Genetically Modified Human Bone Marrow Derived Mesenchymal Stem Cells for Improving the Outcome of Human Islet Transplantation

<div><p>The objective of this study was to determine the potential of human bone marrow derived mesenchymal stem cells (hBMSCs) as gene carriers for improving the outcome of human islet transplantation. hBMSCs were characterized for the expression of phenotypic markers and transduced with Adv-hVEGF-hIL-1Ra to overexpress human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra). Human islets were co-cultured with hBMSCs overexpressing hVEGF and hIL-1Ra. Islet viability was determined by membrane fluorescent method and glucose stimulation test. Transduced hBMSCs and human islets were co-transplanted under the kidney capsule of NOD.Cg-<i>Prkdc^scid Il2rg^tm1Wjl</i>/SzJ (NSG) diabetic mice and blood glucose levels were measured over time to demonstrate the efficacy of genetically modified hBMSCs. At the end of study, immunofluorescent staining of kidney section bearing islets was performed for insulin and von Willebrand Factor (vWF). hBMSCs were positive for the expression of CD73, CD90, CD105, CD146 and Stro-1 surface markers as determined by flow cytometry. Transduction of hBMSCs with adenovirus did not affect their stemness and differentiation potential as confirmed by mRNA levels of stem cell markers and adipogenic differentiation of transduced hBMSCs. hBMSCs were efficiently transduced with Adv-hVEGF-hIL-1Ra to overexpress hVEGF and hIL-1Ra. Live dead cell staining and glucose stimulation test have shown that transduced hBMSCs improved the viability of islets against cytokine cocktail. Co-transplantation of human islets with genetically modified hBMSCs improved the glycemic control of diabetic NSG mice as determined by mean blood glucose levels and intraperitoneal glucose tolerance test. Immunofluorescent staining of kidney sections was positive for human insulin and vWF. In conclusion, our results have demonstrated that hBMSCs may be used as gene carriers and nursing cells to improve the outcome of islet transplantation.</p></div

Intraperitoneal glucose tolerance test after 15 days of islet transplantation.

<p>Mice were fasted overnight and then injected intraperitoneally with glucose (2 g/kg of body weight). Blood glucose levels were measured by tail pricking at indicated time points with a glucometer. Data are presented as the mean ± SD (n = 5).</p

Effect of Adv-hVEGF-hIL-1Ra transduced hBMSCs on human serum insulin and C-peptide levels of diabetic NSG.

<p>Mice transplanted with either islet alone or with hBMSCs before or after transduction with Adv-hVEGF-hIL-1Ra at 35 days post transplantation (A) Human serum insulin (B) Human serum C-peptide. Data are presented as the mean ± SD, n = 5. p<0.05 under t-test.</p

Synthesis and Characterization of a Novel Mycophenolic Acid–Quinic Acid Conjugate Serving as Immunosuppressant with Decreased Toxicity

Mycophenolic acid (MPA) is one of the most commonly used immunosuppressive drugs for improving the outcome of cell and organ transplantations. However, an undesired adverse effect of MPA impedes its application in the clinics for post-transplant patients. By conjugating MPA to quinic acid (QA) via amide bonds, we synthesized a novel immunosuppressant, <i>N</i>-[2-[[(4<i>E</i>)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-1-oxo-4-hexen-1-yl]­amino]­ethyl]-(1α,3R,4α,5R)-1,3,4,5-tetrakis­(acetyloxy)­cyclohexanecarboxamide (abbreviated as MQ4), which exhibits improved stability demonstrated by its incubation in vitro with human plasma, suggesting its better resistance to hydrolytic degradation induced by plasma enzyme. While the immunosuppressive effect of MQ4 on human lymphocyte proliferation was partially compromised as shown by flow cytometry, significant decrease in cytotoxicity of MQ4 to insulin producing β cells could compensate this drawback to some degree. There was a decreased level of apoptotic mediator caspase-3, which may contribute to the decreased toxicity of MQ4 to INS-1E cells. MQ4 could further improve insulin stimulation index and downregulate NFκB expression compared to physical mixing of QA to MPA. Taken together, MQ4 is a promising immunosuppressive agent for preventing and minimizing post-transplanted immune rejection

Immunofluorescence staining of the kidney section bearing human islets 35 days after islet transplantation.

<p>Insulin was stained in red to indicate the functional human islets after transplantation. Kidney section of mice bearing human islets; human islets co-transplanted with hBMSCs; human islets co-transplanted with Adv-hVEGF-hIL-1Ra transduced hBMSCs.</p

Immunofluorescence staining of the kidney sections bearing human islets 35 days after islet transplantation.

<p>Insulin was stained in red to indicate the functional human islets after transplantation. vWF was stained in green to indicate the revascularization of transplanted human islets. Kidney section of mice bearing human islets; human islets co-transplanted with hBMSCs; human islets co-transplanted with Adv-hVEGF-hIL-1Ra transduced hBMSCs.</p

Expression of stem cell markers and differentiation of hBMSCs after transduction with Adv-hVEGF-hIL-1Ra at 200 MOI.

<p>(A) Real Time RT-PCR analysis to quantify change in the expression of Nanog and Oct4 stemness markers. Data are represented as the mean ± SD, n = 3. (B) Oil Red and alizarin red S staining of hBMSCs after culture in adipogenic and osteogenic differentiation media, respectively.</p

Stimulation index (SI) of human islets after incubation with inflammatory cytokine cocktail of 5 ng/ml IL-1β, 10 ng/ml TNF-α and 50 ng/ml IFN-γ for 10 days.

<p>Non-transduced islets were used as controls. SI was determined as the ratio of insulin released from islets when they were incubated in Kreb’s buffer containing 22 mM and 2.2 mM glucose. Data are presented as the mean ± SE (n = 3).</p

Protection of islets against cytokine cocktail as shown by Calcein AM and PI staining.

<p>(A) Islets, (B) Islets with hBMSCs, (C) Islets with hBMSCs transduced with Adv-hVEGF-hIL-1Ra at the ratio of 1 islet equivalent to 100 hBMSCs.</p

Effect of Adv-hVEGF-hIL-1Ra transduced hBMSCs on the outcome of human islet transplantation.

<p>(A, B, C) The blood glucose level of every mouse after receiving 2000 human islet equivalent transplanted either alone or hBMSCs before or after transduction with Adv-hVEGF-hIL-1Ra (A–C). (D) Diabetes reversal ratio of the diabetic mice after human islet transplantation. Blood glucose ≤250 mg/dl (dashed line) was identified as reversed-diabetes (dashed line in A, B, C). Black triangles indicate mice receiving islets with Adv-hVEGF-hIL-1Ra transduced hBMSCs; gray squares indicate mice receiving islets with hBMSCs; black diamond’s indicate mice receiving islets only.</p