Name: mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis
Uploaded: 2014-09-09T15:00:00.000+00:00
Duration: 6 min 40 s
Description: Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

이 연구는 NIH R00HL102154 지원 및 P 지행 혈액 학자 수상의 미국 사회되었다.

이 프로토콜에 기술 된 실험은 노스 웨스턴 대학 기관 동물 관리 및 사용위원회에 의해 명시된 지침과 규정에 따라 수행되었다. 문화 매체의 1 준비 <ol><li> 피브로넥틴 솔루션을 준비합니다. 인간 피브로넥틴 (1 mg)을 중 하나를 유리 병에 물 1 ㎖를 추가합니다. 교반없이 30 분 동안 조직 문화 후드에서 솔루션을 둡니다. 20 μg / ML의 최종 농도를 만들어 PBS 50 ㎖로 전체 액체를 전송하고 부드럽게 잘 섞는다. 판 (아래 참조)을 코팅하기 전에 피브로넥틴에 신선한 용액을합니다. </li><li> EPO 배지 (SCF 매체) 50 ㎖를 준비합니다. 신선한했을 때 표 1에 열거 된 성분을 결합합니다. 매체 1 달 동안 4 ° C에 저장할 수 있습니다. </li><li> EPO 함유 배지 50 ㎖를 준비합니다. 신선한했을 때 표 2에 나열된 성분을 결합합니다. 매체 1 달 동안 4 ° C에 저장할 수 있습니다. </li></올&gt; 2 피브로넥틴 코팅 플레이트 <ol><li> 6 - 웰 플레이트 (또는 12 웰 플레이트의 각 웰에 0.5 ml)을 각 웰에 1 ml의 피브로넥틴 용액을 추가한다. 1 시간 동안 조직 문화 후드에서 판을 둡니다. </li><li> 두 번 멸균 물 우물을 씻어 공기 판을 건조. </li><li> 접시를 싸서 4 ° C의 추운 방에 저장합니다. </li></ol> 태아 간 적혈구 모세포의 3 정화 참고 : E15에 E13에서 사용 태아의 간은 임신 한 쥐 (C57BL / 6) 시간이 초과되었습니다. E13.5 간은이 CFU-E 전구 세포의 수율을 최대화하기 위해 바람직하다. 시간이 초과 임신 한 쥐를 얻으려면 6~8주 세 남성과 여성의 마우스는 한 케이지 (하나 또는 두 개의 여성 대개 한 남성)에 배치되었다. 여성 질 플러그 결합이 성공적인지 여부를 판별하기 위해 다음 날 아침을 조사 하였다. 플러그가 발견 된 후 임신의 첫 번째 날 일이었다. <ol><li> 총 태아 간 세포의 준비 <ol><li> 양해 각서 (MOU)를 희생CO 2 흡입 자궁 전위에 의해 그 자체. 소독 용 70 % 에탄올로 복부를 스프레이. 자궁을 제거하기 위해 가위를 해부와 복부를 엽니 다. 1X PBS에서 자궁을 씻으십시오. </li><li> 조직 문화 후드 및 멸균 도구를 사용하여 멸균 조건에서 태아를 해부하고 1X PBS에 세척. </li><li> 태아에서 태아의 간을 해부하다. 조심스럽게 다른 집게와 떨어져 몸에서 태아의 간 (컬러 핑크)를 당겨, 한 집게로 태아의 몸체를 잡고. 관련 섬유 성 조직을 제거하기 위해 집게로 태아의 간을 청소합니다. 10 % 소 태아 혈청을 함유하는 신선한 1X PBS로 모든 태아 간을 놓습니다. </li><li> 기계적 피펫 팅 아래에 의해 태아의 간을 방해. </li><li> 50ml의 원뿔형 튜브에 40 μm의 셀 스트레이너를 통해 세포 현탁액을 필터. 5 분 동안 800 XG에서 세포 펠렛. </li><li> FBS를 뜨는을 기음 10 %로 1 ml의 PBS에 세포를 재현 탁 (약 8 E13.5 태아 간) ML 당. </li></ol></li><li> TER119 네거티브 적혈구 모세포의 정화 <ol><li> 15 분 동안 얼음에 쥐 IgG를 가진 세포 (0.2 ㎎ / ㎖)을 막기. </li><li> 세탁기 또는 원심 분리없이 세포 현탁액 (1 μg / ㎖)에 비 오티 닐화 항-TER119 항체를 추가한다. 얼음에 15 분 동안 품어. </li><li> 5 분 동안 800 XG에 10 % FBS (1시 10분)와 원심 분리기 1X PBS에 세척 할 것. 10 % FBS와 1 ㎖의 PBS에 세포를 재현 탁. </li><li> 추가 75 μL은 자성 입자가 결합과 얼음에 10 분 동안 품어 스트렙 타비 딘. </li><li> PBS는 10 % FBS를 함유하는 2.5 ml의 볼륨을 추가합니다. 바닥 튜브 라운드 5 ㎖의 폴리 프로필렌에 세포 현탁액을 전송합니다. </li><li> 자기 세포 정렬 장치에 튜브를 삽입하고 10 분 동안 배양한다. TER119 양성 세포는 관의 측면에 부착된다. 무소속 TER119 부정적인 적혈구 모세포는 서스펜션에 남아있을 것이다. </li><li> 바닥 튜브 라운드 새로운 5 ㎖의 폴리 프로필렌에 세포 현탁액을 붓고. </li><li><html> 반복 잔류 TER119 양성 세포를 제거하는 3.2.6와 3.2.7 단계를 반복합니다. </li><li> 5 분 동안 800 XG에서 세포 펠렛. 뜨는을 대기음. 10 % FBS를 함유하는 1 ml의 PBS에 세포를 재현 탁하고 트리 판 블루 생균 카운트. </li></ol></li></ol> 바이러스 인코딩 된 cDNA 나 shRNA를 가진 4 형질 도입 <ol><li> 3-5에서 정제 TER119 네거티브 태아 간세포 판형 × 105 / 웰의 12 - 웰 플레이트를 코팅 피브로넥틴 (다른 플레이트를 사용할 경우 셀 수를 조정)한다. </li><li> 바이러스의 형질 도입을 촉진하기 위해 10 μg / ㎖의 최종 농도로 폴리 브렌을 추가한다. 회전은 37 ° C에서 1.5 시간 동안 800 XG에, lentiviruses 또는 레트로 바이러스, 인코딩하는 cDNA 나 shRNA를 가진 세포를 감염. </li></ol> 형질 TER119 네거티브 마우스 태아 간 적혈구 모세포의 5 문화 <ol><li> (그림 1은 점선) 단자 적혈구의 초기 단계에서 유전자의 기능을 테스트합니다.&quot;&gt; </ol.><ol><li><ol. li=""> 농경 12 시간 동안 EPO 배지 (SCF 배지)에 세포를 형질 도입 형질 도입 유전자와 전구의 유지 상태의 발현을 허용한다. </ol.></li><li> 5 분 동안 800 XG에서 세포를 원심 분리기. 뜨는을 대기음. 12 - 웰 플레이트의 각 웰에서 배지를 함유하는 1 ml의 EPO에서 세포를 재현 탁. </li><li> 24 또는 48 시간 동안 배양 한 후, 추가 분석을 위해 세포를 수확. </li></ol></li><li> 터미널 적혈구 (그림 1 실선)의 후반 단계에서 테스트 유전자의 기능을합니다. <ol><li> 문화 EPO 포함하는 배지에서 바로 형질 세포. </li><li> 24 또는 48 시간 동안 배양 한 후, 추가 분석을 위해 세포를 수확. </li></ol></li></ol> 유세포 분석을위한 배양 된 태아 간 세포의 스테 이닝 (6) <ol><li> 세척하고 1X PBS에 세포를 재현 탁. 수확 유세포 분석을 위해 2 × 105 세포를 배양 하였다. </li><li> 품다어둠 속에서 실온에서 20 분 동안 형광 공역 항체와 세포. 차별화를 테스트하려면 APC 복합 항 CD71 및 PE 복합 항 TER119를 사용합니다. 안구 적출술을 테스트하려면 다른 항체에 추가 훽스트 33342 얼룩을 사용합니다. </li><li> 1X PBS로 세포를 씻으십시오. 죽은 세포를 염색 및 분석에서 제외하는 1 % FBS 및 요오드화 프로피 듐 (PI) (1 μg / ㎖)를 함유하는 0.5 ml의 PBS에 세포를 재현 탁. </li><li> 행동 흐름 cytrometric 분석. 흐름 cytometer의 조정 및 보상을위한 최초의 단일 컬러 염색 흠없는 대조군 세포를 분석 할 수 있습니다. </li></ol>

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저자는 그들이 더 경쟁 금전적 이해 관계가 없다고 선언합니다.

여기에서 우리는 시간 순으로 마우스 태아 간 터미널 적혈구를 분석 할 수있는 독특한 시스템을 제시한다. 다른 배양 조건의 적용을 통해, 우리는 성공적으로 초기와 후기 단계에서 터미널 적혈구 해부. 이는 다양한 기능을 가진 유전자의 메카니즘을 결정하는데 중요하다. 예를 들어, 라 세미 GTPases 단자 적혈구 생성의 다른 단계에서 중요한 역할을한다. 터미널 적혈구에 영향 세포 분화와 증식의...

적혈구는 적혈구 성숙 다 능성 조혈 줄기 세포의 분화 과정이다. 이 단계적인 과정은 최선을 다하고 적혈구 버스트 형성 단위 (BFU-ES), 빠른 속도로 나누어 적혈구 콜로니 형성 단위 (CFU-ES) 및 형태 학적 인식 적혈구 모세포 1,2의 형성을 포함한다. CFU-E의 전구 세포에서 터미널 적혈구는 순차적 에리트로 포이 에틴에 의존하고 독립적 인 단계 2,3을 포함한다. 단말기 적혈구 생성의 초기 단계에서, 에리트로 포이 에틴 (EPO)가 세포 표면 수용체에 결합하고 세포 사멸을 방지하고 빠른 세포 분열 및 유전자 발현을 촉진 1,4 하류 신호 전달 경로의 시리즈를 유도한다. 터미널 적혈구의 후반 단계에서 적혈구 모세포는 매우 응축 핵 5 단자 세포주기의 종료, 크로 마틴과 핵 응축, 압출을받을. 터미널 우리의 이해적혈구가 크게 때문에 체외에서 여러 가지의 성공적인 사용에 생체 마우스 모델 6-9에서 크게이다 지난 수십 년에 향상되었습니다. 이러한 모델 중 마우스 태아 간 적혈구 모세포의 체외 배양 세포 정화, 빠른 증식과 분화의 용이성 등 많은 장점을 제공하며, 인간의 적혈구 10, 11에 가까운 모방. 이 시스템에서, 마우스 태아 간에서 적혈구 전구 세포 다수 용이 TER119의 단일 단계 정제 (성숙한 적혈구 세포에 대한 마커) 네거티브 적혈구 모세포에 의해 단리 될 수있다. 적혈구 모세포의 이틀 동안 배양이 세포의 분화가 트랜스페린 수용체 (CD71) 및 TER119 항원 (12)의 표면 발현에 기초하여 유세포 분석으로 모니터 할 수있다. 또한, 말기 분화 적혈구 모세포 적출술 1 DNA 머신 (훽스트 33342)에 의해 검출 될 수있다3. 또한, 정제 된 전구 세포는 유전자의 cDNA 또는 적혈구 11,13,14에 유전자 발현 기능 역학적 연구를 용이 관심 유전자에 대한 작은 헤어핀 RNA들 (shRNA를)의 외인성 발현에 의해 변형 될 수있다. 그것이 단말 적혈구 생성의 다른 단계에서 유전자의 기능을 특성화하는 것이 곤란하기 때문에, 다른 한편으로는, 고속 셀 성장률 양면성 수있다. 대부분의 경우에, 그것의 cDNA 또는 shRNA를 발현 시간까지 보낸 단말 적혈구 생성의 초기 단계에서 특정 유전자의 기능, 세포가 이미 초기 단계를 통과했는지 여부를 결정하는 것은 곤란하다. 이 문제를 해결하기 위해서, 우리는 터미널 적혈구 생성의 초기와 후기 단계를 해부하는 독특한 시스템을 개발 하였다. 단말기 적혈구 생성의 초기 단계를 들면, 유전자 변형 TER119 네거티브 적혈구 모세포는 EPO 배지 함유하지만 줄기 세포 인자 (SCF)에서 IL-6 및 FLT3 배양리간드는 그들의 선조 상태를 유지하고, 식 (13)로 형질 도입 된 cDNA 또는 shRNA를 허용한다. 세포는 세포 증식 및 분화를 유도하기 위하여 12 시간 후에 EPO 함유 배지로 변경 하였다. 세포를 구별하기 시작할 때 이러한 방법으로, 형질 도입 된 cDNA 또는 shRNA를 이미 발현되었다. 터미널 적혈구의 후기를 들어, TER119 부정적인 적혈구 모세포는 EPO가 전달 후 즉시 매체를 포함에서 배양 하였다. 따라서, 하나의 단말은 적혈구 생성의 후반 단계에서의 관심의 유전자의 기능을 분석 할 수있다. 요약하면, 이러한 시스템의 광범위한 애플리케이션 단말 적혈구 생성의 다른 단계에서의 해부 유전자 기능을 도울 것이다.

<table><tbody><tr><td>Iscove&#039;s Modified Dulbecco&#039;s MediumIMDM (IMDM)</td><td>Gibco</td><td>12440-053</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GEMINI Bio-product</td><td>700-102P</td><td></td></tr><tr><td>Penicillin-Streptomycin solution</td><td>Hyclone</td><td>SV30010</td><td>100x</td></tr><tr><td>L-Glutamine</td><td>Hyclone</td><td>SH30034.01</td><td>200 mM</td></tr><tr><td>Mouse FIT3 Ligand (Flt-3L)</td><td>BD Biosciences&amp;nbsp;</td><td>14-8001-80</td><td></td></tr><tr><td>Mouse Recombinant Interleukin-6 (IL-6)&amp;nbsp;</td><td>GEMINI Bio-product</td><td>300-327P</td><td></td></tr><tr><td>Fibronectin</td><td>BD Biosciences&amp;nbsp;</td><td>354008</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>STEMCELL TECH</td><td>9300</td><td>10% stock in IMDM</td></tr><tr><td>Erythropoietin(Epo)</td><td>PROCRIT</td><td>NOC 59676-303-00</td><td>3,000 U/ml stock</td></tr><tr><td>Streptavidin particles Plus</td><td>BD Pharmingen</td><td>557812</td><td></td></tr><tr><td>EasySep Magnet</td><td>STEMCELL TECH</td><td>18000</td><td></td></tr><tr><td>Polypropylene Round-Bottom Tube, 5 ml</td><td>FALCON</td><td>352063</td><td></td></tr></tbody></table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

적혈구가 빠르게 형성 단위 적혈구 식민지 (CFU-ES)를 나누어 다음에 최선을 다하고 적혈구 버스트 형성 단위 (BFU-ES)로 시작하는 역동적 인 과정을 포함한다. CFU-ES 후, 세포는 형태 학적으로 인식 할 수 있으며 일반적으로 터미널 적혈구 모세포라고합니다. 단말기 적혈구의 연구 과제 중 하나는 연대순 방식 유전자 기능을 해부 실험 방법의 부족이다. 이 프로토콜에서는 단말 적혈구 생성의 초기와 후기 단계에서 유전자의 기능을 결정하기 위해 고유의 전략을 설명한다. 이 시스템에서 마우스 태아 간 TER119 (성숙한 적혈구 세포 마커) 부정적인 적혈구 모세포는 정제하고 cDNA를하거나 관심있는 유전자의 작은 머리 핀의 자형의 RNA (shRNA를)의 외생 적 표현 형질 도입. 세포를이어서 cDNA를 외인성 또는 shRNA를 허용하면서 12 시간 동안 그들의 전구 스테이지를 유지하는 에리트로 포이 에틴 (EPO) 이외의 다른 매체를 포함하는 성장 인자에서 배양 하였다표현. 세포는 외인성 유전 물질이 이미 발현 동안 세포 분화 및 증식을 유도하기 위하여 12 시간 후에 EPO 배지로 변경 하였다. 이 프로토콜은 단말 적혈구 생성의 초기 단계에서 유전자의 기능 분석을 용이하게한다. 후기 단말 적혈구 생성을 연구하기 위해, 세포를 형질 도입 후 즉시 EPO 배지에서 배양 하였다. 형질의 유전 물질이 발현 될 때 이러한 방법으로, 세포는 이미 단말기 적혈구의 말기로 분화되었다. 우리는 터미널 적혈구의 다른 단계에서 세부적인 유전자 기능을 이해하는 데 도움이 될 것이다이 전략의 일반적인 응용 프로그램을 권장합니다.

그림 1은 실험적인 전략을 설명합니다. 프로토콜은 단말 적혈구 생성의 초기와 후기 단계에서 시그널링 분자의 기능을 대상으로 두 가지 독립적 인 상태로 구성된다. TER119 부정적인 태아 간 적혈구 모세포는 E13.5 마우스의 태아에서 정제 하였다. 태아 간 적혈구 세포 전과 정제 후의 유세포 분석은 정제 (도 2a 및도 2b) 효율적임을 보여 주었다. 적혈구 단말 (도 1의</st...

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

우리는 터미널 적혈구의 초기와 후기 단계를 해부 체외 마우스 태아 간 erythroblast 문화 시스템을 제시한다. 이 시스템은 서로 다른 개발 단계에서 특정 유전자의 기능 분석을 용이하게한다.

마우스 태아 간 문화 시스템은 터미널 적혈구의 초기와 후기 단계에서 대상 유전자의 기능을 해부하기

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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A Hierarchical Stem-Cell System: Blood Cell Formation

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14.9K 조회수.  Northwestern University.  우리는 터미널 적혈구의 초기와 후기 단계를 해부 체외 마우스 태아 간 erythroblast 문화 시스템을 제시한다. 이 시스템은 서로 다른 개발 단계에서 특정 유전자의 기능 분석을 용이하게한다. 

비디오: Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

쥐 배아 Hemogenic 내피 세포의 분리

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

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Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

전사 인자와 마우스 배아 섬유 아 세포를 재 프로그램하는 것은 Hemogenic 프로그램을 유도하는

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

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 ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

리니지 Reprogramming 성인 마우스 구와 Erythroid 창시자에의 직접

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

유세포분석에 의한 마우스 조직으로부터 파열 형성 단위 및 콜로니 형성 단위 적혈구 전구체의 식별 및 분리

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

CD71/TER119 흐름 cytometric 어세를 사용하여 마우스 Erythroid Progenitors의 제품 식별 및 분석

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

생물학

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral - 중재 최저 동안 예 VIVO</em인간 조혈 줄기 세포의> 적혈구 조혈

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

인간 배아 줄기 세포 인구에서 Hepatocyte 같은 전지의 강력한 생성

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

멀티 스펙트럼 이미징 유동 세포 계측법에 의한 탈핵 이벤트에 쥐의 적혈구 모세포 모집단 인리 치드의 확인

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

마우스 간에서 세포 유형 별 유전자 발현 프로파일링

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...