The authors acknowledge funding sources responsible for this work: 5K12HD055929 (HHR), 5R01NS080180-02 (DDF).

참고 :이 프로토콜의 모든 동물 절차는 플로리다 기관 동물 관리 및 사용위원회 (IACUC)의 대학의 승인을 실시하고 &#39;실험 동물의 관리 및 사용을위한 안내서&#39;을 준수하고 있습니다. 1. 기본 실험 세트까지 간헐적 저산소증 관리하기 전에 <ol><li> 다른 실험 목적을위한 5,13,14 성인 설치류만큼 동일하게 전신 마우스 plethysmograph 챔버를 사용하여 상이한 가스 혼합물에 새끼 12을 노출. 챔버는 3-4 마우스 새끼 또는 1-2 래트 새끼를 수용하기 충분한 크기 인 4 인치 직경 (부피 = 450 ml)에,이. 여기에, AIH 프로토콜은 출생 후 하루 1-8 (P1 ~ P8) 세 신생아에게 전달됩니다. </li><li> 21 %에서 챔버 산소를 유지 21 % &quot;기준&quot;가스를 제공하기 위해 압축 공기 가스 탱크를 사용합니다. &quot;상대적 저산소증&quot;을 달성하기 위해, 10 % 산소 / 90 %의 질소 가스 탱크를 사용한다. 하지E : 플라스틱 배관 (3/16 &quot;직경)는 유량계 가스 탱크를 연결한다. 따라서, 유량계에 대한 입력은 하나의 튜브 및 저산소증 기준선 기류 각 이루어져있다. </li><li> 다음과 같이 가스 배관을 구성합니다. <ol><li> 각 챔버에 1 L / 분의 흐름을 가능하게하는 바이어스 유동 조절기로 유량계에서 플라스틱 배관 (1/8 &quot;직경)을 실행 (즉, 4 L / 분 (4) 챔버가 사용되는 경우). </li><li> 혈량 측정법 챔버에 바이어스 유량 조절기에서 플라스틱 튜브 (1/8 &quot;직경)를 실행합니다. 기류는 1에서 L / 분으로 각각의 챔버는 가스 교환이 비 생리적 스트레스 유발 및 행동의 관측에 기초하여 동물에 것으로 간주되는 소정 유량이다. </li></ol></li><li> 어떠한 가스 유동이 프로토콜 동안 사용되지 않을, 또는 혈량 측정법 챔버 중 다른 챔버로 빠져 나가지 않도록 캡 챔버 및 상기 바이어스 부에서 유동하지 않는 모든 연결 및 모든 미사용 개구부를 밀봉. </li><li> (실을 배치의) 37 ° C로 평형화 또는 체온을 허용하도록 챔버에 동물을 성공시키는 종래 인큐베이터. </li></ol> 간헐적 저산소증에 대한주기 시간 2. 교정 <ol><li> 공기 탱크 및 유량계 사이, 모든 배관을 검사 적절한 연결 m 바이어스 흐름 부, 그리고 바이어스 흐름 부와 혈량 측정법 챔버 (들)를 흐른다. 예를 들어, 사용하지 않는 미사용 바이어스 유동 부의 아웃 라인 및 챔버 개구를 체크 : 모든 설명 선과 클로저 단계적 수표 접속 확인 단계 1에서 설명한 바와 같이 접속이 설정된다. </li><li> 휴대용 O 2m로 주변 O2 센서를 연결 한 후 혈량 측정법 챔버 뚜껑에 센서를 부착합니다. </li><li> 모두 가스 탱크를 열고 가스의 오프 사이클 흐름을 고정하는 플라스틱 튜브로 절연 수술, 긴 처리 지혈 한 쌍을 연결합니다. 이러한 방식으로, 한 번에 하나의 가스 탱크 챔버 당 1 L / min의 가스 흐름을 제공한다. &quot;저산소증&quot;가스 믹스가 반대로 챔버로 전달되고, 반면 &quot;기준선&quot;가스 믹스 클램프. </li><li> 10 %의 목표 레벨에 도달하고 21 %로 돌아 챔버 O 2에 필요한 시간을 기록한다. 다음 프로토콜 전달을위한 기록 된 시간을 활용합니다. </li></ol> 급성 간헐적 저산소증 3. 관리 <ol><li> 2.1 단계에 설명 된대로 모든 배관 및 연결을 검사합니다. </li><li> 혈량 측정법 챔버 (들)에서 신생아를 놓고 챔버베이스로 혈량 측정법 챔버 뚜껑을 수용. 사용하지 않는 실 연결의 적절한 O- 링 씰 씰입니다뿐만 아니라 폐쇄를 확인합니다. 이 단계는 가스 혼합물의 적절한 공급을 보장하는 데 중요합니다. </li><li> 새끼를 보유 챔버가 37 ° C 배양기로 처리 할 수​​ 놓고 문을 닫습니다. 챔버는 이전 프로토콜을 시작으로 37 ° C의 평형을 허용합니다. incubat 37 ° C에서 제어 동물을 유지또는 실내 공기의 산소에서. </li><li> 모두 가스 탱크를 열고 가스의 오프 사이클 흐름을 고정하는 플라스틱 튜브로 절연 수술, 긴 처리 지혈 한 쌍을 사용합니다. 이러한 방식으로, 한 번에 하나의 가스 탱크 챔버 당 1 L / min의 가스 흐름을 제공한다. </li><li> 정밀 시간주기에 휴대용 타이머를 사용함으로써 챔버로의 가스 유동의 교호 결과, 튜브 사이에 지혈을 전환하는 시간을 표시. </li><li> 이러한 2 단계, 20 사이클 프로토콜의 모든 단계를 표시하기 위해도 1에 제공된 것과 같은 처리 로그를 이용하여 각주기의 완료를 기록한다. </li><li> 동물이 지정된 범위 (37 ° C 결과 인큐베이터에있는 설정입니다 33 ~ 35 ° C)에서의 유지를 보장하기 위해 모든 사이클 변화에 인큐베이터 온도를 모니터링합니다. </li><li> 밀접하게 동물은 발성의 변경된 수준으로 볼 수 고통없이 사이클을 견딜 수 있도록 프로토콜을 통해 강아지의 활동을 모니터링사지 활동이나 압연. </li></ol> 뇌실 영역에서 줄기 / 전구 세포의 4. 분리 및 문화 주 : 대조군과 비교 AIH 다음 neurosphere 포메이션에 변화는 조직 - 및 세포 수준의 변화를 유발하는이 프로토콜의 효능을 입증 엔드의 일례이다. <ol><li> 세포를 형성 neurosphere를 격리하기 위해, AIH가 완료된 직후 챔버에서 마우스 또는 쥐 새끼를 제거합니다. 프로토콜 종료 30 분 이내에 IACUC 프로토콜에 따라 동물을 희생. </li><li> 이전 방법 장 (15, 16)과 별도의 비디오 프로토콜 (8)에 발표 된 바와 같이, SVZ 조직을 수확 neurosphere 문화에 배치하고 표준 통로 파생 neurosphere를의 확장을 실시하고 있습니다. </li></ol>

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The authors have nothing to disclose.

This work reports the development of a protocol to expose neonatal rodents to AIH. The parameters described here effectively alter in situ neural stem cell biology, which is observable over several rounds of cell passage. Specifically, AIH increases the number of non-adherent neurospheres, the expansion of cells within each neurosphere (refected by sphere diameter), the expansion of adherent NPC populations, and the presence of neuroblasts in both non-adherent and adherent populations. It should be emph...

이 방법의 목적은 재현성과 효과적으로 신생아 설치류에 전신 낮아 주변 산소의 간헐적 인 관찰을 제공하는 것입니다. 줄기 세포 생물학 조작 간헐적 저산소증 (IH)를 사용하는 이론적 근거는 성장 배지 2 O의 함유량이 변경되는 시험 관내 세포 배양 실험에서 유래. 20 %의 &quot;표준&quot;조건 O 2, 증가 확산의 3 % O 2 결과 줄기 / 전구 세포 집단 세포의 확장 된 문화에 비해 특히, 세포 사멸을 감소 신경 수익률 1,2 증가했다. 이 그룹은 전신 IH의 관리에 상당한 경험을 가지고 있으며, 호흡 소성 3-7 IH의 역할에 대한 광범위한 연구를 수행하고있다. 이 작품, 만성 IH은 설치류 CNS 8-10에 신경을 증가하는 최근의 연구 결과는 생체 내에서 급성의 탐사를위한 기초를 형성전처리 자극으로 저산소증 (예., 조직 수확 이전에) 신경 줄기 / 전구 세포 (NPC들) (11)의 연속 문화. 마우스 새끼가 급성 간헐적 저산소증 (AIH)의 개요 (&lt;1 시간) 기간에 노출되었을 때 놀랍게도, 뇌실 영역 (SVZ)에서 수확 된 세포는 크게 neurosphere를 또는 부착 단층 세포와 같은 확장을위한 용량을 증가했다. AIH 프로토콜은 또한 &quot;신경 운명&quot;전사 인자 (PAX6)의 발현 증가와 관련이 있었다. 따라서, 생체 내에서 AIH 프로토콜이 &quot;소수&quot;의 NPC 전에 문화 할 수있는 수단을 제공 할 수있다. 예를 들어,이 방법에 대한 응용 프로그램은 손상된 중추 신경계에 이전에 이식 세포 집단을 확장하거나 이전의 시험 관내 실험에 배양 된 세포의 신경 세포 분화를 증가 포함 할 수있다. 또한,이 전신 전달을하기 때문에, 어떠한기관, 조직 또는 세포는 유사한 연구를위한 후보입니다. 따라서 작성된 프로토콜은 작은 포유류 간헐적 산소 조작에 관한 연구의 다양한 잠재적으로 적용될 수있다. 이 방법의 특정 이점이있다. 게시 된 다른 연구에서 신생아가 치료에 앞서, 만성 투여 덜 처리를 할 수 있습니다 기압 성 챔버의 댐과 쓰레기로 취급하고, 치료 9시 산모의 접촉을 유지 하였다. 현재의 접근법은 사육 여성, 또는 각각의 실험에 대해 다른 댐의 사용을 반복 치료를 무시. 이 프로토콜은 또한 정확한 쓰레기 일치 및 연령대 신생아의 연구를 할 수 있습니다. 대표적인 데이터는이 프로토콜의 또 다른 키 강도, AIH는 전달과 같은 신경 줄기 세포 생물학에서 강력하고 일관된 생물학적 반응을 이끌어있는 즉 신속성을 보여줍니다. 이 조직 - 및 세포 수준 biologi을 유도하기 위해이 프로토콜에 대한 선례를 확립세포 생물학을 변경 CAL 변경됩니다. 이 보고서에 AIH 설치류뿐만 아니라 새끼 neurosphere를 성장 중 SVZ 세포의 집단 분석을 노광에 사용되는 세부 과정을 개설 할 것이다.

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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:243px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Mouse plethysmography chambers</td>
			<td style="width:147px;">Buxco</td>
			<td style="width:136px;">PLY4211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Flow meter&#160;</td>
			<td style="width:147px;">Porter</td>
			<td style="width:136px;">F150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bias flow unit</td>
			<td style="width:147px;">AFPS</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Baseline Gas Mix</td>
			<td style="width:147px;">Airgas</td>
			<td style="width:136px;">AIZ300</td>
			<td>Compressed Air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hypoxic Gas Mix</td>
			<td style="width:147px;">Airgas</td>
			<td style="width:136px;">X03NI72C2000189</td>
			<td>10% Oxygen, balance nitrogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Oxygen Meter</td>
			<td style="width:147px;">Teledyne</td>
			<td style="width:136px;">AX-300</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of in vivo acute intermittent hypoxia in neonatal rodents to prime subventricular zone-derived neural progenitor cell cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

기록 데이터에 기초하여 초기의 실험은, 1 분의 사이클 길이를 이용하여 수행 하였다. 상기 단계 2에서 수행되는 후속 캘리브레이션에 기초하여, 그것이 21 % 기준선으로 돌아 유사한 시간 걸렸다, 챔버 내의 O 2 레벨이 1 분 후 저산소증 플러싱에서 13 %라고 결정 하였다. 그러나, 2 분주기의 10 %를 달성 산소 및 &quot;기준&quot;사이클 동안 21 %로 복귀시키기에 충분한 양이었다. 이어서, 2 분주기...

Watch this Scientific Journal Video about Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures at JoVE.com

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

이 문서에서는 출생 후 하루 1-8 마우스 또는 쥐 새끼에 간헐적 저산소증의 짧은 기간을 관리하는 방법을 설명합니다. 이러한 접근은 효과적 저산소증 노출 30 분 이내에 수확 배양 신경 전구 세포에 대한 강력한 조직 수준 &quot;프라이밍 효과&quot;를 도출한다.

배달<em&gt; 생체</em&gt; 급성 간헐적 저산소증 신생아 설치류에서 국무 뇌실 지역 유래 신경 전구 세포 배양에

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

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JoVE Journal

Developmental Biology

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

7.2K 조회수.  University of Florida.  이 문서에서는 출생 후 하루 1-8 마우스 또는 쥐 새끼에 간헐적 저산소증의 짧은 기간을 관리하는 방법을 설명합니다. 이러한 접근은 효과적 저산소증 노출 30 분 이내에 수확 배양 신경 전구 세포에 대한 강력한 조직 수준 "프라이밍 효과"를 도출한다. 

비디오: Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

말초 혈액 조혈 전구 세포에서 인간 신경 줄기 세포의 직접 유도

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

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Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

줄기 세포처럼<em&gt; Xenopus의</em&gt; 배아의 Explants는 초기 신경 발달의 특징을 연구하는<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

불멸화 된 다 능성 귀의 전구 세포의 분화를 초기화

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

개발에 미세 주입을 대상으로 기술<em&gt; Xenopus의</em&gt; 신장

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

차 섬모를 연구에 차 Neurosphere 문화를 사용하여

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

살아있는 개구리와 Zebrafish 태아의 저산소증 유도

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

혈관 내피 줄기 세포의 혈관 생성을 시각화하기위한 유방 팻 패드 이식 기술

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

마우스 배아에서 심장 조상 인구의 양적 전체 마운트 면역 형광 분석

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

칼슘 유입, 기 공 형성 및 성숙한 신경 조상 세포 수용 체 P2X7에 의해 식 균 작용을 조사 하기 위해 실시간 라이브 셀 Cytometry

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

인간 유도 만능 줄기 세포 유래 신경 세포의 치료 이식으로 마우스 뇌 손상의 제어 피질 영향 모델

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...