We thank Kwon laboratory members for helpful discussions. This work was supported by grants from NHLBI/NIH (R01HL111198) and Maryland Stem Cell Research Fund (MSCRF).

모든 마우스는 존스 홉킨스 대학의 동물 시설을 -accredited 실험 동물 관리의 인증을위한 미국 협회 (AAALAC) 유지 및 실험 동물의 관리 및 사용에 대한 가이드에 설명 된 절차에 따라 수용했다. 기관 동물 관리 및 사용위원회 (IACUC)는 모든 실험 프로토콜을 승인했다. 1. 실험 준비 <ol><li> 60 분 동안 인산염 완충 용액에 10 % 태아 소 혈청 (FBS) (PBS)와 코트 12 웰 플레이트. </li><li> 코팅 용액을 제거하고 혈청이없는 미디어 (SFM)의 200 μl를 추가합니다. 미디어 막이 전체 표면 영역을 커버하는지 확인 접시 돌리기. </li></ol> 2. 외과 적 치료 <ol><li> 완전한 안락사을 보장하기 위해 자궁 경부 전위 다음에 기관의 동물 관리위원회 승인 프로토콜을 사용하여 임신 쥐를 안락사. </li><li> 스프레이 70 %와 마우스의 배꼽 지역을 청소에탄올. 오염을 방지하기 위해 엄격한 무균 기술을 유지한다. </li><li> 사용 집게와 가위는 배꼽 부위에 0.5 cm (2) 절개를합니다. </li><li> / 핀치 절개 위 아래 피부를 잡아 부드럽게 반대 방향 (머리와 꼬리)에서 피부를 당겨 손가락을 사용합니다. </li><li> 배꼽 부위에 막에서 초기 절개를 집게와 가위를 사용합니다. 부드럽게하여 자궁을 드러내는, 각면 (2 × 1.5 cm 2)에 난소에 배꼽에서 V 자형의 막을 잘라. </li><li> 따라서 난소에서 자궁을 확보, 난소 측의 난관을 끼지 난소와 가위로 자궁을 연결하는 난관이 끼지 집게를 사용합니다. </li><li> 부드럽게 집게로 난관에 의한 자궁을 당겨 가위로 방광 지역에서 무료로 자궁을 잘라. </li><li> 집게로 난관에 의해 추가로 최대 자궁를 당기고하여 M에서 자궁을 해제, 다른 측면에서 가위로 난관을 잘라여러개. </li><li> 50 ML 튜브에 자궁을 전송하고 칼슘과 마그네슘 2+와 살균 차가운 PBS의 20 ㎖를 추가합니다. PBS는 배아 해부하는 동안 칼슘과 마그네슘을 2 + 포함해야합니다. </li><li> 조심스럽게 피를 자궁을 씻어 10 초 동안 튜브를 흔들. </li><li> 집게와 튜브에서 자궁을 전송하고 10 ml의 접시에 배치하고, 자궁의 상단에 차가운 PBS 5 mL를 추가 할 수 있습니다. </li><li> 실체 현미경 하에서 자궁과 접시를 놓습니다. 참고 : 2.13-2.19는 실체 현미경을 필요로 단계. </li><li> 조심스럽게 하나 하나 자궁에서 태아와 양수 주머니를 자궁을 열고 밖으로 해부 포셉 두 쌍을 사용합니다. <ol><li> 조심스럽게 배아를 둘러싸고있는 양막을 열 포셉 두 쌍을 사용하여 하나의 집게로 주머니를 꼬집어 부드럽게 배아에서 분리, 절단 및 배아에서 양막 낭을 해제하기 위해 다른 집게를 사용합니다. 특정 유전자형이 필요한 경우, 유전자형에 대한 양막 낭을 유지. </li></ol></li><li> 측면에있는 배아를 놓고 아치와 인두 주머니 (그림 1A &#39;&#39;) 사이에 마음에 후방 아치 차 1 차 및 2를 잘라 집게를 사용합니다. </li><li> 조심스럽게 다른 측면으로 배아를 뒤집어 뒤쪽 아치와 인두 주머니 사이의 심장에 1, 2 아치를 잘라 집게를 사용합니다. </li><li> 아직 1 차 및 배아에 2 차 인두 아치를 연결하는 심장 유출 관을 잘라 집게를 사용합니다. 여전히 배아에서 나비 (그림 1B)의 형태로 함께 부착 된 아치를 제거합니다. </li><li> 핀치 집게를 사용하고 나머지 심장 유출 관 및 foregut 내배엽에서 1 차 인두 아치를 해제 (그림 1B &#39;&#39;* 점선으로 표시하고). </li><li> 핀치 집게를 사용하고 나머지 심장 유출 관에서 2 차 인두 아치를 해제및 foregut의 내배엽 (그림 1B &#39;&#39;점선과 **로 표시). </li><li> 별도로 집게를 사용하여 아치의 각 쌍을 전송하고 잘 지정의 중간에 두 아치를 배치합니다. 별도의 우물에 플레이트 1 차와 2 차의 인두 아치. 아치 단계 1.2에 추가 된 미디어의 필름과 접촉에 있는지 확인하십시오. 그것은 그들이이 기간 동안 부착을위한 우물의 표면과 접촉을해야으로 아치가 매체에 의해 포함되지 않는 것이 중요합니다. </li></ol> 3. 보육 및 이미지 <ol><li> 아치 웰 표면적에 첨부 2 시간 동안 37 ° C / 5 % CO 2에서 12 웰 플레이트에 도금 아치를 부화. </li><li> 부드럽게 잘 측면 아래로 37 ° C SFM 200 μl를 추가합니다. 아치가 부착 된 상태를 유지하는지 확인하고 / N을 O를 품어. </li><li> 다음 날, 시각적으로 인두 아치가 연결 부드럽게의 측면 아래로 37 ° C SFM 200 μl를 추가하고 있는지 확인잘. 아치가 부착되지 않은 부동 남아있는 경우, 부드럽게 전용 미디어의 필름이 남아 때까지 아치를 제거하지 않고 피펫 용지를 제거합니다. 잘 단계를 반복 3.1의 중앙에 아치를 배치 피펫 팁을 사용합니다. </li><li> 매일 모니터; 어떤 미디어를 증발 대체하기 위해 모든 2 일 미디어 ~ 100 ~ 200 μl를 추가합니다. 참고 : 셀을 마이그레이션 한 후 24 ~ 48 시간이 아치 주위에 표시하고 증식하고 다음 5-8일을 통해 아치에서 마이그레이션합니다. 구타 심근의 36-72 시간의 형성은 2 차 인두 아치 (그림 2B)에서 관찰 할 수 후. Myotube 형성은 첨부 파일 (그림 2A) 후 1 차 인두 아치 3~7일에서 관찰 할 수있다. </li></ol>

<ol>
	<li>Kelly, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+heart+field.">The second heart field.</a> Curr Top Dev Biol. 100, 33-65 (2012).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+congenital+heart+disease.">The developmental genetics of congenital heart disease.</a> Nature. 451, 943-948 (2008).</li><li>Tzahor, E., Evans, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharyngeal+mesoderm+development+during+embryogenesis:+implications+for+both+heart+and+head+myogenesis.">Pharyngeal mesoderm development during embryogenesis: implications for both heart and head myogenesis.</a> Cardiovasc Res. 91, 196-202 (2011).</li><li>Uosaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Contact+with+Endoderm-like+Cells+Efficiently+Induces+Cardiac+Progenitors+from+Mouse+and+Human+Pluripotent+Stem+Cells.">Direct Contact with Endoderm-like Cells Efficiently Induces Cardiac Progenitors from Mouse and Human Pluripotent Stem Cells.</a> PLoS One. 7 (10), e46413 (2012).</li><li>Cheng, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+mediates+mesendodermal+cell+fate+decisions.">Fibronectin mediates mesendodermal cell fate decisions.</a> Development. 140, 2587-2596 (2013).</li><li>Kwon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+pathway+involving+Notch1/beta-catenin/Isl1+determines+cardiac+progenitor+cell+fate.">A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitor cell fate.</a> Nature cell biology. 11, 951-957 (2009).</li><li>Shenje, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precardiac+deletion+of+Numb+and+Numblike+reveals+renewal+of+cardiac+progenitors.">Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors.</a> Elife. 3, e02164 (2014).</li><li>Grevellec, A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharyngeal+pouches+and+clefts:+Development,+evolution,+structure+and+derivatives.">The pharyngeal pouches and clefts: Development, evolution, structure and derivatives.</a> Semin Cell Dev Biol. 21, 325-332 (2010).</li><li>Kwon, C., Cordes, K. R., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+acts+at+multiple+developmental+stages+to+promote+mammalian+cardiogenesis.">Wnt/beta-catenin signaling acts at multiple developmental stages to promote mammalian cardiogenesis.</a> Cell Cycle. 7, 3815-3818 (2008).</li><li>Cho, G. S., Fernandez, L., Kwon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+heart:+perspectives+on+stem-cell+therapy.">Regenerative medicine for the heart: perspectives on stem-cell therapy.</a> Antioxid Redox Signal. 21, 2018-2031 (2014).</li></ol>

The authors have nothing to disclose.

이 비디오에서는, 우리는 분리하고 9.5 일 이전 마우스 배아의 첫 번째 문화와 두 번째 인두 아치하는 방법을 보여줍니다. 인두 아치, 첫 번째 아치 8에서 두 번째 아치와 머리 근육 전구 세포 -in 배아 10, 11시에 마음을 만들기 위해 멀티 강력한 심장 전구 세포 - 빌딩 블록을 포함하는 개발 배아 (9)의 craniolateral 측면에 표시 부푼를 과도 분할된다 . 인두 ...

인두 중배엽 세포는 심장의 부분과 인두 근육 일으킨다. 배아 개발하는 동안, 두 번째 심장 분야에서 다 능성 심장 전구 세포는 인두 중배엽에서 마이그레이션 및 심장 유출 관과 우심실을 채우고, 그 이상 개발은 밀접하게 출생 결함과 출생 defect-의 선천성 심장 질환의 주요 원인과 관련된 인간 1-3 관련 사망. 최근의 연구는 인두 중배엽은 중배엽 심장 - 두개 안면 개발 4의 중요한 부분 만들기, 마음뿐만 아니라, 근육을 머리에 기여하고 있음을 증명하고있다. 따라서, 유도, 증식 및 인두 중배엽에서 머리와 심장 전구 세포의 분화를 포함하여 개발 프로세스가 활성화 조사 5-7 받고있다. 최근까지, 심장 전구 세포가 팽창 witho를 받아야하는지 여부 알려지지그들의 셀룰러 환경에 대한 정보의 부족으로 인해 부분적 UT 분화. 우리의 최근의 연구는 인두 아치가 심장과 근육 조상의 갱신을위한 미세 역할을하고 몇 주에 걸쳐 8 생체 전 배양 될 수 있음을 시사한다. 이 이식편 방법은 심장 - 두개 안면 전구 세포 생체의 개발을 연구하는 새롭고 독특한 기회를 제공합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS + Ca2 + Mg2</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10cm petri plates</td>
			<td>Fishersci</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Dissection forceps</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Serum Free Media:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Cellgro</td>
			<td>15-016-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s F12</td>
			<td>Cellgro</td>
			<td>10-080-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>N2-SUPPLEMENT</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27-retinoic acid</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>10% BSA (in PBS) &#224; (Invitrogen Cat#. P2489)</td>
			<td>Life Sciences</td>
			<td>P2489</td>
			<td></td>
		</tr>
		<tr>
			<td>100X Glutamine (Gibco Cat.# 25030-081)</td>
			<td>Gibco</td>
			<td>35050-61</td>
			<td></td>
		</tr>
		<tr>
			<td>100X Pen/Strep (Gibco Cat#. 15070-063)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo culture of pharyngeal arches to study heart and muscle progenitors and their niche

개발 배아의 인두​​ 중배엽은 머리와 심장 근육의 넓은 지역에 기여한다. 우리는 (또한 아가미 아치로 알려진) 인두 아치와 머리와 심장 전구 세포 개발 생체를 연구하는 새로운 방법을 개발했다. 이 방법을 사용하여, 우리는 최근 두 번째 인두 아치 자기 갱신 심장 전구 세포를 포함하고 마우스 심장 개발하는 동안 조상의 확장을위한 미세 역할을한다는 설명했다. 전구 세포는 아치 내부 분화와 광대 남아 있지만 신속하게 아치에서 마이그레이션 할 기능 심근된다. 우리는 또한 첫 번째 인두 아치는 아치를 떠난 후 myotubes에 상승을주는 근육 전구 세포를 포함보고했다. 여기, 우리는 해부와 마우스 배아 개발에서 제 1 및 제 2 인두 아치의 체외 배양 과정을 보여줍니다. 이 방법은 머리와 심장 전구 / 근육 dev에 공부를 할 수 있습니다심근 및 세부 생체에 myotube 형성을 포함 elopment.

그들은 증식과 머리와 심장 근육, 각각 (그림 1A, &#39;과&#39;을 &#39;)되고, 1 차와 2 차의 인두 아치에서 마이그레이션으로 개발하는 동안, 얼굴 근육과 심장 전구 세포는 추적 할 수 있습니다. 인두 아치를 배양하는 세부 생체 심장과 근육 발달을 연구하는 독특한 방법을 제공합니다. 해부 및 인두 아치의 부착 후, 부착 아치에서 마이그레이션 세포 배양의 24 ~ 48 시...

Watch this Scientific Journal Video about Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche at JoVE.com

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

인두 아치 전의 VIVO 문화는 심장과 근육 전구 세포와 그 틈새를 연구

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

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Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

7.7K 조회수.  Johns Hopkins University School of Medicine. Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

비디오: Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

사용<em&gt; 전의 VIVO</em&gt; 전체 태아 기관의 강직 한 방울 문화 마우스 기관 형성 기간 동안 발달 프로세스를 연구

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

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Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

예 생체 병아리 소뇌 조각의 문화가 공간적으로 Electroporation에 과립 세포 전구체의 표적

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

심 외막 파생물 문화 분석 및<em&gt; 전의 VIVO</em&gt; 심 외막 유래 세포 마이그레이션 평가

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Flow Cytometry에 의한 마우스 태아의 적혈구 전구 세포 (Erythromyeloid Progenitors)와 그 자손의 동정

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

키메라 Zebrafish의 태아의 개별 망막 전구 세포의 형질 전환 유전자 발현의 생체 내 이미징에서 세포 비 자립적 영향을 연구하기 위해

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

절연, 문화, 및 골 수 기질 세포와 생쥐에서 Osteoclast 창시자의 차별화

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

수축 성 활동 유도 된 쥐 골격 근육 Phenotypic 적응 연구 만성 자극에의 응용

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Vivo에서 근육-힘 줄 Morphogenesis 초파리 번데기에의 이미징

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

첫 번째 심장 필드 같은 심장 창시자와 같은 심 실 Cardiomyocytes 인간 만능 줄기 세포에서의 세대

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

설치류 태 반의 전 생체 관류

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...