我们承认美国国立卫生研究院拨款R01 DE021683，R01 DE019434，U01 HL099776，橡树基金会和Hagey实验室小儿再生医学的支持。

注:伦理声明:在批准的斯坦福大学行政专家组对实验室动物护理（APLAC）按照协议进行的所有涉及脊椎动物的研究。 1.准备<ol><li>允许10毫升市售的密度梯度的细胞分离介质（包含多聚蔗糖和泛影酸钠，调整至10.77克/毫升的密度）来RT在50ml锥形管中。 </li><li>制备流式细胞术（FACS）缓冲液用1×磷酸盐缓冲盐水（PBS）和2％胎牛血清（FCS）中。取50毫升的等分试样，并且在密度梯度的细胞分离介质步骤保持这种等分在RT下使用。 </li><li>有冰准备保持在分离过程中分离的组织和细胞的桶。 </li></ol> 2.准备文化传媒<ol><li>制备基础培养基:MEM（最低必需培养基），不含酚红（500毫升），谷氨酸（1×），FCS（10倍），10,000单位/ ml的青霉N（1X）。 </li><li>过滤基础培养基用0.22微米的过滤器。 </li><li>准备骨髓巨噬细胞诱导培养基: <ol><li>取50毫升步骤2.1准备的基础培养基中。 </li><li>添加前列腺素E2（PGE2，先生352.465克/摩尔）在10 -7 M终浓度。 </li><li>添加巨噬细胞集落刺激因子（M-CSF）在10纳克/毫升。 </li><li>不过滤骨髓巨噬细胞刺激媒体的最终解决方案。 </li></ol></li><li>准备破骨细胞诱导培养基: <ol><li>取50毫升步骤2.1准备的基础培养基中。 </li><li>添加前列腺素E2（PGE2，先生352.465克/摩尔）在10 -7 M终浓度</li><li>添加巨噬细胞集落刺激因子（M-CSF）在10纳克/毫升。 </li><li>添加RANKL在10纳克/毫升。 </li><li>不过滤破骨细胞诱导培养基的最终解决方案。 </li></ol></li></ol> 3.骨髓隔离<ol><li>安乐死1 C57BL / 6小鼠用二氧化碳吸入法，随后通过颈脱位。 注:鼠害必须由经过培训的人员使用适当的技术，设备和代理进行安乐死。 </li><li>位置并固定在鼠标上的夹层板，并喷以70％的酒精。 </li><li>剖析了股骨，胫骨，肱骨和脊柱。 <ol><li>首先用鼠标在仰卧位。做一个切口垂直地沿着下肢并通过除以软组织的附件沿股骨和胫骨的长度解剖开始。最后，脱臼从膝关节和髋关节的骨头从骨架完全释放的骨头。 </li><li>继续使沿上肢分离肱骨长度的切口。通过解剖软组织囊附着脱位的肱骨肩和肘关节。 </li><li>最后，把鼠标，让鼠标趴着。使皮肤切口沿着脊柱的长度，在中线。观察椎旁软组织对脊柱和切割沿脊柱的骨边缘的每一侧，通过椎旁软组织肿块解剖。使用剪刀，使整个脊柱的水平切口在颈部的基部，并在脊柱，刚好接近尾部插入，释放从软组织附着在脊柱的末端。 </li></ol></li><li>一旦骨头收获，让他们在FACS缓冲冰上。 </li><li>用薄纸从骨子里清除肌肉和软组织。 </li><li>将洗净的骨头成杵和研钵用3ml FACS缓冲液。 </li><li>在研钵轻轻粉碎的骨头。 </li><li>吸脱血染色流体和转移到50ml锥形管中通过使溶液通过70微米的细胞过滤网。 </li><li>加入5 ml FACS缓冲的碎骨头，并进一步挤压。再次，用移液管取出流体，并通过一个70微米的过滤器转移到50ml锥形管中。 </li><li>通常，粉碎的骨头TWI策，以新鲜FACS缓冲液三次，用研钵和研杵，每次加入另外5毫升新鲜的FACS缓冲液中，直到该流体不染色的红色。 </li><li>离心机在200×g离心骨髓溶液中5分钟，在4℃下，得到的细胞沉淀。 </li></ol>骨髓细胞4.梯度分离<ol><li>吸去上清，重悬在10ml的FACS缓冲液（保持在室温下）。 </li><li>以50毫升锥形管含有10ml市售的密度梯度细胞分离介质（RT）的。 </li><li>层的骨髓液涂布到密度梯度的细胞分离介质。慢慢地使用电动移液器做到这一点。角对着附近的锥形管顶部的内表面上的移液管尖和倾斜的锥形管中，以大约30°。上吸管用慢速设置，轻轻驱逐骨髓溶液，以便不打扰密度梯度的细胞分离介质。 注:最终会有是上述两种溶液（蜂窝溶液和密度梯度的细胞分离介质溶液）之间的明确定义。 </li><li>使用RT离心机，离心密度梯度细胞分离培养基和细胞溶液中的平衡离心机在200×g离心15分钟。此外，除去在离心机上的加速和减速，以确保有足够的相分离使用密度梯度的细胞分离介质梯度。 </li><li>离心后，吸断其包含感兴趣的骨髓细胞的混浊的中间层。 </li><li>转移这些细胞到新的离心管中。加入20毫升的附加FACS缓冲液（保持在冰上）洗涤细胞。 </li><li>离心机在200×g离心5分钟，在4℃下，得到的细胞沉淀。 </li><li>重悬在巨噬细胞刺激介质，以允许使用血球计数器进行细胞计数1毫升的最终的细胞沉淀。 </li></ol> 5.细胞计数<ol><li>取10微升的细胞溶液和混用10微升台盼蓝。负载10微升所得的混合物到血细胞计数器，将获得每毫升溶液中的细胞计数。 </li></ol> 6.细胞培养<ol><li>放置2毫升巨噬细胞刺激介质到24孔板的每个孔中。 </li><li>加入200,000细胞，以每孔中。 注意:根据我们的经验，细胞铺板密度是足够的破骨细胞的体外培养中最关键的要求之一。 </li><li>轻轻搅动板和放置在一个常规的培养箱中在37℃。 </li><li>不要更改媒体3天。 </li><li>后在巨噬细胞刺激媒体3天，改变媒体破骨细胞培养基。 </li><li>此后，为5-7天，在该点，大，多核破骨细胞应该在培养板上的可见每日更换介质。 </li></ol> 7.染色用抗酒石酸酸性磷酸酶（TRAP） <ol><li>经过5-7天的体外 CULTURE，从培养物中吸出培养基良好。 </li><li>用1×PBS三次，轻轻地洗井。 </li><li>通过加入4％多聚甲醛溶液固定细胞，并在4℃下离开1小时。 </li><li>在此期间，准备为TRAP染色（其可商购）通过预热到37℃。 </li><li> 1小时的多聚甲醛后，吸过和多聚甲醛轻轻洗净用1×PBS 3倍。 </li><li>加入1毫升的预温热的TRAP染色到板的每个孔被染色。 </li><li>将板在培养箱中在37℃下1小时，从光屏蔽。 </li><li> 1小时后，吸脱TRAP染色。 </li><li>用预热的去离子水洗涤各孔3次。 </li><li>染液井与吉尔的苏木（注意）1-2分钟。 </li><li>用亮视野显微镜图像的破骨细胞。 </li><li>洗孔用离子交换水，直至污迹的适当颜色强度来实现，典型地当晶核出现蓝Ë。 </li></ol> 8，破骨细胞骨吸收分析<ol><li>使用市售的24孔聚苯乙烯培养皿是预先涂有骨基质或无机晶体磷酸钙涂层。 </li><li>放置2毫升巨噬细胞刺激介质到每个孔中。 </li><li>添加在步骤2至各孔的端部获得200000新鲜分离骨髓细胞。 </li><li>轻轻之前搅动板以将其放置在培养箱中在37℃下3天。 </li><li>第3天，改变媒体破骨细胞分化的媒体。 </li><li> 5-7天执行与破骨细胞分化的媒体每日介质的变化。 </li></ol> 9.定量破骨细胞骨吸收活动的<ol><li> 5-7天后的体外培养破骨细胞的分化培养基上的矿化基质的，分析的矿化的表面，如下面所述，以评估破骨细胞的形成resorpti的能力上的凹坑，破骨细胞活性的指标。 </li><li>从再吸收测定板抽吸介质，并添加100微升10％的漂白剂溶液到每个孔中。 </li><li>孵育细胞15分钟，在室温下的10％漂白溶液中。 </li><li>用蒸馏水洗涤各孔3次。 </li><li>吸掉任何剩余的水，并从板甩掉多余的水，并允许在空气中干燥3-5小时。 </li><li>染液在板与市售冯Kossa染色试剂盒，以允许将未再吸收衬底更容易可视化。 </li><li>用亮视野显微镜采取的再吸收表面的代表性部分图像。 </li><li>利用图像分析软件，计算出再吸收表面的百分比，以允许破骨细胞骨吸收活性的定量。 </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+and+targeting+osteoclastic+activity+in+prostate+cancer+bone+metastases.">Understanding and targeting osteoclastic activity in prostate cancer bone metastases.</a> Current molecular medicine. 13, 626-639 (2013).</li></ol>

没有一个作家有披露或有利益冲突申报。

一个很容易分离，培养大量的破骨细胞在体外的能力一直负责帮助推动骨生物学和破骨细胞介导的疾病的认识。这是RANKL的识别，导致此，当它最近被鉴定为破骨细胞的形成，分化和存活16-18的主要调节剂。 据我们的经验是， 在体外培养骨髓破骨细胞在很大程度上依赖于接种密度。我们观察到，该协议得到的破骨细胞的失败通常是由于一个不理想的接种?...

骨重建是动态的，并且涉及骨形成与骨吸收1的耦合。这个严格调节的过程是负责维持正常的体内平衡中的骨架，并响应于损伤和疾病。 破骨细胞是独特的，多核细胞，其能够再吸收二者的有机和无机骨基质。破骨细胞是来源于骨髓的2-5的单核细胞/巨噬细胞系。在破骨细胞的功能，或形成的异常可导致多种临床病症，包括骨质疏松症象常见的情况的。 在体外产生的破骨细胞的能力已经允许在我们的骨生物学6的理解显著进步。其结果是，新的治疗剂的新兴治疗破骨细胞有关的疾病，这是负责显著发病率和死亡率7 </sUP&gt;。稳态维持骨量和骨强度需要骨形成的成骨细胞和骨吸收破骨细胞8,9的一致行动人。骨骼平衡被改变中的一些疾病，包括绝经后骨质疏松症，其中增加的破骨细胞活性导致骨质量和密度10的致病损失。随着人类疾病的转基因小鼠模型的可用性，有更多的机会来破译人骨疾病11-13的破骨细胞的作用。 对破骨细胞的培养技术大量协议，出现在文献中，有许多变型描述9,12,14。性和同事描述了类似的方法，以如下所述，在小鼠骨髓细胞中破骨测定中对它们的描述的协议。然而，释放以下长骨收获骨髓细胞，性等冲洗骨髓腔的α-MEM完全培养基14。Catalfamo检查高血糖对破骨细胞功能的影响，并描述了其中的所有细胞动员骨髓潮红的方法进行培养24小时，在该点处的非贴壁细胞被丢弃12，一种技术也可用于由Boyle 等人的9，这些先前公布的协议必要冲洗骨髓，一个繁琐的实践中，其中还引入了针刺伤害和有价值的骨髓损失的风险，因为一个人必须切骨的两端部的做法。的协议中，我们描述了，实现了使用研钵和研杵的分离的破骨细胞，其类似于由Weischenfeldt 等人所述的巨噬细胞分离的方法。15 我们的经验，但是，是使用在破骨细胞的生产方面先前公布的技术的结果变量的结果即破骨细胞的分离和体外培养，常常导致在无法培养的破骨细胞。因此，我们设计了一个协议，该协议允许对小鼠骨髓的一贯隔离，以产生大量的体外多核破骨细胞，具有细胞最初镀形成的巨噬细胞和破骨细胞随后的70-80％的产率约，在存在破骨细胞诱导培养基。

<table border="0" cellpadding="0" cellspacing="0" width="1017">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:503px;">Name of the Material/Equipment</td>
			<td style="width:165px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM, no glutamine, no phenol red</td>
			<td>Gibco</td>
			<td>51200-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-CSF, recombinant mouse</td>
			<td>Gibco</td>
			<td>PMC2044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Mouse TRANCE/RANK L/TNFSF11 (E. coli expressed)</td>
			<td>R&#38;D Systems</td>
			<td>462-TEC-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prostaglandin E2</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Phosphatase, Lekocyte (TRAP) kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osteoassay bone resorption plates, 24 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3987</td>
			<td></td>
		</tr>
	</tbody>
</table>

osteoclast derivation from mouse bone marrow

<html>破骨细胞是从骨髓中的单核细胞/巨噬细胞系衍生的高度特化的细胞。其重吸收两者的有机和无机骨基质的独特能力意味着它们在调节骨骼重塑中发挥关键作用。在一起，成骨细胞和破骨细胞是负责动态耦合的过程，涉及既骨吸收和骨形成的共同行动，以维持健康和疾病过程中的正常骨架。 如在体内主要骨吸收细胞，改变破骨细胞的分化或功能可能导致在体内产生深远的影响。具有改变破骨细胞功能有关的疾病的范围可以在严重程度从致命新生儿疾病由于不能形成对造血骨髓的空间，更通常观察到的疾病，如骨质疏松症，其中过度的破骨细胞骨吸收易患断裂形成。 ENT"&gt;一个孤立的破骨细胞的数量高在体外的能力也允许在骨重建周期的理解显著的进展，并铺平了道路，以应对这些疾病的新的治疗策略的发现。 在这里，我们描述了一个协议，分离培养小鼠骨髓，将产生大量的破骨细胞的破骨细胞。

<html>该方法的目的是要在一个星期容易分离大量破骨细胞的体外培养 ，典型。大量破骨细胞的成功分离用抗酒石酸酸性磷酸酶染色（ 图1A）中得到证实。大破骨细胞被可视化为紫色大细胞细胞核多个（通常≥3核）。使用该协议，它是常见的，分离的破骨细胞与破骨细胞每多达30细胞核（ 图1B）。 使用矿化的表面被认为是评估在体外破骨...

Watch this Scientific Journal Video about Osteoclast Derivation from Mouse Bone Marrow at JoVE.com

Osteoclast Derivation from Mouse Bone Marrow

破骨细胞是体内的主要骨吸收细胞。一个分离的破骨细胞中大量的能力已经导致破骨细胞生物学的理解显著进步。在这个协议中，我们描述了一种用于分离，培养和定量破骨细胞的体外活性。

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

osteoclast-derivation-from-mouse-bone-marrow

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Fibroblast Transformation and Muscle Stem Cells

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24.0K 次观看.  Stanford University School of Medicine. 破骨细胞是体内的主要骨吸收细胞。一个分离的破骨细胞中大量的能力已经导致破骨细胞生物学的理解显著进步。在这个协议中，我们描述了一种用于分离，培养和定量破骨细胞的体外活性。 

视频: Osteoclast Derivation from Mouse Bone Marrow

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.

从小鼠胚胎干细胞的造血干细胞的推导

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 ...

科研

生物学

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

归巢的骨髓造血细胞

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

鼠标滋养层干细胞从胚泡的推导

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.