Current Protocols in Stem Cell Biology最新文献

{"title":"Deriving Keratinocyte Progenitor Cells and Keratinocytes from Human-Induced Pluripotent Stem Cells","authors":"Michel R. Ibrahim,&nbsp;Walid Medhat,&nbsp;Hasan El-Fakahany,&nbsp;Hamza Abdel-Raouf,&nbsp;Evan Y. Snyder","doi":"10.1002/cpsc.119","DOIUrl":"10.1002/cpsc.119","url":null,"abstract":"<p>Skin or hair loss (alopecia) may occur due to a wide variety of causes ranging from trauma to pathological processes including acquired or congenital causes. It would be ideal to replace them with immunologically compatible cells to avoid potentially exacerbating the condition. Deriving the replacement cells from human-induced pluripotent stem cells (hiPSCs) allows for sufficient scale up and using hiPSCs as the choice of human pluripotent stem cells (hPSC) will ensure immunocompatibility. Here we offer a protocol for differentiating hiPSCs into keratinocyte progenitor cells (KPC) and keratinocytes employing all-trans retinoic acid (ATRA) and <span>L</span>-ascorbic acid, (L-AA), bone morphogenic protein-4 (BMP4), and epidermal growth factor (EGF). We observed that the hiPSC-derived KPCs express the same panel of markers as primary hair follicle bulge stem cells (HFBSCs), including CD200, integrin α-6 (ITGA6), integrin β-1 (ITGB1), the transcription factor P63, keratin 15 (KRT15), and keratin 19 (KRT19). If permitted to differentiate further, the hiPSC-derived KPC lose CD200 expression and rather come to express keratin 14 (KRT14) indicating emergence of more mature terminally-differentiated keratinocytes. The HFBSCs are transplantable for hair follicle (HF) restoration, and the keratinocytes may be transplantable for therapy for large burns or ulcers. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Reprogramming of normal human skin fibroblasts into normal hiPSCs using episomal DNA cocktail</p><p><b>Basic Protocol 2</b>: Differentiation of hiPSCs into KPCs and keratinocytes</p><p><b>Alternate Protocol 2</b>: EBS formation protocol using AggreWell™ plates (Antonchuk, 2013)</p><p><b>Support Protocol 1</b>: Passage hiPSC-KPC</p><p><b>Support Protocol 2</b>: Immunocytochemistry (ICC)</p><p><b>Support Protocol 3</b>: Immunofluorescence staining of cells for flow cytometry (FC)</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.119","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38220265","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human-Induced Pluripotent Stem Cell Culture Methods Under cGMP Conditions","authors":"Teresa Rivera,&nbsp;Yuanyuan Zhao,&nbsp;Yuhui Ni,&nbsp;Jiwu Wang","doi":"10.1002/cpsc.117","DOIUrl":"10.1002/cpsc.117","url":null,"abstract":"<p>The discovery of induced pluripotent stem cells (iPSCs) revolutionized the approach to cell therapy in regenerative medicine. Reprogramming of somatic cells into an embryonic-like pluripotent state provides an invaluable resource of patient-specific cells of any lineage. Implementation of procedures and protocols adapted to current good manufacturing practice (cGMP) requirements is critical to ensure robust and consistent high-quality iPSC manufacturing. The technology developed at Allele Biotechnology for iPSC generation under cGMP conditions is a powerful platform for derivation of pluripotent stem cells through a footprint-free, feeder-free, and xeno-free reprogramming method. The cGMP process established by Allele Biotechnology entails fully cGMP compliant iPSC lines where the entire manufacturing process, from tissue collection, cell reprogramming, cell expansion, cell banking and quality control testing are adopted. Previously, we described in this series of publications how to create iPSCs using mRNA only, and how to do so under cGMP conditions. In this article, we describe in detail how to culture, examine and storage cGMP-iPSCs using reagents, materials and equipment compliant with cGMP standards. © 2020 The Authors.</p><p><b>Basic Protocol 1</b>: iPSC Dissociation</p><p><b>Support Protocol 1</b>: Stem cell media</p><p><b>Support Protocol 2</b>: ROCK inhibitor preparation</p><p><b>Support Protocol 3</b>: Vitronectin coating</p><p><b>Basic Protocol 2</b>: iPSC Cryopreservation</p><p><b>Basic Protocol 3</b>: iPSC Thawing</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.117","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38145758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation of Complete Multi−Cell Type Lung Organoids From Human Embryonic and Patient-Specific Induced Pluripotent Stem Cells for Infectious Disease Modeling and Therapeutics Validation","authors":"Sandra L. Leibel,&nbsp;Rachael N. McVicar,&nbsp;Alicia M. Winquist,&nbsp;Walter D. Niles,&nbsp;Evan Y. Snyder","doi":"10.1002/cpsc.118","DOIUrl":"10.1002/cpsc.118","url":null,"abstract":"<p>The normal development of the pulmonary system is critical to transitioning from placental-dependent fetal life to alveolar-dependent newborn life. Human lung development and disease have been difficult to study due to the lack of an in vitro model system containing cells from the large airways and distal alveolus. This article describes a system that allows human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to differentiate and form three-dimensional (3D) structures that emulate the development, cytoarchitecture, and function of the lung (“organoids”), containing epithelial and mesenchymal cell populations, and including the production of surfactant and presence of ciliated cells. The organoids can also be invested with mesoderm derivatives, differentiated from the same human pluripotent stem cells, such as alveolar macrophages and vasculature. Such lung organoids may be used to study the impact of environmental modifiers and perturbagens (toxins, microbial or viral pathogens, alterations in microbiome) or the efficacy and safety of drugs, biologics, and gene transfer. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: hESC/hiPSC dissection, definitive endoderm formation, and lung progenitor cell induction</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.118","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38138118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Design and Derivation of Multi-Reporter Pluripotent Stem Cell Lines via CRISPR/Cas9n-Mediated Homology-Directed Repair","authors":"Rabea Dettmer,&nbsp;Ortwin Naujok","doi":"10.1002/cpsc.116","DOIUrl":"10.1002/cpsc.116","url":null,"abstract":"<p>During the past decade, RNA-guided Cas9 nuclease from microbial clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) has become a powerful tool for gene editing of human pluripotent stem cells (PSCs). Using paired CRISPR/Cas9 nickases (CRISPR/Cas9n) it is furthermore possible to reduce off-target effects that may typically occur with traditional CRISPR/Cas9 systems while maintaining high on-target efficiencies. With this technology and a well-designed homology-directed repair vector (HDR), we are now able to integrate transgenes into specific gene loci of PSCs in an allele conserving way. In this protocol we describe CRISPR/Cas9n design and homology directed repair vector design, transfection of human pluripotent stem cells and selection and expansion of generated cell clones. © 2020 The Authors.</p><p><b>Basic Protocol 1</b>: Repair template design and CRISPR/Cas9n construction</p><p><b>Basic Protocol 2</b>: Transfection of human pluripotent stem cells by electroporation</p><p><b>Basic Protocol 3</b>: Genotyping of generated cell clones</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.116","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38123894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Humanized Mouse Models for Evaluation of PSC Immunogenicity","authors":"Jack Hermsen,&nbsp;Matthew E. Brown","doi":"10.1002/cpsc.113","DOIUrl":"10.1002/cpsc.113","url":null,"abstract":"<p>New human pluripotent stem cell (hPSC)-derived therapies are advancing to clinical trials at an increasingly rapid pace. In addition to ensuring that the therapies function properly, there is a critical need to investigate the human immune response to these cell products. A robust allogeneic (or autologous) immune response could swiftly eliminate an otherwise promising cell therapy, even in immunosuppressed patients. In coming years, researchers in the regenerative medicine field will need to utilize a number of in vitro and in vivo assays and models to evaluate and better understand hPSC immunogenicity. Humanized mouse models—mice engrafted with functional human immune cell types—are an important research tool for investigating the mechanisms of the adaptive immune response to hPSC therapies. This article provides an overview of humanized mouse models relevant to the study of hPSC immunogenicity and explores central considerations for investigators seeking to utilize these powerful models in their research. © 2020 Wiley Periodicals LLC.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.113","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38093544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-throughput Preparation of DNA, RNA, and Protein from Cryopreserved Human iPSCs for Multi-omics Analysis","authors":"Jeffrey X. Zhang,&nbsp;Edward Lau,&nbsp;David T. Paik,&nbsp;Yan Zhuge,&nbsp;Joseph C. Wu","doi":"10.1002/cpsc.114","DOIUrl":"10.1002/cpsc.114","url":null,"abstract":"<p>We describe the procedure to isolate genomic DNA, RNA, and protein directly from cryopreserved induced pluripotent stem cell (iPSC) vials using commercially available solid-phase extraction kits, and we report the relationship between macromolecule yields and experimental and storage factors. Sufficient quantities of DNA, RNA, and protein are recoverable from as low as 1 million cryopreserved cells across 728 distinct iPSC lines suitable for whole-genome sequencing, RNA sequencing, and mass spectrometry experiments. Nucleic acids extracted from iPSC stocks cryopreserved up to 4 years maintain sufficient quantity and integrity for downstream analysis with minimal genomic DNA fragmentation. An expected positive correlation exists between cell count and DNA or RNA yield, with comparable yields recovered between cells across different cryostorage timespans. This article provides an effective way to simultaneously isolate iPSC biomolecules for multi-omics investigations. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: QIAshredder and AllPrep DNA/RNA/protein mini kit extraction and subsequent DNA quantification and quality analysis</p><p><b>Basic Protocol 2</b>: Broad-range RNA quantification and quality assay using QuBit 4 Fluorometer and associated kits</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.114","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38083962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DNA Fiber Assay for the Analysis of DNA Replication Progression in Human Pluripotent Stem Cells","authors":"Jason A. Halliwell,&nbsp;Polly Gravells,&nbsp;Helen E. Bryant","doi":"10.1002/cpsc.115","DOIUrl":"10.1002/cpsc.115","url":null,"abstract":"<p>Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cell-based regenerative medicine. The rapid proliferation of pluripotent cells leads to constitutive replication stress, hindering the progression of DNA replication forks and in some cases leading to replication-fork collapse. Failure to overcome replication stress can result in incomplete genome duplication, which, if left to persist into the subsequent mitosis, can result in structural and numerical chromosomal instability.</p><p>We have recently applied the DNA fiber assay to the study of replication stress in human PSC and found that, in comparison to somatic cells states, these cells display features of DNA replication stress that include slower replication fork speeds, evidence of stalled forks, and replication initiation from dormant replication origins. These findings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA fiber assay has enabled the development of an advanced nucleoside-enriched culture medium that increases replication fork progression and decreases DNA damage and mitotic errors in human PSC cultures.</p><p>The DNA fiber assay allows for the study of replication fork dynamics at single-molecule resolution. The assay relies on cells incorporating nucleotide analogs into nascent DNA during replication, which are then measured to monitor several replication parameters. Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: DNA fiber labeling</p><p><b>Basic Protocol 2</b>: DNA fiber spreading</p><p><b>Basic Protocol 3</b>: Immunostaining</p><p><b>Support Protocol 1</b>: Microscopy/data acquisition</p><p><b>Support Protocol 2</b>: Data analysis</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.115","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38084325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reprogramming of Human Hepatic Non-Parenchymal Cells: Step-by-Step Protocol","authors":"Varvara A. Kirchner,&nbsp;Kirk Twaroski,&nbsp;Kelli Wysoglad,&nbsp;Jenna Algoo,&nbsp;Edward L. LeCluyse,&nbsp;Gi-Won Song,&nbsp;Eunyoung Tak,&nbsp;Weili Chen,&nbsp;Sung-Gyu Lee,&nbsp;Timothy L. Pruett,&nbsp;Jakub Tolar","doi":"10.1002/cpsc.112","DOIUrl":"10.1002/cpsc.112","url":null,"abstract":"<p>Human induced pluripotent stem cells (h-iPSCs) represent a potentially unlimited source for the generation of human hepatocyte-like cells (h-iHLCs) for the establishment of platforms to study drug-induced hepatotoxicity, liver disease modeling, and ultimately the application of h-iHLCs in treatment of patients with end-stage liver disease. To understand the impact of donor-specific factors on the generation of h-iHLCs, the model for the direct comparison of h-iHLCs and primary human hepatocytes (PHHs) from the same human donor is needed. This study proposes a step-by-step protocol for plating, expansion, and characterization of primary human hepatic non-parenchymal cells (h-NPCs) isolated from the human liver, the reprogramming of generated h-NPCs into h-iPSCs and subsequent differentiation of reprogrammed h-iPSCs into h-iHLCs. The ultimate goal is to compare the gene expression involved in hepatocyte metabolism between h-iHLCs and PHHs from the same human donor thus eliminating interdonor variability. This newly developed protocol of h-NPC culture, expansion, and reprogramming into h-iPSCs allows: (1) utilization of a single organ source (i.e., liver) for isolation of PHHs and h-NPCs and (2) the in-depth study of donor factors involved in the generation of h-iHLCs with direct comparison to PHHs from the same donor. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Plating and expansion of human hepatic NPCs in culture</p><p><b>Basic Protocol 2</b>: Reprogramming of h-NPCs to h-NPC-derived induced pluripotent stem cells (h-iPSCs)</p><p><b>Basic Protocol 3</b>: Culture, passaging, and freezing of h-iPSCs</p><p><b>Support Protocol 1</b>: Confirmation of h-NPC ability to uptake Sendai virus: GFP-Sendai infection</p><p><b>Support Protocol 2</b>: Characterization of h-NPCs amenable to transduction with Sendai particles</p><p><b>Support Protocol 3</b>: Characterization of h-iPSCs: Clearance of viral reprogramming vectors</p><p><b>Support Protocol 4</b>: Preparation of Matrigel-coated plates</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.112","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86100441","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purification of Live Stem-Cell-Derived Islet Lineage Intermediates","authors":"Niloofar Rasouli,&nbsp;Douglas A. Melton,&nbsp;Juan R. Alvarez-Dominguez","doi":"10.1002/cpsc.111","DOIUrl":"10.1002/cpsc.111","url":null,"abstract":"<p>Stem-cell-derived tissues offer platforms to study organ development, model physiology during health and disease, and test novel therapies. We describe methods to isolate cells at successive stages during in vitro differentiation of human stem cells into the pancreatic endocrine lineage. Using flow cytometry, we purify live lineage intermediates in numbers not available by fetal biopsy. These include pancreatic and endocrine progenitors, isolated based on known surface markers. We further report a strategy that leverages intracellular zinc content and DPP4/CD26 expression to separate monohormonal insulin⁺ β cells from polyhormonal counterparts. These methods enable comprehensive molecular profiling during human islet lineage progression. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: In vitro isolation of human islet developmental intermediates</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.111","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38029935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generating a Cost-Effective, Weekend-Free Chemically Defined Human Induced Pluripotent Stem Cell (hiPSC) Culture Medium","authors":"Hananeh Fonoudi,&nbsp;Davi M. Lyra-Leite,&nbsp;Hoor A. Javed,&nbsp;Paul W. Burridge","doi":"10.1002/cpsc.110","DOIUrl":"10.1002/cpsc.110","url":null,"abstract":"<p>We have previously developed a cost-effective chemically defined medium formula for weekend-free culture of human induced pluripotent stem cells (hiPSCs), costing ∼3% of the price of commercial medium. This medium, which we termed B8, is specifically optimized for robust and fast growth of hiPSCs and for a weekend-free medium change regimen. We demonstrated that this medium is suitable for reprogramming of somatic cells into hiPSCs and for differentiation into a variety of lineages. Here, we provide a protocol for simple generation of the most cost-effective variant of this medium, along with a protocol for making Matrigel-coated plates and culturing, passaging, cryopreserving, and thawing hiPSCs. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of a highly optimized, robust, and cost-effective human induced pluripotent stem cell culture medium</p><p><b>Basic Protocol 2</b>: Weekend-free maintenance and passaging of human induced pluripotent stem cells in B8 medium</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.110","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37982332","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}