Distinction Between Two Populations of Islet-1-Positive Cells in Hearts of Different Murine Strains
Patricia KhattarFelix W. FriedrichGisèle BonneLucie CarrierThomas EschenhagenSylvia Μ. EvansKetty SchwartzMarc FiszmanJean‐Thomas Vilquin
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Abstract:
Islet-1 expression identifies populations of progenitor cells in embryonic, fetal, and newborn murine hearts that are able to give rise to all cardiac cell lineages ex vivo and in vivo. Using systematic immunohistochemistry, we investigated whether islet-1-positive cells are present in adult mouse heart from the perspective of their potential therapeutic utility. The presence, localization, and nature of islet-1-positive cells were assessed in mice of different strains, ages, and conditions. Islet-1-positive cells were present in mouse heart from postnatal day 1 to young adulthood. Depending on the strain, these cells were organized in either 1 or 2 types of clusters localized to restricted areas, at a distance of 6%-35% of the heart length from the base. The first type of cluster was present in all strains and consisted of neural crest-derived cells that formed cardiac ganglia. The number of cells remained stable (a few hundred) from neonatal up to adult ages, and variations were noted between strains regarding their long-term persistency. The second type of cluster was essentially present in 129SvJ or Balb/C strains and absent from the other strains tested (C57BL/6J, C3H, SJL). It consisted of cells expressing highly ordered sarcomeric actin, consistent with their having cardiomyocyte identity. These cells disappeared in animals older than 4 months. Neither the number nor the type of islet-1-positive cells varied with time in a mouse model of dilated cardiomyopathy. Our studies demonstrate that islet-1-positive cells are relatively few in number in adult murine heart, being localized in restricted and rather inaccessible areas, and can represent both neural crest and cardiomyocyte lineages.Keywords:
Cell type
Many factors influence the outcome of islet transplantation. As islets in the early posttransplant setting are supplied with oxygen by diffusion only and are in a hypoxic state in the portal system, we tested whether small human islets are superior to large islets both in vitro and in vivo. We assessed insulin secretion of large and small islets and quantified cell death during hypoxic conditions simulating the intraportal transplant environment. In the clinical setting, we analyzed the influence of transplanted islet size on insulin production in patients with type 1 diabetes. Our results provide evidence that small islets are superior to large islets with regard to in vitro insulin secretion and show a higher survival rate during both normoxic and hypoxic culture. Islet volume after 48 h of hypoxic culture decreased to 25% compared with normoxic culture at 24 h due to a preferential loss of large islets. In human islet transplantation, the isolation index (islet volume as expressed in islet equivalents/islet number), or more simply the islet number, proved to be more reliable to predict stimulated C-peptide response compared with islet volume. Thus, islet size seems to be a key factor determining human islet transplantation outcome.
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It has been proposed that islet transplants comprised primarily of small rather than large islets may provide better graft function, due to their lower susceptibility to hypoxic damage. Our aim was to determine whether islet size correlated with in vivo graft function in islet transplant recipients with C peptide-negative type 1 diabetes when islets have undergone pretransplant islet culture.Human pancreatic islets were isolated, cultured for 24 hours and infused by standardized protocols. Ninety-minute stimulated C-peptide concentrations were determined during a standard meal tolerance test 3 months posttransplant. The islet isolation index (IEq/islet number) was determined immediately after isolation and again before transplantation (after tissue culture). This was correlated with patient insulin requirement or stimulated C-peptide.Changes in insulin requirement did not significantly correlate with islet isolation index. Stimulated C-peptide correlated weakly with IEq at isolation (P = 0.40) and significantly with IEq at transplantation (P = 0.018). Stimulated C-peptide correlated with islet number at isolation (P = 0.013) and more strongly with the islet number at transplantation (P = 0.001). In contrast, the correlation of stimulated C-peptide and islet isolation index was weaker (P = 0.018), and this was poorer at transplantation (P = 0.034). Using linear regression, the strongest association with graft function was islet number (r = 0.722, P = 0.001). Islet size was not related to graft function after adjusting for islet volume or number.These data show no clear correlation between islet isolation index and graft function; both small and large islets are suitable for transplantation, provided the islets have survived a short culture period postisolation.
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Introduction: The challenging process of obtaining pure islet fractions from donor pancreata hampers the widespread use of allogenic islet transplantation for patients with complicated diabetes mellitus. Limited evidence suggests that transplantation of islet products with a higher percentage of non-islet cells is associated with improved long-term metabolic outcomes. However, conclusive evidence is lacking due to indirect metabolic outcome measurements, the small number of islet recipients, and the lack of an objective and reproducible assessment of islet purity. We aimed to retrospectively evaluate the effect of islet purity on long-term graft function using robust measurement methods to determine islet purity and graft function. Method: In a cohort of islet recipients that underwent an allogenic islet transplantation procedure at Leiden University Medical Center, digitalized microscopic images of the dithizone-stained transplanted islet graft were analyzed using a reproducible computerized deep learning method (IsletNet, version 2020-01-20) to calculate islet purity (expressed as %), islet size index (estimate of the average islet size, index <1 indicates an average islet size <150 μm) and the percentage of embedded islets. The cohort was divided into tertiles based on the purity: low purity (0–38%), intermediate purity (39–58%) and high purity (59–100%). Short- and long-term graft function was evaluated by calculating the area-under-the-curve (AUC) of C-peptide measurements that were obtained with mixed meal tests three months after transplantation, and subsequently yearly up to 5 years. Results: Forty-one islet transplantation patients were included. Twenty-eight grafts consisted of islets derived from 1 donor pancreas and 13 grafts from 2 donor pancreata. The purity (mean±sd) in the low, intermediate and high group was 26±10, 46±6 and 67±7%, respectively. A higher islet purity was positively associated with islet size index (R2=0.47, p<0.0001) and negatively associated with the percentage of embedded islets (R2=0.56, p<0.0001). The C-peptide AUC (mean±sd) at three months after transplantation was 125.4±74.0 (low purity), 107.7±57.1 (intermediate purity), and 172.4±73.0 nmol/L (high purity; high vs low purity p=0.11). Also at 5 years there was no clear difference in C-peptide AUC (mean±sd): 55.5± 65.7 (low purity), 115.2±62.3 (intermediate purity) and 96.0±84.2 nmol/L (high purity). Conclusion: The use of higher purity islet grafts for transplant is not associated with better long-term graft function. Further investigations are required to elucidate the mechanisms underlying the effect of non-islet cells on long-term islet function.
Allotransplantation
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After islet isolation, diffusion has become the main mechanism to transport oxygen and nutrients into the core of islets. However, diffusion has limitations, by which nutrients cannot effectively reach the core of large islets and can eventually cause core cell death and islet loss. This problem can be resolved by dispersing islets into single islet cells, but single islet cells do not exhibit insulin release function in in vitro culture. In this study, we intended to establish a new islet engineering approach by forming islet cell clusters to improve islet survival and function. Therefore, alginate gels were used to encapsulate islet cells to form artificial islets after dispersion of islets into single cells. The shape of the islet cell clusters was similar to native islets, and the size of the islet cell clusters was limited to a maximum diameter of 100 μm. By limiting the diameter of this engineered islet cell cluster, cell viability was nearly 100%, a significant improvement over natural islets. Importantly, islet cell clusters express the genes of islets, including Isl-1, Gcg, and insulin-1, and insulin secretion ability was maintained in vitro.
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Clinical islet allotransplantation is dependent on the ability to achieve a high yield and purity of islets isolated from human cadaver pancreas donors. The aim of this study was to determine the factors influencing the pancreas prior to islet isolation that may alter yield and purity. The results of 50 consecutive islet isolations from cadaver donor human pancreati at the University of Chicago Medical Center from December 1991 to April 1993 were analyzed. All pancreati were first offered for whole pancreas transplantation before being considered for islet isolation. Human pancreatic islet isolation was accomplished by a modified automated method. Some islet isolations resulted in a high islet yield but low islet purity. Other resulted in well-purified islets, but a low yield. Arbitrarily, successful islet isolation is defined as that yielding over 250,000 islet equivalents (EQN) with a purity of at least 80%. The success rate of human pancreatic islet isolation was 70%. The mean final islet yield obtained from these 50 pancreati was 300,000±131,000 islet EQN. The mean purity of the final preparation was 73%±25%. By univariate analysis, five factors were found to affect significantly the yield, purity, or overall success rate of islet isolation: organ cold ischemic time, donor age, donor plasma glucose levels, donor body weight, and cause of donor death. Even when islet isolation was successful, the function of islets from hyperglycemic and older donors appear to be impaired both in vitro and in vivo. These results suggest that islet yield and purity are affected by multiple donor-related factors. Even when adequate yield and purity are obtained, islet function is also dependent on donor variables.
Allotransplantation
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Biocompatibility
Pancreatic Islets
Cell Survival
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This report confirms reproducible methods to isolate and assess viability and function of pig and human islets. We processed 10 pig and five human pancreata. The pancreata were digested by a modification of Ricordi's automated method for islet isolation. The number of islet equivalents (150 μm diameter islets) was 335,190 ± 79,345 islets per pig pancreas (5,146 ± 1,274 islets/g pig pancreas) and 323,630 ± 147,810 per human pancreas (6,252 ± 2,572 islets/g human pancreas). The majority of islets were in the range of 50–200 μm diameter, and 20% of the islet population had a size distribution of 200 pm diameter in both porcine and human models. The purity of the final preparations exceeded 90%. The secretory response of perifused islets showed a biphasic insulin release pattern in both species. Perifused fresh pig islets released 2.5 pmol/L islet−1 min−1 at 2.0 mM glucose and 6.2 pmol/L islet−1 min−1 at 16.7 mM glucose. After 7 days culture at 37°C, human islets released 1.32 pmol/L islet−1 min−1 at 2.0 mM and 12.24 pmol/L islet−1 min−1 at 16.7 mM. These results indicate that this procedure is useful to obtain pure, large, and functional islets from pig and human pancreata.
Isolation
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