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Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells

Featured protocol,  Authors: Daisuke Yamada
Daisuke YamadaAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: daisuke.yamada@riken.jp
Bio-protocol author page: a4503
Tomonori Iyoda
Tomonori IyodaAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4497
Kanako Shimizu
Kanako ShimizuAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4498
Yusuke Sato
Yusuke SatoAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4501
Haruhiko Koseki
Haruhiko KosekiAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4502
 and Shin-ichiro Fujii
Shin-ichiro FujiiAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: shin-ichiro.fujii@riken.jp
Bio-protocol author page: a4504
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2277.

Brief version appeared in Stem Cells, Dec 2016
Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24+iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8+ cytotoxic T lymphocytes to eliminate MHC- and MHC+ tumor cells, respectively. Here we describe a robust protocol to reprogram human Vα24+iNKT cells into iPSC, and then to re-differentiate them into Vα24+iNKT cells (iPS-Vα24+iNKT). We further provide a protocol to measure the activity of iPS-Vα24+iNKT cells.

Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice

Featured protocol,  Authors: Lubna Hindi
Lubna HindiAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a4417
Joseph D. McMillan
Joseph D. McMillanAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a4418
Dil Afroze
Dil AfrozeAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a4419
Sajedah M. Hindi
Sajedah M. HindiAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a3536
 and Ashok Kumar
Ashok KumarAffiliation 1: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Affiliation 2: Professor and Distinguished University Scholar, Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
For correspondence: ashok.kumar@louisville.edu
Bio-protocol author page: a3537
date: 5/5/2017, 243 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2248.

Brief version appeared in Nat Commun, Dec 2015
Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been suggested as the most physiologically relevant model for studying myogenesis in vitro. However, due to their low abundance in adult skeletal muscle, isolation of primary myoblasts is technically challenging. In this article, we describe an improved protocol for the isolation of primary myoblasts from adult skeletal muscle of mice. We also describe methods for their culturing and differentiation into myotubes.

Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC)

Featured protocol,  Authors: Megan V. Jackson
Megan V. JacksonAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
Bio-protocol author page: a4447
 and Anna D. Krasnodembskaya
Anna D. KrasnodembskayaAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
For correspondence: a.krasnodembskaya@qub.ac.uk
Bio-protocol author page: a4449
date: 5/5/2017, 189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2255.

Brief version appeared in Stem Cells, Aug 2016
Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms of MSC immunomodulatory effects are not fully understood yet. We sought to investigate MSC cell contact-dependent communication with alveolar macrophages (AM), professional phagocytes which play an important role in the lung inflammatory responses and anti-bacterial defence. With the use of a basic direct co-culture system, confocal microscopy and flow cytometry we visualised and effectively quantified MSC mitochondrial transfer to AM through tunnelling nanotubes (TNT). To model the human AM, primary monocytes were isolated from human donor blood and differentiated into macrophages (monocyte derived macrophages, MDM) in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), thus allowing adaptation of an AM-like phenotype (de Almeida et al., 2000; Guilliams et al., 2013). Human bone-marrow derived MSC, were labelled with mitochondria-specific fluorescent stain, washed extensively, seeded into the tissue culture plate with MDMs at the ratio of 1:20 (MSC/MDM) and co-cultured for 24 h. TNT formation and mitochondrial transfer were visualised by confocal microscopy and semi-quantified by flow cytometry. By using the method we described here we established that MSC use TNTs as the means to transfer mitochondria to macrophages. Further studies demonstrated that mitochondrial transfer enhances macrophage oxidative phosphorylation and phagocytosis. When TNT formation was blocked by cytochalasin B, MSC effect on macrophage phagocytosis was completely abrogated. This is the first study to demonstrate TNT-mediated mitochondrial transfer from MSC to innate immune cells.

Explant Methodology for Analyzing Neuroblast Migration

Featured protocol,  Authors: Kirsty J. Dixon
Kirsty J. DixonAffiliation: Department of Transplant Surgery, Virginia Commonwealth University, Richmond, USA
Bio-protocol author page: a4427
Alisa Turbic
Alisa TurbicAffiliation: Department of Epidemiology and Preventive Medicine, Monsah University, Melbourne, Australia
Bio-protocol author page: a4428
Ann M. Turnley
Ann M. TurnleyAffiliation: Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, Australia
Bio-protocol author page: a4429
 and Daniel J. Liebl
Daniel J. LieblAffiliation: The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami, Miami, USA
For correspondence: dliebl@miami.edu
Bio-protocol author page: a4430
date: 5/5/2017, 144 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2249.

Brief version appeared in Stem Cell Res, Nov 2016
The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we describe the isolation of SVZ-derived RMS explants from the neonatal mouse brain, and the conditions required to culture and evaluate their migration.

Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells

Authors: Daisuke Yamada
Daisuke YamadaAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: daisuke.yamada@riken.jp
Bio-protocol author page: a4503
Tomonori Iyoda
Tomonori IyodaAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4497
Kanako Shimizu
Kanako ShimizuAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4498
Yusuke Sato
Yusuke SatoAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4501
Haruhiko Koseki
Haruhiko KosekiAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4502
 and Shin-ichiro Fujii
Shin-ichiro FujiiAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: shin-ichiro.fujii@riken.jp
Bio-protocol author page: a4504
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2277.

[Abstract] Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an ...

Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice

Authors: Lubna Hindi
Lubna HindiAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a4417
Joseph D. McMillan
Joseph D. McMillanAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a4418
Dil Afroze
Dil AfrozeAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a4419
Sajedah M. Hindi
Sajedah M. HindiAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a3536
 and Ashok Kumar
Ashok KumarAffiliation 1: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Affiliation 2: Professor and Distinguished University Scholar, Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
For correspondence: ashok.kumar@louisville.edu
Bio-protocol author page: a3537
date: 5/5/2017, 243 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2248.

[Abstract] Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps ...

Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC)

Authors: Megan V. Jackson
Megan V. JacksonAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
Bio-protocol author page: a4447
 and Anna D. Krasnodembskaya
Anna D. KrasnodembskayaAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
For correspondence: a.krasnodembskaya@qub.ac.uk
Bio-protocol author page: a4449
date: 5/5/2017, 189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2255.

[Abstract] Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms ...

Explant Methodology for Analyzing Neuroblast Migration

Authors: Kirsty J. Dixon
Kirsty J. DixonAffiliation: Department of Transplant Surgery, Virginia Commonwealth University, Richmond, USA
Bio-protocol author page: a4427
Alisa Turbic
Alisa TurbicAffiliation: Department of Epidemiology and Preventive Medicine, Monsah University, Melbourne, Australia
Bio-protocol author page: a4428
Ann M. Turnley
Ann M. TurnleyAffiliation: Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, Australia
Bio-protocol author page: a4429
 and Daniel J. Liebl
Daniel J. LieblAffiliation: The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami, Miami, USA
For correspondence: dliebl@miami.edu
Bio-protocol author page: a4430
date: 5/5/2017, 144 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2249.

[Abstract] The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we describe ...

Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels

Authors: Markus Auler
Markus AulerAffiliation 1: Medical Faculty, Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Cologne, Germany
Affiliation 2: Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany
Bio-protocol author page: a4408
Lena Pitzler
Lena PitzlerAffiliation 1: Medical Faculty, Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Cologne, Germany
Affiliation 2: Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany
Bio-protocol author page: a4409
Ernst Pöschl
Ernst PöschlAffiliation: Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK
Bio-protocol author page: a4410
Zhigang Zhou
Zhigang ZhouAffiliation 1: Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK
Affiliation 2: Medical Faculty, University of East Anglia, Norwich Research Park, Norwich, UK
Bio-protocol author page: a4411
 and Bent Brachvogel
Bent BrachvogelAffiliation 1: Medical Faculty, Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Cologne, Germany
Affiliation 2: Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany
For correspondence: bent.brachvogel@uni-koeln.de
Bio-protocol author page: a4412
date: 4/20/2017, 258 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2247.

[Abstract] Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. Endothelial ...

Melanoma Stem Cell Sphere Formation Assay

Authors: Alessandra Tuccitto
Alessandra TuccittoAffiliation: Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Bio-protocol author page: a4374
Valeria Beretta
Valeria BerettaAffiliation: Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Bio-protocol author page: a4375
Francesca Rini
Francesca RiniAffiliation: Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Bio-protocol author page: a4376
Chiara Castelli
Chiara CastelliAffiliation: Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
For correspondence: chiara.castelli@istitutotumori.mi.it
Bio-protocol author page: a4377
 and Michela Perego
Michela PeregoAffiliation: The Wistar Institute, Philadelphia, USA
For correspondence: mperego@wistar.org
Bio-protocol author page: a4378
date: 4/20/2017, 292 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2233.

[Abstract] Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem cells ...

A Co-culture Model for Determining the Target Specificity of the de novo Generated Retinal Ganglion Cells

Authors: Pooja Teotia
Pooja TeotiaAffiliation: Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, USA
Bio-protocol author page: a4276
Matthew J. Van Hook
Matthew J. Van HookAffiliation: Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, USA
Bio-protocol author page: a4277
 and Iqbal Ahmad
Iqbal AhmadAffiliation: Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, USA
For correspondence: iahmad@unmc.edu
Bio-protocol author page: a4278
date: 4/5/2017, 264 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2212.

[Abstract] In glaucoma, the output neurons of the retina, the retinal ganglion cells (RGCs), progressively degenerate, leading to irreversible blindness (Ahram et al., 2015). The ex vivo stem cell method to replace degenerated RGCs remains a potentially viable approach (Levin et al., 2004). However, the success of the approach depends upon the ability of the ...

Reprogram Murine Epiblast Stem Cells by Epigenetic Inhibitors

Authors: Hui Zhang
Hui ZhangAffiliation: Department of Pathology, University of Michigan, Ann Arbor, USA
Bio-protocol author page: a4176
 and Yali Dou
Yali DouAffiliation: Department of Pathology, University of Michigan, Ann Arbor, USA
For correspondence: yalid@med.umich.edu
Bio-protocol author page: a4171
date: 3/5/2017, 446 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2168.

[Abstract] Pluripotent stem cells in the naïve state are highly useful in regenerative medicine and tissue engineering. A robust reprogramming of the primed murine Epiblast Stem Cells (EpiSCs) to naïve pluripotency is feasible via chemical-only approach. This protocol described a method to reprogram murine EpiSCs by MM-401 treatment, which blocks histone H3K4 ...

Isolation and Primary Culture of Adult Human Adipose-derived Stromal/Stem Cells

Authors: Robert B. Jones
Robert B. JonesAffiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, USA
Bio-protocol author page: a4155
Amy L. Strong
Amy L. StrongAffiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, USA
Bio-protocol author page: a4156
Jeffrey M. Gimble
Jeffrey M. GimbleAffiliation 1: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, USA
Affiliation 2: LaCell LLC, New Orleans, USA
Affiliation 3: Department of Surgery, Tulane University School of Medicine, New Orleans, USA
Bio-protocol author page: a4154
 and Bruce A. Bunnell
Bruce A. BunnellAffiliation 1: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, USA
Affiliation 2: Department of Pharmacology, Tulane University School of Medicine, New Orleans, USA
For correspondence: bbunnell@tulane.edu
Bio-protocol author page: a4157
date: 3/5/2017, 571 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2161.

[Abstract] Adipose-derived stromal/stem cells (ASCs) are multipotent cells that can be isolated from adipose tissue. Studies have shown that cells have the capacity to self-renew and differentiate into adipocyte, chondrocyte, myocyte, and osteoblast lineages. Thus, significant interest regarding their use for regenerative purposes to restore aging or damaged ...

Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells

Authors: Seymen Avci
Seymen AvciAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
Bio-protocol author page: a4075
Shiri Gur-Cohen
Shiri Gur-CohenAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
Bio-protocol author page: a4060
Francesca Avemaria
Francesca AvemariaAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
Bio-protocol author page: a4059
 and Tsvee Lapidot
Tsvee LapidotAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
For correspondence: Tsvee.Lapidot@weizmann.ac.il
Bio-protocol author page: a4121
date: 2/20/2017, 579 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2135.

[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional abilities to self-renew and to give rise to all mature blood and immune cell types throughout life. Most HSCs are retained in a non-motile quiescent state within a specialized protective microenvironment in the bone marrow (BM) termed the niche. HSCs are typically distinguished from other ...
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Synchronize Human Embryonic Stem Cells at Different Cell Cycle Stage

Author: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
date: 6/5/2012, 11459 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.193.

[Abstract] Pluripotency and the capability for self-renewal are essential characteristics of human embryonic stem cells (hESCs), which hold great potential as a cellular source for tissue replacement. Short cell cycle (15-16 h) compared to somatic cells is another property of hESCs. Efficient synchronization of ...

Generation of Mouse Bone Marrow-Derived Dendritic Cells (BM-DCs)

Authors: Francesca Granucci
Francesca GranucciAffiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
For correspondence: francesca.granucci@unimib.it
Bio-protocol author page: a59
Renato Ostuni
Renato OstuniAffiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
Bio-protocol author page: a58
 and Ivan Zanoni
Ivan ZanoniAffiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
For correspondence: ivan.zanoni@unimib.it
Bio-protocol author page: a54
date: 6/20/2012, 9660 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.226.

[Abstract] Generating mouse dendritic cells from bone-marrow progenitor cells is a useful tool to study biological functions of mouse dendritic cells. Dendritic cells are one of the major populations of phagocytes able to activate both innate and adaptive immune cells. ...

Sphere Formation (Osteosphere/Sarcopshere) Assay

Authors: Upal Basu-Roy
Upal Basu-RoyAffiliation: Department of Microbiology, NYU School of Medicine, New York, USA
For correspondence: upal.basuroy@nyumc.org
Bio-protocol author page: a190
Claudio Basilico
Claudio BasilicoAffiliation: Department of Microbiology, NYU School of Medicine, New York, USA
Bio-protocol author page: a191
 and Alka Mansukhani
Alka MansukhaniAffiliation: Department of Microbiology, NYU School of Medicine, New York, USA
For correspondence: alka.mansukhani@med.nyu.edu
Bio-protocol author page: a192
date: 12/20/2012, 9480 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.307.

[Abstract] Self-renewing cells from adult tissue (such as bone) that represent a progenitor population can be grown in suspension cultures in the presence of defined serum-free medium. Progenitor cells can be identified by this property of anchorage-independent growth in suspension cultures. These spherical clusters ...

[Bio101] Mouse Embryonic Stem Cell Maintenance for Differentiation

Author: Hogune Im
Hogune ImAffiliation: Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical Schoo, Madison, USA
For correspondence: hoguneim@stanford.edu
Bio-protocol author page: a18
date: 10/20/2011, 8757 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.145.

[Abstract] Embryonic stem cells are derived from inner cell mass of an embryo that can differentiate into every cell type in the body. Clinically, cultured red blood cell supply is of great interest. However, some of the hurdles need to be overcome. This protocol describes a protocol to maintain mouse stem cells ...

[Bio101] Culturing of C57BL/6 Mouse Embryonic Stem (ES) Cell Line

Author: Yuqiong Pan date: 10/5/2011, 8481 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.142.

[Abstract] Using ATCC ES C57BL/6 as an example, it is shown here how to culture mouse embryonic stem (ES) cell line. The clonal embryonic stem cell line #693 ES C57BL/6 was derived from a strain C57BL/6J (B6) mouse blastocyst [PubMed: 11730008]. The ES cells were shown to populate the germ line of two host blastocyst ...

Wnt Reporter Activity Assay

Author: Chen Zhao
Chen ZhaoAffiliation: Department of Developmental Biology, Institute for Stem Cell and Regenerative Medicine, Stanford University, Stanford, USA
For correspondence: chenzhao@stanford.edu
Bio-protocol author page: a1513
date: 7/20/2014, 7848 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1183.

[Abstract] This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt ...

Mouse Embryonic Stem Cell Differentiation to Hematopoietic Precursors

Author: Hogune Im
Hogune ImAffiliation: Department of Pharmacology, University of Wisconsin Medical School, Madison, USA
For correspondence: hoguneim@stanford.edu
Bio-protocol author page: a18
date: 4/5/2012, 7825 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.144.

[Abstract] Embryonic stem cells are derived from inner cell mass of an embryo that can differentiate into every cell type in the body. Clinically, cultured red blood cell supply is of great interest. However, some of the hurdles need to be overcome. This protocol describes a two step protocol to form embryoid ...

Isolation and 3-dimensional Culture of Primary Murine Intestinal Epithelial Cells

Authors: Agnieszka Pastuła
Agnieszka PastułaAffiliation: II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany
For correspondence: agnieszka.pastula@tum.de
Bio-protocol author page: a1359
 and  Michael Quante
Michael QuanteAffiliation: II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany
Bio-protocol author page: a1360
date: 5/20/2014, 7700 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1125.

[Abstract] The intestine, together with skin and blood, belongs to the organs with the highest cell turnover, which makes it a perfect model to study cellular processes, such as proliferation and differentiation. Epithelial cell turnover in intestine is possible due to the presence of intestinal stem cells, which ...

Alkaline Phosphatase Staining

Author: Pearl A. Campbell
Pearl A. CampbellAffiliation: Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, Canada
For correspondence: pcampbell@ohri.ca
Bio-protocol author page: a1193
date: 3/5/2014, 7191 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1060.

[Abstract] Two main features characterize pluripotent cells; self-renewal (unlimited cell division) and the ability to give rise to all cells of the adult organism. Given the recent impact of induced pluripotent stem cells (iPSCs) and ongoing use of pluripotent embryonic stem cells ESCs (ESCs) in basic discovery, ...

Single-cell Gene Expression Profiling of Mouse Stem Cells With Fluidigm BiomarkTM Dynamic Array

Author: Ana Sevilla
Ana SevillaAffiliation: Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, USA
For correspondence: ana.sevilla7@gmail.com
Bio-protocol author page: a351
date: 5/5/2013, 7191 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.692.

[Abstract] This protocol describes how to use Fluidigm BiomarkTM 96.96 dynamic arrays for high-throughput expression profiling from single mouse stem cells, assaying up to 96 independent samples with up to 96 quantitative PCR (qPCR) probes (equivalent to 9,216 reactions) in a single experiment. This Dynamic Array ...
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