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Current Protocols in Stem Cell Biology

Devang K Thakor, Lei Wang, Darcy Benedict, Serdar Kabatas, Ross D Zafonte, Yang D Teng
Human mesenchymal stromal stem cells (hMSCs) hold regenerative medicine potential due to their availability, in vitro expansion readiness, and autologous feasibility. For neural repair, hMSCs show translational value in research on stroke, spinal cord injury (SCI), and traumatic brain injury. It is pivotal to establish multimodal in vitro systems to investigate molecular mechanisms underlying neural actions of hMSCs. Here, we describe a platform protocol on how to set up organotypic co-cultures of hMSCs (alone or polymer-scaffolded) with explanted adult rat dorsal root ganglia (DRGs) to determine neural injury and recovery events for designing implants to counteract neurotrauma sequelae...
July 18, 2018: Current Protocols in Stem Cell Biology
Masayo Kondo, Michihiko Sugimoto, Kuniya Abe
Epiblast stem cells (EpiSCs) are primed pluripotent stem cells (PSCs) derived from mouse postimplantation embryos. Interestingly, EpiSCs share many characteristics with human PSCs such as human embryonic stem cells (hESCs) and human induced PSCs (hiPSC). Thus, EpiSCs can serve as a model for studying primed states of pluripotency. This article describes a simple yet highly efficient protocol for EpiSC derivation and maintenance of homogenous EpiSCs using an inhibitor of WNT secretion. Using this method, EpiSCs can be readily derived from mouse strains with different genetic background including C57BL/6N...
August 2018: Current Protocols in Stem Cell Biology
Wei Lian, Yuning Jia, Lingyun Li, Zhong Huang, Jianyong Xu
Cardiospheres represent a more effective cell-based therapy for treatment of myocardial infarction than stem cells of non-cardiac origin. Unfortunately, their therapeutic application is limited by low yield of cell harvesting, declining quality and quantity during the aging process, and the need for highly invasive heart biopsy. Therefore, there is an emerging interest in generating cardiosphere-like stem cells from somatic cells via somatic reprogramming. This novel approach would provide an unlimited source of stem cells with cardiac differentiation potential...
August 2018: Current Protocols in Stem Cell Biology
Joseph J Kim, Prabhas V Moghe
This unit describes a protocol for acquiring and analyzing high-content super-resolution images of human stem cell nuclei for the characterization and classification of the cell differentiation paths based on distinct patterns of epigenetic mark organization. Here, we describe the cell culture, immunocytochemical labeling, super-resolution imaging parameters, and MATLAB-based quantitative image analysis approaches for monitoring human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cells (hiPSCs) as the cells differentiate towards various lineages...
August 2018: Current Protocols in Stem Cell Biology
Dan C Wilkinson, Michael Mellody, Luisa K Meneses, Ashley C Hope, Bruce Dunn, Brigitte N Gomperts
This unit describes a protocol for generation of lung organoids. A lung organoid is a 3D cell/hydrogel composite that resembles the morphology and cellular composition of the human distal lung. These tissue-engineered constructs provide an in vitro model of human lung and are best suited for disease modeling applications. The organoid generation methodology is flexible, allowing for easy scalability in the number of organoids produced and in the ability to accommodate a wide range of cell types. © 2018 by John Wiley & Sons, Inc...
August 2018: Current Protocols in Stem Cell Biology
Claudia Lo Sicco, Daniele Reverberi, Luisa Pascucci, Roberta Tasso
The unit describes protocols for isolating and characterizing extracellular vesicles (EVs) derived from human adipose tissue-derived mesenchymal stromal cells (MSCs). EVs are a mixed population of membrane-surrounded structures with overlapping composition and size. Advances made in recent years have led to a better understanding of the biological role of EVs. In particular, they can be considered key factors responsible for MSC-paracrine activity, mediating their anti-inflammatory effects towards innate immune cells, such as macrophages...
August 2018: Current Protocols in Stem Cell Biology
Arja Ray, Rachel K Morford, Paolo P Provenzano
Cell migration is strongly influenced by the organization of the surrounding 3-D extracellular matrix. In particular, within fibrous solid tumors, carcinoma cell invasion may be directed by patterns of aligned collagen in the extra-epithelial space. Thus, studying the interactions of heterogeneous populations of cancer cells that include the stem/progenitor-like cancer stem cell subpopulation and aligned collagen networks is critical to our understanding of carcinoma dissemination. Here, we describe a robust method to generate aligned collagen matrices in vitro that mimic in vivo fiber organization...
August 2018: Current Protocols in Stem Cell Biology
Marie Fernandes, Brian McArdle, Lauren Schiff, Timothy A Blenkinsop
An adult human retinal pigment epithelial layer (ahRPE) model derived from stem cells isolated from native RPE monolayers (ahRPE-SCs) exhibits key physiological characteristics of native tissue and therefore provides the means to create a human "disease in a dish" model to study RPE diseases. Traditionally, RPE lines are established from whole globes dedicated to research. Here we describe a new technique for establishing primary RPE lines from the posterior poles of globes used for corneal transplants...
May 2018: Current Protocols in Stem Cell Biology
Katherine B McCauley, Finn Hawkins, Darrell N Kotton
New protocols to efficiently generate functional airway epithelial organoids from human pluripotent stem cells (PSCs) would represent a major advance towards effective disease modeling, drug screening and cell based therapies for lung disorders. This unit describes an approach using stage-specific signaling pathway manipulation to differentiate cells to proximal airway epithelium via key developmental intermediates. Cells are directed via definitive endoderm (DE) to anterior foregut, and then specified to NKX2-1+ lung epithelial progenitors...
May 2018: Current Protocols in Stem Cell Biology
Navin Gupta, Koichiro Susa, Yoko Yoda, Joseph V Bonventre, M Todd Valerius, Ryuji Morizane
Human pluripotent stem cells (hPSCs) represent a formidable tool for disease modeling, drug discovery, and regenerative medicine using human cells and tissues in vitro. Evolving techniques of targeted genome editing, specifically the CRISPR/Cas9 system, allow for the generation of cell lines bearing gene-specific knock-outs, knock-in reporters, and precise mutations. However, there are increasing concerns related to the transfection efficiency, cell viability, and maintenance of pluripotency provided by genome-editing techniques...
May 2018: Current Protocols in Stem Cell Biology
Yoshinobu Takahashi, Takanori Takebe, Hideki Taniguchi
Despite the promise of emerging organoid-based approaches, building additional complexity, such as the vascular network, remains a major challenge toward regenerative therapy. Recently, we developed a complex organoid engineering method by "self-condensation," wherein mesenchymal cell-dependent contraction enables large-scale condensation from heterotypic multiple progenitors. Here, we describe the adaptation of this protocol for generating three-dimensional (3D) pancreatic condensates from dissociated β cell lines (MIN6) together with blood vessel-forming progenitors...
May 2018: Current Protocols in Stem Cell Biology
Jessica R Perez, Norma Ybarra, Frederic Chagnon, Olivier Lesur, Jan Seuntjens, Issam El Naqa
Stem cell therapy has shown great promise for organ repair and regeneration. In the context of lung disease, such as radiation-induced lung damage (RILD) in cancer radiotherapy, mesenchymal stem cells (MSCs) have shown the ability to reduce damage possibly due to their immunomodulatory properties and other unknown mechanisms. However, once MSCs are transplanted into the body, little is known as to their localization or their mechanisms of action. In this work, we proposed, implemented, and validated a fluorescence endomicroscopy (FE) imaging technique that allows for the real-time detection and quantification of transplanted pre-labeled MSCs in vivo and tracking in a rat model...
May 2018: Current Protocols in Stem Cell Biology
Seonmi Park, Gustavo Mostoslavsky
Human induced pluripotent stem cells (hiPSCs) offer great opportunities for the study of human development and disease modeling and have enormous potential for use in future clinical cell-based therapies. However, most current systems to create hiPSCs often expose the cells to animal feeder layers or xenogeneic reagents; this raises safety concerns about using hiPSC-derived cells for therapeutic purposes. Here, we describe protocols to generate hiPSCs without exposing the cells to xenogeneic materials that uses a defined, feeder-free reprogramming system...
May 2018: Current Protocols in Stem Cell Biology
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
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
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
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
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
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
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
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