Characterization of the Nonendocrine Cell Populations in Human Embryonic Stem Cell–Derived (hESC) Islet-Like Clusters Posttransplantation

2021 ◽  
pp. 019262332110363
Author(s):  
Nikolai K. Jensen ◽  
Camilla Ingvorsen ◽  
Dorthe R. Petersen ◽  
Maria J. Pereira ◽  
Tess T. H. Lu ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Embryonic Stem ◽  
Immunodeficient Mice ◽  
Differentiated Cells ◽  
Process Characterization ◽  
Poorly Differentiated ◽  
Teratoma Formation

Islet-like clusters derived from human embryonic stem cells (hESC) hold the potential to cure type 1 diabetes mellitus. Differentiation protocols of islet-like clusters lead to the generation of minor fractions of nonendocrine cells, which are mainly from endodermal and mesodermal lineages, and the risk of implanting these is unclear. In the present study, the histogenesis and the tumorigenicity of nonendocrine cells were investigated in vivo. Immunodeficient mice were implanted under the kidney capsule with islet-like clusters which were derived from differentiation of cells batches with either an intermediate or poor cell purity and followed for 8 or 26 weeks. Using immunohistochemistry and other techniques, it was found that the intermediate differentiated cell implants had limited numbers of small duct-like cysts and nonpancreatic tissue resembling gastrointestinal and retinal pigmented epithelium. In contrast, highly proliferative cystic teratomas were found at a high incidence at the implant site after 8 weeks, only in the animals implanted with the poorly differentiated cells. These findings indicate that the risk for teratoma formation and the amount of nonpancreatic tissue can be minimized by careful in-process characterization of the cells and thus highlights the importance of high purity at transplantation and a thorough ex-vivo characterization during cell product development.

scholarly journals Prunellae Spica Extract Suppresses Teratoma Formation of Pluripotent Stem Cells through p53-Mediated Apoptosis

Nutrients ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 721 ◽  
Author(s):  
Aeyung Kim ◽  
Seo-Young Lee ◽  
Chang-Seob Seo ◽  
Sun-Ku Chung
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Apoptotic Cell ◽  
Embryonic Stem ◽  
M Cell ◽  
In Ovo ◽  
Differentiated Cells ◽  
Teratoma Formation

Induced pluripotent stem cells (iPSCs) have similar properties to embryonic stem cells in terms of indefinite self-renewal and differentiation capacity. After in vitro differentiation of iPSCs, undifferentiated iPSCs (USCs) may exist in cell therapy material and can form teratomas after in vivo transplantation. Selective elimination of residual USCs is, therefore, very important. Prunellae Spica (PS) is a traditional medicinal plant that has been shown to exert anti-cancer, antioxidant, and anti-inflammatory activities; however, its effects on iPSCs have not been previously characterized. In this study, we find that ethanol extract of PS (EPS) effectively induces apoptotic cell death of USCs through G2/M cell cycle arrest, generation of intracellular reactive oxygen species, alteration of mitochondrial membrane potentials, and caspase activation of USCs. In addition, EPS increases p53 accumulation and expression of its downstream targets. In p53 knockout (KO) iPSCs, the EPS did not induce apoptosis, indicating that EPS-mediated apoptosis of USCs was p53-dependent. In addition, EPS was not genotoxic towards iPSCs-derived differentiated cells. EPS treatment before injection efficiently prevented in ovo teratoma formation of p53 wild-type (WT) iPSCs but not p53KO iPSCs. Collectively, these results indicate that EPS has potent anti-teratoma activity and no genotoxicity to differentiated cells. It can, therefore, be used in the development of safe and efficient iPSC-based cell therapies.

scholarly journals Consequences of SUR2[A478V] Mutation in Skeletal Muscle of Murine Model of Cantu Syndrome

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1791
Author(s):  
Rosa Scala ◽  
Fatima Maqoud ◽  
Nicola Zizzo ◽  
Giuseppe Passantino ◽  
Antonietta Mele ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Katp Channel ◽  
Ex Vivo ◽  
Channel Modulation ◽  
Flexor Digitorum Brevis ◽  
Inflammatory Edema ◽  
Flexor Digitorum ◽  
Channel Currents

(1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K+ (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2wt/AV) and homozygous SUR2[A478V] (SUR2AV/AV) CS mice. (3) Results: In SUR2wt/AV and SUR2AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2wt/AV, dramatically so in SUR2AV/AV mice; IC50 for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10−5 M in SUR2wt/AV and 8.6 ± 0.4 × 10−6 M in WT mice and was not measurable in SUR2AV/AV. A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] “knock-in” mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2AV/AV, with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.

scholarly journals Characterization of Intrinsically Radiolabeled Poly(lactic-co-glycolic acid) Nanoparticles for ex Vivo Autologous Cell Labeling and in Vivo Tracking

2021 ◽  
Author(s):  
Massis Krekorian ◽  
Gerwin G. W. Sandker ◽  
Kimberley R. G. Cortenbach ◽  
Oya Tagit ◽  
N. Koen van Riessen ◽  
...  
Keyword(s):  
Glycolic Acid ◽  
Ex Vivo ◽  
Cell Labeling ◽  
Autologous Cell

scholarly journals Early Spatial and Temporal Events of Human T-Lymphotropic Virus Type 1 Spread following Blood-Borne Transmission in a Rabbit Model of Infection

2010 ◽  
Vol 84 (10) ◽  
pp. 5124-5130 ◽  
Author(s):  
Rashade A. H. Haynes ◽  
Bevin Zimmerman ◽  
Laurie Millward ◽  
Evan Ware ◽  
Christopher Premanandan ◽  
...  
Keyword(s):  
Virus Type ◽  
Ex Vivo ◽  
Rabbit Model ◽  
Virus Detection ◽  
Mononuclear Leukocytes ◽  
T Lymphotropic Virus ◽  
Viral Spread ◽  
Temporal Events

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia/lymphoma (ATL) and is associated with a variety of lymphocyte-mediated disorders. HTLV-1 transmission occurs by transmission of infected cells via breast-feeding by infected mothers, sexual intercourse, and contaminated blood products. The route of exposure and early virus replication events are believed to be key determinants of virus-associated spread, antiviral immune responses, and ultimately disease outcomes. The lack of knowledge of early events of HTLV-1 spread following blood-borne transmission of the virus in vivo hinders a more complete understanding of the immunopathogenesis of HTLV-1 infections. Herein, we have used an established animal model of HTLV-1 infection to study early spatial and temporal events of the viral infection. Twelve-week-old rabbits were injected intravenously with cell-associated HTLV-1 (ACH-transformed R49). Blood and tissues were collected at defined intervals throughout the study to test the early spread of the infection. Antibody and hematologic responses were monitored throughout the infection. HTLV-1 intracellular Tax and soluble p19 matrix were tested from ex vivo cultured lymphocytes. Proviral copy numbers were measured by real-time PCR from blood and tissue mononuclear leukocytes. Our data indicate that intravenous infection with cell-associated HTLV-1 targets lymphocytes located in both primary lymphoid and gut-associated lymphoid compartments. A transient lymphocytosis that correlated with peak virus detection parameters was observed by 1 week postinfection before returning to baseline levels. Our data support emerging evidence that HTLV-1 promotes lymphocyte proliferation preceding early viral spread in lymphoid compartments to establish and maintain persistent infection.

P.3.d.011 In vitro and in vivo characterization of glycine transporter type-1 by two inhibitors, Org-24461 and NFPS

2006 ◽  
Vol 16 ◽  
pp. S435 ◽  
Author(s):  
B. Marko ◽  
G. Szabo ◽  
S. Udvari ◽  
M. Agoston ◽  
E. Szabo ◽  
...  
Keyword(s):  
Glycine Transporter ◽  
In Vivo Characterization

About Puncture Testing Applied for Mechanical Characterization of Fetal Membranes

2014 ◽  
Vol 136 (11) ◽  
Author(s):  
Wilfried Bürzle ◽  
Edoardo Mazza ◽  
John J. Moore
Keyword(s):  
Deformation Behavior ◽  
Mechanical Characterization ◽  
Ex Vivo ◽  
Fetal Membrane ◽  
Membrane Rupture ◽  
Resistance To Deformation ◽  
Ex Vivo Testing ◽  
Insight Into

Puncture testing has been applied in several studies for the mechanical characterization of human fetal membrane (FM) tissue, and significant knowledge has been gained from these investigations. When comparing results of mechanical testing (puncture, inflation, and uniaxial tension), we have observed discrepancies in the rupture sequence of FM tissue and significant differences in the deformation behavior. This study was undertaken to clarify these discrepancies. Puncture experiments on FM samples were performed to reproduce previous findings, and numerical simulations were carried out to rationalize particular aspects of membrane failure. The results demonstrate that both rupture sequence and resistance to deformation depend on the samples' fixation. Soft fixation leads to slippage in the clamping, which reduces mechanical loading of the amnion layer and results in chorion rupturing first. Conversely, the stiffer, stronger, and less extensible amnion layer fails first if tight fixation is used. The results provide a novel insight into the interpretation of ex vivo testing as well as in vivo membrane rupture.

Abstract 290: Haemogenic Endocardium Contribute To Definitive Hematopoiesis During Cardiogenesis

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Haruko Nakano ◽  
Xiaoqian Liu ◽  
Armin Arshi ◽  
Ben van Handel ◽  
Rajkumar Sasidharan ◽  
...  
Keyword(s):  
De Novo ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Circulatory System ◽  
Lineage Tracing ◽  
Mouse Heart ◽  
Heart Tube ◽  
Cardiac Progenitors ◽  
Definitive Hematopoiesis

The circulatory system is the first functional organ system that develops during mammalian life. Accumulating evidences suggest that cardiac and endocardial cells can arise from a single common progenitor cell during mammalian cardiogenesis. Notably, these early cardiac progenitors express multiple hematopoietic transcription factors, consistent with previous reports. Indeed, a close relationship among cardiac, endocardial and hematopoietic lineages has been suggested in fly, zebrafish, and embryonic stem cell in vitro differentiation models. However, it is unclear when, where and how this hematopoietic gene program is in operation during in vivo mammalian cardiogenesis. Hematopoietic colony assay suggests that mouse heart explants generate myeloids and erythroids in the absence of circulation, suggesting that the heart tube is a de novo site for the definitive hematopoiesis. Lineage tracing revealed that putative cardiac-derived Nkx2-5+/Isl1+ endocardial cells give rise to CD41+ hematopoietic progenitors that contribute to definitive hematopoiesis in vivo and ex vivo during embryogenesis earlier than in the AGM region. Furthermore, Nkx2-5 and Isl1 are both required for the hemogenic activity of the endocardium. Together, identification of Nkx2-5/Isl1-dependent hemogenic endocardial cells (1) adds hematopoietic component in the cardiogenesis lineage tree, (2) changes the long-held dogma that AGM is the only major source of definitive hematopoiesis in the embryo proper, and (3) represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway during the development of circulatory system.

Abstract 129: Embryonic Stem Cell Derived Exosomes Revive Endogenous Repair Mechanisms In Failing Heart

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Mohsin Khan ◽  
Suresh K Verma ◽  
Alexander R Mackie ◽  
Erin Vaughan ◽  
Srikanth Garikipati ◽  
...  
Keyword(s):  
Stem Cells ◽  
Therapeutic Potential ◽  
Embryonic Stem ◽  
Cardiac Regeneration ◽  
Great Promise ◽  
Target Cells ◽  
Free System ◽  
Teratoma Formation

Rationale: Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to ethical concerns, lack of autologous donors and teratoma formation. Recently, it has been observed that beneficial effects of stem cells are mediated by exosomes secreted out under various physiological conditions. ESCs have the ability to produce exosomes however their effect in the context of the heart is unknown. Objective: Determine the effect of ESC derived exosomes for cardiac repair and modulation of CPCs functions in the heart following myocardial infarction. Methods and Results: Exosomes were isolated from murine ESCs (mES Ex) or embryonic fibroblasts (MEFs) by ultracentrifugation and verified by Flotillin-1 immunoblot analysis. Induction of pluripotent markers, survival and in vitro tube formation was enhanced in target cells receiving ESC exosomes indicating therapeutic potential of mES Ex. mES Ex administration resulted in enhanced neovascularization, cardiomyocyte survival and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex mediated considerable enhancement of cardiac progenitor cell (CPC) survival, proliferation and cardiac commitment concurrent with increased c-kit+ CPCs in vivo 4 weeks after mES Ex transfer. miRNA Array analysis of ESC and MEF exosomes revealed significantly high expression of miR290-295 cluster in the ESC exosomes compared to MEF exosomes. The underlying beneficial effect of mES Ex was tied to delivery of ESC miR-294 to the heart and in particular CPCs thereby promoting CPC survival and proliferation as analyzed by FACS based cell death analysis and CyQuant assay respectively. Interestingly, enhanced G1/S transition was observed in CPCs treated with miR-294 in conjunction with significant reduction of G1 phase. Conclusion: In conclusion, mES Ex provide a novel cell free system for cardiac regeneration with the ability to modulate both cardiomyocyte and CPC based repair programs in the heart thereby avoiding the risk of teratoma formation associated with ESCs.

In vivo characterization of the transitional bronchioles by aerosol-derived airway morphometry

1999 ◽  
Vol 87 (3) ◽  
pp. 920-927 ◽  
Author(s):  
Kirby L. Zeman ◽  
Gerhard Scheuch ◽  
Knut Sommerer ◽  
James S. Brown ◽  
William D. Bennett
Keyword(s):  
Ex Vivo ◽  
Normal Subjects ◽  
Characteristic Line ◽  
Chronic Obstructive ◽  
Obstructive Pulmonary Disease ◽  
Lung Capacity ◽  
Single Breath ◽  
In Vivo Characterization

Effective airway dimensions (EADs) were determined in vivo by aerosol-derived airway morphometry as a function of volumetric lung depth (VLD) to identify and characterize, noninvasively, the caliber of the transitional bronchiole region of the human lung and to compare the EADs by age, gender, and disease. By logarithmically plotting EAD vs. VLD, two distinct regions of the lung emerged that were identified by characteristic line slopes. The intersection of proximal and distal segments was defined as VLDtransand associated EADtrans. In our normal subjects ( n = 20), VLDtrans [345 ± 83 (SD) ml] correlated significantly with anatomic dead space (224 ± 34 ml) and end of phase II of single-breath nitrogen washout (360 ± 53 ml). The corresponding EADtranswas 0.42 ± 0.07 mm, in agreement with other ex vivo measurements of the transitional bronchioles. VLDtrans was smaller (216 ± 64 ml) and EADtrans was larger (0.83 ± 0.04 mm) in our patients with chronic obstructive pulmonary disease ( n = 13). VLDtrans increased with age for children (age 8–18 yr; P = 0.006, n = 26) and with total lung capacity for age 8–81 yr ( P < 0.001, n = 61). This study extends the usefulness of aerosol-derived airway morphometry to in vivo measurements of the transitional bronchioles.

scholarly journals Generation and Characterization of Smac/DIABLO-Deficient Mice

2002 ◽  
Vol 22 (10) ◽  
pp. 3509-3517 ◽  
Author(s):  
Hitoshi Okada ◽  
Woong-Kyung Suh ◽  
Jianping Jin ◽  
Minna Woo ◽  
Chunying Du ◽  
...  
Keyword(s):  
Physiological Function ◽  
Es Cells ◽  
Embryonic Stem ◽  
Caspase Activation ◽  
Proapoptotic Protein ◽  
Apoptosis Proteins ◽  
Deficient Mice

ABSTRACT The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac−/− ) mice by using homologous recombination in embryonic stem (ES) cells. Smac−/− mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac−/− cells, all types of cultured Smac−/− cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.

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