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Do H.V.T.,Humphrey Oei Institute of Cancer Research | Loke W.T.,Humphrey Oei Institute of Cancer Research | Kee I.,SingHealth Experimental Medicine Center | Liang V.,SingHealth Experimental Medicine Center | And 11 more authors.
Cell Transplantation | Year: 2015

Cell therapy could potentially meet the need for pancreas and islet transplantations in diabetes mellitus that far exceeds the number of available donors. Bone marrow stromal cells are widely used in clinical trials mainly for their immunomodulatory effects with a record of safety. However, less focus has been paid to developing these cells for insulin secretion by transfection. Although murine models of diabetes have been extensively used in gene and cell therapy research, few studies have shown efficacy in large preclinical animal models. Here we report optimized conditions for ex vivo expansion and characterization of porcine bone marrow stromal cells and their permissive expression of a transfected insulin gene. Our data show that these cells resemble human bone marrow stromal cells in surface antigen expression, are homogeneous, and can be reproducibly isolated from outbred Yorkshire–Landrace pigs. Porcine bone marrow stromal cells were efficiently expanded in vitro to >1010 cells from 20 ml of bone marrow and remained karyotypically normal during expansion. These cells were electroporated with an insulin expression plasmid vector with high efficiency and viability, and secreted human insulin and C-peptide indicating appropriate processing of proinsulin. We showed that autologous insulin-secreting bone marrow stromal cells implanted and engrafted in the liver of a streptozotocin-diabetic pig that modeled type 1 diabetes resulted in partial, but significant, improvement in hyperglycemia that could not be ascribed to regeneration of endogenous β-cells. Glucose-stimulated insulin secretion in vivo from implanted cells in the treated pig was documented by a rise in serum human C-peptide levels during intravenous glucose tolerance tests. Compared to a sham-treated control pig, this resulted in significantly reduced fasting hyperglycemia, a slower rise in serum fructosamine, and prevented weight loss. Taken together, this study suggests that bone marrow stromal cells merit further development as autologous cell therapy for diabetes. © 2015 Cognizant Comm. Corp.


Wang W.,Cellular and Molecular Research | Iyer N.G.,Cellular and Molecular Research | Iyer N.G.,The Surgical Center | Tay H.T.,Singapore General Hospital | And 10 more authors.
BMC Cancer | Year: 2015

Background: Despite advances in therapeutics, outcomes for hepatocellular carcinoma (HCC) remain poor and there is an urgent need for efficacious systemic therapy. Unfortunately, drugs that are successful in preclinical studies often fail in the clinical setting, and we hypothesize that this is due to functional differences between primary tumors and commonly used preclinical models. In this study, we attempt to answer this question by comparing tumor morphology and gene expression profiles between primary tumors, xenografts and HCC cell lines. Methods: Hep G2 cell lines and tumor cells from patient tumor explants were subcutaneously (ectopically) injected into the flank and orthotopically into liver parenchyma of Mus Musculus SCID mice. The mice were euthanized after two weeks. RNA was extracted from the tumors, and gene expression profiling was performed using the Gene Chip Human Genome U133 Plus 2.0. Principal component analyses (PCA) and construction of dendrograms were conducted using Partek genomics suite. Results: PCA showed that the commonly used HepG2 cell line model and its xenograft counterparts were vastly different from all fresh primary tumors. Expression profiles of primary tumors were also significantly divergent from their counterpart patient-derived xenograft (PDX) models, regardless of the site of implantation. Xenografts from the same primary tumors were more likely to cluster together regardless of site of implantation, although heat maps showed distinct differences in gene expression profiles between orthotopic and ectopic models. Conclusions: The data presented here challenges the utility of routinely used preclinical models. Models using HepG2 were vastly different from primary tumors and PDXs, suggesting that this is not clinically representative. Surprisingly, site of implantation (orthotopic versus ectopic) resulted in limited impact on gene expression profiles, and in both scenarios xenografts differed significantly from the original primary tumors, challenging the long-held notion that orthotopic PDX model is the gold standard preclinical model for HCC. © 2015 Wang et al.


PubMed | Cellular and Molecular Research, The Surgical Center, Nanyang Technological University, 11 Hospital Drive and 3 more.
Type: | Journal: BMC cancer | Year: 2015

Despite advances in therapeutics, outcomes for hepatocellular carcinoma (HCC) remain poor and there is an urgent need for efficacious systemic therapy. Unfortunately, drugs that are successful in preclinical studies often fail in the clinical setting, and we hypothesize that this is due to functional differences between primary tumors and commonly used preclinical models. In this study, we attempt to answer this question by comparing tumor morphology and gene expression profiles between primary tumors, xenografts and HCC cell lines.Hep G2 cell lines and tumor cells from patient tumor explants were subcutaneously (ectopically) injected into the flank and orthotopically into liver parenchyma of Mus Musculus SCID mice. The mice were euthanized after two weeks. RNA was extracted from the tumors, and gene expression profiling was performed using the Gene Chip Human Genome U133 Plus 2.0. Principal component analyses (PCA) and construction of dendrograms were conducted using Partek genomics suite.PCA showed that the commonly used HepG2 cell line model and its xenograft counterparts were vastly different from all fresh primary tumors. Expression profiles of primary tumors were also significantly divergent from their counterpart patient-derived xenograft (PDX) models, regardless of the site of implantation. Xenografts from the same primary tumors were more likely to cluster together regardless of site of implantation, although heat maps showed distinct differences in gene expression profiles between orthotopic and ectopic models.The data presented here challenges the utility of routinely used preclinical models. Models using HepG2 were vastly different from primary tumors and PDXs, suggesting that this is not clinically representative. Surprisingly, site of implantation (orthotopic versus ectopic) resulted in limited impact on gene expression profiles, and in both scenarios xenografts differed significantly from the original primary tumors, challenging the long-held notion that orthotopic PDX model is the gold standard preclinical model for HCC.


Hennedige T.,National Cancer Center | Koh T.S.,National Cancer Center | Hartono S.,National Cancer Center | Yan Y.Y.,National Cancer Center | And 9 more authors.
Magnetic Resonance Imaging | Year: 2015

Purpose: To evaluate non-invasive imaging biomarkers for assessing renal fibrosis. DWI is used to assess renal function; intravoxel incoherent motion (IVIM) provides additional measures of perfusion-related diffusion (D*, blood flow; f, perfusion fraction). We aim to determine if reduced ADC seen in renal fibrosis is attributable to perfusion-related diffusion changes or to known reduction in tissue diffusivity (D). Materials and methods: Unilateral ureteral obstruction (UUO) was created in six mice to induce renal fibrosis. DWI was performed the day before and 7 days post-UUO. A range of b-values from 0 to 1200 s/mm2 were used. IVIM parameters were obtained using region of interests drawn over the renal parenchyma. Histopathological analysis of both kidneys was performed in all mice. Results were analyzed using the paired t-test with P<0.05 considered statistically significant. Results: D and f were significantly lower in the ligated kidneys at Day 7 compared to before ligation and no significant difference was found for D*. Comparing non-ligated and ligated kidneys within the same mouse at Day 7, significantly lower D values were observed in the ligated kidneys, while no significant difference was found for f and D*, although the values of f were generally lower. Histopathological analysis confirmed development of fibrosis and reduction in glomeruli in all the ligated kidneys at Day 7. Conclusion: Our study shows that the reduction in ADC seen in renal fibrosis is attributable not only to reduced D as previously encountered but also a decrease in vascularity as assessed by f. Reduction in f is possibly related to a reduction in glomeruli. © 2015.


Mehta A.,Third Hospital Avenue | Chung Y.Y.,Third Hospital Avenue | Ng A.,Third Hospital Avenue | Iskandar F.,SingHealth Experimental Medicine Center | And 6 more authors.
Cardiovascular Research | Year: 2011

Aims Generation of human induced pluripotent stem cell (hiPSC) lines by reprogramming of fibroblast cells with virus-free methods offers unique opportunities for translational cardiovascular medicine. The aim of the study was to reprogramme fibroblast cells to hiPSCs and to study cardiomyogenic properties and ion channel characteristics of the virus-free hiPSC-derived cardiomyocytes. Methods and resultsThe hiPSCs generated by episomal vectors generated teratomas in severe combined immunodeficient mice, readily formed embryoid bodies, and differentiated into cardiomyocytes with comparable efficiency to human embryonic stem cells. Temporal gene expression of these hiPSCs indicated that differentiation of cardiomyocytes was initiated by increasing expression of cardio/mesodermal markers followed by cardiac-specific transcription factors, structural, and ion channel genes. Furthermore, the cardiomyocytes showed characteristic cross-striations of sarcomeric proteins and expressed calcium-handling and ion channel proteins, confirming their cardiac ontogeny. Microelectrode array recordings established the electrotonic development of a functional syncytium that responded predictably to pharmacologically active drugs. The cardiomyocytes showed a chronotropic doseresponse (0.110 M) to isoprenaline and Bay K 8644. Furthermore, carbamycholine (5 M) suppressed the response to isoprenaline, while verapamil (2.5 M) blocked Bay K 8644-induced inotropic activity. Moreover, verapamil (1 M) reduced the corrected field potential duration by 45, tetrodotoxin (10 M) shortened the minimal field potential by 40, and E-4031 (50 nM) prolonged field repolarization. ConclusionVirus-free hiPSCs differentiate efficiently into cardiomyocytes with cardiac-specific molecular, structural, and functional properties that recapitulate the developmental ontogeny of cardiogenesis. These results, coupled with the potential to generate patient-specific hiPSC lines, hold great promise for the development of an in vitro platform for drug pharmacogenomics, disease modelling, and regenerative medicine. © 2011 The Author.


Ramachandran S.,KK Womens and Childrens Hospital | Ong Y.-S.,Singapore General Hospital | Chin A.Y.H.,Singapore General Hospital | Song I.-C.,Singhealth Experimental Medicine Center | And 2 more authors.
Archives of Plastic Surgery | Year: 2014

Microsurgery training in Singapore began in 1980 with the opening of the Experimental Surgical Unit. Since then, the unit has continued to grow and have held microsurgical training courses biannually. The road to becoming a full-fledged reconstructive surgeon requires the mastering of both microvascular as well as flap raising techniques and requires time, patience and good training facilities. In Singapore, over the past 2 decades, we have had the opportunity to develop good training facilities and to refine our surgical education programmes in reconstructive microsurgery. In this article, we share our experience with training in reconstructive microsurgery. © 2014 The Korean Society of Plastic and Reconstructive Surgeons.


PubMed | National Cancer Center, Royal Marsden Hospital, Roche Holding AG, Singapore General Hospital and 2 more.
Type: Journal Article | Journal: Magnetic resonance imaging | Year: 2015

To evaluate non-invasive imaging biomarkers for assessing renal fibrosis. DWI is used to assess renal function; intravoxel incoherent motion (IVIM) provides additional measures of perfusion-related diffusion (D*, blood flow; f, perfusion fraction). We aim to determine if reduced ADC seen in renal fibrosis is attributable to perfusion-related diffusion changes or to known reduction in tissue diffusivity (D).Unilateral ureteral obstruction (UUO) was created in six mice to induce renal fibrosis. DWI was performed the day before and 7 days post-UUO. A range of b-values from 0 to 1200 s/mm(2) were used. IVIM parameters were obtained using region of interests drawn over the renal parenchyma. Histopathological analysis of both kidneys was performed in all mice. Results were analyzed using the paired t-test with P<0.05 considered statistically significant.D and f were significantly lower in the ligated kidneys at Day 7 compared to before ligation and no significant difference was found for D*. Comparing non-ligated and ligated kidneys within the same mouse at Day 7, significantly lower D values were observed in the ligated kidneys, while no significant difference was found for f and D*, although the values of f were generally lower. Histopathological analysis confirmed development of fibrosis and reduction in glomeruli in all the ligated kidneys at Day 7.Our study shows that the reduction in ADC seen in renal fibrosis is attributable not only to reduced D as previously encountered but also a decrease in vascularity as assessed by f. Reduction in f is possibly related to a reduction in glomeruli.

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