Of the 47 completed patients, 4 required retinal laser photocoagulation
or vitrectomy and 5 patients with microalbuminuria developed macroproteinuria. The need for multiple antihypertensive medications
increased from 6% pretransplant to 42% posttransplant, while the use of statin therapy increased from 23 to 83% posttransplant.
There was no change in the neurothesiometer scores pre- versus posttransplant. In conclusion, islet transplantation can relieve
glucose instability and problems with hypoglycemia. C-peptide secretion was maintained in the majority of subjects for up
to 5 years, although most reverted to using some insulin. The results, though promising, still point to the need for further
progress in the availability of transplantable islets, improving islet engraftment, preserving islet function, and reducing
toxic immunosuppression.
CBC, complete blood count
CMV, cytomegalovirus
HOMA, homeostasis model assessment
HYPO score, Hypoglycemic score
LI, lability index
LFT, liver function test
PRA, panel reactive antibody
Footnotes
Accepted April 13, 2005.
Received February 18, 2005.
DIABETES
Previous studies have shown that necrostatin-1 (Nec-1) supplementation improved the viability of murine islets following exposure to nitric oxide, increased the survival of human islets during ...hypoxic culture, and augmented the maturation of pre-weaned porcine islets (PPIs) after 7 days of tissue culture. A limitation of these studies is that only one concentration of Nec-1 was used, and no studies have determined the optimal dose of Nec-1 for PPIs. Thus, the present study examined the effects of Nec-1 on PPIs at four different doses-0, 25, 50, 100, and 200 μM-after 7 days of tissue culture when supplemented on day 3. PPIs were isolated from pancreata of pre-weaned Yorkshire piglets (8-15 days old) and cultured in a specific islet maturation media added with Nec-1 on day 3 of tissue culture at 4 different doses-0, 25, 50, 100, and 200 μM (n = 6 for each dose). After 7 days of tissue culture, islets were assessed for recovery, viability, endocrine cellular content, GLUT2 expression in beta cells, and insulin secretion after glucose challenge. Nec-1 did not affect the viability of both intact islets and dissociated islets cells during tissue culture regardless of doses. Islets cultured in media supplemented with Nec-1 at 100 μM, but not 25, 50, or 200 μM, had a significantly higher recovery, composition of endocrine cells, GLUT2 expression in beta cells, and insulin secretion capacity than control islets cultured in media without Nec-1 supplementation. Moreover, culturing islets in 200 μM Nec-1 supplemented media not only failed to improve the insulin release but resulted in a lower glucose-induced insulin stimulation index compared to islets cultured in media added with 100 μM Nec-1. Xenotransplantation using porcine islets continues to demonstrate scientific advances to justify this area of research. Our findings indicate that Nec-1 supplementation at 100 μM was most effective to enhance the in vitro maturation of PPIs during tissue culture.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In recent years, human umbilical cord blood has emerged as a rich source of stem, stromal and immune cells for cell-based therapy. Among the stem cells from umbilical cord blood, CD45+ multipotent ...stem cells and CD90+ mesenchymal stem cells have the potential to treat type I diabetes mellitus (T1DM), to correct autoimmune dysfunction and replenish β-cell numbers and function. In this review, we compare the general characteristics of umbilical cord blood-derived multipotent stem cells (UCB-SCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and introduce their applications in T1DM. Although there are some differences in surface marker expression between UCB-SCs and UCB-MSCs, the two cell types display similar functions such as suppressing function of stimulated lymphocytes and imparting differentiation potential to insulin-producing cells (IPCs) in the setting of low immunogenicity, thereby providing a promising and safe approach for T1DM therapy.
The limited availability of human islets has led to the examination of porcine islets as a source of clinically suitable tissue for transplantation in patients with diabetes mellitus. Islets from ...porcine donors are commonly used in both in vitro and in vivo experiments studying diabetes mellitus. However, there are significant differences in quality and quantity of islet yield depending on donor pig age, as well as substantial differences in the costs of pancreas procurement in adult versus neonatal and juvenile pigs. In this study, we compared the total cost per islet of juvenile pig pancreata with that of neonatal and adult pigs. Although adult porcine pancreata yield, on average, more than five times the amount of islets than do juvenile and neonatal pancreata, we found that the high price of adult pigs led to the cost per islet being more than twice that of juvenile and neonatal islets (US $0.09 vs $0.04 and $0.02, respectively). In addition, neonatal and juvenile islets are advantageous in their scalability and retention of viability after culture. Our findings indicate that isolating neonatal and juvenile porcine islets is more cost-effective and scalable than isolating adult porcine islets.
Physiologic insulin secretion consists of an oscillating pattern of secretion followed by distinct trough periods that stimulate ligand and receptor activation. Apart from the large postprandial ...bolus release of insulin, β cells also secrete small amounts of insulin every 4-8 min independent of a meal. Insulin resistance is associated with a disruption in the normal cyclical pattern of insulin secretion. In the case of type-2 diabetes, β-cell mass is reduced due to apoptosis and β cells secrete insulin asynchronously. When ligand/receptors are constantly exposed to insulin, a negative feedback loop down regulates insulin receptor availability to insulin, creating a relative hyperinsulinemia. The relative excess of insulin leads to insulin resistance (IR) due to decreased receptor availability. Over time, progressive insulin resistance compromises carbohydrate metabolism, and may progress to type-2 diabetes (T2D). In this review, we discuss insulin resistance pathophysiology and the use of dynamic exogenous insulin administration in a manner consistent with more normal insulin secretion periodicity to reverse insulin resistance. Administration of insulin in such a physiologic manner appears to improve insulin sensitivity, lower HgbA1c, and, in some instances, has been associated with the reversal of end-organ damage that leads to complications of diabetes. This review outlines the rationale for how the physiologic secretion of insulin orchestrates glucose metabolism, and how mimicking this secretion profile may serve to improve glycemic control, reduce cellular inflammation, and potentially improve outcomes in patients with diabetes.
The scourge of type-1 diabetes (T1D) is the morbidity and mortality it and its complications cause at a younger age. This propels the constant search for better diagnostic, treatment, and management ...strategies, with the ultimate quest being a cure for T1D. Recently, the therapeutic potential of exosomes has generated a lot of interest. Among the characteristics of exosomes of particular interest are (a) their regenerative capacity, which depends on their “origin”, and (b) their “content”, which determines the cell communication and crosstalk they influence. Other functional capacities, including paracrine and endocrine homeostatic regulation, pathogenic response ability resulting in insulin secretory defects or β-cell death under normal metabolic conditions, immunomodulation, and promotion of regeneration, have also garnered significant interest. Exosome “specificity” makes them suitable as biomarkers or predictors, and their “mobility” and “content” lend credence to drug delivery and therapeutic suitability. This review aims to highlight the functional capacities of exosomes and their established as well as novel contributions at various pathways in the onset and progression of T1D. The pathogenesis of T1D involves a complex crosstalk between insulin-secreting pancreatic β-cells and immune cells, which is partially mediated by exosomes. We also examine the potential implications for type 2 diabetes (T2D), as the link in T2D has guided T1D exploration. The collective landscape presented is expected to help identify how a deeper understanding of exosomes (and their cargo) can provide a framework for actionable solutions to prevent, halt, or change the very course of T1D and its complications.
Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases such as diabetes mellitus. Similarities ...of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α- and δ-cells followed by transcriptome analysis (RNA-seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features that are not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet β-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to β-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.
Long term function of implantable biomaterials are determined by their integration with the host's body. Immune reactions against these implants could impair the function and integration of the ...implants. Some biomaterial-based implants lead to macrophage fusion and the formation of multinucleated giant cells, also known as foreign body giant cells (FBGCs). FBGCs may compromise the biomaterial performance and may lead to implant rejection and adverse events in some cases. Despite their critical role in response to implants, there is a limited understanding of cellular and molecular mechanisms involved in forming FBGCs. Here, we focused on better understanding the steps and mechanisms triggering macrophage fusion and FBGCs formation, specifically in response to biomaterials. These steps included macrophage adhesion to the biomaterial surface, fusion competency, mechanosensing and mechanotransduction-mediated migration, and the final fusion. We also described some of the key biomarkers and biomolecules involved in these steps. Understanding these steps on a molecular level would lead to enhance biomaterials design and improve their function in the context of cell transplantation, tissue engineering, and drug delivery.
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