This study was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant (371697-2011, AT). The authors want to acknowledge the help of bariatric surgeons Drs S. Biron, F-S. Hould, S. Lebel, O. Lescelleur, P. Marceau as well as Christine Racine and Caroline Gagnon from the IUCPQ Tissue Bank. We thank Mr Jacques Cadorette from the IUCPQ&#8217;s audiovisual services for video shooting and editing.

윤리 문 :이 프로젝트는 이전에 환자 모집에 IUCPQ의 연구 윤리위원회에 의해 승인되었습니다. 이 문서 / 비디오의 목적을 위해, 우리는이 환자의 조직을 얻을 : 1) 57kg의 체질량 지수 50.7 kg / m 2, 2) 35 세 여자 환자의 BMI와 62 세 남자 환자 / 2, m. 실험은 모두 지방 구획하여 수행 할 수 있지만,이 동영상의 목적을 위해 하나의 지방 구획에 한정되어왔다. 비디오의 기술적 인 측면은 환자 1 수행하고 FABP4 면역 형광이 환자 세포로부터 분화를 수행 하였다. 1. 시료 처리 <ol><li> 복강경 비만 수술시의 대망과 피하 지방 구획에서 지방 조직을 수집하는 외과 의사에게 문의하십시오. </li><li> 빠르게 RT에서 실험실로 지방 샘플을 가져 즉시 처리합니다. </li><li> 비 멸균 분위기에서, 실험실에서 소화를 수행합니다.결국 세포 배양실에 옮기고 멸균 조건 하에서 배양한다. 오염을 방지하기 위해, 소화 이전에 증류 여과 물 KRH 버퍼를 준비하고 여과 (0.22μM 필터)에 의해 수행합니다. 플라스크 및 플레이트 제조를위한 세포 배양 후드에서 에탄올 전에 전송으로 깨끗하게 청소 튜브. </li><li> 사전 가중 요리와 기록 체중에 지방 조직을 놓습니다. 파라핀 매립하기 전에 적어도 24 시간 동안 실온에서 10 % 포르말린 버퍼 내의 각 조직 샘플 (이하 1cm 2)의 작은 조각을 고정. 면역 조직 화학 실험이 포함 된 샘플 (도시하지 않음 기술)를 사용합니다. </li><li> (- 기술 도시하지 않은 예는, 유전자 발현)이 전체 지방 조직에 대한 자세한 연구를 위해 -80 ° C에 저장하기 전에 액체 질소에 50 ml의 튜브와 플래시 동결의 다른 조각을 놓습니다. </li></ol> 2. 콜라게나 소화 <ol><li> 50 ㎖ TU의 나머지 지방 조직 조각을 배치소화합니다. </li><li> 소화 관에 시료의 g 당 콜라게나 제 (350 U / ml)로 보충 KRH-WB의 4 ML을 추가합니다. </li><li> 가위로 지방 조직을 말하다. </li><li> 45 분 배양 (최대 1 시간)에 대한 통에 37 ° C, 90 RPM의 최대, 다진 지방 조직 서스펜션을 놓습니다. </li></ol> 지방 세포 Preadipocytes 3. 정제 <ol><li> 플라스틱 비커에 메쉬 400 μm의 나일론을 통해 지방의 몇 덩어리 반투명 솔루션을 붓는다. </li><li> 핀셋, 나일론 메쉬에 셀 준비를 문질러 KRH-WB 5 ㎖로 세척한다. </li><li> 섬세 그것에 플라스틱 배관 및 배관 말단에 부착 된 60cc 주사기에 50 ㎖ 튜브에 여과 세포 현탁액을 전송합니다. </li><li> 성숙한 지방 세포 현탁액으로 세포가 부상하여 버퍼의 상단에 도달 할 수 있도록 약 10 분간 방치하자. </li><li> 천천히 60cc의 syrin를 사용하여 튜브의 하단에있는 버퍼를 대기음GE의 흡입. </li><li> KRH-WB 20 ml의 세척에 추가. 2 추가 세척을위한 단계 3.4에서 반복합니다. </li><li> 셀의 양에 따라, 5 또는 10 ㎖의 최종 부피로 지방 세포 현탁액을 가지고 수집 버퍼. 섹션 5에서 단계를 추구한다. </li><li> 원하는 경우 상기 일차 세포 배양 용 원심 60cc 주사기 (3000 RPM, RT, 5 분)으로 수집 버퍼로부터 간질 혈관 부분을 복구 (기술은 도시 생략). </li></ol> 4. 성숙한 지방 세포의 세포 수 <ol><li> 부하 계산 챔버 (혈구)에 부드럽게 흔들리고 지방 세포 현탁액의 10 μL. 4 번씩 세포 수를 수행합니다. </li><li> 격리 된 성숙한 세포의 수를 계산합니다. </li></ol> T-25 플라스크에 5 성숙한 지방 세포 탈분화 <ol><li> DMEM / F12-20 %의 혈청을 사용하여 볼륨의 3/4 25cm 두 조직 문화 플라스크를 입력합니다. </li><li> 세포 수에 따라, 플라스크 500,000 성숙한 세포를 붓는다. </li&gt; <li> 완전 배지로 50ML 튜브를 사용하여 플라스크를 채우고 가능한 많은 거품을 제거한다. </li><li> 플라스크에 환기가 안되는 캡 나사. </li><li> 오염을 방지하기 위해 배양하기 전에 에탄올로 플라스크를 청소합니다. </li><li> 세포의 흡착을 방해 할 수있는 문화 운동을 방지하기 위해 그것을 건드리지 않고 거꾸로 일주일 동안 플라스크를 품어. </li><li> 역 문화 7 일에서 플라스크를 반전하기 전에 부드럽게 갑작스러운 움직임을 방지, 플라스크를 조작하고 흡인하여 플라스크에 모든 매체를 제거합니다. </li><li> DMEM-F12-20 %의 혈청 12 ML을 추가하고 표준 기술을 가진 세포를 배양. 여과 벤트 캡 플라스크에 첨가 될 수있다. </li></ol> 6 웰 플레이트에 6 성숙한 지방 세포 탈분화 <ol><li> 6- 웰 플레이트의 각 웰의 바닥에 배치 커버 슬립 </li><li> 각 커버 슬립 위에 ½ &quot;플라스틱 부싱을 추가합니다. </li><li> 20 % 송아지 혈청 DMEM 매체의 8 ml로 우물을 입력합니다. </li><li> 각 플라스틱 부싱에 커버 슬립을 넣습니다. </li><li> 슬라이드 및 슬라이드에서 세포를 주입하는 튜브 (물론 당 50,000 세포) 사이의 피펫 팁을 삽입합니다. </li><li> 주 5 % CO 2, 37 ℃에서 표준 세포 배양 인큐베이터에서 인큐베이션 플레이트. </li><li> 각 웰에 20 % 송아지 혈청 미디어 2 ㎖를 포함하고 추구하는 문화에 부착 된 세포와 역 커버 슬립. </li><li> 세포가 면역 등 여러 가지 목적으로 탈분화를 겪고로 커버 슬립을 사용 (기술은 도시하지 않음). </li></ol>

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The authors declare no conflict of interest.

천장 배양법 성숙한 지방 세포의 탈분화 네이티브 지방 조직의 작은 샘플로부터 지방 줄기 세포를 얻기 위해 새로운 방식이다. 경험 등 (2)의 기준, 1g 조직은 25 cm-2 플라스크 판형하고 균질 Poloni 협력자 (3)에 의해 입증되었다 DFAT있는 세포 집단을 수득하기에 충분하다. 지방 세포의 탈분화는 독립적으로 연령, 성별 및 기타 특성, 어떤 기증자 세포 가능한 것 같다. DFAT는 얻?...

In vitro dedifferentiation of mature adipocytes is achieved through a technique called ceiling culture1. Because of their natural tendency to float in aqueous solutions, isolated mature adipocytes adhere to the surface of an inverted flask fully filled with culture medium. Over a few days, cells modify their spherical morphology and become fibroblast-like cells. The resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent2. Research articles on adipocyte dedifferentiation, especially on human cells, are limited. However, they have already provided interesting information regarding multipotency, cell phenotype and replicative capacity of DFAT cells2. Mature adipocytes originating from various fat compartments have been successfully dedifferentiated including those originating from human visceral and subcutaneous adipose tissues2-4. In addition to these depots, Kishimoto and collaborators sampled adipose tissue from the buccal fat pads and dedifferentiated adipocytes into DFAT cells5. Matsumoto and collaborators successfully generated subcutaneous DFAT cells from patients covering a wide range of ages, and the majority of cells had a high proliferative rate and less than 6% of senescence even after 10 passages in culture2.
DFAT cells have been successfully re-differentiated into several lineages, including adipogenic, osteogenic, chondrogenic and neurogenic lineages2,3,6. These cells express several embryonic stem cell markers such as Nanog and the four identified pluripotent factors Oct4, c-myc, Klf4 and Sox23. They also express markers specific to each of the three germ layers7. In addition, DFAT cells are similar to Bone Marrow-derived Mesenchymal Stem Cells (BM-derived MSC) based on their epigenetic signature3. Exploiting the stem cell capacity of DFAT cells, many groups have investigated their potential to treat or improve various diseases8,9. Improvements of pathologic conditions, such as infracted cardiac tissue, spinal cord injury and urethral sphincter dysfunction, have been observed with DFAT cell injections in rat models of disease10-12. 
In addition to the stem cell properties of DFAT cells, they may represent a new cellular model for adipocyte physiology studies. The 3T3-L1 cell line is often used for this purpose as these cells differentiate into adherent, lipid-storing adipocytes under adipogenic stimulation13. However, these cells originate from mouse embryo tissue13. Also, depot-specificity cannot be investigated with this model and it may not fully reflect human adipocyte physiology14. Other laboratories work with isolated adipose cells from murine fat depots, but fat distribution is not dimorphic in mice and anatomical configuration of the rodent's abdominal cavity prevents from extrapolating directly to humans15. In order to study adipocytes in the context of the physiopathology of human obesity, consideration of body fat distribution and fat depot-specific differences has become essential16. Some limitations of primary preadipocyte cultures, including cell quantities obtained from adipose tissue biopsy samples and their senescence after a few passages in culture, created the need for alternate models. Perrini and collaborators investigated depot-specificity in gene expression of DFAT cells originating from visceral and subcutaneous fat and compared them to adipose-derived stem cells (ASC) from the same fat depot. They demonstrated that differences in gene expression and function where mainly found between depots than between cell types, suggesting that DFAT cells are physiologically close to ASC from the same depot. DFAT cells may represent an interesting alternative to available models for studies on fat distribution in the pathophysiology of human obesity. Moreover, ceiling culture is a promising method to obtain adult stem cells for tissue engineering purposes. 
Here, we describe collagenase digestion, a widely-used technique to isolate mature adipocytes from the subcutaneous and/or visceral fat samples17, and the subsequent steps to perform ceiling culture and dedifferentiate these cells into multipotent, fibroblast-like cells.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bovine serum albumine</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma</td>
			<td>A4036</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A0278</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Type I collagenase</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS-004196</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, no phenol red</td>
			<td>Gibco-Life Technologies</td>
			<td>11039-021</td>
			<td>Add to medium : 20% calf serum, gentamicin (50&#181;g/ml) and fungizone (2.5&#181;g/ml)</td>
		</tr>
		<tr>
			<td>Calf Serum, iron supplemented, from formula-fed calves</td>
			<td>Sigma</td>
			<td>C8056-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 In plastic bushing</td>
			<td>Iberville</td>
			<td>2704-CP</td>
			<td>SKU:1000120918 (Home Depot)</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Linde</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin soluton, neutral buffered, 10%</td>
			<td>SIGMA</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60cc syringes</td>
			<td>BD Syringe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs-Ringer-Henseleit stock buffer (KRH)</td>
			<td></td>
			<td></td>
			<td>Prepare stock buffer as following: 25mM HEPES pH7.6, 125mM NaCl, 3.73mM KCl, 5mM CaCl2.2H2O, 2.5mM MgCl2.6H2O, 1mM K2HPO4. Adjust pH to 7.4.</td>
		</tr>
		<tr>
			<td>Krebs-Ringer-Henseleit-Working Buffer (KRH-WB)</td>
			<td></td>
			<td></td>
			<td>Add the following components freshly to KRH buffer: 4% bovine serum albumin, 5mM glucose, 0.1&#181;M adenosine, 560 &#181;M ascorbic acid</td>
		</tr>
		<tr>
			<td>KRH-WB supplemented with Type I collagenase</td>
			<td></td>
			<td></td>
			<td>Add 350U/ml of Type I collagenase</td>
		</tr>
		<tr>
			<td>T25 unvented cap tissue culture flask</td>
			<td>Sarsted or other brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>BD Falcon or other brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass 22x22</td>
			<td>Fisherbrand</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile beakers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

주요 형태 학적 변화는 탈분화 (그림 1) 동안 지방 세포를 성숙 발생합니다. 도 2에 도시 된 바와 같이, 탈분화을받은 세포는 형광 분석을위한 안티 FABP4 항체로 염색 하였다. 섬유 아세포 유사 세포의 대부분은하지 않았다 반면 라운드 형태로 세포 FABP4 단백질 발현. 탈분화 후 DFAT 세포는 여러 통로위한 표준 절차를 재배 할 수있다. 우리는 인간 피하 장간막 DFAT 세포주 대 ?...

Watch this Scientific Journal Video about Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures at JoVE.com

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

천장 문화 성숙한 지방 세포의 탈분화를 통해 인간 된 지방 줄기 세포의 생성

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

generation-human-adipose-stem-cells-through-dedifferentiation-mature

Research

JoVE Journal

Developmental Biology

11.6K 조회수.  IUCPQ Research Center. Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

비디오: Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

분리 및 인간의 농축 향상된 골 연장술에 대한 기질 세포를 지방이 유래

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

연구

발생학

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 ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

피더가없는 조건에 센다이 바이러스를 사용하여 인간 말초 T 세포로부터 유도 된 다 능성 줄기 세포의 생성

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

약화 된 인간의 진피를 사용하여 유전자 변형의 Organotypic 피부 문화의 생성

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

돼지 피하 지방이 조직에서 분리 및 지방이 유래 줄기 세포의 분화

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

인간의 흑색 종 종양 침윤 림프구에서 유도 만능 줄기 세포의 생성

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

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 ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

정의 및 간세포와 같은 세포의 확장 성 생성 인간 다 능성 줄기 세포에서

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

성숙한 슈반 세포 생성을위한 골수 유래 선조 세포의 저산소 전처리

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

꼬마 선 충 생식 핵의 분포, 단백질, 및 골격을 전산 분석

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...