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Current Protocols in Stem Cell Biology

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https://www.readbyqxmd.com/read/28510334/angiogenesis-within-stem-cell-seeded-silk-scaffolds-cultured-on-the-chorioallantoic-membrane-and-visualized-by-3d-imaging
#1
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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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
#12
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
#13
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
#14
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
https://www.readbyqxmd.com/read/27532815/isolation-and-assessment-of-single-long-term-reconstituting-hematopoietic-stem-cells-from-adult-mouse-bone-marrow
#15
David G Kent, Brad J Dykstra, Connie J Eaves
Hematopoietic stem cells with long-term repopulating activity can now be routinely obtained at purities of 40% to 50% from suspensions of adult mouse bone marrow. Here we describe robust protocols for both their isolation as CD45(+) EPCR(+) CD150(+) CD48(-) (ESLAM) cells using multiparameter cell sorting and for tracking their clonal growth and differentiation activity in irradiated mice transplanted with single ESLAM cells. The simplicity of these procedures makes them attractive for characterizing the molecular and biological properties of individual hematopoietic stem cells with unprecedented power and precision...
August 17, 2016: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/27171793/a-method-for-sectioning-and-immunohistochemical-analysis-of-stem-cell-derived-3-d-organoids
#16
Luke A Wiley, David C Beebe, Robert F Mullins, Edwin M Stone, Budd A Tucker
This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types...
May 12, 2016: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/26840227/delivery-of-genome-editing-reagents-to-hematopoietic-stem-progenitor-cells
#17
REVIEW
Megan D Hoban, Zulema Romero, Gregory J Cost, Matthew Mendel, Michael Holmes, Donald B Kohn
This unit describes the protocol for the delivery of reagents for targeted genome editing to CD34(+) hematopoietic stem/progenitor cells (HSPCs). Specifically, this unit focuses on the process of thawing and pre-stimulating CD34(+) HSPCs, as well as the details of their electroporation with in vitro-transcribed mRNA-encoding site-specific nucleases [in this case zinc-finger nucleases (ZFNs)]. In addition, discussed is delivery of a gene editing donor template in the form of an oligonucleotide or integrase-defective lentiviral vector (IDLV)...
February 3, 2016: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/26840226/identifying-adult-stem-cells-using-cre-mediated-lineage-tracing
#18
REVIEW
Diana L Carlone
Lineage-tracing has been used for decades to establish cell fate maps during development. Recently, with the advent of genetic lineage-tracing techniques (employing Cre-lox recombination), it has been possible to permanently mark progenitor/stem cell populations within somatic tissues. In addition, pulse-chase studies have shown that only stem cells are capable of producing labeled progeny after an extensive period of chase. This unit focuses on the protocols used to target putative adult stem cells in vivo...
February 3, 2016: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/26840225/differentiation-of-mouse-embryonic-stem-cells-into-ventral-foregut-precursors
#19
REVIEW
Michaela Rothová, Jurriaan J Hölzenspies, Alessandra Livigni, Santiago Nahuel Villegas, Joshua M Brickman
Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut derivatives...
February 3, 2016: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/26840224/derivation-of-human-skin-fibroblast-lines-for-feeder-cells-of-human-embryonic-stem-cells
#20
REVIEW
Christian Unger, Ulrika Felldin, Sergey Rodin, Agneta Nordenskjöld, Sirac Dilber, Outi Hovatta
After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures...
February 3, 2016: Current Protocols in Stem Cell Biology
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