Name: initiating differentiation in immortalized multipotent otic progenitor cells
Uploaded: 2016-01-02T13:00:00.000+00:00
Duration: 12 min 17 s
Description: Watch this Scientific Journal Video about Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells at JoVE.com

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.

<table><tbody><tr><td>CoolCell LX Alcohol-Free Cell Freezing Containers</td><td>BioCision</td><td>BCS-405</td><td></td></tr><tr><td>Cryogenic Vials (2 ml)&amp;nbsp;</td><td>Corning</td><td>430654</td><td></td></tr><tr><td>1.5 Thickness Glass Coverslip (Round 12 mm)</td><td>Electron Microscopy Sciences</td><td>72230-01</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11320-082</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Life Technologies</td><td>21103</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) pH 7.4</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14025-092</td><td></td></tr><tr><td>B27 Supplement (50X) Serum Free</td><td>Life Technologies</td><td>17504-044</td><td>Stored as 1 ml aliquots</td></tr><tr><td>L-Glutamine(200 mM)&amp;nbsp;</td><td>Life Technologies</td><td>25030-081</td><td>Stored as 5 ml aliquots</td></tr><tr><td>Natural Mouse Laminin</td><td>Life Technologies</td><td>23017-015</td><td>Stored as 1 mg/ml aliquots</td></tr><tr><td>Click-iT EdU Alexa Fluor 488&amp;nbsp;</td><td>Life Technologies</td><td>C10337</td><td></td></tr><tr><td>Synth-A-Freeze Cryopreservation Media</td><td>Life Technologies</td><td>A12542-01</td><td></td></tr><tr><td>Prolong Gold Antifade Mountant</td><td>Life Technologies</td><td>47743-736</td><td>Stored as 10 mg/ml 100 &amp;micro;l aliquots</td></tr><tr><td>Moxi Z Mini Automated Cell Counter</td><td>Orflo&amp;nbsp;</td><td>MXZ001</td><td></td></tr><tr><td>Moxi Z Cassette Type S</td><td>Orflo&amp;nbsp;</td><td>MXC002</td><td></td></tr><tr><td>Recombinant Murine Fibroblast Growth Factor, basic (bFGF)</td><td>Peprotech</td><td>450-33</td><td>Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P7886</td><td>Resuspended in 1X PBS and stored as 10 mg/ml 100 &amp;micro;l aliquots</td></tr><tr><td>Carbenicillin, Disodium Salt</td><td>Thermo Fisher Scientific</td><td>BP2648-1</td><td>Resuspended in 10 mM Hepes pH 7.4 and stored as 100 mg/ml aliquots</td></tr><tr><td>5 ml pipet individually wrapped paperback (200/case)</td><td>Thermo Fisher Scientific</td><td>1367811D</td><td></td></tr><tr><td>10 ml pipet individually wrapped paperback (200/case)</td><td>Thermo Fisher Scientific</td><td>1367811E</td><td></td></tr><tr><td>Tissue Culture Treated Biolite 24-Well Plate</td><td>Thermo Fisher Scientific</td><td>130188</td><td></td></tr><tr><td>Tissue Culture Treated Biolite 6-Well Plate</td><td>Thermo Fisher Scientific</td><td>130184&amp;nbsp;</td><td></td></tr><tr><td>Tissue Culture Treated 6 cm Dish</td><td>Thermo Fisher Scientific</td><td>130181&amp;nbsp;</td><td></td></tr><tr><td>EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22 &amp;mu;m</td><td>Thermo Fisher Scientific</td><td>SLGV033RS</td><td></td></tr><tr><td>TipOne filter pipet tips 0.1 - 10 &amp;mu;l elongated filter tip</td><td>USA Scientific</td><td>1120-3810</td><td></td></tr><tr><td>TipOne filter pipet tips 1 - 20 &amp;mu;l filter tip</td><td>USA Scientific</td><td>1120-1810</td><td></td></tr><tr><td>TipOne filter pipet tips 1 - 200 &amp;mu;l&amp;nbsp; filter tip</td><td>USA Scientific</td><td>1120-8810</td><td></td></tr><tr><td>TipOne filter pipet tips 101 - 1000 &amp;mu;l filter tip</td><td>USA Scientific</td><td>1126-7810</td><td></td></tr><tr><td>15 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td><td>USA Scientific</td><td>1475-0511</td><td></td></tr><tr><td>50 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td><td>USA Scientific</td><td>1500-1211</td><td></td></tr></tbody></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 ...

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Developmental Biology

8.5K 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

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

인간 태아 뇌에서 전구 - 파생 Oligodendrocyte 문화 시스템

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

연구

신경과학

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocyte 엽 성의 전구 물질로 배아 줄기 세포의 분화

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

생물학

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

인간 유도 만능 줄기 세포에서 파골세포의 분화 및 특성화

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

발생학

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

세균과 같은 세포로 신생아 마우스 피부 유래 줄기 세포의 분화<em&gt; 체외</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

뉴런-희소돌기아교세포 상호작용 모델링을 위한 만능줄기세포로부터 인간 뉴런 및 희소돌기아교세포 생성

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Source: Jeon, K. et al., <a href="https://app.jove.com/v/60571">Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.</a> J. Vis. Exp.&#160;(2020)
This video demonstrates the differentiation of human embryonic stem cells, or hESCs, directly into neural progenitor cells without using feeder cells or any intermediate steps. The selective inhibition of bone morphogenetic protein or BMP and transforming growth factor-beta, or TGF-&#946;, signaling pathways leads to hESCs differentiation into neural progenitor cells, or NPCs.

인간 배아 줄기 세포를 신경 전구 세포로 분화

1. Preparation of human embryonic stem cells (hESC)-qualified Basement Membrane Matrix-coated Plates

	Slowly thaw the hESC-qualified basement membrane ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Source: Assetta, B.,et al. <a href="https://app.jove.com/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons&#39; axons.

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days)
	
		Plate ~1 million Human embryonic kidney 293T (HEK293T) ...

Co-culture and Interaction Studies

Differentiation of Neural Progenitor Cells into Neurons

Source: Mao, X., et al. <a href="https://app.jove.com/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.

신경전구세포(Neural Progenitor Cells)를 뉴런(Neuron)으로 분화(Differentiation of Neural Progenitor Cells into Neuron)

1. Differentiation from NPCs to neurons (Phase II)

	Seed about 5 x 105 mouse embryonic stem cells or mESC derivatives within 2 mL of basal ...

Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

Source: Azadeh, J. et al., <a href="https://app.jove.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a>&#160;J. Vis. Exp. (2016).
This video demonstrates the differentiation of immortalized multipotent otic progenitor (iMOP) cells into sensory epithelial cells. It outlines the steps involved in culturing iMOP cells, forming otospheres, inducing differentiation, and visualizing cellular components and differentiation markers through fluorescence microscopy to confirm successful cell differentiation.

이(Otic progenitor) 세포를 감각 상피 세포(sensory epithelial cell)로 분화한 후 면역염색(immunostaining)

1. Maintaining Self-renewal in IMOP Cells

	Prepare iMOP culture media: DMEM/F12, 1X B27 supplement, 25 &#181;g/ml carbenicillin and 20 ng/ml basic ...

Differentiating Immortalized Multipotent Otic Progenitors into Spiral Ganglion Neurons and Evaluating the Differentiation

Source: Azadeh, J., et al. <a href="https://app.jove.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a> J. Vis. Exp. (2016).
The video demonstrates the differentiation of immortalized multipotent otic progenitors (iMOPs) into spiral ganglion neurons (SGNs) and immunofluorescence-based confirmation of the differentiation. The iMOPs are plated onto culture substrate-coated coverslips in a neuronal differentiation medium for differentiation into SGNs. The differentiated cells are labeled with antibodies specific for neuronal differentiation markers and analyzed under a microscope to confirm differentiation.

불멸화된 다능성 귀 전구세포를 나선형 신경절 뉴런으로 분화시키고 분화 평가

1. Differentiating iMOP-Derived Neurons 

	Make 50 ml of neuronal differentiation media: Neurobasal media, 1X B27 supplement, 2 mM L-Glutamine. Thaw ...