The work was supported in part by the Duncan and Nancy MacMillan Faculty Development Chair Endowment Fund (K.Y.K.), Busch Biomedical Research Grant (K.Y.K.) and the Rutgers Faculty Development Grant (K.Y.K.).

1. IMOP 세포의 자기 갱신을 유지 <ol><li> 문화 매체를 iMOP 준비 : DMEM / F12, 1X B27 보충제, 25 μg의 / ㎖ carbenecillin 20 NG / ㎖의 bFGF를. 멸균 시약을 사용하여 iMOP 문화 매체의 50 mL로한다. 37 ° C의 물을 욕조에 50 ML 원뿔에 DMEM / F12의 49 ml의 워밍업. <ol><li> 해동 50X B27 보충제와 37 ° C의 물을 욕조에 5 분 동안 필터 멸균 100 ㎎ / ㎖ carbenecillin 분취. 실온에서 100 μg의 / ㎖의 bFGF 나누어지는을 녹여. 추가 1 ml의 50 배 B27, 100 μg의 / ㎖의 bFGF를 10 μL와 DMEM / F12에 100 ㎎ / ㎖ carbenecillin의 12.5 μL. </li></ol></li><li> 배양 iMOP 세포 60mm 조직 배양 접시에 미디어의 3 ㎖를 사용합니다. 미디어의 볼륨을 두 배로 매일 문화에 신선한 매체를 추가합니다. 의 bFGF의 농도가 5 ng를 / ㎖ 이하로 떨어지지 않도록해야합니다. <ol><li> 아래 단계에 나열된 문화 5~7일 후 5 % CO 2 항로 세포와 37 ° C에서 문화 iMOP 세포. (15)로 전송 문화10 ML의 피펫 팁을 사용하여 ML 원뿔. </li></ol></li><li> HBSS의 50 ㎖에 0.5 M EDTA의 pH 8.0의 0.1 ml에 희석하여 HBSS에 1 mM의 EDTA를 확인합니다. 사전 따뜻한 1 mM의 EDTA는 37 ° C의 물을 욕조에 HBSS로했다. </li><li> 중력 침강 또는 5 분 200 XG에서 원심 분리하여 수확 세포. 실온에서. 10 분 - 중력 침강를 들어, 5 37 ° C 배양기에서 문화를 포함하는 15 ML 원뿔을 배치합니다. 그 시간 후, otospheres이 15 ml의 원추형의 바닥에 수집 관찰. 참고 : 과도한 원심력 세포 손상의 원인이 될 수 있습니다. </li><li> 조심스럽게 흡인 세포 펠렛을 방해하지 않고 피펫을 흡입 2 ㎖의를 사용하여 미디어를 보냈다. 펠렛을 셀에 미리 예열 1 mM의 EDTA HBSS 용액 0.5ml를 추가합니다. P1000 피펫을 사용하여 부드럽게 피펫 아래로 2-3 회. 37 ° C에서 원추형 배치 및 단일 세포로 분리를 용이하게하기 &lt;5 분 동안 배양한다. </li><li> 부드럽게 otospheres가 해리되어 있는지 확인 셀 솔루션을 소용돌이 친다. 더 OTO 경우구가 원뿔의 바닥에 침전, 세포를 분해한다. 배양 시간은 다를 수 있습니다. 참고 : EDTA와 장기간 배양 과도한 세포 사망을 초래할 것입니다. </li><li> 37 ° C에서 세포를 제거 EDTA를 중화하고 희석 미디어 2 ㎖를 추가합니다. RT에서 5 분 동안 200 XG에서 원심 분리하여 세포를 수집한다. 대기음 아웃 2 ml의 흡입 피펫을 사용하여 세포를 씻어 1X PBS 5 mL를 추가 EDTA를 희석. </li><li> 실온에서 5 분 동안 200 XG에서 세포를 스핀 다운 및 2 ml의 흡입 피펫을 사용하여 1X PBS를 대기음. 3 번 - 부드럽게 내려 2를 피펫 팅에 의해 iMOP 배양액 0.5 ㎖에 P1000 피펫을 사용하여 세포를 재현 탁. </li><li> 해당 카세트 (28)와 미세 입자 계수기를 사용하여 세포를 카운트. iMOP 세포의 합류 판 ~ 6 X10 6 셀이 포함됩니다. 미디어 (100) 및 계산을위한 카세트에 셀 솔루션의 75 UL을 추가 세포 1을 희석. iMOP의 cultu에서 6cm 접시에 플레이트 1 × 10 6 세포미디어 재 (~ 1 : 10 희석). 통로 iMOPs 모든 5-7일. 참고 : 큰 otospheres을 형성하거나 차별화 조직 배양 접시의 바닥에 부착 세포. 문화가 융합을 통해 경우 세포는 죽을 것이다. </li></ol> 2. 동결 융해 IMOP 세포 <ol><li> 해동 전에 동결 세포에 4 ° C에 대한 해결책을 평형화에 의해 합성 동결 매체를 준비합니다. </li><li> iMOP 세포의 합류 6cm 접시에서 세포를 수집합니다. 15 ML 원뿔로 - (8 × 10 6 세포 ~ 6) 10ml를 피펫 및 전송 세포를 사용합니다. </li><li> 중력 침강 또는 실온에서 5 분 200 XG에서 원심 분리하여 수확 세포. otospheres가 작은 경우, 세포가 중력 침강에 의해 해결되지 않습니다. 옵션 : 총 세포 수를 결정하는 단계 1.9을 사용하여 세포를 계산합니다. </li><li> 대기음 뒤에 느슨한 세포 펠렛을 유지하면서 피펫을 흡입 2 ㎖를 사용하여 미디어를 보냈다. </li><li> CE를 재현 탁하는 합성 동결 미디어의 0.25 ML을 추가~ 5 × 5 × 106 3 세포 / ml의 밀도로 LLS. 부드럽게 P1000 피펫으로 세포를 피펫. 팁을 여과 1 mL를 P1000 피펫을 사용하여 극저온 튜브에 세포 현탁액을 전송합니다. </li><li> 알코올 무료 냉​​동 컨테이너에 튜브를 놓고 온도에 도달 할 때까지 -80 ° C ~ 분당 온도 1 ° C를 감소 -80 ℃에서 냉동 컨테이너를 배치합니다. </li><li> 세포를 장기간 저장을 위해, 액체 질소 저장 탱크의 증기 상에 튜브를 전송. 문화에 대한 세포를 해동, -80 ℃에서 냉동 세포를 포함하는 극저온 유리 병을 평형. </li><li> 37 ° C의 물을 욕조에 미리 따뜻한 iMOP 문화 미디어. </li><li> 37 ℃의 수욕 바이알의 저부 소용돌이에 의해 신속하게 냉동 바이알을 해동. 마지막 얼음 결정이 사라지면 해동 세포에 미리 예열 iMOP 문화 미디어의 1 ML을 추가합니다. 전사 15ml의 원뿔형으로 iMOP 세포 배양 배지의 추가적인 4를 가하여 </li><li> 15 ml의 공동 스핀5 분 nical. 200 XG에서 보낸 매체를 대기음. 6cm iMOP 접시에 배양 배지 및 세포 플레이트 2 ㎖의 세포를 재현 탁. 확장을 위해 5 %, 37 ℃에서 이산화탄소를 문화를 품어. </li></ol> 3. 감각 상피에 IMOP 세포를 차별화 <ol><li> DMEM / F12, 1X B27 보충제, 25 μg의 / ㎖ carbenecillin : iMOP 감각 상피 분화 매체의 50 ㎖를 준비합니다. iMOP 감각 상피 분화 매체의 50 mL로한다. 37 ° C의 물을 욕조에 50 ML 원뿔에 DMEM / F12의 49 ml의 워밍업. 해동 50X의 B27 보충제, 5 분 동안 100 mg을 / ㎖ carbenecillin 분취. 37 ° C의 물을 욕조에. 1 ml의 50X B27와 DMEM / F12에 100 ㎎ / ㎖ carbenecillin의 12.5 μl를 추가합니다. </li><li> , 수확, 재현 탁을 해리와 카운트 세포는 1.4-1.9 단계를 반복 </li><li> 일 -3 문화 매체를 iMOP 사용에 6cm 접시에 플레이트 1 × 10 6 세포. 일 0에서 전송 문화는 15 ML 원뿔에 10 ml의 피펫을 사용하여. OTO를 수집중력 침강에 의해 구체로 단계 1.6에서 말했다. 대기음 밖으로 피펫을 흡입 2 ㎖의를 사용하여 미디어를 소비하고, 원뿔 밑면의 otospheres 둡니다. </li><li> 부드럽게 감각 상피 분화 미디어 2 ㎖에 추가 할 수 있습니다. 전송은 큰 구멍 10ml를 피펫을 사용하여 6cm 접시에 otospheres. 참고 : 거친 피펫 팅에서 기계 전단은 otospheres에서 세포를 떼어 놓다 수 있습니다. </li><li> 매일 문화에 신선한 감각 상피 분화 매체의 2 ML을 추가합니다. 필요한 경우, 세포 대체 중력 침강 및 매체에 의해 회수 할 수있다. </li><li> 15 ML 원뿔로 전송하고 otospheres 단계에서 언급 한 바와 같이 보냈다 미디어 피펫을 흡입 2 ml에 사용 1.6.Aspirate 침전물과 방해받지 otospheres을 떠날 수 있도록하여 10 일에 otospheres를 수집합니다. </li><li> 실온에서 15 분 동안 1X PBS에 4 % 포름 알데히드에서 배양하여 otospheres을 수정합니다. 포름 알데히드 용액을 제거 세척 완충액 (PBS 1X, 0.1 % 트리톤-10을 함유하는 세척 otospheres0)를 차단 완충액에서 배양 하였다 (1X PBS 10 % 정상 염소 혈청에서 1 시간 동안 0.1 % 트리톤 X-100)을 함유. <ol><li> 버퍼를 교체하고 희석 항체와 버퍼를 차단에서 샘플을 품어. 4 ° C에서 품어. 1X PBS 27 면역 염색에 0.1 % 트리톤 X-100 대상에게 otosphere를 포함 씻을 샘플. P1000 피펫을 사용하여 유리 슬라이드에 미디어를 장착에 1X PBS로 전송 otospheres 및 장소 otospheres. 샘플을 통해 coverslip에 넣고 설치 미디어가 4 ℃에서 건조 할 수 있습니다. </li></ol></li><li> 16 비트 CCD 카메라와 20 배 0.75 공기 중 또는 40X 1.3 NA 오일 침지 목적이 장착 거꾸로 현미경 설정을 사용하여 표면 형광 이미지를 획득. 블루 (377 ± 25 나노 / 447 ± 30 ㎚), 녹색 (475 ± 25 나노 / 540 ± 25 ㎚), 빨간색 (562 ± 20 나노 / 625 : 상기 (여기 및 방출) 파장을 사용하여 다른 색상 채널에서 형광을 수집 ± 20 나노) 및 적외선 (628 ± 20 나노 / 및 692 ± 20㎚). </li></ol> 4. 시금 에듀 설립 <ol><li> 일 -3에서 6cm 접시에 1 × 10 6 iMOP 세포를 접시. 삼일 후에 일 0에, 수확, 해리, 재현 탁하고 단계 1.4-1.9를 반복하여 세포를 계산합니다. </li><li> 플레이트 iMOP 배양 배지에 2.5 × 10 5 세포와 6 잘 접시의 다른 우물의 감각 상피 분화 미디어 5 X10 5 세포. </li><li> 에듀 통합 분석을 사용하여 3 일에 듀 설립하여 S 단계에서 세포의 비율을 결정 <ol><li> 배양 배지에서 1 μM 듀의 최종 농도를 얻기 위해 iMOP 배양 직접 듀 콘텐츠를 추가한다. </li><li> 2 시간 동안 iMOP 문화에 EDU을 품어 5 % CO 2와 37 ° C에서 품어. 수확, 해리와 수집 세포 단계 1.4-1.9를 반복합니다. 15 분 동안 1X PBS에 4 % 포름 알데히드에 추가합니다. 실온에서 세포를 해결하려면. 실온에서 5 분 200 XG에서 원심 분리하여 수확 세포. 포름 알데히드 용액과 properl 제거Y는 폐기하십시오. </li><li> 라벨 훽스트 (Hoechst)와 세포의 핵 및 제조 업체의 프로토콜에 따라 녹색 형광 염료 아 지드와 에듀를 통합. 참고 : 모든 세척이 1X PBS, 두 번 1X PBS로 3 % BSA, 0.1 % 트윈 20 세척 세포로 이루어집니다. </li><li> 및 antifade 시약을 사용하여 슬라이드에 마운트 세포 샘플을 통해 1.5 커버 유리를 놓습니다. 표면 형광 현미경을 사용하여 표지 된 세포의 형광 이미지를 획득. </li></ol></li></ol> 5. IMOP 유래 신경 세포를 차별화 <ol><li> 로 Neurobasal 미디어, 1X B27 보충제, 2 mM의 L 글루타민 : 신경 세포 분화 매체의 50 mL로한다. 5 분 37 ° C의 물을 욕조에 50 배 B27 200 mM의 L 글루타민의 해동 병. 로 Neurobasal 미디어의 48.5 ml의 1ml를 50X B27 0.5 ml의 200 mM의 L 글루타민을 추가합니다. </li><li> 멸균 10cm 플레이트 라운드 12mm 1.5 커버 글라스를 배치하고 멸균 된 커버를 청소 판에 70 %의 EtOH를 추가로 코트 커브 글라스. </li><li> 부드럽게 코브를 교반rslips은 에탄올에 포함되어 있는지 확인합니다. 실온에서 10 분 동안 접시를 남겨주세요. 나머지 에탄올을 씻어 살균 1X PBS로 된 커버 3 회 씻어. 1X PBS 나머지 씻어 살균 H 2 0 번 커버 슬립을 씻어. </li><li> 피펫을 흡입 2 ㎖의를 사용하여 H 2 0 대기음 된 커버가 건조 할 수 있습니다. 15 분 동안 조직 배양 후드에서 UV 광에 노출 된 커버. 즉시 사용하지 않을 경우 저장소는 무균 환경에서 커버 슬립. </li><li> 24 잘 접시의 각 웰에 한 12mm 라운드 커버 슬립을 놓습니다. 커버 슬립이 잘 바닥에 평평하게 놓여 있는지 확인 부드럽게 판을 흔들어. </li><li> 해동 2 밀리그램 / 1X PBS에서 10 μg의 / ㎖ 폴리 D 라이신 농도 실온에서 폴리 -D- 라이신 원액 mL 및 희석. 10 UG / ㎖ 폴리 D 라이신 0.25 ml의 우물에 추가합니다. 1 시간 동안 37 ° C를 인큐베이터에서 접시를 남겨주세요. </li><li> 멸균 1X PBS로 우물 3 회 반복한다. 실온에서 10 ㎎ / ㎖ 라미닌 원액 및 해동 10 μg의 / M로 희석1X PBS에서 L. 추가 10 μg의 24 멀티 잘 접시의 단일 잘으로 솔루션을 작동 / ㎖ 라미닌 0.25 ml의. 37 ° C 배양기에서 접시를 품어. 2ml의 흡인 피펫을 사용하여 용액을 라미닌 대기음. </li><li> P1000 피펫 1X PBS 1 ml의 추가 및 피펫을 흡입 2 ㎖로 흡입하여 1X PBS를 제거하여 커버 슬립을 1X PBS로 3 회 반복한다. 세포가 도금 될 준비가 될 때까지 잘 마지막 세척에서 1X PBS를 남겨주세요. 해리와 일 -3에서 6cm 접시에 단계 1.4-1.9.Plate 1 × 10 6 세포를 세포를 계산, 수확에 의해 신경 세포의 분화를 시작합니다. </li><li> 일 0, 수확, 해리와 1.4-1.9를 반복하여 세포를 계산합니다. 씨 1 × 10 5 - 잘 24 멀티 잘 접시 당 예열 신경 세포 분화 미디어 0.5 ml의에 × 10 5 iMOP 세포 1.5. 기음과 매일 문화에 미리 예열 신경 세포의 분화 미디어를 추가 할 수 있습니다. </li><li> 15 분 동안 4 % 포름 알데히드에 iMOP 유래 신경 세포를 고정합니다. 에이t RT 7 일째 제거 포름 알데히드 용액에 세척 완충액으로 iMOP 유래의 신경 세포를 세척 버퍼를 차단에 배양 하였다 (1X PBS 0.1 % 트리톤-100을 포함하는) (1X PBS 10 % 정상 염소 혈청과 0.1 %를 함유 트리톤 X-100) 1 시간 동안. <ol><li> 버퍼를 교체하고 희석 항체와 버퍼를 차단에서 샘플을 품어. 4 ° C에서 품어. 1X PBS 0.1 % 트리톤 X-100 주제 세포를 포함 씻을 샘플은 27 면역 염색합니다. 설치 미디어에 배치하기 전에 1X PBS로 커브 글라스를 씻으십시오. 설치 미디어가 단계 3.8에 나와있는 표면 형광 이미지를 획득하기 전에 4 ℃에서 건조하도록 허용합니다. </li></ol></li></ol>

The authors have nothing to disclose.

IMOP 문화 모니터링 신규 iMOP 세포주의 분화를 자기 갱신을 유지 및 촉진시키기위한 프로토콜을 설명하는 추가 도금 포맷 (표 1)을 포함한다. 일상적인 확장과 iMOP 세포의 분화에 도움이되는 몇 가지 중요한 단계가 설명되어 있습니다. 다 능성 줄기 세포 배양과 마찬가지로, iMOP 세포는 이론적으로 불명확 수명을 갖는다. 세포주가 제대로 유지되도록하...

The organs of the inner ear, the cochlea, utricle, saccule and three semicircular canals, mediate the ability to hear and balance. Within the cochlea, hair cells convert sounds into electrical signals that are relayed to the spiral ganglion neurons (SGN). The SGNs fire action potentials to propagate neural signals through the auditory circuit. Genetic mutations, ototoxic drugs and exposure to loud sounds contribute to hair cell and SGN death that result in hearing loss1-4. Once lost, these cells are not replaced. Use of iPSC and ESC to generate nascent hair cells or SGNs holds great promise for inner ear cell replacement therapies5-8. A flurry of progress has shown that pluripotent stem cells and inner ear derived progenitors can differentiate into hair cells and SGNs at various stages of maturity. Mammalian embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can be used to generate functional hair cells and SGNs9-11. Stem cells and progenitor cells derived from the mammalian inner ear have also been shown to form hair cells and neurons with properties of their in vivo cellular counterparts12-16. 
Use of iPSC or ESC-derived otic progenitors to replace lost hair cells and SGNs requires efficient differentiation. Improper differentiation or continued proliferation of engrafted stem-derived progenitors in the inner ear can exacerbate inner ear function and pose a tumorigenic risk such as teratomas formation in the inner ear17. There is a clear need for developing culture conditions and understanding differentiation of otic progenitors. One strategy in developing these methods is to recapitulate cell fate decisions made by neurosensory progenitors during inner ear development. Protocols that prevent proliferation and direct otic progenitors into hair cells or SGNs will help improve safety as well as efficacy of replacement therapies. 
During development, the inner ear begins with the thickening of surface ectoderm in a restricted region between rhombomeres 5 and 6 to become the otic placode. As the otic placode invaginates to form an otic cup, a collection of cells in the anterior region of the otic cup gives rise to the neural-sensory-competent domain (NSD), which contains precursors of hair cells and neurons of the inner ear18. Fate mapping studies from mouse, chicken and zebrafish developing inner ear suggest multiple populations of neurosensory progenitors that give rise to the sensory hair cells, surrounding supporting cells and otic neurons19-22. The high mobility group transcription factor, Sox2, has been implicated in sensory cell specification and used as a marker for inner ear progenitors23,24. Hypomorphic mutations that decrease Sox2 expression levels in the inner ear result in the loss of the hair cells, supporting cells and SGNs in the cochlea25,26. 
To study otic progenitor cells undergoing cell fate decisions, a fate restricted immortalized multipotent otic progenitor (iMOP) cell line from Sox2 expressing cochlear progenitors was previously established. iMOP cells were originally derived from embryonic E12.5-13.5 cochlea and infected with a c-Myc retrovirus27. iMOP cells can continually proliferate as colony forming cells known as otospheres and have the capacity to differentiate into hair cells, supporting cells and SGNs27. Understanding the capacity of iMOP cells to differentiate into distinct otic lineages allows application of these findings to efficiently generate iPSC or ESC-derived hair cells and SGNs. Efficient differentiation protocols will open new avenues for cell replacement therapies of inner ear diseases that are recalcitrant to conventional treatments. A crucial issue in generating otic cells by in vitro cell culture is to have differentiation markers that help determine if cells are undergoing differentiating. Cdkn1b (p27KIP) has been extensively used as an early marker for differentiation in developing inner ear, however, expression of Cdkn1b in iMOP cells and how it correlates to differentiation has not been addressed. In this study, the current culture conditions and how Cdkn1b expression correlates to other markers of iMOP differentiation are described.

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			<td height="20" style="height:20px;width:455px;">50 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td>
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</table>

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.

IMOP 세포에서 bFGF의 탈퇴 감소 확산 iMOP 세포의 증식능을 감소시키고 iMOP 세포의 분화를 개시하기 위해,는 bFGF를 배양으로부터 회수 하였다. 성장 인자 철수 증식을 감소 함을 확인하려면, 에듀 통합은 증식 분석으로 사용 하였다. (bFGF를하지 않고) iMOP 문화 미디어 (bFGF를 함유) 및 감각 상피 미디어에서 배양 otospher...

Watch this Scientific Journal Video about Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells at JoVE.com

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

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 ...

initiating-differentiation-immortalized-multipotent-otic-progenitor

Research

JoVE Journal

Developmental Biology

8.4K 조회수.  Rutgers University. 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.

비디오: Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

배아 줄기 세포에서 심장 전구 세포의 유도

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

연구

발생학

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 ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

에반스 블루 염료와 Zebrafish의 애벌레 골격 근육 무결성 분석

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

사람 골수에서 분리 Mesangiogenic 전구 세포 (MPC와)

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

기본 및 확실 한 조 혈 창시자 인간 만능 줄기 세포에서의 감독

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

인간 다 능성의 체외 분화에서 융모 세포로 줄기 세포

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

생체 외에서 기능 Cardiomyocyte-같은 세포로 인간 중간 엽 줄기 세포의 분화

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

생활 그대로 Zebrafish에 단일 셀 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

낮은 혈 청 조건 하에서 심장 조상 세포에서 내 피 분화의 유도

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

인간 유도에서 Somite 파생 상품의 체 외 발생에서 만능 줄기 세포

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...