No abstract

The use of regional human islet cell processing centers (ICPC) supporting distant clinical islet transplantation programs (CITP) has proven successful in recent clinical trials. Standardization of islet shipping protocols is needed to preserve cell product identity, quantity, quality and sterility, and to meet criteria for transplantation. We evaluated the use of gas-permeable bags for human islet preparation shipment from a single ICPC to two remote CITPs. Product release tests (counts, purity, viability, sterility and potency) were performed at both centers using identical protocols to determine adequacy for transplantation. Thirty-five islet preparations were shipped either immediately after isolation (n = 20) or following culture (n = 15). Islet recovery rate after shipment was higher in cultured preparations, when compared to those not cultured (91.2 ± 4.9% vs. 72.9 ± 4.7%, respectively; p < 0.05), though the overall recovery rate based on isolation and pretransplant counts was comparable (72.9 ± 4.7% vs. 70.4 ± 3.5%, respectively; p = N.S.). All preparations met product release criteria for transplantation. Additional experiments showed that gas-permeable bags led to improved recovery and potency, when compared to 50-mL conical tubes or to non-gas-permeable bags for shipment. Collectively, our data demonstrate that the use of gas-permeable bags is efficient for clinicalgrade and should be preferred also for the shipment of research-grade islet preparations.

We investigated the effects of nicotinamide (NA) supplementation of the processing medium during islet isolation. One hundred and two human pancreata were processed for clinical transplantation after preservation either in the University of Wisconsin (UW) or using the two-layer method (TLM). Pancreata were then divided into four groups and retrospectively analyzed. Group I: UW preservation followed by processing without NA, Group II: UW preservation and processing with NA, Group III: TLM preservation without NA, Group IV: TLM preservation with NA. We observed a significant increase in islet yield in Group II (4343 ± 348 IEQ/g) [mean ± SEM], compared to Group I (2789 ± 348 IEQ/g) (p = 0.005). Similarly, a significant increase in islet yield was observed when NA was used in the processing of organs preserved with TLM (Group IV: 5538 ± 413 vs. Group III: 3500 ± 629; p = 0.02). Furthermore islet yield was higher in Group IV than in Group II (p < 0.05). The percentages of preparations that qualified for transplantation were 25, 47, 45, 69% in Groups I, II, III, IV, respectively. Addition of NA to the processing medium significantly improved islet yields in both the UW and TLM preservation protocols, allowing for a higher percentage of islet preparations to qualify for clinical transplantation.

Clinical trials demonstrate the regenerative potential of cardiac stem cell (CSC) therapy in the post-infarcted heart. Despite these encouraging preliminary clinical findings, the basic biology of these cells remains largely unexplored. The principal requirement for cell transplantation is to effectively prime them for survival within the unfavorable environment of the infarcted myocardium. In the adult mammalian heart, the β-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a unique post-translational modification that confers cardioprotection from various otherwise lethal stressors. It is not known whether this signaling system exists in cardiac stem cells. In the present study, we demonstrate that protein O-GlcNAcylation is an inducible stress response in adult murine Sca-1+/lin− CSCs and exerts an essential pro-survival role. Post-hypoxic CSCs responded by time-dependently increasing protein O-GlcNAcylation upon reoxygenation. We utilized pharmacological interventions for loss- and gain-of-function, i.e., enzymatic inhibition of OGT (adds the O-GlcNAc modification to proteins) by TT04, or inhibition of OGA (removes O-GlcNAc) by thiamet-G (ThG). Reduction in the O-GlcNAc signal (via TT04, or OGT gene deletion using Cre-mediated recombination) significantly sensitized CSCs to post-hypoxic injury, whereas augmenting O-GlcNAc levels (via ThG) enhanced cell survival. Diminished O-GlcNAc levels render CSCs more susceptible to the onset of post-hypoxic apoptotic processes via elevated PARP cleavage due to enhanced caspase-3/7 activation, whereas promoting O-GlcNAcylation can serve as a pre-emptive anti-apoptotic signal regulating the survival of CSCs. Thus, we report the primary demonstration of protein O-GlcNAcylation as an important pro-survival signal in CSCs, which could enhance CSC survival prior to in vivo autologous transfer.

Selection of an optimal donor pancreas is the first key task for successful islet isolation. We conducted a retrospective multicenter study in 11 centers in North America to develop an islet donor scoring system using donor variables. The data set consisting of 1,056 deceased donors was used for development of scoring system to predict islet isolation success (defined as post-purification islet yield >400,000 islet equivalents). With an aid of univariate logistic regression analyses, we developed North American Islet Donor Score (NAIDS) ranging 0 through 100 points. The c-index in the development cohort was 0.73 [95% confidence interval 0.70 - 0.76]. The success rate increased proportionally as NAIDS increased, from 6.8% success in NAIDS < 50 points to 53.7% success in NAIDS ≥ 80 points. We further validated NAIDS using a separate set of data consisting of 179 islet isolations. Comparable outcome of NAIDS was observed in the validation cohort. The NAIDS may be a useful tool for donor pancreas selection in the clinical practice. Apart from its utility in clinical decision-making, the NAIDS may also be used in research setting as a standardized measurement of pancreas quality.