Plasma-derived extracellular matrix for xenofree and cost-effective organoid modeling for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) causes significant cancer mortality worldwide. Cancer organoids can serve as useful disease models by high costs, complexity, and contamination risks from animal-derived products and extracellular matrix (ECM) that limit its applications. On the other hand, synthetic ECM alternatives also have limitations in mimicking native biocomplexity. This study explores the development of a physiologically relevant HCC organoid model using plasma-derived extracellular matrix as a scaffold and nutritive biomatrix with different cellularity components to better mimic the heterogenous HCC microenvironment. Plasma-rich platelet is recognized for its elevated levels of growth factors, which can promote cell proliferation. By employing it as a biomatrix for organoid culture there is a potential to enhance the quality and functionality of organoid models for diverse applications in biomedical research and regenerative medicine and to better replicate the heterogeneous microenvironment of HCC. METHOD: To generate the liver cancer organoids, HUH-7 hepatoma cells were cultured alone (homogenous model) or with human bone marrow-derived mesenchymal stromal cells and human umbilical vein endothelial cells (heterogeneous model) in plasma-rich platelet extracellular matrix (ECM). The organoids were grown for 14 days and analyzed for cancer properties including cell viability, invasion, stemness, and drug resistance. RESULTS: HCC organoids were developed comprising HUH-7 hepatoma cells with or without human mesenchymal stromal and endothelial cells in plasma ECM scaffolds. Both homogeneous (HUH-7 only) and heterogeneous (mixed cellularity) organoids displayed viability, cancer hallmarks, and chemoresistance. The heterogeneous organoids showed enhanced invasion potential, cancer stem cell populations, and late-stage HCC genetic signatures versus homogeneous counterparts. CONCLUSION: The engineered HCC organoids system offers a clinically relevant and cost-effective model to study liver cancer pathogenesis, stromal interactions, and drug resistance. The plasma ECM-based culture technique could enable standardized and reproducible HCC modeling. It could also provide a promising option for organoid culture and scaling up.

Association between hepatitis C virus genotype 4 and renal cell carcinoma: Molecular and virological studies.

Hepatitis C virus (HCV) is the most common infection worldwide. The correlation between HCV and renal cell carcinoma (RCC) is still mysterious. Therefore, the relationship between HCV and RCC was investigated. The study included 100 patients with RCC; 32 with HCV infection, and 68 without HCV infection. Expressions of viral proteins (NS3 and NS5A) were tested using an immune electron-microscope (IEM) and immunohistochemistry (IHC). IHC and quantitative real time-PCR investigated the presentation of human proteins TP53 and p21 genes. Transmission electron (TEM) detected viral-like particles in infected RCC tissues. The gene and protein expression of P53 was higher in HCV positive versus HCV negative patients and p21 was lower in HCV positive versus HCV negative in both tumor and normal tissue samples. Viral like particles were observed by TEM in the infected tumor and normal portion of the RCC tissues and the plasma samples. The IEM showed the depositions of NS3 and NS5A in infected renal tissues, while in noninfected samples, were not observed. The study hypothesizes that a correlation between HCV and RCC could exist through successfully detecting HCV-like particles, HCV proteins, and (p53 and p21) in RCC-infected patients.

Omega-3 polyunsaturated fatty acids: a modified approach for chemo-prevention of bladder cancer in a rat model and molecular studies of antineoplastic mechanisms.

OBJECTIVES: To test the chemo-preventative effects of omega-3 against bladder cancer (BC) induction in a rat model and its potential antineoplastic mechanisms. MATERIAL AND METHODS: Ninety male Fisher rats were divided into three groups during a 22-week protocol: group 1 (control), group 2 (Placebo + N-butyl-N-4- hydroxybutyl nitrosamine (BBN) for induction of BC and group 3 received omega-3 (1200 mg/kg/day) + BBN. At the end, blood samples and bladder tissues were collected and checked for the presence of malignancy, markers of angiogenesis (VEGF relative gene expression), inflammation (IL-6), proliferation (KI-67 expressions), oxidative stress (serum MDA and serum SOD) and epigenetic control (miRNA-145 level). RESULTS: At the end of the study, 60% and 86.6% rats survived in group 2 and 3 with significant weight loss among rats in group 2 when compared with other groups. In group 2, all rats developed visible bladder lesions of which five and 13 developed squamous cell carcinoma (SCC) and transitional cell carcinoma (TCC). In omega3-treated group, only one developed low grade SCC and one developed high grade non- invasive TCC. Bladders from omega-3-treated rats showed lower expression ofKI-67 (p < 0.05), VEGF (p < 0.001) and IL-6 (p < 0.001) and significant higher expression of mi-RNA (p < 0.001). Also, omega-3-treated group showed statistically significant lower MDA level (p < 0.001). CONCLUSION: Omega-3 inhibits bladder tumor growth in the BBN-induced BC rat model, due to anti-inflammatory, antioxidant, anti-proliferative, and anti-angiogenic properties together with epigenetic control.

Effect of low-energy shock wave therapy on intravesical epirubicin delivery in a rat model of bladder cancer.

OBJECTIVES: To study the efficacy of low-energy shock wave therapy (LESW) on enhancing intravesical epirubicin (EPI) delivery in a rat model of bladder cancer (BCa). MATERIALS AND METHODS: A total of 100 female Fischer rats were randomly allocated into five groups: control; BCa; LESW; EPI; and EPI plus LESW. After BCa induction by N-butyl-N-(4-hydroxybutyl)nitrosamine, EPI (0.6 mg/0.3 mL of EPI diluted in 0.3 mL saline) or saline (0.6 mL) was administered and retained in the bladders for 1 h with or without LESW treatment (300 pulses at 0.12 mJ/mm2 ). This was repeated weekly for 6 weeks. Survival was then calculated, rats were weighed and their bladders were harvested for bladder/body ratio estimation, histopathological examination, p53 immunostaining, miR-210, hypoxia-inducible factor (HIF)-1α, tumour necrosis factor (TNF)-α and interleukin (IL)-6 relative gene expression and fluorescence spectrophotometric drug quantification. Heart and blood samples were also collected for assessment of the safety profile and toxicity. RESULTS: The EPI plus LESW group had significantly lower mortality rates, loss of body weight and bladder/body ratio. Histopathological results in terms of grossly visible bladder lesions, mucosal thickness, dysplasia formation and tumour invasion were significantly better in the combined treatment group. The EPI plus LESW group also had statistically significant lower expression levels of p53 , miR-210, HIF-1α, TNF-α and IL-6. LESW increased urothelial concentration of EPI by 5.7-fold (P < 0.001). No laboratory variable exceeded the reference ranges in any of the groups. There was an improvement of the indicators of EPI-induced cardiomyopathy in terms of congestion, hyalinization and microvesicular steatosis of cardiomyocytes (P = 0.068, 0.003 and 0.046, respectively) in the EPI plus LESW group. CONCLUSIONS: The combined use of intravesical EPI and LESW results in less BCa invasion and less dysplasia formation, as LESW increases urothelial permeability of EPI and enhances its delivery into tumour tissues, without subsequent toxicity.

Prophylaxis against renal ischemia-reperfusion injury in canine model: Stem cell approach.

INTRODUCTION: Stem cell therapy at the time of ischemia/reperfusion (I/R) injury has been hypothesized to attenuate the severity of acute kidney injury and to accelerate the regeneration process in lower animal models. Data in higher animal models is limited and discordant. We aimed to explore the reno-protective effects of stem cells on I/R related renal injury in a canine model. MATERIALS AND METHODS: Twenty-seven dogs that were treated with bone marrow-derived mesenchymal stem cells (BM-MSCs) were compared with another 27 dogs treated with adipose tissue-derived MSCs (AT-MSCs) following 90 min of warm ischemia to assess IR injury. Each group was divided into three subgroups (nine dogs each), according to the stem cell dose (5, 10, 15 × 106 in 500 µl volume) injected directly into the renal cortex after reperfusion. All dogs were re-evaluated by renogram, histopathology, and pro-inflammatory markers at 2 weeks, 2, and 3 months. RESULTS: In Group I, there was a mean reduction of creatinine clearance by 78%, 64%, and 74% at the three used doses, respectively, at 2 weeks. At 3 months, these kidneys regained a mean of 84%, 92%, and 72%, respectively, of its basal function. In Group II, the reduction of clearance was much more modest with mean of 14%, 6%, and 24% respectively at 2 weeks with more intense recovery of renal function by mean of 90%, 100%, and 76%, respectively, at 3 months. Group I had significantly more tubular necrosis and delayed regeneration compared with the Group II. Expressions of pro-inflammatory markers were upregulated in both the groups with a higher and more sustained expression in Group I. CONCLUSION: Stem cells protected against ischemic reperfusion injury in a canine model. AT-MSCs provided better protection than BM-MSCs.

Modulation of renal ischemia/reperfusion in rats by a combination of ischemic preconditioning and adipose-derived mesenchymal stem cells (ADMSCs).

The present study investigated the effects of combination of ischemic preconditioning (Ipre) and adipose-derived mesenchymal stem cells (ADMSCs) on renal ischemia-reperfusion (I-R) injury in rats. 90 male Sprague Dawley rats were divided into 5 equal groups; sham operated, control (45 min left renal ischemia), Ipre group as control group with 3 cycles of Ipre just before renal ischemia, ADMSCs-treated group (as control with ADMSCs 10(6) cells in 0.1 mL via penile vein 60 min before ischemia time), and Ipre + ADMSCs group as ADMCs group with 3 cycles of Ipre. Ipre and ADMSCs groups showed significant decrease in serum creatinine and blood urea nitrogen (BUN) and caspase-3 and CD45 expression in kidney and significant increase in HIF-1α, SDF-1α, CD31, and Ki67 expressions in kidney compared with the control group (p < 0.05). Moreover, the Ipre + ADMSCs group showed significant decrease in serum BUN and caspase-3 and CD45 expression in kidney with significant increase in HIF-1α, SDF-1α, CD31, and Ki67 expression in kidney compared with the Ipre and ADMCs groups (p < 0.05). We concluded that Ipre potentiates the renoprotective effect of ADMSCs against renal I/R injury probably by upregulation of HIF-1α, SDF-1α, CD31, and Ki67 and downregulation of caspase-3 and CD45.

Quality by design approach of apocynin loaded clove oil based nanostructured lipid carrier as a prophylactic regimen in hemorrhagic cystitis in vitro and in vivo comprehensive study.

Apocynin (APO) is a naturally occurring acetophenone with eminent anti-inflammatory and anti-oxidant peculiarities. It suffers from poor bioavailability due to low aqueous solubility. Herein, APO was loaded in a Clove oil (CO) based Nanostructured lipid carrier (NSLC) system using a simple method (ultrasonic emulsification) guided by a quality-by-design approach (23 full factorial design) to optimize the formulated NSLCs. The prepared NSLCs were evaluated regarding particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (EE%). The optimal formula (F2) was extensively investigated through transmission electron microscope (TEM), Fourier transform infrared (FT-IR) spectroscopy, Differential scanning calorimetry (DSC), X-ray diffractometry (XRD), in vitro release, and stability studies. Cytotoxicity against human urinary bladder carcinoma (T24) cell line and in vivo activity studies in rats with induced cystitis were also assessed. The results disclosed that the optimal formula (F2) had PS of 214.8 ± 5.8 nm with EE% of 79.3 ± 0.9%. F2 also exhibited a strong cytotoxic effect toward the T24 cancer cells expressed by IC50 value of 5.8 ± 1.3 µg/mL. Pretreatment with the optimal formula (orally) hinted uroprotective effect against cyclophosphamide (CP)-induced hemorrhagic cystitis (HC) in rat models, emphasized by histopathological, immunohistochemical, and biochemical investigations. In consideration of the simple fabrication process, APO-loaded CO-based NSLCs can hold prospective potential in the prophylaxis of oncologic and urologic diseases.

Modulation of naïve mesenchymal stromal cells by extracellular vesicles derived from insulin-producing cells: an in vitro study.

This study was to determine whether extracellular vesicles (EVs) derived from insulin-producing cells (IPCs) can modulate naïve mesenchymal stromal cells (MSCs) to become insulin-secreting. MSCs were isolated from human adipose tissue. The cells were then differentiated to generate IPCs by achemical-based induction protocol. EVs were retrieved from the conditioned media of undifferentiated (naïve) MSCs (uneducated EVs) and from that of MSC-derived IPCs (educated EVs) by sequential ultracentrifugation. The obtained EVs were co-cultured with naïve MSCs.The cocultured cells were evaluated by immunofluorescence, flow cytometry, C-peptide nanogold silver-enhanced immunostaining, relative gene expression and their response to a glucose challenge.Immunostaining for naïve MSCs cocultured with educated EVs was positive for insulin, C-peptide, and GAD65. By flow cytometry, the median percentages of insulin-andC-peptide-positive cells were 16.1% and 14.2% respectively. C-peptide nanogoldimmunostaining providedevidence for the intrinsic synthesis of C-peptide. These cells released increasing amounts of insulin and C-peptide in response to increasing glucose concentrations. Gene expression of relevant pancreatic endocrine genes, except for insulin, was modest. In contrast, the results of naïve MSCs co-cultured with uneducated exosomes were negative for insulin, C-peptide, and GAD65. These findings suggest that this approach may overcome the limitations of cell therapy.

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.

Impact of insulin producing cells derived from adipose tissue mesenchymal stem cells on testicular dysfunction of diabetic rats.

The present study is to clarify the effect of insulin-producing cells (IPCs) derived from adipose tissue mesenchymal stem cells (AT-MSCs) on diabetic-induced impairments as the abnormalities of testicular tissues, oxidative stress of testes, and defects of spermatogenesis. Diabetes was stimulated by streptozotocin (STZ) injection in male adult Sprague Dawley (SD) rats. Diabetes was confirmed by taking two highly consecutive fasting blood sugar readings; more than 300 mg/dl; within one week. Five million of IPCs derived from AT-MSCs; encased in TheraCyte capsule; were then directly transplanted (one implant for each rat) subcutaneously in diabetic rats. Implants were maintained for 3 months and the fasting blood sugar of the transplanted rats was observed every month. At the end of the experiment; serum testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were also estimated. The sperm parameters (count, motility, and abnormality) were recorded. In testicular tissue; GPX4, Bcl2, and Bax levels were evaluated, while oxidative stress and antioxidant enzymes activities were measured in the testes homogenate. Also, histopathological alterations were examined in the testes cross-section. In the results, it was found that IPCs treatment enhanced the serum testosterone, FSH, and LH levels. Diabetic-induced impairments in the sperm parameters were noticeably improved post-IPCs transplantation in the diabetic rats. Moreover, the treatment improved the diabetic-associated testicular oxidative stress. Also, it was recognized that the Bax expression decreased, while, GPX4 and Bcl2 expression increased in the treated rats. Meanwhile, the abnormalities showed in the histopathological studies of the hyperglycemic rat's testes were attenuated post-treatment. So, IPCs transplantation improved diabetes and consequently protected against hyperglycemia-induced testicular damages.

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.

Copper and zinc levels in plasma and cancerous tissues and their relation with expression of VEGF and HIF-1 in the pathogenesis of muscle invasive urothelial bladder cancer: a case-controlled clinical study.

To evaluate Cu and Zn levels in bladder cancer (BC) patients and their relationship with expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1). Plasma levels of Cu and Zn were determined in 66 transitional bladder cell carcinoma patients (BC group) and 60 matched controls. The concentration of Cu and Zn as well as the expressions of both VEGF and HIF-1 were also estimated in cancerous and non-cancerous bladder tissues in the BC group. The results showed that plasma Cu and Cu/Zn ratio were significantly higher in BC group when compared with the control group. In contrast, the plasma Zn in BC group was significantly lower than in the controls. Comparing levels of Cu and Zn in cancerous and non-cancerous bladder tissues among the BC group indicated a significantly higher Cu levels in the cancerous tissues, while Zn levels was significantly lower. There were higher expressions of both VEGF and HIF-1 in the cancerous samples. Moreover, the Cu concentration in cancerous tissues was significantly correlated with expressions of VEGF and HIF-1. Logistic regression analysis revealed that the increase in plasma Cu/Zn ratio and plasma Cu and the decrease in plasma Zn may be risk factors for development of bladder cancer. We concluded that alteration of plasma and bladder tissue levels of both Cu and Zn is correlated with pathogenesis of bladder cancer. The increase in Cu level in cancerous tissues of BC group has an important role in angiogenesis in bladder cancer cells.

Preventive effect of pomegranate juice against chemically induced bladder cancer: An experimental study.

OBJECTIVES: Pomegranate juice (PJ) is rich in important compounds with anti-cancer activities. This study aims to investigate the preventive effect of pomegranate juice (PJ) against bladder cancer (BC). METHODS: Eighty male Sprague Dawley rats were randomly classified into 4 equal groups: (1) Normal controls; (2) PJ group: supplied by PJ for 12 weeks; (3) Cancer-induced group: intake 0.05% v/v N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) for 8 weeks; (4) Cancer-prevented group: BBN + PJ. After 12 weeks, all rats were sacrificed and their urinary bladder tissues were subjected to histopathological and immunohistochemical (p53) examinations, expression of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), hypoxia-inducible factor 1 (HIF-1) and the tumor protein p53 (TP53) and analysis of oxidative stress markers. RESULTS: The development of BC was: 0/20 (0%) in normal, PJ and cancer-prevented groups and 20/20 (100%) in cancer-induced group. Significant neoplastic lesions were observed in cancer-induced group. Mild preneoplastic alterations were noticed in 25% (5/20) of cancer-prevented group. p53 immunostaining were significantly elevated in the cancer-induced group, which was decreased in the cancer-prevented group. The relative expressions of IL-6, TNF-α, HIF-1 and TP53 were significantly lower in the cancer-prevented group compared to the cancer-treated group. Correction in the oxidative stress markers were also observed in the cancer-prevented group. CONCLUSION: PJ possesses a promising inhibitory effect on BC development, probably due to its anti-oxidant and anti-inflammatory properties.

Urinary hyaluronic acid: a versatile marker of bladder cancer.

PURPOSE: To evaluate the role of urinary hyaluronic acid (HA) as a diagnostic marker in urothelial carcinoma (UCC), squamous cell carcinoma (SCC), and adenocarcinoma (ADC) of urinary bladder and compare it with urine cytology. METHODS: HA was estimated in 170 subjects divided into three groups. Group I: UCC 88 patients, 28 with SCC and 12 with ADC; group II: 34 patients with benign bladder tumors; and group III: 10 healthy bladders. HA was estimated in urine and then readjusted to creatinine (HA/Cr) and protein (HA/Pr) in urine. Urine cytology was evaluated. RESULTS: The mean ± SD level HA was higher in UCC (589 ± 72), SCC (637 ± 45), and ADC (526 ± 30) as compared with benign (476 ± 92) and normal (277 ± 44) groups regardless the grade of tumor (p < 0.0001). A cutoff value of 490 ng/ml was calculated to detect malignancy with sensitivity of 98% and specificity of 66%. PPV, NPV, and ACC were 88.6%, 94.1%, and 90%, respectively. Urine cytology showed sensitivity of, specificity, PPV, NPV, and ACC of 52.6%, 90%, 90.45, 50%, and 65.5%, respectively. HA/Pr and HA/Cr, cutoff values for detection of malignancy were 84.9 and 9.6 but with less predictive values. Histopathological type was the only independent factor affecting level of HA on multivariate analysis, (p = 0.012, Exp (B) 14.98, 95% CI 1.8-121). CONCLUSION: Combination of urinary HA and urine cytology provides reliable marker of bladder cancer.

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.

Subcutaneous transplantation of bone marrow derived stem cells in macroencapsulation device for treating diabetic rats; clinically transplantable site.

BACKGROUND/AIM: Diabetes mellitus (DM) is a serious, chronic and epidemic disease. Its effective therapy with exogenous insulin places an overwhelming burden on the patient's lifestyle. Moreover, pancreatic islet transplantation is limited by the scarceness of donors and the need for chronic immunosuppression. Cell-based therapy is considered an alternative source of insulin-producing cells (IPCs); encapsulating such cellular grafts in immunoisolating devices would protect the graft from immune attack without the need for immunosuppression. Herein, we investigate the ability of TheraCyte capsule as an immunoisolating device to promote the maturation of differentiated rat bone marrow derived mesenchymal stem cells (BM-MSCs), transplanted subcutaneously to treat diabetic rats in comparison with intratesticular transplantation. MAIN METHODS: Rat BM-MSC were differentiated into IPCs, and either encapsulated in TheraCyte capsules for subcutaneous transplantation or transplanted intratesticular into diabetic rats. Serum insulin, C-peptide & blood glucose levels of transplanted animals were monitored. Retrieved cells were further characterized by immunofluorescence staining and gene expression analysis. KEY FINDINGS: Differentiated rat BM-MSC were able to produce insulin in vitro, ameliorate hyperglycemia in vivo and survive for 6 months post transplantation. Transplanted cells induced higher levels of insulin and C-peptide, lower levels of blood glucose in the cured animals of both experimental groups. Gene expression revealed a further in vivo maturation of the implanted cells. SIGNIFICANCE: These data suggest that TheraCyte encapsulation of allogeneic differentiated stem cells are capable of reversing hyperglycemia, which holds a great promise as a new cell based, clinically applicable therapies for diabetes.

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.