en Center for Regenerative Medicine

Toronto, Canada

en Center for Regenerative Medicine

Toronto, Canada
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Mohammadi Amirabad L.,Iran National Institute of Genetic Engineering and Biotechnology | Mohammadi Amirabad L.,Shahid Beheshti University of Medical Sciences | Massumi M.,Mount Sinai Hospital | Massumi M.,University of Toronto | And 10 more authors.
ACS Applied Materials and Interfaces | Year: 2017

In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus venosus and appear to be involved in cardiogenesis. In this work, aligned and random polyaniline/polyetersulfone (PANI/PES) nanofibrous scaffolds doped by Camphor-10-sulfonic acid (β) (CPSA) were fabricated via electrospinning and used to conduct electrical impulses in a unidirectional and multidirectional fashion, respectively. A bioreactor was subsequently engineered to apply electrical impulses to cells cultured on PANI/PES scaffolds. We established cardiovascular disease-specific induced pluripotent stem cells (CVD-iPSCs) from the fibroblasts of patients undergoing cardiothoracic surgeries. The CVD-iPSCs were seeded onto the scaffolds, cultured in cardiomyocyte-inducing factors, and exposed to electrical impulses for 1 h/day, over a 15-day time period in the bioreactor. The application of the unidirectional electrical stimulation to the cells significantly increased the number of cardiac Troponin T (cTnT+) cells in comparison to multidirectional electrical stimulation using random fibrous scaffolds. This was confirmed by real-time polymerase chain reaction for cardiac-related transcription factors (NKX2.5, GATA4, and NPPA) and a cardiac-specific structural gene (TNNT2). Here we report for the first time that applying electrical pulses in a unidirectional manner mimicking the unidirectional wave of electrical stimulation in the heart, could increase the derivation of cardiomyocytes from CVD-iPSCs. © 2017 American Chemical Society.

Steinbach S.K.,en Center for Regenerative Medicine | Steinbach S.K.,Toronto General Research Institute | El-Mounayri O.,en Center for Regenerative Medicine | El-Mounayri O.,Toronto General Research Institute | And 9 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2011

Objective-The goal of this study was to characterize the factors and conditions required for smooth muscle cell (SMC)-directed differentiation of Sox2 multipotent rat and human skin-derived precursors (SKPs) and to define whether they represent a source of fully functional vascular SMCs for applications in vivo. Methods and results-We found that rat SKPs can differentiate almost exclusively into SMCs by reducing serum concentrations to 0.5% to 2% and plating them at low density. Human SKPs derived from foreskin required the addition of transforming growth factor-β1 or-β3 to differentiate into SMCs, but they did so even in the absence of serum. SMC formation was confirmed by quantitative reverse transcription-polymerase chain reaction, immunocytochemistry, and fluorescence-activated cell sorting, with increased expression of smoothelin-B and little to no expression of telokin or smooth muscle γ-actin, together indicating that SKPs differentiated into vascular rather than visceral SMCs. Rat and human SKP-derived SMCs were able to contract in vitro and also wrap around and support new capillary and larger blood vessel formation in angiogenesis assays in vivo. Conclusion-SKPs are Sox2 progenitors that represent an attainable autologous source of stem cells that can be easily differentiated into functional vascular SMCs in defined serum-free conditions without reprogramming. SKPs represent a clinically viable cell source for potential therapeutic applications in neovascularization. © 2011 American Heart Association, Inc.

Basford C.L.,Toronto General Research Institute | Prentice K.J.,Toronto General Research Institute | Hardy A.B.,King's College | Sarangi F.,en Center for Regenerative Medicine | And 10 more authors.
Diabetologia | Year: 2012

Aims/hypothesis: Using a novel directed differentiation protocol, we recently generated up to 25% insulin-producing cells from human embryonic stem cells (hESCs) (insulin + cells). At this juncture, it was important to functionally and molecularly characterise these hESC-derived insulin + cells and identify key differences and similarities between them and primary beta cells. Methods: We used a new reporter hESC line with green fluorescent protein (GFP) cDNA targeted to the INS locus by homologous recombination (INS GFP/w ) and an untargeted hESC line (HES2). INS GFP/w allowed efficient identification and purification of GFP-producing (INS:GFP +) cells. Insulin + cells were examined for key features of adult beta cells using microarray, quantitative PCR, secretion assays, imaging and electrophysiology. Results: Immunofluorescent staining showed complete co-localisation of insulin with GFP; however, cells were often multihormonal, many with granules containing insulin and glucagon. Electrophysiological recordings revealed variable K ATP and voltage-gated Ca 2+ channel activity, and reduced glucose-induced cytosolic Ca 2+ uptake. This translated into defective glucose-stimulated insulin secretion but, intriguingly, appropriate glucagon responses. Gene profiling revealed differences in global gene expression between INS:GFP + cells and adult human islets; however, INS:GFP + cells had remarkably similar expression of endocrine-lineage transcription factors and genes involved in glucose sensing and exocytosis. Conclusions/interpretation: INS:GFP + cells can be purified from differentiated hESCs, providing a superior source of insulin-producing cells. Genomic analyses revealed that INS:GFP + cells collectively resemble immature endocrine cells. However, insulin + cells were heterogeneous, a fact that translated into important functional differences within this population. The information gained from this study may now be used to generate new iterations of functioning beta cells that can be purified for transplant. © 2011 Springer-Verlag.

Kubo A.,Nara Medical University | Stull R.,VistaGen Therapeutics | Takeuchi M.,Nara Medical University | Bonham K.,VistaGen Therapeutics | And 6 more authors.
PLoS ONE | Year: 2011

In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit + endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells. © 2011 Kubo et al.

Nostro M.C.,en Center for Regenerative Medicine | Nostro M.C.,Toronto General Research Institute | Nostro M.C.,University of Toronto | Sarangi F.,en Center for Regenerative Medicine | And 8 more authors.
Stem Cell Reports | Year: 2015

Human pluripotent stem cells (hPSCs) represent a renewable source of pancreatic beta cells for both basic research and therapeutic applications. Given this outstanding potential, significant efforts have been made to identify the signaling pathways that regulate pancreatic development in hPSC differentiation cultures. In this study, we demonstrate that the combination of epidermal growth factor (EGF) and nicotinamide signaling induces the generation of NKX6-1+ progenitors from all hPSC lines tested. Furthermore, we show that the size of the NKX6-1+ population is regulated by the duration of treatment with retinoic acid, fibroblast growth factor 10 (FGF10), and inhibitors of bone morphogenetic protein (BMP) and hedgehog signaling pathways. Whentransplanted intoNODscid gamma (NSG) recipients, these progenitors differentiate to give rise to exocrine and endocrine cells, includingmonohormonal insulin+ cells. Together, these findings provide an efficient and reproducible strategy for generating highly enriched populations of hPSC-derived beta cell progenitors for studies aimed at further characterizing their developmental potential in vivoand deciphering the pathways that regulate their maturation in vitro.

Ichim C.V.,University of Toronto | Ichim C.V.,Sunnybrook Research Institute | Atkins H.L.,Ottawa Hospital Research Institute | Iscove N.N.,University of Toronto | And 6 more authors.
Leukemia | Year: 2011

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy. © 2011 Macmillan Publishers Limited All rights reserved.

Duchesneau P.,University of Toronto | Duchesneau P.,Toronto General Research Institute | Duchesneau P.,en Center for Regenerative Medicine | Wong A.P.,University of Toronto | And 5 more authors.
Molecular Therapy | Year: 2010

Cell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis, although rates of engraftment need to be improved. We previously showed improved cell retention in the lung using transtracheal delivery compared to intravenous injection. Here, we optimized other parameters of cell delivery using 7-day cultured bone marrow cells (BMCs). Retention of BMC in the lung was dose-dependent. Naphthalene treatment had maximal effects on BMC retention when given 2 days before cell delivery. Naphthalene treatment of the donor amplified a CCSP population and increased retention efficiency in the recipient. Repeated naphthalene treatment and repeated cell delivery both resulted in greater retention. The contribution of the second cell dose was minimal suggesting that a second delivery of BMC promotes proliferation of the first. Busulfan-induced myelosuppression augmented retention of exogenous BMC by up to 20-fold. These BMC helped CCSP reconstitution. Using the optimal delivery techniques and cytokeratin-18-driven green fluorescent protein (GFP) reporter mice, we detected threefold more GFP suggesting more BMC differentiated to epithelial cells. We propose that improved engraftment in the lung will increase cell replacement and thus be a more efficient therapeutic approach for various lung diseases. © The American Society of Gene & Cell Therapy.

Korytnikov R.,University of Toronto | Korytnikov R.,en Center for Regenerative Medicine | Nostro M.C.,Toronto General Research Institute
Methods | Year: 2015

Generation of pancreatic β-cells from human pluripotent stem cells (hPSCs) has enormous importance in type 1 diabetes (T1D), as it is fundamental to a treatment strategy based on cellular therapeutics. Being able to generate β-cells, as well as other mature pancreatic cells, from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) will also enable the development of platforms that can be used for disease modeling and drug testing for a variety of pancreas-associated diseases, including cystic fibrosis. For this to occur, it is crucial to develop differentiation strategies that are robust and reproducible across cell lines and laboratories. In this article we describe two serum-free differentiation protocols designed to generate specific pancreatic lineages from hPSCs. Our approach employs a variety of cytokines and small molecules to mimic developmental pathways active during pancreatic organogenesis and allows for the in vitro generation of distinct pancreatic populations. The first protocol is designed to give rise to polyhormonal cells that have the potential to differentiate into glucagon-producing cells. The second protocol is geared to generate multipotent pancreatic progenitor cells, which harbor the potential to generate all pancreatic lineages including: monohormonal endocrine cells, acinar, and ductal cells. © 2015 Elsevier Inc.

Mihic A.,Toronto General Research Institute | Li J.,Toronto General Research Institute | Li J.,Guangzhou Medical College | Miyagi Y.,Toronto General Research Institute | And 9 more authors.
Biomaterials | Year: 2014

The goal of cardiac tissue engineering is to restore function to the damaged myocardium with regenerative constructs. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can produce viable, contractile, three-dimensional grafts that function invivo. We sought to enhance the viability and functional maturation of cardiac tissue constructs by cyclical stretch. hESC-CMs seeded onto gelatin-based scaffolds underwent cyclical stretching. Histological analysis demonstrated a greater proportion of cardiac troponin T-expressing cells in stretched than non-stretched constructs, and flow sorting demonstrated a higher proportion of cardiomyocytes. Ultrastructural assessment showed that cells in stretched constructs had a more mature phenotype, characterized by greater cell elongation, increased gap junction expression, and better contractile elements. Real-time PCR revealed enhanced mRNA expression of genes associated with cardiac maturation as well as genes encoding cardiac ion channels. Calcium imaging confirmed that stretched constructs contracted more frequently, with shorter calcium cycle duration. Epicardial implantation of constructs onto ischemic rat hearts demonstrated the feasibility of this platform, with enhanced survival and engraftment of transplanted cells in the stretched constructs. This uniaxial stretching system may serve as a platform for the production of cardiac tissue-engineered constructs for translational applications. © 2014 Elsevier Ltd.

Benveniste P.,Ontario Cancer Institute | Benveniste P.,University of Toronto | Benveniste P.,en Center for Regenerative Medicine | Frelin C.,Ontario Cancer Institute | And 17 more authors.
Cell Stem Cell | Year: 2010

Sustained blood cell production depends on divisions by hematopoietic stem cells (HSCs) that yield both differentiating progeny as well as new HSCs via self-renewal. Differentiating progeny remain capable of self-renewal, but only HSCs sustain self-renewal through successive divisions securely enough to maintain clones that persist life-long. Until recently, the first identified next stage consisted of "short-term" reconstituting cells able to sustain clones of differentiating cells for only 4-6 weeks. Here we expand evidence for a numerically dominant "intermediate-term" multipotent HSC stage in mice whose clones persist for 6-8 months before becoming extinct and that are separable from both short-term as well as permanently reconstituting "long-term" HSCs. The findings suggest that the first step in stem cell differentiation consists not in loss of initial capacity for serial self-renewal divisions, but rather in loss of mechanisms that stabilize self-renewing behavior throughout successive future stem cell divisions. © 2010 Elsevier Inc. All rights reserved.

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