murine 난황과 대동맥 - 생식선 - mesonephros 지역에서 초기 조혈 줄기 세포의 해부 및 절연 <ol><li> 시작하기 전에 프로 시저에 필요한 모든 시약과 도구를 가지고하는 것이 중요합니다 : <ul><li> 가위 </li><li> 집게, 크기 4 및 수술 성적 벤트 - 스쳐 포셉, 크기 45분의 5 (옵션) </li><li> 주사기 30 게이지 바늘 </li><li> 35mm와 100mm 페트리 접시 </li><li> PBS 10 % 20% 태아 소 혈청과 </li><li> 그리고 마지막으로, Collagenase </li></ul></li><li> 절개를 시작하려면, 우리는 10.5 일 우편 개념에서 임신 여성에서 자궁 뿔을 수확해야합니다. </li><li> 동물이 승인 절차에 따라 euthanized 있습니다. 우리는 자궁 경부 전위 다음 이산화탄소 질식을 사용합니다. </li><li> 여성의 복부는 에탄올와 젖은이고 피부는 가위로 열립니다. </li><li> 포셉로 피부를 개최, 추가 인하는 복막을 엽니다 만들어집니다. </li><li> 복부가 열립니다과 자궁 뿔이 시각하실 수 있습니다. 자궁 뿔 잡고 집게를 사용하여, 각 말초 엔드 및 소비세 모두 뿔을를 중심 잘라. </li><li> 자궁 뿔 그런 다음 자궁 조직을 제거 10 % FBS와 차가운 PBS를 포함하는 페트리 접시에 배치됩니다. </li><li> 크기 4 포셉를 사용하여 현미경으로 작동, 부드럽게하지 소중히 태반를주의하고, 각각의 배아 conceptus 주변에서 자궁 내막 조직을 제거합니다. </li><li> 일단 제거, 배아는 차가운 PBS와 10 % FBS와 신선한 접시에 배치됩니다. </li><li> 조심스럽게 배아 떠나 자신의 시점에서 태반에서 태아 - 쌌다는 난황를 잘라 집게를 사용하여 난황으로 ​​쌌다 나타내기 위해 placental 조직을 제거합니다. vitelline의 동맥과 탯줄의 일부를 포함하는 난황은 부드럽게 배아 떨어져 조롱과 분리를위한 얼음에 별도로 설정할 수 있습니다. </li><li> 배아는 10% FBS와 PBS의 최소한의 수준 작은 페트리 접시로 이동 - 단지 충분 배아를 엿먹일 수 있지만 너무 많이는 말고. 접시가 천천히 해부 동안에 안전하게 배아를 유지, 흥분 상태에서 최적의 수준은 배가 정지 유지. </li><li> 앞발 위와 hindlimbs 아래 절단하여 배 (胚)의 상단과 하단 부분을 제거하는 데 사용되는 30 게이지 바늘로 전환합니다. 절삭 악기로 바늘을 사용하여 익숙한되기 위해서는 연습이 걸릴 수 있습니다. 바늘을 횡단하여 반복 작은 상처를 사용하는 방법은 비 지배적인 손에 개최되는 바늘 절단 가이드 역할을 주로 사용하는 손이에서 개최된 바늘과 조직을 해부하다 것입니다. </li><li> 배아의 지느러미 부분을 제거합니다. 이것은 somites을 포함하는 조직입니다. 어떤 닉스를 방지하기 위해 대동맥 (빨간색 선)에서 충분한 거리를 유지하면서 부드럽게 지느러미 - 대부분의 조직을 버려야. 대동맥을 절단하는 것은 혈액의 손실 쉽게 위치를 시각화하는 무능력을 초래합니다. 다시 바늘로 작은 상처를 만드는 것은 정확한 절단 위치를 유지하기 위해 매우 중요합니다. </li><li> 밖으로보기의 거리에 여분의 조직을 푸시하고 ​​다시 그것을 knicking 않고 가능한 대동맥 가까운 절단, 복부 조직을 잘라 배아를 돌아서. 상처는 너무 멀리 최적의 위치에서 AGM가 격리되면 제거할 수 있습니다 존재 복부 조직, 초래 하리라는했다. </li><li> 플레이트에 대해 아래의 지느러미 쪽 또는 다시와 배아를 놓습니다. 부드럽게 측면을 추진하고 gonadal 산등성이의 위치를​​ 시각화하여 열고 배아를 넓히다. 이러한 대동맥의 양쪽에 튜브와 같은 구조로 중심을 벗어난 약간 있습니다. </li><li> 이전과 같은 가공 기술을 사용하여 여분의 측면 조직​​을 제거하여 절개를 완료합니다. 배아의 측면 조직​​이 제거되면 다음 배아가 남아있는 측면 조직​​을 제거하기 위해 거꾸로 켜집니다. </li><li> 이 시점에서, AGM은 대동맥이나 생식선에 부상하지 않고 바늘로 쉽게 제거할 수있는 여분의 조직을위한 검사입니다. 우리가 정화에 대해서만 몇 세포 표면 마커에 의존 이후 줄기 세포 인구의 더 나은 분리 가능 결과 많은 초과로 제거. </li><li> 벤트 - 스쳐 집게를 사용하여 부드럽게 분리 준비까지 10% FBS와 차가운 PBS 각 AGM과 장소를 특종. </li></ol> 분리 및 분석 외과 <ol><li> 별도의 튜브에있는 조직, 첫번째 장소 노른자의 주머 니나와 AGMs을 떼어 놓다합니다. </li><li> 펠렛 조직을 간략하게 스핀와 10 % FBS와 PBS를 버리고. </li><li> 적절하게 조직을 감추기에 충분 할만큼 볼륨, 20 % FBS와 PBS에 0.​​25 %의 collagenase의 조직을 Resuspend. </li><li> 37도에서 조직을 품어. 난황 주머 니나에 대한 AGMs 35 - 30-40 분 25 - 30 분. </li><li> 부화의 끝 부분에서 부드럽게 조직을 피펫 아래로 분리하는 단세포 suspensions에. </li><li> 초과 PBS 위스콘신과 조직 와시일 10% FBS, 펠렛 10% FBS 신선한 멸균 PBS에 세포 resuspend. </li><li> 외과의 분석을 위해, 전지는 제조 업체의 권장 사항에 따라 얼룩이 있습니다. 우리는 유동세포계측법을 수행하는 BD Biosciences에서 FACSAria을 사용합니다. 배아 세포의 크기와 취약성으로 인해, 정렬 조리개 우리가 또한 실험실에서 공부 성인 조혈 세포에 대한보다 큰 직경을 위해 조정됩니다. 마찬가지로, 압력은 성인 조혈 세포에 필요한 조건보다 적은 것으로 조정됩니다. 예를 들어, FACSAria에 정렬, 노즐의 크기는 100 미크론 및 정렬 압력 30 PSI 될 것입니다. </li><li> 줄기 세포 분석은 마커의 cKit, CD34, CD41 표현 및 트리플 긍정적인 세포가 고립되고 난황 더블 반응 (cKit, CD34)의 정렬은 외과 통해 AGM에서 격리됩니다. 전형적인 줄거리는 이런 모양이 될 : cKit 긍정적인 세포는 CD34과 동시에 또는 개별적으로 CD41에 대한 분석입니다. 보통 세포는 긍정적인 인구의 일부로 발견된 줄기 세포 인구 각 마커에 대한 양성의 그라데이션을 표시합니다. 이러한 플롯 우리가 난황과 당 AGM 당 100-300 세포와 좋은 세포 수율을 달성한다는 우리에게 보여줍니다. </li></ol>

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

마우스 배아에서 초기 조혈은 난황과 대동맥 - 생식선 - mesonephros 영역을 포함 여러 장기에 동시에 발생합니다. 이 지역은 태아의 혈액 시스템을 구축에 중요하고 태아 간 다음 bonemarrow의 성인 줄기 세포의 개발에 줄기 세포의 최종 움직임에 이르게한다. 초기 조혈 줄기 세포는 이러한 기관에서보다 순수한 인구를 얻는 흐름 cytometric 정렬 뒤에 배의 microdissection을 통해 격리 수 있습니다. 그것은 이러한 줄기 세포 인구가 태아와 성인 줄기 세포 수영장에 기여하는 방법을 명확 남아있다. 또한, 우리 연구소는 초기 줄기 세포가 기능 태아 및 성인 조혈 줄기 세포 차이가 어떻게 조사. 또한, 우리 실험실 종류의 조혈 줄기 세포의 다른 인구 및 유전자 모델의 다양한 맥락에서 자신의 기능 역할을 테스트합니다. 콜로니 assays 및 골수 이식 :이 비디오는, 우리가 일반적으로 사용하고 이러한 희귀한 인구를 분리 전형적인 외과의 plotfter의 결과를 보여 마이크로 절개 절차를 보여줍니다, 그것은 포함한 기능 assays 다양한 수행할 수 있습니다.

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

이 비디오는 태아의 난황과 대동맥 - 생식선 - mesonephros 영역을 미세 절개하다 및 조혈 줄기 세포를 정렬 유동세포계측법를 사용하는 방법을 보여줍니다.

Murine 난황과 AGM에서 조기 조혈 줄기 세포의 분리

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

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Biology

14.6K 조회수.  Brigham and Women's Hospital and Harvard Medical School. 이 비디오는 태아의 난황과 대동맥 - 생식선 - mesonephros 영역을 미세 절개하다 및 조혈 줄기 세포를 정렬 유동세포계측법를 사용하는 방법을 보여줍니다.

비디오: 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

분리 및 조혈 줄기 세포의 이식 (HSCs)

연구

생물학

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.

Murine 배아 줄기 세포로부터 조혈 줄기 세포의 유도

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.

태반에서 조혈 줄기 세포의 분리 및 분석

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.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

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.

Murine 조혈 줄기 세포와 리니지 - 헌신 Progenitors의 Phenotypic 분석 및 절연

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.

쥐과 배아의 레트로 바이러스 감염은 조혈 분화 분석을위한 세포 유래 배아 바디 줄기 세포

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.

성인 뮤 린 눈물과 턱 밑 동맥에서 Myoepithelial 세포의 고립

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+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

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.

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.

장기 및 단기 조혈모세포의 분리 방법

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