This research was supported by the S-Innovation project of the Japan Science and technology Agency (JST), the Graduate Program for Leaders in Life Innovation (GPLLI) of the University of Tokyo, and the Research Fellowship for Young Scientists of Japan Society for the Promotion of Science. We thank nac Image Technology Inc. for taking movies using a hi-speed camera.

1. 준비 재료 <ol><li> 10 mM의 HEPES 버퍼를 준비합니다. 실온에서 7.0으로 pH를 조정하고 0.9 %의 염화나트륨을 추가합니다. </li><li> 1.1 준비 HEPES 완충 식염수를 혼합하여 5 % 알긴산 용액 10 mM의 EDTA 솔루션을 준비합니다. 실온에서 7.0으로 pH를 조절합니다. </li><li> 121 ℃에서 20 분 동안 시약 (1.1, 1.2)를 압력솥. </li><li> DMEM 10 % ES-자격 FBS와 90 마이 토마 이신 C의 10 ㎍ / ㎖의를 포함하는 내 배양 피더 세포로 처리됩니다 2.0 × 10 6 마우스 배아 섬유 아세포 (MEF) 세포, - 1.2 계층화 젤라틴 코팅 60mm 요리를 준비합니다 - 120 분. </li><li> (ES-정규화 FBS 20 % 2- 머 캅토 에탄올, 비 필수 아미노산 및 항생제 50 μM을 함유하는 5 ㎖의 DMEM 고 글루코오스의 존재 하에서 피더 세포와 접시에 세포 (IPS) 마우스 유도 된 다 능성 줄기 유지 페니실린 G 나트륨, 황산 스트렙토 마이신 100 μg의 / ㎖의 100 단위 / ㎖ 및 250 ng를 amphoteri / ㎖CIN의 B). </li><li> 시드 4 × 10 5 만능 줄기 젤라틴 코팅 60mm 접시에 세포 및 CO 2를 37 ° C에서 그들을 품어 5 %. 배양시 배양 배지 매일을 변경합니다. 3 후 - 문화의 4 일, 37 ° C에서 0.05 % 트립신은 0.02 %의 EDTA를 포함하는 500 μL와 1 분 5 % CO 2 세포를 Trypsinize. 그 후, 배양 접시에 10 번을 두드려서 세포를 분리. </li><li> 3 분 동안 160 × g에서 원심 분리기 세포 현탁액 (세 번 1,000 μL 마이크로 피펫과 피펫 팅하여 단일 세포로 분리 마우스 IPS 세포 다음) FBS와 항생제 ES-자격의 10 % DMEM의 2.5 ML을 추가하고 상층 액을 제거하고 세포를 계산 . 셀 카운팅 후, 젤라틴 코팅 접시에 세포를 수집하고 시드 MEF 세포로부터 만능 줄기 세포를 분리하기 위해 30 분 동안 배양한다. 세포를 카운팅 한 후 (일반적으로 1 - × 106 세포 / 접시가 수집 될 수 있음), 4 × 105 IPS 세포를 시드요리. </li></ol> 알긴산 하이드로 겔 캡슐에 2 셀 캡슐화 <ol><li> 1.5 및 1.6에 기재된 방법과 동일한 방법에 의해 세포를 수집. 3 분 동안 160 × g에서 원심 분리 후, 세 번 HEPES 완충 식염수와 유도 만능 줄기 세포 펠렛을 씻는다. HEPES 완충 식염수에 세포를 다시 현탁 한 후, 별도로 노즐을 막히게 할 수있는 큰 응집체를 제거하기 위해 40 μm의 셀 스트레이너를 통해 세포 현탁액을 필터. </li><li> HEPES 완충 식염수 내에서 세포 현탁액 2 ㎖ 및 5 % Δ1g의-나트륨 용액 3 mL를 섞는다. 마지막으로 3 % Δ1g의-나트륨 용액 내 세포 현탁액 5 ml를 얻을 수있다. 셀 밀도는 바람직하게 10 개 이상 6 세포 / ml이다. </li><li> 5 ML의 주사기로 세포 현탁액을 수집 한 후, 주사기에 동축 노즐 (그림 1A, B)를 설치하고 주사기 펌프를 설정합니다. 발광 N 2 가스 유동 (저급 1L / 분 이하) 동축 노즐의 외 바늘 통해내부 노즐을 통해 세포 현탁액을 추방. </li><li> 0.5 % CaCl2를 250 ml의 용액에 방울을 모아서 10 기다려 - 60-90 rpm에서 교반하면서 겔을 20 분. </li></ol> 알긴산 캡슐 3. 치료 (선택 사항) <ol><li> 0.05 %에서 Δ1g의 - 칼슘 캡슐 부화 폴리 -L- 라이신 (PLL) 37 ° C에서 5 분 동안 5 % 용액 (/ V w) CO 2. </li><li> HEPES 완충 식염수와 캡슐을 세척 한 후, DMEM는 그 표면에 전하를 중화하기 위해 10 % FBS를 함유하는 배양에서 그들을. 코팅 캡슐 (그림 1C)로 활용. </li><li> 5 분 동안 10 mM의 EDTA를 함유하는 HEPES 완충 식염수에서 캡슐 부화. EDTA 처리 된 캡슐 빈 캡슐 (그림 1C)로 사용된다. </li></ol> 알긴산 캡슐 4. 문화 및 수집 세포 <ol><li> 37 ℃ 및 5 % CO 2에서 진탕 75 rpm에서 캡슐화 된 세포를 부화십일합니다. </li><li> 수집하고 10 분 동안 CO 2, 37 ℃에서 10 mM의 EDTA에 캡슐을 배양하고, 5 %. 3 분 1,000 × g에서 원심 분리하여 PLL을 처리하지 않고 알긴산 캡슐에서 세포를 수집합니다. </li><li> 5 분 동안 5,000 ×의 G - 알지네이트 캡슐이 PLL로 처리하는 경우, 최대 피펫 아래로 열 배 1,000에서 주사기와 원심 분리기를에 부착 된 바늘에 의해 ALG 파일-PLL 막을 휴식. 니들 직경은 캡슐의 크기 및 25 G (260 μm의) 바늘이 실험에서 사용되는보다 낮아야한다 (600 μm의) </li><li> 당신이 세포에서 mRNA의 샘플을 수집 할 경우 원심 분리 후, 깨진 Δ1g의-PLL 막으로부터 엄격하게 세포 펠렛을 수집합니다. 남은 Δ1g의-PLL 막은 가능한 mRNA의 정제를 방지한다. </li></ol>

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The authors have nothing to disclose.

<html> 캡슐화 배양 직접 현탁 배양 물과 비교 될 수있다. 현탁 배양은 캡슐화 방법보다 다 능성 줄기 세포를 대량으로 얻을 수있는 간단한 방법이다. 그러나, 현탁 배양 세포의 집계를 제어하는​​ 것은 여전히​​ 어렵습니다. 밀봉 방법에서, 세포 응집은 캡슐에 제한되며, 따라서 잘 제어 할 수있다. 이전 간행물은 큰 세포 덩어리가 자유 현탁 배양 7에 등장하는 반면 캡슐화 된 세포, 균?...

Induced pluripotent stem cells (iPS cells) are currently the source of intense research due to their role in regenerative medicine. However, huge amounts of cells are required for tissue regeneration. For instance approximately one billion pancreatic cells required for a type 1 diabetic patient1. However, conventional dish culture is only able to obtain 1 &#215; 105 cells/cm2, thus requiring 1 m2 of culture area to obtain enough stem cell-derived pancreatic cells to treat a type 1 diabetic patient. The development of a system for the mass-culture of pluripotent stem cells, such as microcarrier2 and suspension culture is therefore required for regenerative medicine. Suspension culture represents a promising method of mass culture but controlling the aggregation of cells is challenging in direct suspension cultures of human iPS cells3. Indeed, suspended cells are exposed to shear stress, which causes cell damage3 or differentiation4.
Research into hydrogel-based encapsulation has been conducted to solve problems associated with suspension culture. In hydrogel capsules, cells are protected from the flow of the medium. Previous reports have documented the use of various types of hydrogel, including agarose5, PEG6, and alginate (Alg), for cellular encapsulation. Alg-Ca hydrogel is one of the most useful hydrogels for cell encapsulation because Alg&#8722;Ca hydrogel is formed immediately after dropping alginate solution into a CaCl2 solution and is also readily digested by enzymes or chelating reagents. 
Here, we have established a stable alginate encapsulation process for iPS cells using a co-axial nozzle. By using N2 gas flow for forming droplets, it is possible to encapsulate cells into uniform capsules without the need for other reagents such as oil. In this method, the flow rate of N2 and concentration of both CaCl2 and alginate are the major operating conditions affecting the size, shape, and uniformity of capsules. This report demonstrates the optimization of these operating conditions through the use of a hi-speed camera and a microscope.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mouse embryo fibroblast</td>
			<td>Cell Biolabs</td>
			<td>SNL 76/7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse induced pluripotent stem cell</td>
			<td>RIKEN Bio resorce centre</td>
			<td>iPS-MEF-Ng-20D-17</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high-glucose</td>
			<td>GIBCO</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>ES qualified&#160; FBS</td>
			<td>GIBCO</td>
			<td>16141079</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibacterial Antibiotics</td>
			<td>GIBCO</td>
			<td>15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential Amino Acid</td>
			<td>GIBCO</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>GIBCO</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>ESGRO Leukemia Inhibitory Factor</td>
			<td>Merck Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>GIBCO</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr>
			<td>26G/16G needle</td>
			<td>Hoshiseido</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>TERUMO</td>
			<td>SS-10ESZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium&#160; Chloride</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Alginate</td>
			<td>Wako</td>
			<td>194-09955</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Wako</td>
			<td>039-00475</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (Mw=15,000-30,000)</td>
			<td>SIGMA</td>
			<td>P7890</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>DOJINDO</td>
			<td>345-01865</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylinge pump</td>
			<td>AS ONE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM IRB</td>
			<td></td>
		</tr>
		<tr>
			<td>Hispeed camera</td>
			<td>nac image technology</td>
			<td>Memrecam HX-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

alginate encapsulation of pluripotent stem cells using a co-axial nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

프로토콜 2.5에서 추방 알지네이트 솔루션은 추방 후 즉시 구형 (- H 그림 2A)를 형성한다. 현탁액을 1L / 분 이하 N 2 유량으로 배출되는 경우, 액 적의 크기가 균일 한 (도 2I)이다. N 2 흐름은 1L / 분보다 큰 경우, 액체 방울 (도 2G, 흰색 화살표로)을 분해 및 액 적의 크기가 불균일 (도 2J)를하게된다. 이 주어지면,이 방법을 사용하여 500 ...

Watch this Scientific Journal Video about Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle at JoVE.com

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

동축 노즐을 사용하여 다 능성 줄기 세포의 알긴산 캡슐화

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

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Research

JoVE Journal

Developmental Biology

12.5K 조회수.  The University of Tokyo. We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

비디오: Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

비 통합 에피 솜 플라스미드를 사용하여 냉동 버피 코트에서 유도 만능 줄기 세포의 생성

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

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Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

헤어 유래 각질 세포를 사용하여 통합없는 인간 유도 만능 줄기 세포의 생성

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

BMP 길항제 Grem2를 사용하여 다 능성 줄기 세포에서 심방 심근의 차별화

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

고도의 재현 작은 분자 기반의 차별화 프로토콜을 사용하여 인간 다 능성 줄기 세포로부터 심근 세포의 대규모 생산

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

트랜스포존 시스템을 사용하여 마우스 양수 세포로부터 다 능성 줄기 세포의 제조

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

에피 솜 벡터를 이용하여 인간 말초 혈액 단핵 세포로부터의 통합없이 유도 다 능성 줄기 세포의 생성

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

수평 한 전체적인 거치 : 피부의 두껍고, 3 차원 조직 단면도를위한 비발 한 가공 그리고 화상 진찰 의정서

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Laminin 521 매트릭스를 이용한 인간 유래 다 능성 줄기 세포의 통합 된 자유 유도

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

약물 발견에 대 한 만능 줄기 세포 유래 Hepatocyte 같은 세포를 유도 하는 인간을 사용 하 여

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

가족성 콜레스테롤 혈 증 인간의 간 공상 마우스 모델을 사용 하 여 유도 만능 줄기 세포 유래 Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...