Transplantation of insulin-producing cells derived from human mesenchymal stromal/stem cells into diabetic humanized mice.

BACKGROUND: The purpose of this study was to investigate allogenic immune responses following the transplantation of insulin-producing cells (IPCs) differentiated from human adipose tissue-derived stem cells (hAT-MSCs) into humanized mice. METHODS: hAT-MSCs were isolated from liposuction aspirates obtained from HLA-A2-negative healthy donors. These cells were expanded and differentiated into IPCs. HLA-A2-positive humanized mice (NOG-EXL) were divided into 4 groups: diabetic mice transplanted with IPCs, diabetic but nontransplanted mice, nondiabetic mice transplanted with IPCs and normal untreated mice. Three million differentiated cells were transplanted under the renal capsule. Animals were followed-up to determine their weight, glucose levels (2-h postprandial), and human and mouse insulin levels. The mice were euthanized 6-8 weeks posttransplant. The kidneys were explanted for immunohistochemical studies. Blood, spleen and bone marrow samples were obtained to determine the proportion of immune cell subsets (CD4+, CD8+, CD16+, CD19+ and CD69+), and the expression levels of HLA-ABC and HLA-DR. RESULTS: Following STZ induction, blood glucose levels increased sharply and were then normalized within 2 weeks after cell transplantation. In these animals, human insulin levels were measurable while mouse insulin levels were negligible throughout the observation period. Immunostaining of cell-bearing kidneys revealed sparse CD45+ cells. Immunolabeling and flow cytometry of blood, bone marrow and splenic samples obtained from the 3 groups of animals did not reveal a significant difference in the proportions of immune cell subsets or in the expression levels of HLA-ABC and HLA-DR. CONCLUSION: Transplantation of IPCs derived from allogenic hAT-MSCs into humanized mice was followed by a muted allogenic immune response that did not interfere with the functionality of the engrafted cells. Our findings suggest that such allogenic cells could offer an opportunity for cell therapy for insulin-dependent diabetes without immunosuppression, encapsulation or gene manipulations.

From Mesenchymal Stromal/Stem Cells to Insulin-Producing Cells: Immunological Considerations.

Mesenchymal stem cell (MSC)-based therapy for type 1 diabetes mellitus (T1DM) has been the subject matter of many studies over the past few decades. The wide availability, negligible teratogenic risks and differentiation potential of MSCs promise a therapeutic alternative to traditional exogenous insulin injections or pancreatic transplantation. However, conflicting arguments have been reported regarding the immunological profile of MSCs. While some studies support their immune-privileged, immunomodulatory status and successful use in the treatment of several immune-mediated diseases, others maintain that allogeneic MSCs trigger immune responses, especially following differentiation or in vivo transplantation. In this review, the intricate mechanisms by which MSCs exert their immunomodulatory functions and the influencing variables are critically addressed. Furthermore, proposed avenues to enhance these effects, including cytokine pretreatment, coadministration of mTOR inhibitors, the use of Tregs and gene manipulation, are presented. As an alternative, the selection of high-benefit, low-risk donors based on HLA matching, PD-L1 expression and the absence of donor-specific antibodies (DSAs) are also discussed. Finally, the necessity for the transplantation of human MSC (hMSC)-derived insulin-producing cells (IPCs) into humanized mice is highlighted since this strategy may provide further insights into future clinical applications.

Impact of Therapeutic Dose Monitoring of Mycophenolic Acid on the Outcome of Live-Donor Kidney Transplant Recipients - A Prospective Controlled Study.

Immunosuppressive therapy is the backbone to renal transplantation. Although an adequate level of immunosuppression is required to dampen the immune response to the allograft, the level of chronic immunosuppression is slowly decreased over time (as the risk of acute rejection decreases) to help lower the overall risk of infection and malignancy. Several studies have discussed the clinical use of therapeutic drug monitoring of mycophenolic acid (MPA) in kidney transplant recipients. This prospective single-center study included 88 patients with end-stage renal disease who were transplanted in Mansoura Urology and Nephrology Center from living related donors, from the beginning of February 2016 to the end of December 2016. Eight patients were excluded, the remaining 80 patients were divided into two groups; the study group (40 patients) who were followed up using therapeutic trough level monitoring of MPA and, control group (40 patients) who were followed up using the fixed-dose of Mycophenolate according to our local immunosuppressive protocol. These patients were followed up for one year posttransplantation with regard to graft function, rejection episodes, gastrointestinal (GI), and hematological side effects, the incidence of infection or malignancy, patient survival, and graft survival. Fifteen patients from the study group (37.5%) needed dose reduction of MPA, no patients needed to increase the dose. Our study showed insignificant differences regarding the patients' characteristics and demographic data. Significantly higher incidence of GI manifestations was noted in the control group (P = 0.001). Although the higher frequency of incidence of infection, anemia, leukopenia and thrombocytopenia was seen in the fixed- dose group, the difference was statistically insignificant. Regarding proteinuria and post-transplant diabetes mellitus, comparable data were obtained. Significantly higher percentage of recipients in the study group is still having normally functioning grafts (P = 0.02). Furthermore, higher percent of recipients in the control group died with functioning graft after one year of follow-up (P = 0.04). There were insignificant differences as regarding patient and graft survival. The decrease in the dose of MPA reduced the annual cost by around six thousand US dollars. Our results suggest that adopting therapeutic dose monitoring strategy during follow-up of kidney transplant recipients is adequate. Longer-term studies with a larger sample size may be needed to support this policy.

Evaluation of serum fatty acid binding protein-4 (FABP-4) as a novel biomarker to predict biopsy outcomes in prostate biopsy naïve patients.

OBJECTIVES: To investigate the role of serum fatty acid-binding protein-4 (FABP-4) as a surrogate of obesity and metabolic syndrome in the prediction of the outcome of prostate biopsy. METHODS: A prospective pilot study was conducted for patients undergoing prostate needle biopsy (PNB) for clinically suspected prostate cancer (PCa) between June 2016 and August 2017. Fifty consecutive patients with biopsy-proven PCa were included as study group and 50 consecutive patients with negative biopsy were included as a control group. Receiver Operating Characteristic (ROC) curve was used to calculate the area under the curve (AUC) to compare the accuracy of the different parameters in the diagnosis as well as the presence of high-grade PCa (Gleason score 8-9) at PNB. Predictors of the outcome were analyzed using univariate and multivariate logistic regression analysis. RESULTS: FABP-4 (AUC: 0.75; P < 0.001) and PSA-density (AUC: 0.84; P < 0.001) were the most accurate to detect PCa at PNB. On multivariate analysis, FABP-4 > 22.5 ng/ml (OR: 16.6; 95% CI 2.8-98; P = 0.002) and PSA-density > 0.38 ng/ml/ml OR: 17.7; 95% CI 5.3-59; P < 0.001) were independent predictors of PCa detection. Regarding high-grade PCa at PNB, FABP-4 (AUC: 0.79; P < 0.001) and %Free-PSA (AUC: 0.75; P < 0.001) were the most accurate. Independent predictors of high-grade PCa were FABP-4 > 32.3 ng/ml OR: 9.2; 95% CI 1.8-45; P = 0.006) and %Free-PSA ≤ 21.9 (OR: 5.5; 95% CI 1.1-27; P = 0.03). CONCLUSIONS: FABP-4 is an independent predictor for both the diagnosis and high-grade Gleason score at PNB. This novel biomarker might have a promising role in optimizing PNB outcomes.

PRDX6 Promotes the Differentiation of Human Mesenchymal Stem (Stromal) Cells to Insulin-Producing Cells.

Mesenchymal stem cells (MSCs) can be differentiated in vitro to form insulin-producing cells (IPCs). However, the proportion of induced cells is modest. Extracts from injured pancreata of rodents promoted this differentiation, and three upregulated proteins were identified in these extracts. The aim of this study was to evaluate the potential benefits of adding these proteins to the differentiation medium alone or in combination. Our results indicate that the proportion of IPCs among the protein(s)-supplemented samples was significantly higher than that in the samples with no added proteins. The yield from samples supplemented with PRDX6 alone was 4-fold higher than that from samples without added protein. These findings were also supported by the results of fluorophotometry. Gene expression profiles revealed higher levels among protein-supplemented samples. Significantly higher levels of GGT, SST, Glut-2, and MafB expression were noted among PRDX6-treated samples. There was a stepwise increase in the release of insulin and c-peptide, as a function of increasing glucose concentrations, indicating that the differentiated cells were glucose sensitive and insulin responsive. PRDX6 exerts its beneficial effects as a result of its biological antioxidant properties. Considering its ease of use as a single protein, PRDX6 is now routinely used in our differentiation protocols.

Serum lipids might improve prostate-specific antigen sensitivity in patients undergoing transrectal ultrasonography-guided biopsy for suspected prostate cancer: A pilot study.

Objectives: To investigate the potential use of body mass index (BMI) and serum lipids in improving prostate-specific antigen (PSA) sensitivity in patients undergoing biopsy for suspicion of prostate cancer, as there is an established relationship between metabolic syndrome, obesity and serum lipids with prostate cancer. Patients and methods: A pilot study was conducted in a tertiary referral centre between June 2016 and August 2017 of patients undergoing transrectal ultrasonography (TRUS)-guided biopsy. After the result of TRUS-biopsy, the first 50 patients diagnosed with prostate cancer (study group) and those with no prostate cancer (control group) were enrolled. BMI, serum PSA level, fasting blood sugar and lipid profile (e.g. cholesterol, triglycerides, low-density lipoprotein [LDL] and high-density lipoprotein [HDL]), were compared between the groups. Results: Higher BMI, cholesterol, LDL and lower HDL together with PSA were significantly associated with a positive biopsy. On multivariate analysis, LDL (odds ratio [OR] 5.3, 95% confidence interval [CI] 1.2-24.9; P = 0.03) and total PSA level (OR 12.9, 95% CI 4.7-35; P < 0.001) were independent predictors of a positive biopsy. A combination of LDL <80 mg/dL and PSA level <26 ng/mL threshold values determined by receiver operating characteristic curve analysis, had a sensitivity and specificity of 94% and 28%, respectively; whilst, the negative (NPV) and positive predictive values were 82.4% and 56.6%, respectively. The sensitivity and NPV of the combination was significantly higher than that of PSA level alone (94% vs 72% and 82.4% vs 75%, respectively; P < 0.001). Conclusions: Serum lipids might have a role in the diagnosis of prostate cancer and could be used as an adjunct to PSA measurement to improve sensitivity and avoid unnecessary biopsies. Abbreviations: AUC: area under the curve; BMI: body mass index; FBS: fasting blood sugar; HDL: high-density lipoprotein; LDL: low-density lipoprotein; LOX-1: lectin-like oxidised LDL receptor-1; OR: odds ratio; ROC: receiver operating characteristic; RP: radical prostatectomy; TG: triglyceride.

Differentiation Potential of Nestin (+) and Nestin (-) Cells Derived from Human Bone Marrow Mesenchymal Stem Cells into Functional Insulin Producing Cells.

The feasibility of isolating and manipulating mesenchymal stem cells (MSCs) from human patients provides hope for curing numerous diseases and disorders. Recent phenotypic analysis has shown heterogeneity of MSCs. Nestin progenitor cell is a subpopulation within MSCs which plays a role in pancreas regeneration during embryogenesis. This study aimed to separate nestin (+) cells from human bone marrow MSCs, and differentiate these cells into functional insulin producing cells (IPCs) compared with nestin (-) cells. Manual magnetic separation was performed to obtain nestin (+) cells from MSCs. Approximately 91±3.3% of nestin (+) cells were positive for anti-nestin antibody. Pluripotent genes were overexpressed in nestin (+) cells compared with nestin (-) cells as revealed by quantitative real time-PCR (qRT-PCR). Following in vitro differentiation, flow cytometric analysis showed that 2.7±0.5% of differentiated nestin (+) cells were positive for anti-insulin antibody in comparison with 0.08±0.02% of nestin (-) cells. QRT-PCR showed higher expression of insulin and other endocrine genes in comparison with nestin (-) cells. While immunofluorescence technique showed the presence of insulin and C-peptide granules in nestin (+) cells. Therefore, our results introduced nestin (+) cells as a pluripotent subpopulation within human MSCs which is capable to differentiate and produce functional IPCs.

Insulin-producing Cells from Adult Human Bone Marrow Mesenchymal Stromal Cells Could Control Chemically Induced Diabetes in Dogs: A Preliminary Study.

Ten mongrel dogs were used in this study. Diabetes was chemically induced in 7 dogs, and 3 dogs served as normal controls. For each diabetic dog, 5 million human bone marrow-derived mesenchymal stem cells/kg were differentiated to form insulin-producing cells using a trichostatin-based protocol. Cells were then loaded in 2 TheraCyte capsules which were transplanted under the rectus sheath. One dog died 4 d postoperatively from pneumonia. Six dogs were followed up with for 6 to 18 mo. Euglycemia was achieved in 4 dogs. Their glucose tolerance curves exhibited a normal pattern demonstrating that the encapsulated cells were glucose sensitive and insulin responsive. In the remaining 2 dogs, the fasting blood sugar levels were reduced but did not reach normal values. The sera of all transplanted dogs contained human insulin and C-peptide with a negligible amount of canine insulin. Removal of the transplanted capsules was followed by prompt return of diabetes. Intracytoplasmic insulin granules were seen by immunofluorescence in cells from the harvested capsules. Furthermore, all pancreatic endocrine genes were expressed. This study demonstrated that the TheraCyte capsule or a similar device can provide adequate immunoisolation, an important issue when stem cells are considered for the treatment of type 1 diabetes mellitus.

In vitro differentiation of human multilineage differentiating stress-enduring (Muse) cells into insulin producing cells.

Mesenchymal stem cells (MSCs) is a heterogeneous population. Muse cells is a rare pluripotent subpopulation within MSCs. This study aims to evaluate the pulirpotency and the ability of Muse cells to generate insulin producing cells (IPCs) after in vitro differentiation protocol compared to the non-Muse cells. Muse cells were isolated by FACSAria III cell sorter from adipose-derived MSCs and were evaluated for its pluripotency. Following in vitro differentiation, IPCs derived from Muse and non-Muse cells were evaluated for insulin production. Muse cells comprised 3.2 ±â€¯0.7% of MSCs, approximately 82% of Muse cells were positive for anti stage-specific embryonic antigen-3 (SSEA-3). Pluripotent markers were highly expressed in Muse versus non-Muse cells. The percentage of generated IPCs by flow cytometric analysis was higher in Muse cells. Under confocal microscopy, Muse cells expressed insulin and c-peptide while it was undetected in non-Muse cells. Our results introduced Muse cells as a new adult pluripotent subpopulation, which is capable to produce higher number of functional IPCs.

From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells.

The aim of this study is to compare human bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs), for their differentiation potentials to form insulin-producing cells. BM-MSCs were obtained during elective orthotopic surgery and AT-MSCs from fatty aspirates during elective cosmetics procedures. Following their expansion, cells were characterized by phenotyping, trilineage differentiation ability, and basal gene expression of pluripotency genes and for their metabolic characteristics. Cells were differentiated according to a Trichostatin-A based protocol. The differentiated cells were evaluated by immunocytochemistry staining for insulin and c-peptide. In addition the expression of relevant pancreatic endocrine genes was determined. The release of insulin and c-peptide in response to a glucose challenge was also quantitated. There were some differences in basal gene expression and metabolic characteristics. After differentiation the proportion of the resulting insulin-producing cells (IPCs), was comparable among both cell sources. Again, there were no differences neither in the levels of gene expression nor in the amounts of insulin and c-peptide release as a function of glucose challenge. The properties, availability, and abundance of AT-MSCs render them well-suited for applications in regenerative medicine. Conclusion. BM-MSCs and AT-MSCs are comparable regarding their differential potential to form IPCs. The availability and properties of AT-MSCs render them well-suited for applications in regenerative medicine.

A randomised controlled trial evaluating renal protective effects of selenium with vitamins A, C, E, verapamil, and losartan against extracorporeal shockwave lithotripsy-induced renal injury.

OBJECTIVE: To evaluate the protective effects of selenium with vitamins A, C and E (selenium ACE, i.e. antioxidants), verapamil (calcium channel blocker), and losartan (angiotensin receptor blocker) against extracorporeal shockwave lithotripsy (ESWL)-induced renal injury. PATIENTS AND METHODS: A randomised controlled trial was conducted between August 2012 and February 2015. Inclusion criteria were adult patients with a single renal stone (<2 cm) suitable for ESWL. Patients with diabetes, hypertension, congenital renal anomalies, moderate or marked hydronephrosis, or preoperative albuminuria (>300 mg/L) were excluded. ESWL was performed using the electromagnetic DoLiS lithotripter. Eligible patients were randomised into one of four groups using sealed closed envelopes: Group1, control; Group 2, selenium ACE; Group 3, losartan; and Group 4, verapamil. Albuminuria and urinary neutrophil gelatinase-associated lipocalin (uNGAL) were estimated after 2-4 h and 1 week after ESWL. The primary outcome was differences between albuminuria and uNGAL. Dynamic contrast-enhanced magnetic resonance imaging was performed before ESWL, and at 2-4 h and 1 week after ESWL to compare changes in renal perfusion. RESULTS: Of 329 patients assessed for eligibility, the final analysis comprised 160 patients (40 in each group). Losartan was the only medication that showed significantly lower levels of albuminuria after 1 week (P < 0.001). For perfusion changes, there was a statistically significant decrease in the renal perfusion in patients with obstructed kidneys in comparison to before ESWL (P = 0.003). These significant changes were present in the control or antioxidant group, whilst in the losartan and verapamil groups renal perfusion was not significantly decreased. CONCLUSIONS: Losartan was found to protect the kidney against ESWL-induced renal injury by significantly decreasing post-ESWL albuminuria. Verapamil and losartan maintained renal perfusion in patients with post-ESWL renal obstruction.

Impact of dialyzer membrane flux on metal clearance in hemodialysis patients.

Deficiency of essential trace elements (such as Cu or Zn) and accumulation of potentially toxic trace elements (as Cd or Pb) are both known to have adverse effects in hemodialysis (HD) patients. Up to our knowledge, no studies about the permeability of low and high flux polysulfone membranes on metal ions during hemodialysis are available. Therefore, the aim of the present study was to address this issue. Forty one hemodialysis patients (19 were using high flux polysulfone membrane while the remaining were using low flux one) participated in the study. Blood levels of Cu, Zn, Cd and Pb were determined by graphite furnace atomic absorption spectrometry among HD patients, before and after dialysis session, as well as among matched 40 healthy persons. Blood concentrations of Cu and Zn in the whole hemodialysis group was significantly lower than those of the healthy control group, on the other hand the toxic metals (Cd and Pb) levels were observed to be significantly higher among HD patients compared to the normal persons. Among the hemodialysis group, there were no significant differences between the low and high flux dialyzer groups in terms of pre-dialysis blood levels of Cu, Zn, Cd and Pb. In addition, significantly decreased levels of all metal ions were observed after dialysis sessions using either low or high flux membranes. An exception was Pb which did not show any difference between pre-dialysis and post-dialysis values in the low flux groupIn conclusion Zn and Cu deficiencies should be considered in the treatment of these patients. High flux membranes are more efficient than low flux ones in removing excess Cd and Pb. Therefore, when high flux membranes are used, chelation therapy might not be required for Cd and Pb overload.

Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells into Insulin-Producing Cells: Evidence for Further Maturation In Vivo.

The aim of this study was to provide evidence for further in vivo maturation of insulin-producing cells (IPCs) derived from human bone marrow-derived mesenchymal stem cells (HBM-MSCs). HBM-MSCs were obtained from three insulin-dependent type 2 diabetic volunteers. Following expansion, cells were differentiated according to a trichostatin-A/GLP protocol. One million cells were transplanted under the renal capsule of 29 diabetic nude mice. Blood glucose, serum human insulin and c-peptide levels, and glucose tolerance curves were determined. Mice were euthanized 1, 2, 4, or 12 weeks after transplantation. IPC-bearing kidneys were immunolabeled, number of IPCs was counted, and expression of relevant genes was determined. At the end of in vitro differentiation, all pancreatic endocrine genes were expressed, albeit at very low values. The percentage of IPCs among transplanted cells was small (≤3%). Diabetic animals became euglycemic 8 ± 3 days after transplantation. Thereafter, the percentage of IPCs reached a mean of ~18% at 4 weeks. Relative gene expression of insulin, glucagon, and somatostatin showed a parallel increase. The ability of the transplanted cells to induce euglycemia was due to their further maturation in the favorable in vivo microenvironment. Elucidation of the exact mechanism(s) involved requires further investigation.

Determination of Cu(2+), Zn(2+) and Pb(2+) in biological and food samples by FAAS after preconcentration with hydroxyapatite nanorods originated from eggshell.

Hydroxyapatite nanorods (HAPNRs) were prepared from recycled eggshell by using precipitation method. The structure of the HAPNRs was physicochemically and morphologically characterized by X-ray diffraction, transmission electron microscopy and Fourier transform infrared spectroscopy. The resulting HAPNRs were used for solid phase extractive preconcentration of Cu(2+), Zn(2+) and Pb(2+) prior to its determination by flame atomic absorption spectrometry. Experimental variables that influence the quantitative extraction of metal ions were optimized by both batch and column methods. The analytes were quantitatively sorbed on the matrix between pHs6 and 9. The maximum sorption capacity of the HAPNRs has been found to be 2.43, 2.37 and 2.53 mmol g(-1) for Cu(2+), Zn(2+) and Pb(2+), respectively, with the preconcentration factor of 250. The 3σ detection limit and 10σ quantification limit for Cu(2+), Zn(2+) and Pb(2+) were found to be 0.72, 0.55 and 5.12 µg L(-1) and 2.40, 1.83 and 17.06 µg L(-1), respectively. The calibration curves were linear up to 250 µg L(-1) for Cu(2+), 300 µg L(-1) for Zn(2+) and 400 µg L(-1) for Pb(2+). Accuracy of the proposed method was verified using certified reference materials (NCS ZC85006 Tomato, Seronorm Trace Elements Whole Blood L-1, Seronorm Trace Elements Whole Blood L-3 and Seronorm Trace Elements Urine). The present method was successfully applied to the analysis of these metal ions in sea water, biological and food samples.

Structure of Yin Yang 1 oligomers that cooperate with RuvBL1-RuvBL2 ATPases.

Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood. Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1. Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination.

Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin.

Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity.

Long term prospective assessment of living kidney donors: single center experience.

Virtually, all studies reporting the outcomes of living kidney donation beyond the first year from donation were retrospective. In this prospective study, the outcome of 81 consecutive living kidney donors was thoroughly evaluated. Clinical, laboratory, and radiological assessments were carried out at predonation (basal), 3, 6, 12, and 24 months after donation. The mean age at time of donation was 37.8 ± 9.8 years and the majority was females (75.3%). The mean BMI increased significantly after donation (P < 0.04). The mean serum creatinine levels (mg/dl) were 0.75 ± 0.14, 1.01 ± 0.22, 0.99 ± 0.21, 0.98 ± 0.20, and 0.94 ± 0.20 (P < 0.0001). Likewise, the mean levels of measured creatinine clearance (mL/min) were 148.8 ± 35.7, 94.7 ± 26.6, 95.5 ± 24.6, 96.7 ± 20.2, and 101.6 ± 26.2 (P < 0.0001). The mean 24 hours urinary protein excretion (gm/dL) were 0.09 ± 0.03, 0.19 ± 0.18, 0.16 ± 0.09, 0.18 ± 0.25, and 0.17 ± 0.12 (P < 0.0001). There were significant increases in the means of the longitudinal and transverse diameters of the remaining kidney over time (P < 0.001). Out of 42 female donors, eleven female donors have got successful postdonation pregnancies. There were no reported surgical complications, either intra- or postoperative. Long-term follow-up is necessary for all living kidney donors through local institutional and world registries. This trial is registered with ClinicalTrials.gov NCT00813579.

Generation of insulin-producing cells from human bone marrow-derived mesenchymal stem cells: comparison of three differentiation protocols.

INTRODUCTION: Many protocols were utilized for directed differentiation of mesenchymal stem cells (MSCs) to form insulin-producing cells (IPCs). We compared the relative efficiency of three differentiation protocols. METHODS: Human bone marrow-derived MSCs (HBM-MSCs) were obtained from three insulin-dependent type 2 diabetic patients. Differentiation into IPCs was carried out by three protocols: conophylline-based (one-step protocol), trichostatin-A-based (two-step protocol), and ß -mercaptoethanol-based (three-step protocol). At the end of differentiation, cells were evaluated by immunolabeling for insulin production, expression of pancreatic endocrine genes, and release of insulin and c-peptide in response to increasing glucose concentrations. RESULTS: By immunolabeling, the proportion of generated IPCs was modest ( ≃ 3%) in all the three protocols. All relevant pancreatic endocrine genes, insulin, glucagon, and somatostatin, were expressed. There was a stepwise increase in insulin and c-peptide release in response to glucose challenge, but the released amounts were low when compared with those of pancreatic islets. CONCLUSION: The yield of functional IPCs following directed differentiation of HBM-MSCs was modest and was comparable among the three tested protocols. Protocols for directed differentiation of MSCs need further optimization in order to be clinically meaningful. To this end, addition of an extracellular matrix and/or a suitable template should be attempted.

DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities.

MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.