Name: initiating differentiation in immortalized multipotent otic progenitor cells
Uploaded: 2016-01-02T13:00:00
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 ...

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 border="0" cellpadding="0" cellspacing="0" width="1075">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">CoolCell LX Alcohol-Free Cell Freezing Containers</td>
			<td style="width:196px;">BioCision</td>
			<td style="width:145px;">BCS-405</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Cryogenic Vials (2 ml)&#160;</td>
			<td style="width:196px;">Corning</td>
			<td style="width:145px;">430654</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">1.5 Thickness Glass Coverslip (Round 12 mm)</td>
			<td style="width:196px;">Electron Microscopy Sciences</td>
			<td style="width:145px;">72230-01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">DMEM/F12</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">11320-082</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Neurobasal Medium</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">21103</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Phosphate Buffered Saline (PBS) pH 7.4</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">10010-023</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">14025-092</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">B27 Supplement (50X) Serum Free</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">17504-044</td>
			<td style="width:280px;">Stored as 1 ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">L-Glutamine(200 mM)&#160;</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">25030-081</td>
			<td style="width:280px;">Stored as 5 ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Natural Mouse Laminin</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">23017-015</td>
			<td style="width:280px;">Stored as 1 mg/ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Click-iT EdU Alexa Fluor 488&#160;</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">C10337</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Synth-A-Freeze Cryopreservation Media</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">A12542-01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Prolong Gold Antifade Mountant</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">47743-736</td>
			<td style="width:280px;">Stored as 10 mg/ml 100 &#181;l aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Moxi Z Mini Automated Cell Counter</td>
			<td style="width:196px;">Orflo&#160;</td>
			<td style="width:145px;">MXZ001</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Moxi Z Cassette Type S</td>
			<td style="width:196px;">Orflo&#160;</td>
			<td style="width:145px;">MXC002</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Recombinant Murine Fibroblast Growth Factor, basic (bFGF)</td>
			<td style="width:196px;">Peprotech</td>
			<td style="width:145px;">450-33</td>
			<td style="width:280px;">Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Poly-D-Lysine</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:145px;">P7886</td>
			<td style="width:280px;">Resuspended in 1X PBS and stored as 10 mg/ml 100 &#181;l aliquots</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Carbenicillin, Disodium Salt</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">BP2648-1</td>
			<td style="width:280px;">Resuspended in 10mM Hepes pH 7.4 and stored as 100 mg/ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">5 ml pipet individually wrapped paperback (200/case)</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">10 ml pipet individually wrapped paperback (200/case)</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>1367811E</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated Biolite 24 -Well Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130188</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated Biolite 6 -Well Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130184&#160;</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated 6 cm Dish</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130181&#160;</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22&#956;m</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 0.1-10 ul elongated filter tip</td>
			<td>USA Scientific</td>
			<td>1120-3810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 1-20 ul filter tip</td>
			<td>USA Scientific</td>
			<td>1120-1810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 1-200 ul&#160; filter tip</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 101-1000 ul filter tip</td>
			<td>USA Scientific</td>
			<td>1126-7810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">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 height="20">
			<td height="20" style="height:20px;width:455px;">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 ...

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

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

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

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

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

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

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

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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

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

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 ...

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

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

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 ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

골격 근 생검 절차에서 인간 근육 선조 세포의 격리, 배양, 특성화 및 분화

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...