マウス卵黄嚢と大動脈 - 生殖腺 - 中腎領域からの初期の造血幹細胞の解離と分離<ol><li>開始する前に、手続に必要なすべての試薬とツールを持つことが重要です。 <ul><li>はさみ</li><li>鉗子、サイズ4、および外科グレードの曲がった先端鉗子、サイズ5 / 45（オプション） </li><li>シリンジと30ゲージの針</li><li> 35mmと100ミリメートルペトリ皿</li><li> 10％、20％ウシ胎児血清を含むPBS </li><li>そして最後に、コラゲナーゼ</li></ul></li><li>解剖を開始するには、我々は、10.5日後に懐妊​​した時に妊娠中の女性から子宮角を収穫する必要があります。 </li><li>動物は、承認された手順に従って安楽死させる。我々は、頸椎脱臼に続く二酸化炭素の窒息を、使用してください。 </li><li>女性の腹部にはエタノールを用いてウェットであり、皮膚をはさみで開かれます。 </li><li>ピンセットで皮膚を押し、追加のカットは、腹膜を開くように作られています。 </li><li>腹部が開かれ、子宮角を可視化することができる。子宮角を保持するための鉗子を用いて、それぞれの遠位端と消費税の両方の角をに中心をカット。 </li><li>子宮角をして子宮組織を削除するには、10％FBSを冷PBSを含むペトリ皿に配置されます。 </li><li>サイズ4鉗子を使用して、顕微鏡の下で働いて、静かに胎盤ではない穿刺に注意して、各胚の受胎前後から子宮内膜組織を除去。 </li><li>一度削除された、胚を冷PBSと10％FBSと新鮮な皿に置かれます。 </li><li>慎重に胚がその時点で胎盤から胎児の箱に入った卵黄嚢をカットするために鉗子を使用して卵黄嚢に包まれて明らかにするために胎盤組織を削除します。卵黄動脈、および臍帯の一部を含む卵黄嚢が、静かに胚から離れてからかったと解離し、氷上で積み立てることができます。 </li><li>胚は、10％FBSを含むPBSの最小限のレベルの小さなペトリ皿に移されている - だけで十分な胚を湿らせるだけではないが多すぎる。プレートがゆっくりと攪拌しながら、最適なレベルでは、解剖時に所定の位置にしっかり胚を保ち、胚が静止してください。 </li><li>前肢の上と後肢下切断することによって胚の上部と下部を除去するために使用される30ゲージの針、に切り替えます。最先端の楽器として針を使用してに精通することはある程度の慣れがかかる場合があります。針を交配することによって繰り返される小さなカットを使用しての方法は、非利き手で開催されている針のためのカッティングガイドとして機能するように利き手で開催された針で、組織を細かく分析します。 </li><li>胚の背側の部分を削除します。これは、体節が含まれている組織です。どんなニックスを防ぐために、大動脈（赤線）から十分な距離を保ちながら優しく背ほとんどの組織を切り取る。大動脈を切断すると、血液の損失と、簡単にその場所を可視化することができないことになります。もう一度、針で小さな切り傷を作ることは、正確な切断位置を維持するために不可欠です。 </li><li>視界が遮られて離れて余分な組織を押して、再度knickingすることなく可能な限り大動脈の近くにカット、腹側組織を切断するために胚を好転させる。カットに最適な場所から離れすぎて作られたがAGMが分離されれば除去することができる、存在している腹部の組織になるでしょう。 </li><li>プレートと下にその背側またはそのバックで胚を置きます。ゆっくりと両側を一度押し下げ、性腺尾根の位置を視覚化することによって胚が開いているスプレー。これらは、わずかに中心から外れて大動脈の両側にチューブ状の構造として配置されています。 </li><li>以前と同じ最先端技術を使用して過剰側の組織を除去することによって解剖を完了します。胚の側の組織が削除されると、その後、胚は、残りの側の組織を除去するために上下逆さまになっています。 </li><li>この時点で、AGMは、大動脈や生殖腺に損傷することなく針で容易に除去できる余分な組織のために検査されます。我々が精製のための少数の細胞表面マーカーに依存しているため、幹細胞集団のよりよい分離の可能性が結果として非常に過剰に除去する。 </li><li>曲がった先端鉗子を使用して、静かに解離するための準備が整うまで、10％FBSを冷PBSで各AGMと場所をすくい上げる。 </li></ol>解離およびFACS分析<ol><li>組織の関連付けを解除するには、別々のチューブに最初の場所の卵黄嚢と年​​次総会。 </li><li>ペレットの組織に簡単にスピンし、10％FBSを含むPBSを捨てます。 </li><li>十分に組織をカバーするのに十分なボリュームで、20​​％FBSを含むPBS中0.25％コラゲナーゼで組織を再懸濁します。 </li><li> 37℃で組織をインキュベートする。卵黄嚢のための年次総会および35 - 40分のための25 - 30分。 </li><li>インキュベーションの終わりに、静かに単一の細胞懸濁液に解離するために上下の組織をピペット。 </li><li>過剰PBSウィスコンシン州で組織を洗う目10％FBS、ペレット10％FBSと新鮮な滅菌PBSで細胞を再懸濁します。 </li><li> FACS分析のために、細胞は、製造業者の推奨に従って染色される。我々は、フローサイトメトリーを実行するためにBD Biosciences社からFACSAriaを使用してください。胚細胞の大きさと脆さのために、並べ替え、絞り値は、我々はまた、実験室で勉強成人造血細胞に対するよりも大径に調整されます。同様に、圧力が成人造血細胞で必要とされる条件よりも小さくなるように調整されます。例えば、FACSAriaにソート、ノズルのサイズが100ミクロンになるとソートの圧力は30 psiのだろう。 </li><li>幹細胞の分析のために、マーカーのcKit、CD34、およびCD41を発現している三重陽性細胞が単離され、卵黄嚢と二重陽性（cKit、CD34）からソートをFACSを介してAGMから分離されています。典型的なプロットは次のようになります。cKit陽性細胞は、CD34と同時または個別にCD41で解析されます。通常、細胞は正の集団のサブセットとして見られる幹細胞集団で、各マーカーの陽性のグラデーションが表示されます。これらのプロットは、我々は卵黄嚢と当たりAGM当たり100〜300細胞との良好な細胞収率を達成しているということを示しています。 </li></ol>

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

マウス胚では、初期の造血は卵黄嚢と大動脈性腺中腎領域を含む複数の臓器に同時に発生します。これらの領域は、胚の血液系の確立に極めて重要であり、胎児の肝臓及びその後bonemarrowにおける成体幹細胞の開発に幹細胞の最終的な動きにつながる。初期の造血幹細胞はこれらの器官から、より純粋な集団を得るために、フローサイトメトリー選別が続く胚の顕微解剖によって単離することができる。これらの幹細胞集団は、胎児および成体幹細胞のプールに貢献する方法、それは不明なままである。また、私たちの研究室では、初期の幹細胞が機能的に胎児と成人の造血幹細胞からどのように異なるか調査する。さらに、私たちの研究室をソート造血幹細胞の異なる集団と遺伝的モデルのさまざまな文脈でそれらの機能的役割をテストします。コロニーアッセイと骨髄移植：このビデオでは、我々は我々が一般的に使用し、また、これらの貴重な個体群を隔離typical FACSのplotfterの結果を示す顕微解剖の手順を紹介する、それが含む機能的アッセイのさまざまな実行することが可能です。

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

このビデオでは、胚から卵黄嚢と大動脈 - 生殖腺 - 中腎領域をマイクロ細かく分析し、造血幹細胞をソートするには、フローサイトメトリーを使用する方法を示します。

マウス卵黄嚢とAGMから早期の造血幹細胞の分離

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 ビュー.  Brigham and Women's Hospital and Harvard Medical School. このビデオでは、胚から卵黄嚢と大動脈 - 生殖腺 - 中腎領域をマイクロ細かく分析し、造血幹細胞をソートするには、フローサイトメトリーを使用する方法を示します。

ビデオ: 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

造血幹細胞の分離と移植（造血幹細胞）

生物学

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.

マウス胚性幹細胞から造血幹細胞の導出

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.

プラセンタから造血幹細胞の単離と解析

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.

ヒト皮膚から大人上皮幹細胞の単離と培養

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.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

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.

マウス胚のレトロウイルス感染は、造血分化の解析のための細胞由来の胚様体幹細胞

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.

成人ネズミ涙腺および顎下腺からの筋上皮細胞の単離

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.

Ca2+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

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.

マウス骨髄造血幹・前駆細胞および間質ニッチ細胞のフローサイトメトリー解析

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.

長期および短期造血幹細胞の単離方法

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