scholarly journals Two novel direct SPIO labels and in vivo MRI detection of labeled cells after acute myocardial infarct

2017 ◽  
Vol 6 (8) ◽  
pp. 205846011771840 ◽  
Author(s):  
Riikka M Korpi ◽  
Kirsi Alestalo ◽  
Timo Ruuska ◽  
Eveliina Lammentausta ◽  
Ronald Borra ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Myocardial Infarct ◽  
Superparamagnetic Iron Oxide ◽  
Large Animal Model ◽  
Large Animal ◽  
Differentiation Potential ◽  
Acute Myocardial Infarct ◽  
Incubation Method

Background Acute myocardial infarction (AMI) is a leading cause of morbidity and mortality worldwide. Cellular decay due hypoxia requires rapid and validated methods for possible therapeutic cell transplantation. Purpose To develop direct and rapid superparamagnetic iron oxide (SPIO) cell label for a large-animal model and to assess in vivo cell targeting by magnetic resonance imaging (MRI) in an experimental AMI model. Material and Methods Bone marrow mononuclear cells (BMMNCs) were labeled with SPIO particles using two novel direct labeling methods (rotating incubation method and electroporation). Labeling, iron incorporation in cells and label distribution, cellular viability, and proliferation were validated in vitro. An AMI porcine model was used to evaluate the direct labeling method (rotating incubation method) by examining targeting of labeled BMMNCs using MRI and histology. Results Labeling (1 h) did not alter either cellular differentiation potential or viability of cells in vitro. Cellular relaxation values at 9.4 T correlated with label concentration and MRI at 1.5 T showing 89 ± 4% signal reduction compared with non-labeled cells in vitro. In vivo, a high spatial correlation between MRI and histology was observed. The extent of macroscopic pathological myocardial changes (hemorrhage) correlated with altered function detected on MRI. Conclusion We demonstrated two novel direct SPIO labeling methods and demonstrated the feasibility of clinical MRI for monitoring targeting of the labeled cells in animal models of AMI.

scholarly journals Light-degradable hydrogels as dynamic triggers for gastrointestinal applications

2020 ◽  
Vol 6 (3) ◽  
pp. eaay0065 ◽  
Author(s):  
Ritu Raman ◽  
Tiffany Hua ◽  
Declan Gwynne ◽  
Joy Collins ◽  
Siddartha Tamang ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Implantable Devices ◽  
Large Animal Model ◽  
Large Animal ◽  
Gi Tract ◽  
Biomedical Device ◽  
Precise Tool ◽  
And Performance

Triggerable materials capable of being degraded by selective stimuli stand to transform our capacity to precisely control biomedical device activity and performance while reducing the need for invasive interventions. Here, we describe the development of a modular and tunable light-triggerable hydrogel system capable of interfacing with implantable devices. We apply these materials to two applications in the gastrointestinal (GI) tract: a bariatric balloon and an esophageal stent. We demonstrate biocompatibility and on-demand triggering of the material in vitro, ex vivo, and in vivo. Moreover, we characterize performance of the system in a porcine large animal model with an accompanying ingestible LED. Light-triggerable hydrogels have the potential to be applied broadly throughout the GI tract and other anatomic areas. By demonstrating the first use of light-degradable hydrogels in vivo, we provide biomedical engineers and clinicians with a previously unavailable, safe, dynamically deliverable, and precise tool to design dynamically actuated implantable devices.

The Wnt Inhibitor sFRP2 Enhances Mesenchymal Stem Cell Engraftment, Granulation Tissue Formation and Myocardial Repair

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1365-1365
Author(s):  
Maria Paula Alfaro ◽  
Matthew Pagni ◽  
Alicia Vincent ◽  
Michael F. Hill ◽  
Ethan Lee ◽  
...  
Keyword(s):  
Infarct Size ◽  
Granulation Tissue ◽  
Myocardial Infarct ◽  
Left Ventricular ◽  
Human Mscs ◽  
Myocardial Repair ◽  
Acute Myocardial Infarct ◽  
Intramyocardial Injection

Abstract Cell-based therapies using bone marrow-derived mesenchymal stem cells (MSCs) for organ regeneration are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. In an attempt to elucidate a way to strengthen the regenerative potential of MSCs, preliminary studies in our lab were performed comparing MSCs isolated from wildtype and regenerative mouse strains. The MRL/MpJ mouse has been described as a “super healer” mouse that is able to repair soft tissue with minimal scaring. MSCs were isolated from the MRL/MpJ mouse (MRL-MSCs) and from C57/Bl6 mice (WT-MSCs) and their differing qualities assessed. Compared to WT-MSCs, MRL-MSCs demonstrated increased proliferation in vitro. We utilized a Poly-vinyl alcohol (PVA) sponge model of repair stimulation to assess their capacity to generate wound repair tissue. We observed that the MRL-MSCs demonstrated increased in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity. The MRL-MSCs also reduced infarct size and improved cardiac function as compared to WT-MSCs in a murine acute myocardial infarct model. Genomic and functional analyses indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized specifically by upregulation of secreted frizzled related proteins (sFRPs). In vitro proliferation studies confirmed that recombinant sFRP2 mediated enhanced proliferation of both mouse and human MSCs. Based on these observations, we hypothesized that sFRP2 served an important role in MSC-mediated repair and regeneration. We generated WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction to test this hypothesis. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and proliferated faster than the vector only control MSCs (GFP-MSCs). When implanted in vivo in the PVA sponge model, the sFRP2-MSCs recapitulated the MRL phenotype by mediating greater, more vascularized granulation tissue. Moreover, periinfarct intramyocardial injection of sFRP2-MSCs resulted in reduced infarct size, favorable remodeling and better preserved left ventricular function following acute myocardial infarct in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype of MSCs.

ZFN-Mediated Gene Targeting at the Albumin Locus in Liver Results in Therapeutic Levels of Human FIX in Mice and Non-Human Primates

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 200-200 ◽  
Author(s):  
Thomas Wechsler ◽  
Kathleen E. Meyer ◽  
S. Kaye Spratt ◽  
Judith Greengard ◽  
Yolanda Santiago ◽  
...  
Keyword(s):  
Hemophilia B ◽  
Large Animal Model ◽  
Zinc Finger Nucleases ◽  
Clotting Factor ◽  
Large Animal ◽  
Spontaneous Bleeding ◽  
Virus Serotype ◽  
Donor Template

Abstract Hemophilia is an attractive target for gene therapy, since activity levels as low as 1% to 2% of normal are beneficial and levels of ~5% prevent spontaneous bleeding. Our goal was to provide a single treatment that permanently enables hepatic production of therapeutic levels of hFIX activity to decrease or potentially eliminate the need for prophylactic treatment in hemophilia B patients. We performed targeted in vivo genome editing using 1) two zinc finger nucleases (ZFNs) targeting intron 1 of the albumin locus, and 2) a human F9 donor template construct. The ZFNs and donor template are encoded on separate hepatotropic adeno-associated virus serotype 2/6 (AAV2/6) vectors injected intravenously, resulting in targeted insertion of a corrected copy of the hF9 gene into the albumin locus in a proportion of liver hepatocytes. The albumin locus was selected as a "safe harbor" as production of this most abundant plasma protein exceeds 10 g/day, and moderate reductions in those levels are well-tolerated. These genome edited hepatocytes produce normal hFIX in therapeutic quantities, rather than albumin, driven by the highly active albumin enhancer/promoter, to treat hemophilia B; the genetic modification is expected to be sustained even in the face of hepatocyte turnover, making this approach attractive for treating young children with hemophilia before the appearance of significant organ damage. Transformed and primary human hepatocytes transduced in vitro with AAV2/6 encoding human albumin ZFNs and a promoterless hF9 transgene were shown to secrete hFIX. Extensive molecular analyses demonstrated that this was due to targeted integration of the hF9 transgene at the albumin locus and splicing of this gene into the albumin transcript. By employing AAV2/6 delivery of murine-specific ZFNs in vivo, stable levels of hFIX were observed in blood of mice injected with the albumin ZFNs and hF9 transgene donor. C57BL/6 mice were administered vehicle (n=20) or AAV2/6 vectors (n=25) encoding mouse surrogate reagents at 1.0 x1013 vector genome (vg)/kg via tail vein injection. ELISA analysis of plasma hFIX in the treated mice showed peak levels of 50-1053 ng/mL that were sustained for the duration of the 6-month study. Analysis of FIX activity from mouse plasma confirmed bioactivity commensurate with expression levels. Next, we report the feasibility of this approach in non-human primates (NHPs), showing that a single intravenous co-infusion of AAV2/6 vectors encoding the NHP targeted albumin-specific ZFNs and a human F9 donor at 1.2x1013 vg/kg (n=5/group) resulted in >50 ng/mL (>1% of normal) in this large animal model. The use of higher AAV2/6 doses (up to 1.5x1014 vg/kg) yielded plasma hFIX levels up to 1000 ng/ml (or 20% of normal) in several animals and up to 2000 ng/ml (or 50% of normal) in a single animal, for the duration of the study (3 months). The treatment was well tolerated in mice and NHPs, with no significant toxicological findings related to AAV2/6 ZFN + donor treatment in either species at therapeutic doses. Together, these data support a clinical trial to determine if a single co-administration of ZFN and donor AAV vectors is sufficient to enable therapeutic and potentially lifelong production of the clotting factor for the treatment of Hemophilia B. Disclosures Wechsler: Sangamo BioSciences: Employment. Meyer:Sangamo Biosciences Inc: Employment. Spratt:Sangamo Biosciences Inc: Employment. Greengard:Sangamo Biosciences Inc: Employment. Santiago:Sangamo Biosciences Inc: Employment. Sproul:Sangamo Biosciences Inc: Employment. Surosky:Sangamo Biosciences Inc: Employment. Paschon:Sangamo Biosciences Inc: Employment. Dubois-Stringfellow:Sangamo Biosciences Inc: Employment. Ando:Sangamo Biosciences Inc: Employment. Nichol:Sangamo Biosciences Inc: Employment. Rebar:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment.

scholarly journals A Novel Approach for the Isolation and Long-Term Expansion of Pure Satellite Cells Based on Ice-Cold Treatment

2020 ◽  
Author(s):  
Anna Benedetti ◽  
Gianluca Cera ◽  
Daniele De Meo ◽  
Ciro Villani ◽  
Marina Bouche ◽  
...  
Keyword(s):  
Satellite Cells ◽  
Mononuclear Cells ◽  
Cold Treatment ◽  
Cell Manipulation ◽  
Minimal Cell ◽  
Differentiation Potential ◽  
Novel Approach ◽  
In Vitro Expansion

Abstract Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15-30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.

scholarly journals A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna Benedetti ◽  
Gianluca Cera ◽  
Daniele De Meo ◽  
Ciro Villani ◽  
Marina Bouche ◽  
...  
Keyword(s):  
Satellite Cells ◽  
Mononuclear Cells ◽  
Cold Treatment ◽  
Cell Manipulation ◽  
Minimal Cell ◽  
Differentiation Potential ◽  
Novel Approach ◽  
In Vitro Expansion

AbstractSatellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15–30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.

scholarly journals Effect of cariporide on ram sperm pH regulation and motility: possible role of NHE1

Reproduction ◽  
2018 ◽  
Vol 155 (5) ◽  
pp. 433-445 ◽  
Author(s):  
Stefania Muzzachi ◽  
Lorenzo Guerra ◽  
Nicola Antonio Martino ◽  
Maria Favia ◽  
Giuseppe Punzi ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Prolonged Exposure ◽  
Ph Regulation ◽  
Equatorial Region ◽  
Large Animal Model ◽  
Large Animal ◽  
Significant Drop

Sperm motility, a feature essential forin vivofertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHiregulation, among which sodium–hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+for H+across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparentKmfor external Na+of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHirecovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation.In vitrofertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHiand motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.

scholarly journals KRASG12D and TP53R167H Cooperate to Induce Pancreatic Ductal Adenocarcinoma in Sus Scrofa Pigs

2018 ◽  
Author(s):  
Daniel R. Principe ◽  
Nana Haahr Overgaard ◽  
Alex J. Park ◽  
Andrew M. Diaz ◽  
Carolina Torres ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Sus Scrofa ◽  
Cancer Biology ◽  
Ductal Adenocarcinoma ◽  
Large Animal Model ◽  
Large Animal ◽  
Desmoplastic Stroma ◽  
Immunocompromised Mice

AbstractAlthough survival has improved in recent years, the prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite substantial differences in anatomy, physiology, genetics, and metabolism, the overwhelming majority of preclinical testing relies on transgenic mice. Hence, while mice have allowed for tremendous advances in cancer biology, they have been a poor predictor of drug performance/toxicity in the clinic. Given the greater similarity of sus scrofa pigs to humans, we engineered transgenic sus scrofa expressing a LSL-KRASG12D-TP53R167H cassette. By applying Adeno-Cre to pancreatic duct cells in vitro, cells self-immortalized and established tumors in immunocompromised mice. When Adeno-Cre was administered to the main pancreaticduct in vivo, pigs developed extensive PDAC at the injection site hallmarked by excessive proliferation and desmoplastic stroma. This serves as the first large animal model of pancreatic carcinogenesis, and may allow for insight into new avenues of translational research not before possible in rodents.

scholarly journals Generation of tumorigenic porcine pancreatic ductal epithelial cells: toward a large animal model of pancreatic cancer

2018 ◽  
Author(s):  
Neeley Remmers ◽  
Jesse L. Cox ◽  
James A. Grunkemeyer ◽  
Shruthi Aravind ◽  
Christopher K. Arkfeld ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Animal Model ◽  
Cell Lines ◽  
Large Animal Model ◽  
Large Animal ◽  
Murine Models ◽  
Transformed Cell ◽  
Transformed Cell Lines

AbstractBackground. A large animal model of pancreatic cancer would permit development of diagnostic and interventional technologies not possible in murine models, and also would provide a more biologically-relevant platform for penultimate testing of novel therapies, prior to human testing. Here, we describe our initial studies in the development of an autochthonous, genetically-defined, large animal model of pancreatic cancer, using immunocompetent pigs.Methods. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs; epithelial origin was confirmed with immunohistochemistry. Three transformed cell lines subsequently were generated from these primary cells using expression of oncogenic KRAS and dominant negative p53, with/without knockdown of p16 and SMAD4. We tested these cell lines using in vitro and in vivo assays of transformation and tumorigenesis.Results. The transformed cell lines outperformed the primary cells in terms proliferation, population doubling time, soft agar growth, 2D migration, and Matrigel invasion, with the greatest differences observed when all four genes (KRAS, p53, p16, and SMAD4) were targeted. All three transformed cell lines grew tumors when injected subcutaneously in nude mice, demonstrating undifferentiated morphology, mild desmoplasia, and staining for both epithelial and mesenchymal markers. Injection into the pancreas of nude mice resulted in distant metastases, particularly when all four genes were targeted.Conclusions. Tumorigenic porcine pancreatic cell lines were generated. Inclusion of four genetic “hits” (KRAS, p53, p16, and SMAD4) appeared to produce the best results in our in vitro and in vivo assays. The next step will be to perform autologous or syngeneic implantation of these cell lines into the pancreas of immunocompetent pigs. We believe that the resultant large animal model of pancreatic cancer could supplement existing murine models, thus improving preclinical research on diagnostic, interventional, and therapeutic technologies.

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