Dissection und Isolierung von frühen hämatopoetischen Stammzellen aus der Maus Dottersack und Aorta-Gonaden-Mesonephros Region <ol><li> Bevor Sie beginnen, ist es wichtig, alle Reagenzien und Instrumenten für die das Verfahren haben: <ul><li> Schere </li><li> Zange, Größe 4 und chirurgische grade gebogen Spitze Pinzette, Größe 5 / 45 (optional) </li><li> 30-Gauge-Nadel mit der Spritze </li><li> 35mm und 100mm Petrischalen </li><li> PBS mit 10% und 20% fötalem Rinderserum </li><li> und schließlich, Collagenase </li></ul></li><li> Zu Beginn der Dissektion, müssen wir die Uterushörner von einer schwangeren Frau auf 10,5 Tage post Konzeption Ernte. </li><li> Die Tiere sind gemäß den vereinbarten Verfahren eingeschläfert. Wir verwenden Kohlendioxid Ersticken, durch Genickbruch folgte. </li><li> Die weiblichen Bauch ist nass mit Ethanol und die Haut wird mit einer Schere geöffnet. </li><li> Halten Sie die Haut mit einer Pinzette, sind weitere Kürzungen vorgenommen, um das Peritoneum zu eröffnen. </li><li> Der Bauch wird geöffnet und der Uterushörner visualisiert werden können. Mit einer Pinzette die Uterushörner halten, schneiden Sie jeden distalen Ende und der Mitte der Verbrauchsteuer beiden Hörner. </li><li> Uterushörner werden dann in eine Petrischale mit kaltem PBS mit 10% FBS auf uterine Gewebe zu entfernen platziert. </li><li> Mit Größe 4 Zangen und Arbeiten unter dem Mikroskop, entfernen Sie vorsichtig das Endometrium aus um jeden Embryo conceptus, darauf achtend, nicht Punktion der Plazenta. </li><li> Nach dem Herausnehmen werden die Embryonen in einer frischen Schüssel mit kaltem PBS und 10% FBS platziert. </li><li> Entfernen Sie vorsichtig das Plazentagewebe zu offenbaren, den Embryo umhüllt den Dottersack mit der Pinzette auf den Embryo-ummantelt Dottersack weg von der Plazenta bei ihrer Stelle geschnitten. Der Dottersack enthält vitelline Arterien, und ein Teil der Nabelschnur, kann sanft gehänselt werden vom Embryo und beiseite stellen auf Eis Dissoziation. </li><li> Der Embryo wird zu einer kleinen Petrischale mit minimalem Niveau von PBS mit 10% FBS bewegt - gerade genug, um den Embryo nass, aber nicht zu viel. Optimal Niveau zu halten den Embryo stationär, während die Platte langsam bewegt wird, hält der Embryo sicher an seinem Platz beim Präparieren. </li><li> Wechseln Sie auf die 30-Gauge-Nadeln, mit denen die oberen und unteren Teilen des Embryos, indem oberhalb der Vorderbeine und unterhalb der hinteren Gliedmaßen zu entfernen sind. Kennenlernen mit den Nadeln als schneidenden Instrumenten kann einige Übung. Ein Verfahren zur Verwendung wiederholt kleine Schnitte durch Kreuzung der Nadeln wird sezieren das Gewebe mit der Nadel in die dominante Hand als Schnittführung für die Nadel, die in der nicht-dominanten Hand gehalten wird, zu handeln. </li><li> Entfernen Sie den hinteren Teil des Embryos. Dies ist das Gewebe, das die Somiten enthält. Vorsichtig wegschneiden dorsal-die meisten Gewebe, während verbleibenden einen ausreichenden Abstand von der Aorta (rote Linie) auf einen Knicks zu verhindern. Schneiden der Aorta würde in den Blutverlust und die Unfähigkeit, einfach visualisieren seiner Lage führen. Auch hier macht kleine Schnitte mit den Nadeln ist wichtig für die Erhaltung einer präzisen Schneiden Lage. </li><li> Drücken Sie die zusätzlichen Gewebe entfernt aus der Sicht und drehen Sie den Embryo in der ventralen Gewebe geschnitten, wieder Schnitt möglichst nahe an der Aorta wie möglich, ohne knicking es. Cuts gemacht zu weit von der optimalen Lage wäre in Bauch-Gewebe vorhanden, die entfernt werden, sobald die Hauptversammlung ist isoliert werden können führen. </li><li> Position der Embryo mit seiner dorsalen Seite nach unten oder dem Rücken gegen die Platte. Gently splay des Embryos öffnen, indem Sie die Seiten nach unten und Visualisierung der Lage der Gonaden Grate. Diese sind leicht aus der Mitte, wie Rohr-ähnliche Strukturen auf beiden Seiten der Aorta befindet. </li><li> Füllen Sie die Dissektion durch das Entfernen der überschüssigen Seite Geweben unter Verwendung der gleichen Schnitt-Techniken wie zuvor. Sobald die Seite Gewebe des Embryos entfernt wird, dann wird der Embryo auf den Kopf, um die restlichen Seiten Gewebe zu entfernen eingeschaltet. </li><li> An diesem Punkt ist die Hauptversammlung für überschüssiges Gewebe, das leicht mit den Nadeln ohne Verletzung der Aorta oder Keimdrüsen entfernt werden inspiziert. Entfernen so viel wie möglich über Ergebnisse in eine bessere Isolierung der Stammzellen Bevölkerung, da wir nur auf ein paar Zelloberflächenmarker für die Reinigung zu verlassen. </li><li> Mit der gebogenen Spitze Pinzette vorsichtig schöpfen jeder Hauptversammlung und in kaltem PBS mit 10% FBS, bis sie zur Dissoziation. </li></ol> Dissoziation und FACS-Analyse <ol><li> Um distanzieren dem Gewebe, den ersten Platz Dottersäcke und Hauptversammlungen in getrennten Röhren. </li><li> Spin kurz Pellet Geweben und entsorgen PBS mit 10% FBS. </li><li> Resuspendieren Gewebe in 0,25% Kollagenase in PBS mit 20% FBS, mit genügend Volumen, um angemessen abdecken Gewebe. </li><li> Inkubieren des Gewebes bei 37 Grad. 25-30min für Hauptversammlungen und 35-40min für Dottersäcke. </li><li> Am Ende der Inkubationszeit, sanft Pipette Gewebe nach oben und unten, um die Dissoziation in einzelne Zell-Suspensionen. </li><li> Waschen Sie die Gewebe mit einem Überschuss an PBS with 10% FBS, Pellet der Zellen und Resuspension in frischem sterilem PBS mit 10% FBS. </li><li> Für FACS-Analyse werden die Zellen nach den Empfehlungen des Herstellers gefärbt. Wir verwenden eine FACSAria von BD Biosciences auf der Durchflusszytometrie durchgeführt. Aufgrund der Größe und Fragilität der embryonalen Zellen, wird die Sortierung Öffnung für einen größeren Durchmesser als bei Erwachsenen hämatopoetischen Zellen, die wir auch im Labor-Studie angepasst. Ebenso wird der Druck so eingestellt, daß weniger als die Bedingungen in der Erwachsenenbildung hämatopoetischen Zellen erforderlich. Zum Beispiel, Sortierung auf dem FACSAria, würde die Düsengröße 100 Mikrometer und die Art Druck 30 psi werden würde. </li><li> Für Stammzell-Analysen-, Dreibett-positive Zellen, die Marker CKIT, CD34, CD41 und werden isoliert und sortiert aus dem Dottersack und doppelt positiven (CKIT, CD34) sind von der Hauptversammlung via FACS isoliert. Ein typisches Grundstück würde wie folgt aussehen: CKIT positive Zellen sind für CD34 und CD41 gleichzeitig oder einzeln analysiert. Normalerweise Zellen zeigen eine Abstufung der Positivität für jeden Marker mit der Stammzell-Population als eine Teilmenge der positiven Populationen gefunden. Diese Diagramme zeigen uns, dass wir eine gute Zellausbeute mit 100-300 Zellen pro Dottersack und pro AGM erreicht. </li></ol>

isolation of early hematopoietic stem cells from murine yolk sac and agm

In der Maus-Embryo, tritt frühen Hämatopoese gleichzeitig in mehreren Organen, die den Dottersack und Aorta-Gonaden-Mesonephros Region umfasst. Diese Regionen sind entscheidend bei der Festlegung der Blutkreislauf in die Embryonen und führt zu den eventuellen Bewegung von Stammzellen in der fetalen Leber und dann die Entwicklung von adulten Stammzellen in der bonemarrow. Frühe hämatopoetischen Stammzellen können aus diesen Organen durch Mikrodissektion des Embryos durch durchflusszytometrische Sortierung gefolgt, um eine reine Population zu erhalten, isoliert werden. Es bleibt unklar, wie diese Stammzell-Populationen der fetalen und adulten Stammzellen-Pool beitragen. Auch untersucht unser Labor, wie früh Stammzellen funktionell unterscheiden sich von fetalen und adulten hämatopoetischen Stammzellen. Außerdem testen unsere Labor möglichen verschiedenen Populationen von hämatopoetischen Stammzellen und deren funktionelle Rolle im Kontext einer Vielzahl von genetischen Modellen. In diesem Video haben wir die Mikro-Dissektion Verfahren, das wir verwenden häufig und zeigen auch die Ergebnisse eines typischen FACS plotfter Isolierung dieser seltenen Populationen zeigen, ist es möglich, eine Vielzahl von funktionellen Assays darunter: Kolonie-Assays und Knochenmarktransplantationen.

Watch this Scientific Journal Video about Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM at JoVE.com

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

Dieses Video zeigt, wie Mikro-sezieren den Dottersack und Aorta-Gonaden-Mesonephros Region aus Embryonen und die Verwendung der Durchflusszytometrie, um hämatopoetische Stammzellen zu sortieren.

Isolierung von Anfang hämatopoetischen Stammzellen aus Murine Dottersack und AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

isolation-early-hematopoietic-stem-cells-from-murine-yolk-sac

Research

JoVE Journal

Biology

14.6K Aufrufe.  Brigham and Women's Hospital and Harvard Medical School. Dieses Video zeigt, wie Mikro-sezieren den Dottersack und Aorta-Gonaden-Mesonephros Region aus Embryonen und die Verwendung der Durchflusszytometrie, um hämatopoetische Stammzellen zu sortieren.

Serie: Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolierung und Transplantation von hämatopoetischen Stammzellen (HSZ)

Forschung

Biologie

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Ableitung von hämatopoetischen Stammzellen aus embryonalen Stammzellen der Maus

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolierung und Analyse von hämatopoetischen Stammzellen aus der Plazenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Phänotypische Analyse und Isolierung von murinen hämatopoetischen Stammzellen und Lineage-festgelegte Vorläuferzellen

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Retrovirale Infektion von murinen embryonalen Stammzellen abgeleitete Embryoidkörper Zellen für die Analyse von hämatopoetischen Differenzierung

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

Isolierung von myepithelialen Zellen aus der Urmorine, Lacrimal und Submandibular Drüsen

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolierung von hochwertigen atrialen und ventrikulären Myozyten der Maus für die simultane Messung von Ca2+ Transienten und L-Typ Calciumstrom

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

Durchflusszytometrische Analyse von hämatopoetischen Stamm- und Vorläuferzellen des murinen Knochenmarks und stromalen Nischenzellen

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs),&#160;defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter system.

Isolationsmethode für lang- und kurzfristige hämatopoetische Stammzellen

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells ...