Pharmacological approach to understanding the control of insulin secretion in human islets.

AIMS: To understand better the control of insulin secretion by human ß cells and to identify similarities to and differences from rodent models. METHODS: Dynamic insulin secretion was measured in perifused human islets treated with pharmacological agents of known modes of action. RESULTS: Glucokinase activation (Ro28-1675) lowered the glucose threshold for stimulation of insulin secretion to 1 mmol/L (G1), augmented the response to G3-G5 but not to G8-G15, whereas tolbutamide remained active in G20, which indicates that not all KATP channels were closed by high glucose concentrations. An almost 2-fold greater response to G15 than to supramaximal tolbutamide in G3 or to KCl+diazoxide in G15 vs G3 quantified the contribution of metabolic amplification to insulin secretion. Both disruption (latrunculin-B) and stabilization (jasplakinolide) of microfilaments augmented insulin secretion without affecting metabolic amplification. Tolbutamide-induced insulin secretion was consistently greater in G10 than G3, with a threshold at 1 and maximum at 10 µmol/L tolbutamide in G10, vs 10 and 25 µmol/L in G3. Sulphonylurea effects were thus clearly glucose-dependent. Insulin secretion was also increased by inhibiting K channels other than KATP channels: Kv or BK channels (tetraethylammonium), TASK-1 channels (ML-365) and SK4 channels (TRAM-34). Opening KATP channels with diazoxide inhibited glucose-induced insulin secretion with half maximum inhibitory concentrations of 9.6 and 24 µmol/L at G7 and G15. Blockade of L-type Ca channels (nimodipine) abolished insulin secretion, whereas a blocker of T-type Ca channels (NNC-55-0396) was ineffective at specific concentrations. Blockade of Na channels (tetrodotoxin) did not affect glucose-induced insulin secretion. CONCLUSIONS: In addition to sharing a KATP channel-dependent triggering pathway and a metabolic amplifying pathway, human and rodent ß cells were found to display more similarities than differences in the control of insulin secretion.

Dynamics of glucose-induced insulin secretion in normal human islets.

The biphasic pattern of glucose-induced insulin secretion is altered in type 2 diabetes. Impairment of the first phase is an early sign of ß-cell dysfunction, but the underlying mechanisms are still unknown. Their identification through in vitro comparisons of islets from diabetic and control subjects requires characterization and quantification of the dynamics of insulin secretion by normal islets. When perifused normal human islets were stimulated with 15 mmol/l glucose (G15), the proinsulin/insulin ratio in secretory products rapidly and reversibly decreased (∼50%) and did not reaugment with time. Switching from prestimulatory G3 to G6-G30 induced biphasic insulin secretion with flat but sustained (2 h) second phases. Stimulation index reached 6.7- and 3.6-fold for the first and second phases induced by G10. Concentration dependency was similar for both phases, with half-maximal and maximal responses at G6.5 and G15, respectively. First-phase response to G15-G30 was diminished by short (30-60 min) prestimulation in G6 (vs. G3) and abolished by prestimulation in G8, whereas the second phase was unaffected. After 1-2 days of culture in G8 (instead of G5), islets were virtually unresponsive to G15. In both settings, a brief return to G3-G5 or transient omission of CaCl2 restored biphasic insulin secretion. Strikingly, tolbutamide and arginine evoked immediate insulin secretion in islets refractory to glucose. In conclusion, we quantitatively characterized the dynamics of glucose-induced insulin secretion in normal human islets and showed that slight elevation of prestimulatory glucose reversibly impairs the first phase, which supports the view that the similar impairment in type 2 diabetic patients might partially be a secondary phenomenon.

Local complications of massive bone allografts: an appraisal of their prevalence in 128 patients.

Bone allografts were used in our department since twenty-five years to reconstruct segmental bone loss and our data were retrospectively reviewed to assess the complications related to the use of a bone allograft. A consecutive series of 128 patients who received a bone allograft was analyzed. The minimal follow-up was 18 months. Fracture, nonunion, infection and explantation were investigated using a multivariate analysis and logistical regression. Kaplan-Meier survival of the allograft was performed, using allograft removal as the end point. Tumour disease was excluded from this study. Patients were followed up for an average 103 months. Bone tumour occurred in 78% of the patients whereas revision arthroplasty was the cause of implantation in 15% of them. Nonunion was the most prevalent complication, occurring in 35% of the grafts. For nonunion occurrence, the type of reconstruction was found to be a significant variable, the intercalary allograft being the most exposed. Primary bone autografting at the anastomotic site was not significant to prevent nonunion. Fracture of the allograft was the second most frequent complication with a prevalence of 16.4%. The length of the allograft and an osteoarticular allograft were two significant variables in that occurrence. Infection of the allograft was present with a rate of 5.4% of patients. Explantation of failed allografts occurred in 30% of them. The duration of the frozen storage of the allograft and the donor age of the allograft were not significant on any local complication occurrence. Bone allografts are a reliable material but a high rate of local complications must be anticipated.

Bioengineered sites for islet cell transplantation.

Although islet transplantation has demonstrated its potential use in treating type 1 diabetes, this remains limited by the need for daily immunosuppression. Islet encapsulation was then proposed with a view to avoiding any immunosuppressive regimen and related side effects. In order to obtain a standard clinical procedure in terms of safety and reproducibility, two important factors have to be taken into account: the encapsulation design (which determines the graft volume) and the implantation site. Indeed, the implantation site should meet certain requirements: (1) its space must be large enough for the volume of transplanted tissues; (2) there must be proximity to abundant vascularization with a good oxygen supply; (3) there must be real-time access to physiologically representative blood glucose levels; (4) there must be easy access for implantation and the reversibility of the procedure (for safety); and finally, (5) the site should have minimal early inflammatory reaction and promote long-term survival. The aim of this article is to review possible preclinical/clinical implantation sites (in comparison with free islets) for encapsulated islet transplantation as a function of the encapsulation design: macro/microcapsules and conformal coating.

Beware of the commercialization of human cells and tissues: situation in the European Union.

With this analysis we would like to raise some issues that emerge as a result of recent evolutions in the burgeoning field of human cells, tissues, and cellular and tissue-based product (HCT/P) transplantation, and this in the light of the current EU regulatory framework. This paper is intended as an open letter addressed to the EU policy makers, who will be charged with the review and revision of the current legislation. We propose some urgent corrections or additions to cope with the rapid advances in biomedical science, an extensive commercialization of HCT/Ps, and the growing expectation of the general public regarding the ethical use of altruistically donated cells and tissues. Without a sound wake-up call, the diverging interests of this newly established 'healthcare' industry and the wellbeing of humanity will likely lead to totally unacceptable situations, like some of which we are reporting here.

Formalin fixation could interfere with the clinical assessment of the tumor-free margin in tumor surgery: magnetic resonance imaging-based study.

OBJECTIVES: The tumor-free margin in bone and soft-tissue cancer is a key factor for subsequent treatment. While flattening and shrinkage of specimens after formalin fixation have been described in breast cancer, there are no data for bone and soft tissue sarcoma. Fixation could interfere with the accuracy of the assessment of the tumor-free margin. METHODS: The influence of formalin fixation was assessed on forelimb specimens in a preclinical porcine model. The specimens were subjected to magnetic resonance imaging before and after formalin fixation. Weight, width and height of the specimen were measured and different consecutive volumes (total, muscles, bones and fatty tissue) were obtained by segmentation. RESULTS: After formalin fixation, the weight increased, total volume and muscle volume slightly increased while bone did not change and fatty tissue decreased. The width of the specimens increased while their height decreased. CONCLUSIONS: Formalin fixation caused slight muscle expansion, fatty tissue shrinkage and flattening of the specimen. These changes could interfere with the assessment of the tumor-free margin in clinical practice.

Beta-5 score to evaluate pig islet graft function in a primate pre-clinical model.

BACKGROUND: We developed a composite scoring system to accurately assess pig islet function in pre-clinical primate studies. METHODS: Two scoring methods that have been clinically validated in human islet allotransplantation were tested in six non-diabetic and nine streptozotocin (STZ)-induced diabetic primates: (i) SUITO index=[1500 × fasting C-peptide (ng/ml)]/[fasting blood glucose (FBG, mg/dl) - 63] and (ii) CP/G ratio =[fasting C-peptide (ng/ml) × 100]/FBG (mg/dl). Both scores were analysed as a function of the ß-cell mass of the native primate pancreas. Next, a proposed ß5 score based on FBG values, daily glycosuria, post-prandial glycosuria, polydipsia, and polyuria was validated on the same primates. Ranges of normal and pathologic values for each parameter were assessed during 5 months in non-diabetic and diabetic primates, respectively. Finally, scores were tested on the nine STZ-induced diabetic primates, four of which were transplanted with microencapsulated pig islets and five with macroencapsulated pig islets. All parameters required for each score were measured prior to transplantation and up to 12 weeks post-transplantation. For the CP/G ratio after transplantation, primate C-peptide was replaced by porcine C-peptide. RESULTS: The Suito index was not correlated with the pancreatic ß-cell mass in contrast to the CP/G ratio (R(2) = 0.17, P = 0.645 vs. R(2) = 0.76, P = 0.003; respectively). The internal consistency of the parameters implied by the ß5 score was confirmed by a Cronbach's alpha test of 0.97. Diabetes was confirmed by a significant decrease in the CP/G ratio and the ß5 score before and after diabetes induction, respectively. After transplantation, a significant correlation was found between the CP/G ratio and the ß5 score, which reflected the functionality of pig islet xenografts and diabetes control. In addition, the CP/G ratio and ß5 score were correlated with the glycosylated hemoglobin course after transplantation and diabetes correction with macroencapsulated pig islets. CONCLUSION: The proposed ß5 score provides a valid tool to accurately assess islet transplantation in a primate pre-clinical model.

Dispersion of ceramic granules within human fractionated adipose tissue to enhance endochondral bone formation.

Engineering of materials consisting of hypertrophic cartilage, as physiological template for de novo bone formation through endochondral ossification (ECO), holds promise as a new class of biological bone substitutes. Here, we assessed the efficiency and reproducibility of bone formation induced by the combination of ceramic granules with fractionated human adipose tissue ("nanofat"), followed by in vitro priming to hypertrophic cartilage. Human nanofat was mixed with different volumetric ratios of ceramic granules (0.2-1 mm) and cultured to sequentially induce proliferation (3 weeks), chondrogenesis (4 weeks), and hypertrophy (2 weeks). The resulting engineered constructs were implanted ectopically in nude mouse. The presence of ceramic granules regulated tissue formation, both in vitro and in vivo. In particular, their dispersion in nanofat at a ratio of 1:16 led to significantly increased cell number and glycosaminoglycan accumulation in vitro, as well as amount and inter-donor reproducibility of bone formation in vivo. Our findings outline a strategy for efficient utilization of nanofat for bone regeneration in an autologous setting, which should now be tested at an orthotopic site. STATEMENT OF SIGNIFICANCE: In this study, we assessed the efficiency and reproducibility of bone formation by a combination of ceramic granules and fractionated human adipose tissue, also known as nanofat, in vitro primed into hypertrophic cartilage. The resulting engineered cartilaginous constructs, when implanted ectopically in nude mouse, resulted in bone and bone marrow formation, more reproducibly and strongly that nanofat alone. This project evaluates the impact of ceramic granules on the functionality and chondrogenic differentiation of mesenchymal progenitors inside their native adipose tissue niche and outlines a novel strategy for an efficient application of nanofat for bone regeneration in an autologous setting.

Pig islets for clinical islet xenotransplantation.

PURPOSE OF REVIEW: Allogeneic islet transplantation faces difficulties because organ shortage is recurrent; several pancreas donors are often needed to treat one diabetic recipient; and the intrahepatic site of islet implantation may not be the most appropriate one. Another source of insulin-producing cells, therefore, would be of major interest, and pigs represent a possible and serious source for obtaining such cells. RECENT FINDINGS: Pig islet grafts may appear difficult because of the species barrier, but recent studies demonstrate that pig islets may function in diabetic primates for at least 6 months. SUMMARY: Pig islet xenotransplantation, however, must still overcome the selection of a suitable pig donor to translate preclinical findings into clinical applications. This review summarizes the actual acquired knowledge of pig islet transplantation in primates to select the 'ideal' pig donor.

Native pancreatic alpha-cell adaptation in streptozotocin-induced diabetic primates: importance for pig islet xenotransplantation.

BACKGROUND: Metabolic compatibility between donor and recipient species is an important matter for pig islet xenotransplantation. Glucagon is a key hormone for the function of pig islets as well as control of hypoglycemia in the recipients of the islets. Because a discrepancy exists in the composition of glucagon cells of pig and human/primate islets, the present study was designed to determine the role of native recipient glucagon cells in the treatment of diabetes by islet transplantation in a "pig-to-primate" model. METHODS: Streptozotocin-treated (50 mg/kg) monkeys (n = 12, follow-up of 6 to 231 days) were compared with non-diabetic animals (n = 5; follow-up, 180 days). Metabolic [fasting and intravenous glucose tolerance tests (IVGTTs) for serum levels of glucose, insulin, glucagon] and morphologic (endocrine volume density and cell mass for insulin and glucagon) were compared between non-diabetic and diabetic animals. Six additional diabetic primates were given transplants of 15 000 adult pig islet equivalents without immunosuppression to monitor glucose, glucagon, insulin, and porcine C-peptide levels until 48 h after transplantation. RESULTS: Elevated fasting blood glucose, pathologic IVGTT, destruction of 95% of beta-cell mass, and glycosylated hemoglobin (>13%) were assessed in diabetic monkeys. The serum glucagon levels and glucagon cell mass correlated significantly with diabetes time course of diabetes (R = 0.940, p = 0.005; R = 0.663, p = 0.019, respectively). A mean increase of 89% in glucagon cell mass was observed for primates suffering from diabetes >53 days. No response of glucagon secretion was observed for diabetic animals during IVGTT, because no increase of serum insulin levels followed glucose loading. Blood glucose levels dropped after pig islet xenografts in diabetic primates. This reduction was maintained by an insulin level >20 microU/ml over the period of time of xenograft function (porcine C-peptide >0.1 ng/ml). A total restoration of native primate glucagon sensitivity to insulin was found after pig islets xenotransplantation as revealed by a reduction of 80% of the glucagon level. When graft dysfunction (>24 h post-transplantation), the insulin level dropped and glucagon levels rose again (>50 pg/ml). CONCLUSIONS: Native glucagon cells provide morphologic and functional plasticity to diabetes. Adult pig islet xenotransplantation can restore the sensitivity of primate glucagon to insulin but cannot protect the diabetic recipient against hypoglycemia.

Regeneration of abdominal wall musculofascial defects by a human acellular collagen matrix.

This work studied the reconstruction of an abdominal wall defect by a human acellular collagen matrix. The abdominal wall defect was cured in 40 rats by implanting (i) polypropylene (Pro), (ii) polyester (Mers) meshes, and (iii) human acellular collagen matrix with two orientations: fibres in parallel (fascia lata longitudinal [FLL]) or perpendicular (fascia lata transversal [FLT]) to native rats' abdominal walls. Hernia recurrence, adhesions, and histology (for inflammation and remodelling) were assessed at 4 and 8 weeks after implantation. Two large abdominal eventrations were cured by a human acellular matrix in human patients. A higher hernia recurrence rate was observed for rats transplanted with FLL than with FLT/Pro/Mers at 4 and 8 weeks after implantation. A lower adhesion rate was achieved for FLL/FLT than for Pro/Mers meshes (p<0.05). A decrease in immunologic cell infiltrations in FLL/FLT was observed between day 30 and day 60 (p<0.05). Collagen, elastin, and muscular tissues were found only in FLL/FLT matrix; a weaker muscular cell infiltration for FLL occurred at 8 weeks. Human abdominal eventrations were totally cured by using FLT as confirmed by computed tomography scanning at 12 and 16 months after implantation. In conclusion, human acellular collagen matrix, placed in an FLT position, can induce an abdominal wall reconstitution without adhesions and hernia recurrence.

Dissecting the instant blood-mediated inflammatory reaction in islet xenotransplantation.

BACKGROUND: A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non-human primates. The detrimental instant blood-mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen. MATERIALS AND METHODS: Low molecular weight dextran sulfate (LMW-DS) and the complement inhibitor Compstatin were used in a combination of in vitro and in vivo studies designed to dissect the xenogeneic IBMIR in a non-human primate model of pancreatic islet transplantation. Adult porcine islets (10,000 IEQs/kg) were transplanted intraportally into three pairs of cynomolgus monkeys that had been treated with LMW-DS or heparin (control), and the effects on the IBMIR were characterized. Porcine islets were also incubated in human blood plasma in vitro to assess complement inhibition by LMW-DS and Compstatin. RESULTS: Morphological scoring and immunohistochemical staining revealed that the severe islet destruction and macrophage, neutrophilic granulocyte, and T-cell infiltration observed in the control (heparin-treated) animals were abrogated in the LMW-DS-treated monkeys. Both coagulation and complement activation were significantly reduced in monkeys treated with LMW-DS, but IgM and complement fragments were still found on the islet surface. This residual complement activation could be inhibited by Compstatin in vitro. CONCLUSIONS: The xenogeneic IBMIR in this non-human primate model is characterized by an immediate binding of antibodies that triggers deleterious complement activation and a subsequent clotting reaction that leads to further complement activation. The effectiveness of LMW-DS (in vivo and in vitro) and Compstatin (in vitro) in inhibiting this IBMIR provides the basis for a protocol that can be used to abrogate the IBMIR in pig-human clinical islet transplantation.

Nutrient control of insulin secretion in isolated normal human islets.

Pancreatic islets were isolated from 16 nondiabetic organ donors and, after culture for approximately 2 days in 5 mmol/l glucose, were perifused to characterize nutrient-induced insulin secretion in human islets. Stepwise increases from 0 to 30 mmol/l glucose (eight 30-min steps) evoked concentration-dependent insulin secretion with a threshold at 3-4 mmol/l glucose, K(m) at 6.5 mmol/l glucose, and V(max) at 15 mmol/l glucose. An increase from 1 to 15 mmol/l glucose induced biphasic insulin secretion with a prominent first phase (peak increase of approximately 18-fold) and a sustained, flat second phase ( approximately 10-fold increase), which were both potentiated by forskolin. The central role of ATP-sensitive K(+) channels in the response to glucose was established by abrogation of insulin secretion by diazoxide and reversible restoration by tolbutamide. Depolarization with tolbutamide or KCl (plus diazoxide) triggered rapid insulin secretion in 1 mmol/l glucose. Subsequent application of 15 mmol/l glucose further increased insulin secretion, showing that the amplifying pathway is operative. In control medium, glutamine alone was ineffective, but its combination with leucine or nonmetabolized 2-amino-bicyclo [2,2,1]-heptane-2-carboxylic acid (BCH) evoked rapid insulin secretion. The effect of BCH was larger in low glucose than in high glucose. In contrast, the insulin secretion response to arginine or a mixture of four amino acids was potentiated by glucose or tolbutamide. Palmitate slightly augmented insulin secretion only at the supraphysiological palmitate-to-albumin ratio of 5. Inosine and membrane-permeant analogs of pyruvate, glutamate, or succinate increased insulin secretion in 3 and 10 mmol/l glucose, whereas lactate and pyruvate had no effect. In conclusion, nutrient-induced insulin secretion in normal human islets is larger than often reported. Its characteristics are globally similar to those of insulin secretion by rodent islets, with both triggering and amplifying pathways. The pattern of the biphasic response to glucose is superimposable on that in mouse islets, but the concentration-response curve is shifted to the left, and various nutrients, in particular amino acids, influence insulin secretion within the physiological range of glucose concentrations.

Impact of Age on Human Adipose Stem Cells for Bone Tissue Engineering.

Bone nonunion is a pathological condition in which all bone healing processes have stopped, resulting in abnormal mobility between 2 bone segments. The incidence of bone-related injuries will increase in an aging population, leading to such injuries reaching epidemic proportions. Tissue engineering and cell therapy using mesenchymal stem cells (MSCs) have raised the possibility of implanting living tissue for bone reconstruction. Bone marrow was first proposed as the source of stem cells for bone regeneration. However, as the quantity of MSCs in the bone marrow decreases, the capacity of osteogenic differentiation of bone marrow stem cells is also impaired by the donor's age in terms of reduced MSC replicative capacity; an increased number of apoptotic cells; formation of colonies positive for alkaline phosphatase; and decreases in the availability, growth potential, and temporal mobilization of MSCs for bone formation in case of fracture. Adipose-derived stem cells (ASCs) demonstrate several advantages over those from bone marrow, including a less invasive harvesting procedure, a higher number of stem cell progenitors from an equivalent amount of tissue harvested, increased proliferation and differentiation capacities, and better angiogenic and osteogenic properties in vivo. Subcutaneous native adipose tissue was not affected by the donor's age in terms of cellular senescence and yield of ASC isolation. In addition, a constant mRNA level of osteocalcin and alkaline phosphatase with a similar level of matrix mineralization of ASCs remained unaffected by donor age after osteogenic differentiation. The secretome of ASCs was also unaffected by age when aiming to promote angiogenesis by vascular endothelial growth factor (VEGF) release in hypoxic conditions. Therefore, the use of adipose cells for bone tissue engineering is not limited by the donor's age from the isolation of stem cells up to the manufacturing of a complex osteogenic graft.

Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: proof of concept.

BACKGROUND: Pig islets xenotransplantation remains associated with a strong humoral and cellular xenogeneic immune responses. The aim of this study was to assess the long-term biocompatibility of alginate encapsulated pig islets after transplantation in primates. METHODS: Adult pig islets encapsulated in alginate under optimal conditions (n=7) or not (n=5) were transplanted under the kidney capsule of nondiabetic Cynomolgus maccacus. Additional primates received empty capsules (n=1) and nonencapsulated pig islets (n=2) as controls. Capsule integrity, cellular overgrowth, pig islet survival, porcine C-peptide and anti-pig IgM/IgG antibodies were examined up to 6 months after implantation. RESULTS: Nonencapsulated islets and islets encapsulated in nonoptimal capsules were rapidly destroyed. In seven primates receiving perfectly encapsulated pig islets, part of the islets survived up to 6 months after implantation without immunosuppression. Porcine C-peptide was detected after 1 month in 71% of the animals. The majority of grafts (86%) were intact and completely free of cellular overgrowth or capsule fibrosis. Explanted capsules, after 135 (n=2/2) and 180 (n=2/3) days, demonstrated residual insulin content and responses to glucose challenge (stimulation index of 2.2). Partial islet survival was obtained despite an elicited anti-pig IgG humoral response. CONCLUSIONS: Optimal alginate encapsulation significantly prolonged adult pig islet survival into primates for up to 6 months, even in the presence of antibody response.

Streptozotocin-induced diabetes in large animals (pigs/primates): role of GLUT2 transporter and beta-cell plasticity.

BACKGROUND: To induce irreversible diabetes in large animals, the efficiency of streptozotocin (STZ) was evaluated in pigs, primates and compared to the gold standard model in rats. METHODS: Low (50 mg/kg) and high (150 mg/kg) doses of STZ were tested. Hepatic/renal function, glucose metabolism (intravenous glucose tolerance tests, fasting blood glucose) and histomorphometry were evaluated prior to, 1, and 4 weeks after STZ treatment. RESULTS: In rats and primates, expressing a high level of GLUT2 expression on beta cells, a dose of 50 mg/kg STZ induced irreversible diabetes (due to the 97% destruction of beta cell mass) without provoking liver or renal failure. In pigs, despite the use of high STZ dose, partial correction of hyperglycaemia was observed four weeks after STZ injection (decreased fasting blood glucose and intravenous glucose tolerance tests; increased insulin production). The correction of hyperglycaemia was associated with significant hypertrophy of immature pig beta-cell clusters (+30%, P<0.05), whereas no hypertrophy was observed in rats/primates. CONCLUSION: These results demonstrated that STZ might be used to induce irreversible diabetes in rats and primates. In contrast, the low STZ sensitivity in pigs related to a low expression of GLUT2, higher number of immature beta cells and compensatory beta-cell hypertrophy, renders STZ-induced diabetes inappropriate for studying islet allografts in swine.

The influence of implantation site on the biocompatibility and survival of alginate encapsulated pig islets in rats.

This work investigated the impact of implantation sites on the biocompatibility of alginate encapsulated pig islets. Non-diabetic rats were implanted with adult pig islets encapsulated in alginate either intraperitoneally (IP; n=25), subcutaneously (SC; n=37) or under the kidney capsule (KC; n=34). Capsule biocompatibility (retrieval rate, capsule diameter, degree of capsule broken and cellular overgrowth, CD68/CD3 staining) as well as islets viability and functionality were assessed until 30 days after transplantation. Implantation site did not significantly influence the biocompatibility of empty alginate capsules after transplantation (n=48). Most of the empty capsules (>90%) were retrieved after harvesting and were free of cellular overgrowth until day 30 post-transplantation. Three days after implantation, no significant difference for encapsulated pig islets was observed in terms of capsule biocompatibility and islet functionality in peritoneum, KC or subcutaneously. However, between days 5 and 30 after transplantation, explanted capsules from IP demonstrated a higher degree of broken capsules (>13%) and capsules with severe cellular overgrowth (>50%, CD68+ infiltration) than capsules removed from SC and KC (p<0.05). This was associated with a significant reduction of islet viability, insulin content and insulin secretion. In rats, the peritoneum site seems not appropriate for promoting the engraftment of encapsulated pig islets. Kidney subcapsular and subcutaneous spaces represent an interesting alternative.

[Treatment diabetes by transplantation of insulin secreting cells. Part I]. / Leczenie cukrzycy poprzez przeszczep komórek wydzielajacych insuline. Czesc I.

Treatment of diabetes mellitus poses a huge medical problem. Both the transplantation of pancreas and insulin treatment is connected with numerous complications. The method of treatment which would entirely relieve patients from the necessity of insulin applications and complications of diabetes mellitus has yet not been found. The paper addresses to the issues related to procurement, preservation and culture of pancreatic islets. In this review, the current state of knowledge as well as perspectives of treatment of diabetes through the transplantation of insulin secreting cells was presented.

[Treatment diabetes by transplantation of insulin secreting cells. Part II--prevention of rejection of the transplanted cells]. / Leczenie cukrzycy poprzez przeszczep komórek wydzielajacych insuline. Czesc II--zapobieganie reakcji odrzucenia przeszczepionych komórek.

Transplantation of pancreatic islets is the treatment method for type 1 diabetes mellitus permitting maintenance of stable glycemia. Due to the progress of immunosuppression, the results of transplantation of the pancreatic islets are better. Although applied immunosuppression drugs prevent graft rejection, they have many side effects. The latest methods of modulation of the immunological tolerance, an alternative for immunosuppression, were introduced in this paper. The methods of pancreatic islets encapsulation and induction of allogenic chimerism were described.

A Simple Method to Determine the Purity of Adipose-Derived Stem Cell-Based Cell Therapies.

: It is important to standardize methods to quantify the purity of adipose tissue-derived cells for regenerative medicine. We developed a simple and robust tool to discriminate fibroblasts and adipose stem cells (ASCs) by testing release of specific growth factors. ASCs and dermal fibroblasts (DFs) were isolated from human donors (n = 8). At passage 4, cultures were prepared with progressive ASC/DF ratios of 100%/0%, 75%/25%, 50%/50%, 25%/75%, and 0%/100% for each donor and incubated in hypoxic chambers at 0.1% and 5% O2 and hyperglycemia at 1.0 and 4.5 g/l. After incubation for 24 hours, cell survival, proliferation, and growth factor release (vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], insulin-like growth factor 1 [IGF-1], stromal cell-derived factor 1α [SDF-1α], and basic fibroblast growth factor [bFGF]) were assessed for each condition. The proliferation and viability of ASCs and DFs were not impacted by the oxygen tension conditions. No significant difference in HGF, IGF-1, bFGF, and keratinocyte growth factor secretome was found across the various ASC/DF ratios. Interestingly, a negative relation for VEGF secretion was found when ASCs were contaminated by fibroblasts, especially when cells were exposed to 4.5 g/l glucose and 0.1% O2 (R = -0.521; p < .001). In contrast, secretion of SDF-1α was positively correlated with the fibroblast ratio, more prominently in low glucose and low oxygen tension (r = .657; p < .001). Above and beyond these previously unreported metabolic features, these results (a) allow us to discriminate fibroblasts and ASCs specifically and (b) allow new tools be developed for the rapid testing (a response within 24 hours) for the release of ASC-based therapies. SIGNIFICANCE: In order to provide direction to academia, industry, and regulatory authorities regarding purity assessment for adipose tissue-derived cells, this report describes a simple tool to facilitate development of international standards based on reproducible parameters and endpoints that may systematize cellular products across boundaries and accelerate the delivery of safe and effective adipose stem cell (ASC)-based tools to the medical community and the patients it serves. This tool (a) can discriminate specifically fibroblasts and ASCs and (b) can be rapidly implemented and performed before the release of the ASC-based therapy (a response within 24 hours).