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Current Protocols in Stem Cell Biology

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https://www.readbyqxmd.com/read/28806856/direct-cell-labeling-to-image-transplanted-stem-cells-in-real-time-using-a-dual-contrast-mri-technique
#1
Ethel J Ngen, Yoshinori Kato, Dmitri Artemov
Exogenous direct cell labeling with superparamagnetic iron oxide nanoparticles (SPIONs) is currently the most employed cell-labeling technique for tracking transplanted cells using magnetic resonance imaging (MRI). Although SPION-based cell labeling is effective for monitoring cell delivery and migration, monitoring cell survival is still a challenge. This unit describes an MRI technique that permits detection of the delivery, migration, and death of transplanted cells. This dual-contrast technique involves labeling cells with two different classes of MRI contrast agents, possessing different diffusion coefficients: SPIONs (T2 /T2(*) contrast agents, with lower diffusion coefficients) and gadolinium chelates (T1 contrast agents, with higher diffusion coefficients)...
August 14, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28806855/functional-stem-cell-integration-into-neural-networks-assessed-by-organotypic-slice-cultures
#2
David Forsberg, Charoensri Thonabulsombat, Johan Jäderstad, Linda Maria Jäderstad, Petri Olivius, Eric Herlenius
Re-formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell-mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host...
August 14, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28806854/generation-of-xeno-free-cgmp-compliant-patient-specific-ipscs-from-skin-biopsy
#3
Luke A Wiley, Kristin R Anfinson, Cathryn M Cranston, Emily E Kaalberg, Malia M Collins, Robert F Mullins, Edwin M Stone, Budd A Tucker
This unit describes protocols for the generation of clinical-grade patient-specific induced pluripotent stem cell (iPSC)-derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how, using xeno-free reagents in an ISO class 5 environment, one can isolate and culture dermal fibroblasts, generate iPSCs, and derive autologous retinal cells via 3-D differentiation. The universal methods described herein for the isolation of dermal fibroblasts and generation of iPSCs can be employed regardless of disease, tissue, or cell type of interest...
August 14, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28806853/development-of-hepatocyte-like-cell-derived-from-human-induced-pluripotent-stem-cell-as-a-host-for-clinically-isolated-hepatitis-c-virus
#4
Khanit Sa-Ngiamsuntorn, Suradej Hongeng, Adisak Wongkajornsilp
This unit describes protocols to develop hepatocyte-like cells (HLCs) starting from mesenchymal stem cells (MSCs) as a natural host for hepatitis C virus (HCV). These include the preparation of MSCs from bone marrow, the reprogramming of MSCs into induced pluripotent stem cells (iPSCs), and the differentiation of iPSCs into HLCs. This unit also incorporates the characterization of the resulting cells at each stage. Another section entails the preparations of HCV. The sources of HCV are either the clinically isolated HCV (HCVser) and the conventional JFH-1 genotype...
August 14, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28806852/generation-of-oligodendrogenic-spinal-neural-progenitor-cells-from-human-induced-pluripotent-stem-cells
#5
Mohamad Khazaei, Christopher S Ahuja, Michael G Fehlings
This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o-NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem cell-derived neural progenitor cells (hiPS-NPCs), and human induced pluripotent stem cell-oligodendrogenic neural progenitor cells (hiPSC-o-NPCs) with the final products being suitable for in vitro experimentation or in vivo transplantation...
August 14, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28806851/production-of-single-contracting-human-induced-pluripotent-stem-cell-derived-cardiomyocytes-matrigel-mattress-technique
#6
Adrian G Cadar, Tromondae K Feaster, Matthew D Durbin, Charles C Hong
This unit describes the published Matrigel mattress method. Briefly, we describe the preparation of the mattress, replating of the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) on the Matrigel mattress, and hiPSC-CM mattress maintenance. Adherence to this protocol will yield individual, robustly shortening hiPSC-CMs, which can be used for downstream applications. © 2017 by John Wiley & Sons, Inc.
August 14, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28510334/angiogenesis-within-stem-cell-seeded-silk-scaffolds-cultured-on-the-chorioallantoic-membrane-and-visualized-by-3d-imaging
#7
Anna Woloszyk, Thimios A Mitsiadis
The long-term survival and successful integration of implants for tissue replacement and regeneration highly depends upon the fast ingrowth of blood vessels from the surrounding tissues. Before selecting potential biomaterials for clinical applications, they must be thoroughly tested with proper analytical tools. This unit provides a protocol for studying the potential of cell-seeded scaffolds to attract vessels that will form vascular networks within biomaterials. It includes seeding of stem cells into silk fibroin scaffolds, angiogenesis assay on the chorioallantoic membrane (CAM) of fertilized chicken eggs, a procedure for perfusion with MicroFil, and finally microcomputed tomography (µCT) scanning...
May 16, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28510333/tetracycline-inducible-and-reversible-stable-gene-expression-in-human-ipsc-derived-neural-progenitors-and-in-the-postnatal-mouse-brain
#8
Aslam Abbasi Akhtar, Joshua J Breunig
The pB-tet-GOI plasmid system allows for stable piggyBac transposition-mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox to the cell culture or diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain...
May 16, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28510332/human-umbilical-cord-mesenchymal-stromal-cell-isolation-expansion-cryopreservation-and-characterization
#9
J Robert Smith, Adrienne Cromer, Mark L Weiss
Revised methods to derive, expand, and characterize mesenchymal stromal cells (MSCs) from the umbilical cord are provided. Several considerations are taken for GMP compliance including using a closed system isolation method and eliminating several xenogenic components. With this method cells are isolated using mechanical and enzymatic digestion and then expanded with high viabilities that retain >90% viability after cryopreservation. Lastly, characterization methods have been optimized to identify these cells as MSCs according to the ISCT minimal criteria...
May 16, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28510331/isolation-of-ready-to-use-adipose-derived-stem-cell-asc-pellet-for-clinical-applications-and-a-comparative-overview-of-alternate-methods-for-asc-isolation
#10
Edoardo Raposio, Nicolò Bertozzi
Current literature does not offer a standardized method to isolate adipose-derived stem cells (ASCs) for clinical applications and hence clinical studies using ASCs often show inconsistent results. Most of these studies borrow laboratory or benchside-derived protocols, which are complex, time consuming, and involve the use of chemical, animal-derived reagents. In this unit we describe a relatively simple and faster isolation protocol that allows collection of a ready-to-use ASC pellet for clinical application...
May 16, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28510330/rhesus-macaque-ipsc-generation-and-maintenance
#11
Ravi Chandra Yada, So Gun Hong, Yongshun Lin, Thomas Winkler, Cynthia E Dunbar
The rhesus macaque (Macaca mulatta) is physiologically and phylogenetically similar to humans, and therefore represents an invaluable model for the pre-clinical assessment of the safety and feasibility of iPSC-derived cell therapies. The use of an excisable polycistronic lentiviral STEMCCA vector to reprogram rhesus fibroblasts or bone marrow stromal cells (BMSCs) into RhiPSCs is described. After reprogramming, the pluripotency transgenes can be removed by transient expression of Cre, leaving a residual genetic tag that may be useful for identification of RhiPSC-derived tissues in vivo...
May 16, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28510329/isolation-of-stem-cells-and-progenitors-from-mouse-epidermis
#12
Lana Kostic, Egor Sedov, Despina Soteriou, Yahav Yosefzon, Yaron Fuchs
The epidermis consists of several distinct compartments including the interfollicular epidermis (IFE), sweat glands, sebaceous glands (SGs), and the hair follicle (HF). While the IFE and SGs are in a constant state of self-renewal, the HF cycles between phases of growth, destruction, and rest. The hair follicle stem cells (HFSCs) that fuel this perpetual cycle have been well described and are located in a niche termed the bulge. These bulge SCs express markers such as CD34 and Keratin 15 (K15), enabling the isolation of these cells...
May 16, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28152183/efficient-generation-of-cardiac-purkinje-like-cells-from-embryonic-stem-cells-by-activating-camp-signaling
#13
Su-Yi Tsai, Shuibing Chen, Todd Evans
Strategies to derive cardiac conduction system (CCS) cells including Purkinje cells (PC) would facilitate models for mechanistic studies and drug discovery, and also provide new cellular materials for regenerative therapies. However, using current cardiac differentiation protocols, the differentiation efficiency of CCS cells is extremely low, typically below 1% of the culture. High-throughput chemical screening is a powerful strategy for identifying small molecules that can activate signaling pathways to enhance embryonic stem cell (ESC) differentiation...
February 2, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28152182/reprogramming-of-pancreatic-acinar-cells-to-functional-beta-cells-by-in-vivo-transduction-of-a-polycistronic-construct-containing-pdx1-ngn3-mafa-in-mice
#14
C Cavelti-Weder, A Zumsteg, W Li, Q Zhou
To generate new beta cells after birth is a key focus of regenerative medicine, which could greatly aid the major health burden of diabetes. Beta-cell regeneration has been described using four different approaches: (1) the development of beta cells from putative precursor cells of the adult pancreas, which is termed neogenesis, (2) replication of existing beta cells, (3) differentiation from embryonic or induced pluripotent stem cells, and (4) reprogramming of non-beta cells to beta cells. Studies from the authors' laboratory have shown that beta-cell reprogramming can be achieved by transduction of adult pancreatic tissues with viral constructs containing the three developmentally important transcription factors Pdx1, Ngn3, and MafA...
February 2, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28152181/purification-of-definitive-endoderm-generated-from-pluripotent-stem-cells-by-magnetic-cell-sorting
#15
Ulf Diekmann, Claudia Davenport, Jasmin Kresse, Ortwin Naujok
Pluripotent stem cells have the capability to differentiate into any somatic cell type of the human body. The generation of surrogate cells for the treatment of liver, lung, and pancreatic diseases is of great medical interest. First, the in vitro formation into cells of the definitive endoderm is required. Upon commitment into this lineage, the cells express transcription factors such as FOXA2, SOX17, HNF1B; GATA family members; and the surface protein CXCR4. Unfortunately, some pluripotent stem cells resist the differentiation and contaminate the culture...
February 2, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28152180/magnetic-nanoparticle-mediated-gene-delivery-to-two-and-three-dimensional-neural-stem-cell-cultures-magnet-assisted-transfection-and-multifection-approaches-to-enhance-outcomes
#16
Mark R Pickard, Christopher F Adams, Divya M Chari
Neural stem cells (NSCs) have high translational potential in transplantation therapies for neural repair. Enhancement of their therapeutic capacity by genetic engineering is an important goal for regenerative neurology. Magnetic nanoparticles (MNPs) are major non-viral vectors for safe bioengineering of NSCs, offering critical translational benefits over viral vectors, including safety, scalability, and ease of use. This unit describes protocols for the production of suspension (neurosphere) and adherent (monolayer) murine NSC cultures...
February 2, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/27532820/comprehensive-protocols-for-crispr-cas9-based-gene-editing-in-human-pluripotent-stem-cells
#17
David P Santos, Evangelos Kiskinis, Kevin Eggan, Florian T Merkle
Genome editing of human pluripotent stem cells (hPSCs) with the CRISPR/Cas9 system has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field...
August 17, 2016: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/27532819/self-cloning-crispr
#18
Mandana Arbab, Richard I Sherwood
CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines...
August 17, 2016: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/27532817/neural-stem-cell-or-human-induced-pluripotent-stem-cell-derived-gaba-ergic-progenitor-cell-grafting-in-an-animal-model-of-chronic-temporal-lobe-epilepsy
#19
Dinesh Upadhya, Bharathi Hattiangady, Geetha A Shetty, Gabriele Zanirati, Maheedhar Kodali, Ashok K Shetty
Grafting of neural stem cells (NSCs) or GABA-ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug-resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self-renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). On the other hand, GPCs could be generated from the medial and lateral ganglionic eminences of the embryonic brain and from hESCs and hiPSCs...
August 17, 2016: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/27532816/efficient-generation-of-viral-and-integration-free-human-induced-pluripotent-stem-cell-derived-oligodendrocytes
#20
Araceli Espinosa-Jeffrey, Bruno Blanchi, Juan Carlos Biancotti, Shalini Kumar, Megumi Hirose, Berhan Mandefro, Dodanim Talavera-Adame, Nissim Benvenisty, Jean de Vellis
Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation-our protocol generates viral- and integration-free OLs that efficiently commit and move forward in the OL lineage, recapitulating all the steps known to occur during in vivo development; (2) a method for the isolation, propagation and maintenance of neural stem cells (NSCs); and (3) a protocol for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs...
August 17, 2016: Current Protocols in Stem Cell Biology
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