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Current Protocols in Stem Cell Biology

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https://www.readbyqxmd.com/read/29512130/conditional-manipulation-of-gene-function-in-human-cells-with-optimized-inducible-shrna
#1
Alessandro Bertero, Loukia Yiangou, Stephanie Brown, Daniel Ortmann, Matthias Pawlowski, Ludovic Vallier
The difficulties involved in conditionally perturbing complex gene expression networks represent major challenges toward defining the mechanisms controlling human development, physiology, and disease. We developed an OPTimized inducible KnockDown (OPTiKD) platform that addresses the limitations of previous approaches by allowing streamlined, tightly-controlled, and potent loss-of-function experiments for both single and multiple genes. The method relies on single-step genetic engineering of the AAVS1 genomic safe harbor with an optimized tetracycline-responsive cassette driving one or more inducible short hairpin RNAs (shRNAs)...
February 28, 2018: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29512129/controlling-the-effective-oxygen-tension-experienced-by-cells-using-a-dynamic-culture-technique-for-hematopoietic-ex-vivo-expansion
#2
Abhilasha Tiwari, Cynthia S Wong, Lakshmi P Nekkanti, James A Deane, Courtney McDonald, Jingang Li, Yen Pham, Amy E Sutherland, Graham Jenkin, Mark A Kirkland
Clinical hematopoietic stem/progenitor cell (HSPC) transplantation outcomes are strongly correlated with the number of cells infused. Hence, to generate sufficient HSPCs for transplantation, the best culture parameters for expansion are critical. It is generally assumed that the defined oxygen (O2 ) set for the incubator reflects the pericellular O2 to which cells are being exposed. Studies have shown that low O2 tension maintains an undifferentiated state, but the expansion rate may be constrained because of limited diffusion in a static culture system...
February 28, 2018: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29512111/derivation-of-multipotent-mesenchymal-stromal-cells-from-ovine-bone-marrow
#3
Daniel Vivas, Marta Caminal, Irene Oliver-Vila, Joaquim Vives
In the field of orthopedics, translational research of novel therapeutic approaches involves the use of large animal models (such as sheep, goat, pig, dog, and horse) due to the similarities with humans in weight, size, joint structure, and bone/cartilage healing mechanisms. Particularly in the development of cell-based therapies, the lack of manageable immunocompromised preclinical large animal models prevents the use of human cells, which makes it necessary to produce equivalent homologous cell types for the study of their pharmacodynamics, pharmacokinetics, and toxicology...
February 28, 2018: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29512108/direct-conversion-of-human-pluripotent-stem-cells-to-osteoblasts-with-a-small-molecule
#4
Heemin Kang, Yu-Ru V Shih, Shyni Varghese
Human pluripotent stem cells (hPSCs), which exhibit unlimited self-renewal ability and can differentiate into all cell types in the human body, are a promising cell source for cell-based therapies and regenerative medicine. Small molecules hold great potential in the derivation of tissue-specific cells from hPSCs owing to their cost-effectiveness and scalability. Here, we describe a protocol for deriving osteoblasts from hPSCs by using a single, natural small molecule: adenosine. This simple and effective experimental protocol allows one to obtain large numbers of osteoblasts or osteoprogenitor cells, with the ability to form functional bone tissues, from hPSCs, including human embryonic stem cells and induced pluripotent stem cells...
February 28, 2018: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29512106/crispr-cas9-based-genome-editing-of-human-induced-pluripotent-stem-cells
#5
Joseph C Giacalone, Tasneem P Sharma, Erin R Burnight, John F Fingert, Robert F Mullins, Edwin M Stone, Budd A Tucker
Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome...
February 28, 2018: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29512105/hpsc-derived-midbrain-dopaminergic-neurons-generated-in-a-scalable-3-d-biomaterial
#6
Maroof M Adil, David V Schaffer
Human pluripotent stem cell (hPSC)-derived midbrain dopaminergic (mDA) neurons may facilitate the development of therapies for Parkinson's disease via disease modeling, drug screening, and cell replacement therapy. However, large numbers of cells are typically needed for these applications, and 2-D culture-based approaches typically used for mDA differentiation are difficult to scale up and require a long time for mDA maturation. Here we present a protocol to rapidly generate functional mDA neurons in a fully defined, scalable, thermoresponsive 3-D biomaterial...
February 28, 2018: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29140570/rapid-screening-of-the-endodermal-differentiation-potential-of-human-pluripotent-stem-cells
#7
Richard Siller, Gareth J Sullivan
Human pluripotent stem cells (hPSCs) hold tremendous promise for regenerative medicine, disease modeling, toxicology screening, and developmental biology. These applications are hindered due to inherent differences in differentiation potential observed among different hPSC lines. This is particularly true for the differentiation of hPSCs toward the endodermal lineage. Several groups have developed methods to screen hPSCs for their endodermal differentiation potential (EP). Particularly notable studies include (i) the use of WNT3A expression as a predictive biomarker, (ii) an embryoid body-based screen, and (iii) a transcriptomics-based approach...
November 15, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29140569/single-cell-functional-analysis-of-stem-cell-derived-cardiomyocytes-on-micropatterned-flexible-substrates
#8
Jan David Kijlstra, Dongjian Hu, Peter van der Meer, Ibrahim J Domian
Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC-CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC-CM contractile function...
November 15, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29140568/crispr-cas9-based-safe-harbor-gene-editing-in-rhesus-ipscs
#9
Ravi Chandra Yada, John W Ostrominski, Ilker Tunc, So Gun Hong, Jizhong Zou, Cynthia E Dunbar
NHP iPSCs provide a unique opportunity to test safety and efficacy of iPSC-derived therapies in clinically relevant NHP models. To monitor these cells in vivo, there is a need for safe and efficient labeling methods. Gene insertion into genomic safe harbors (GSHs) supports reliable transgene expression while minimizing the risk the modification poses to the host genome or target cell. Specifically, this protocol demonstrates targeting of the adeno-associated virus site 1 (AAVS1), one of the most widely used GSH loci in the human genome, with CRISPR/Cas9, allowing targeted marker or therapeutic gene insertion in rhesus macaque induced pluripotent stem cells (RhiPSCs)...
November 15, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29140567/human-adipose-derived-stromal-cell-isolation-methods-and-use-in-osteogenic-and-adipogenic-in-vivo-applications
#10
Elizabeth Brett, Ruth Tevlin, Adrian McArdle, Eun Young Seo, Charles K F Chan, Derrick C Wan, Michael T Longaker
Adipose tissue represents an abundant and easily accessible source of multipotent cells, which may serve as excellent building blocks for tissue engineering. This article presents a newly described protocol for isolating adipose-derived stromal cells (ASCs) from human lipoaspirate, compared to the standard protocol for harvesting ASCs established in 2001. Human ASC isolation is performed using two methods, and resultant cells are compared through cell yield, cell viability, cell proliferation and regenerative potential...
November 15, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29140566/mesenchymal-stem-cell-preparation-and-transfection-free-ferumoxytol-labeling-for-mri-cell-tracking
#11
Li Liu, Chien Ho
Mesenchymal stem cells (MSCs) are multipotent cells and are the most widely studied cell type for stem cell therapies. In vivo cell tracking of MSCs labeled with an FDA-approved superparamagnetic iron-oxide (SPIO) particle by magnetic resonance imaging (MRI) provides essential information, e.g., MSC engraftment, survival, and fate, thus improving cell therapy accuracy. However, current methodology for labeling MSCs with Ferumoxytol (Feraheme® ), the only FDA-approved SPIO particle, needs transfection agents...
November 15, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29140565/visualization-and-modeling-of-the-in-vivo-distribution-of-mesenchymal-stem-cells
#12
Haolu Wang, Camilla A Thorling, Zhi Ping Xu, Darrell H G Crawford, Xiaowen Liang, Xin Liu, Michael S Roberts
This unit describes a protocol for elucidating the in vivo disposition of administered mesenchymal stem cells (MSCs). Specifically, direct visualization of donor cell spatiotemporal distribution and assessment of donor cell quantity in recipient organs are described. Protocols for data analysis are suggested, with the goal of developing a model to characterize and predict the physiological kinetics of administered MSCs. The use of this model is described, suggesting that it can be applied to abnormal conditions and has potential interspecies and inter-route predictive capability...
November 15, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29081882/derivation-of-telencephalic-oligodendrocyte-progenitors-from-human-pluripotent-stem-cells
#13
Tamara Major, Ann Powers, Viviane Tabar
Oligodendrocytes are the main myelinating cell of the adult CNS and are vulnerable to injury in diverse disorders such as spinal cord injury, stroke, trauma, pharmacological and radiation toxicity, as well as neuroinflammation. Human pluripotent stem cells are attractive sources of oligodendrocyte lineage cells and provide a promising treatment strategy for exogenous myelin repair through transplantation. This unit describes a protocol for the step-wise differentiation of forebrain late oligodendrocyte progenitor cells (OPCs) from human pluripotent stem cells in defined chemical in vitro culture conditions...
November 8, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/28806856/direct-cell-labeling-to-image-transplanted-stem-cells-in-real-time-using-a-dual-contrast-mri-technique
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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