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Reprogram Murine Epiblast Stem Cells by Epigenetic Inhibitors

Featured protocol,  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, 170 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2168.

Brief version appeared in Cell Stem Cell, Apr 2016
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 methylation by MLL1/KMT2A.

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

Featured protocol,  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, 177 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2161.

Brief version appeared in Stem Cells, Mar 2016
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 tissue has grown in recent decades. These cells have also been shown to immunomodulate the microenvironment and secrete abundant growth factors, which minimize inflammation and aid repair and regeneration. ASCs can be readily isolated from the stromal vascular fraction (SVF) of lipoaspirates. Given their ease of accessibility, bountiful source, and potential application in regenerative medicine and tissue engineering, there is growing interest in the characterization and utilization of ASCs. This protocol describes the isolation of ASCs from adult human adipose tissue as well as methods for culture maintenance including expansion and cryopreservation.

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, 170 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, 177 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, 277 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 ...

VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells

Authors: Francesca Avemaria
Francesca AvemariaAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4059
Shiri Gur-Cohen
Shiri Gur-CohenAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4060
Seymen Avci
Seymen AvciAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4075
 and Tsvee Lapidot
Tsvee LapidotAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
For correspondence: Tsvee.Lapidot@weizmann.ac.il
Bio-protocol author page: a4121
date: 2/20/2017, 233 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2134.

[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional ability to self-renew and to differentiate into all blood cell lineages. The majority of HSC reside in specific anatomical locations in the bone marrow (BM) microenvironment, in a quiescent non motile mode. Adhesion interactions between HSCs and their supporting BM microenvironment cells ...

A Streamlined Method for the Preparation of Growth Factor-enriched Thermosensitive Hydrogels from Soft Tissue

Authors: Christopher J. Poon
Christopher J. PoonAffiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
For correspondence: christopher.poon@alumni.unimelb.edu.au
Bio-protocol author page: a4044
Shaun S. Tan
Shaun S. TanAffiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
Bio-protocol author page: a4045
Sholeh W. Boodhun
Sholeh W. BoodhunAffiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
Bio-protocol author page: a4046
Keren M. Abberton
Keren M. Abberton Affiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
Bio-protocol author page: a4047
 and Wayne A. Morrison
Wayne A. MorrisonAffiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
Bio-protocol author page: a4048
date: 2/5/2017, 315 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2128.

[Abstract] Hydrogels are an ideal medium for the expansion of cells in three dimensions. The ability to induce cell expansion and differentiation in a controlled manner is a key goal in tissue engineering. Here we describe a detailed method for producing hydrogels from soft tissues with an emphasis on adipose tissue. In this method, soluble, extractable proteins ...

Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin

Authors: Eri Shibata
Eri ShibataAffiliation: School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Bio-protocol author page: a4049
Kazunori Ando
Kazunori AndoAffiliation: School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Bio-protocol author page: a4050
 and Atsushi Kawakami
Atsushi KawakamiAffiliation: School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: atkawaka@bio.titech.ac.jp
Bio-protocol author page: a4051
date: 1/20/2017, 369 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2109.

[Abstract] Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies (Shibata et al., 2016; Yoshinari et al., 2012) is a useful approach for tracking and ...

Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors

Authors: Valeria Fernandez Vallone
Valeria Fernandez ValloneAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3957
Morgane Leprovots
Morgane LeprovotsAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3956
Gilbert Vassart
Gilbert VassartAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3958
 and Marie-Isabelle Garcia
Marie-Isabelle GarciaAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
For correspondence: mgarcia@ulb.ac.be
Bio-protocol author page: a3959
date: 1/5/2017, 450 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2089.

[Abstract] Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal ...

Ex vivo Culture of Adult Mouse Antral Glands

Authors: Valeria Fernandez Vallone
Valeria Fernandez ValloneAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3957
Morgane Leprovots
Morgane LeprovotsAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3956
Gilbert Vassart
Gilbert VassartAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3958
 and Marie-Isabelle Garcia
Marie-Isabelle GarciaAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
For correspondence: mgarcia@ulb.ac.be
Bio-protocol author page: a3959
date: 1/5/2017, 409 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2088.

[Abstract] The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids ...

Gene Expression Analysis of Sorted Cells by RNA-seq in Drosophila Intestine

Authors: Jun Chen
Jun ChenAffiliation: National Institute of Biological Sciences, Beijing, China
Bio-protocol author page: a3929
Jia Li
Jia LiAffiliation: National Institute of Biological Sciences, Beijing, China
Bio-protocol author page: a16
Huaiwei Huang
Huaiwei HuangAffiliation: National Institute of Biological Sciences, Beijing, China
Bio-protocol author page: a3931
 and Rongwen Xi
Rongwen XiAffiliation: National Institute of Biological Sciences, Beijing, China
For correspondence: xirongwen@nibs.ac.cn
Bio-protocol author page: a3932
date: 12/20/2016, 640 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2079.

[Abstract] RNA sequencing (RNA-seq) has become a popular method for profiling gene expression. Among many applications, one common purpose is to identify differentially expressed genes and pathways in different biological or pathological conditions. This protocol provides detailed procedure for RNA-seq analysis of ~250,000 sorted Drosophila intestinal cells (Chen ...

Quantitative 3D Time Lapse Imaging of Muscle Progenitors in Skeletal Muscle of Live Mice

Authors: Micah T. Webster
Micah T. WebsterAffiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, USA
Bio-protocol author page: a3892
Tyler Harvey
Tyler HarveyAffiliation 1: Department of Embryology, Carnegie Institution for Science, Baltimore, USA
Affiliation 2: Department of Biology, Johns Hopkins University, Baltimore, USA
Bio-protocol author page: a3893
 and Chen-Ming Fan
Chen-Ming FanAffiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, USA
For correspondence: fan@ciwemb.edu
Bio-protocol author page: a3894
date: 12/20/2016, 485 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2066.

[Abstract] For non-optically clear mammalian tissues, it is now possible to use multi-photon microscopy to penetrate deep into the tissue and obtain detailed single cell images in a live animal, i.e., intravital imaging. This technique is in principle applicable to any fluorescently marked cell, and we have employed it to observe stem cells during the regenerative ...
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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, 11005 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, 9258 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, 9076 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, 8384 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, 8155 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 ...

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, 7523 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, 7216 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 ...

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, 7132 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 ...

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, 6793 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 ...

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, 6499 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, ...
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