이 작품은 NIH / NCI 부여 R01CA - 118916에 의해 투자입니다

1. 사람의 피부에서 상피 줄기 세포를 추출 <ol><li> 상피 줄기 세포를 분리의 절차를 시작하기 전에 하나는 각 medias 및 시약을 (table1 참조) 준비해야합니다. </li><li> facelift 절차 또는 펀치 생검에서 신선한 성인 인간의 두피가 수집된 후, 4 DMEM / 10 % FBS / 숙박 Dispase (4 MG / ML) ° C.에 품어 37 2-4 시간을위한 인큐베이션 ° C도 효과적입니다. 피부 조각에 침투하는 효소 수 있도록 1cm의 최대 너비가되어야합니다. </li><li> 멸균 페트리 접시에 피부를 전송, 피부 표면 근처의 머리 샤프트​​ 쥐고 단단히하고 부드럽게 당겨 피부의 각 머리를 당겨. 해부 현미경으로 자신의 형태에 따라 telogen 단계에서 모공을 선택하고, 전송 모공은 15 ML 멸균 튜브에 (그림 1A) 팽창 영역을 잘라. 뿌리의 1 / 3 상단에 상처가 머리 뿌리 &#39;팽창&#39;영역 (그림 1B)을 구하는 경우 Anagen의 모공도 사용할 수 있습니다. </li><li> 주기적으로 잡고 실온에서 15-20 분 0.05 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) (GIBCO)와 Versene (0.53 MM EDTA (에틸렌 다이아 민 테트라 초산) 4Na, GIBCO) (1:1)의 혼합물에 고립 뿌리 조각을 품어 추가 4 ML DMEM + 10 % FBS는 반응을 중지 800 rpm으로 5 분 스핀 다운합니다. 또는, 소낭 조직 조각의 explants은 단일 모공의 문화를 세포에 트립신 소화하지 않고 문화에 배치하실 수 있습니다. </li><li> 신중하게 뜨는 폐기 (세포가 손실되는 것을 방지하기 위해 ~ 0.2-0.5 ML을 저장)와 EGF없이 한 ML KCM (keratinocyte 중간)에 다시 일시 중지합니다. </li></ol> 2. 문화 차 상피 줄기 세포 <ol><li> culturing 전에 상피 줄기 세포는 3T3 - J2 세포 (피더 세포) 아래와 같이 준비되어야 C - 처리 mitomycin. 3T3 - J2 전지는 치료 전에 10% FBS과 DMEM의 양식입니다. culturing 미디어를 제거하고 PBS로 두 번 세포를 씻어, 37에서 2 시간에 대한 혈청없이 DMEM에 15μg / ML mitomycin C ° C, 5 % CO 2와 세포를 치료. mitomycin C 함유 DMEM를 제거하고 PBS로 두 번 세척, 전지는 사용할 수 있습니다. (Mitomycin은 C - 처리 3T3 - J2 전지는 -80에서 이전에 준비하고 저장할 수 있습니다 ° C.의 해동과 씨앗은 세포 culturing 기본 상피 줄기 세포 하루 전에, 37 품어 ° C, 5 % CO 2. 150,000-200,000 6 - 잘 접시의 잘 당 3T3 - J2 전지 권장합니다. </li><li> mitomycin의 단계 1.5에서 시드 고립 머리 뿌리의 줄기 세포는 37에서 하루 EGF없이 KCM의 3T3 - J2 세포를 C - 대우 다음날 EGF 함유 KCM 변경 ° C 5 % CO 2. 전지는 37 ° 재배되는 CO 2 5 %를 포함한 습한 분위기에서 C. 모든 keratinocytes는 모든 이일 KCM 포함 EGF와 함께 공급하고 14~20일를위한 성장하고 있습니다. 현미경 매일 세포를 확인합니다. 헤어 뿌리의 줄기 세포는 식민지 (그림 2A)을 형성합니다. 헤어 뿌리의 explants은 10-14일 (그림 2B) 이후 outgrowths를 형성합니다. </li></ol> 3. 통로 상피 줄기 세포 <ol><li> 한 번 PBS로 세포를 씻으십시오. Versene (RT 사전 예열) 처리하여 3T3 - J2 피더 세포를 제거합니다. 부화 문화 5 분 Versene에 걸려 다음 부드럽게 흔들 피더 세포를 대기음. </li><li> Versene 또는 PBS 한 번에 하나와 머리 뿌리의 줄기 세포를 씻으십시오. </li><li> 사전 예열 추가 (37 ° C, 중요) 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) (2X) 37에서 품어 ° 7분 이상 분리에 머리 뿌리의 줄기 세포에 필요한 경우를 위해 C. 더 이상보다 15 분 가장 적합합니다. </li><li> , 트립신을 중지 KCM W / O EGF를 추가 부드럽게, 200g에 스핀 다운, 세포를 해산하기 위해 5 분 위아래로 피펫합니다. </li><li> mitomycin C - 3T3 - J2 처리 세포 계층에 EGF, 카운트하고 다시 플레이트없이 KCM과 함께 다시 정지 세포. </li></ol> 4. 상피 줄기 세포를 불멸하게하다 차 전지, 심지어는 성인 줄기 세포는 문화에 시리얼 통과하고 개월 만에 노화에 도달. 기본 줄기 세포의 불멸화이 문제를 완화하는 효과적인 방법입니다. <ol><li> 플레이트 2 일 기본 상피 줄기 피더 계층에 통로 1 셀 (~ 350,000 셀 / 물론 6 자 접시) (3T3 - J2)과 문화를. </li><li> DMEM 10 % FBS를 포함하는으로 기본 상피 줄기 세포을 심는 후 문화 PA317 LXSN 16E6E7 세포주 하루. (이 라인은 HPV16 E6와 E7 오픈 읽기 프레임을 인코딩 amphotropic 레트로 바이러스의 LXSN16E6E7을 생산하고있는이 안정적으로 감염 많은 세포 유형을 불멸하게하다하는 데 사용할 수 있습니다.) </li><li> 2 시간 mitomycin C (3T3 - J2 전지와 동일한 프로토콜)와 PA317 LXSN 16E6E7 세포 라인을 취급. mitomycin C는 EGF와 KCM 6 일 동안 기본 상피 줄기 세포로 PA317 LXSN 16E6E7 세포주, 공동 문화를 처리 추가합니다. 모든 이일 EGF 미디어 신선한 KCM을 갱신. </li><li> 3T3 - J2를 제거하고 Versene와 PA317 LXSN 16E6E7 세포. 추가 mitomycin - C 처리 3T3 - J2 NHP 세포 (네오 마이신, hygromycin, puromycin 내성을 잘 당 ~ 200,000 개 셀) 상피 줄기 세포 수 있습니다. 전지는 추가에 대한 G418 (Gibco)의 0.2 MG / ML 아래에 선택됩니다itional 육일. EGF 미디어 매 2 일 신선한 G418 포함 KCM을 갱신. 살아 상피 줄기 세포는 줄기 세포를 영원히 것입니다. PA317 LXSN 16E6E7 셀과 함께 공동 교양되지 않은 기본 상피 줄기 세포의 잘이 G418의 선택을위한 컨트롤로 설정해야이 잘의 모든 세포가 선택 6 일 후에 G418에 의해 사망​​한다. </li></ol> 5. 대표 결과 피부 상피 줄기 세포와 영원히 상피 줄기 세포의 조기 통과가 피더 세포에 둘러싸인 작은 keratinocytes (그림 2A)로 구성된 꽉 식민지를 형성하고 있습니다. 피더 레이어없이 정의된 보조제와 함께 혈청 무료 미디어 교양 경우, 줄기 세포가 꽉 식민지를 (그들은 단일 세포와 작은 클러스터로 성장) 분산과 양식을하지 않습니다. 불후의 상피 줄기 세포가 지속적으로 통과&gt; 12 개월 동안 안정적인 표현형를 유지하지만, 주요 전지보다 성능이 더 좋은걸 식민지를 형성하는 경향이. 그들은이 불후의 세포가 암 세포의 특성을 가지고하지 않는 것이 의미 소프트 한천 assays에서 앵커리지 독립 식민지를 형성하지 않습니다. 기본 및 상피 줄기 세포를 영원히 둘 다 머리 뿌리의 줄기 세포 마커에게 cytokeratin 15 (그림 3A)를 표현하고, 표피, 헤어 대과 및 피지선 lineages (그림 3B - D)으로 구별 할 수 있습니다. <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="그림 1" /> 그림 1. 머리카락을 끌고. dispase - 치료 피부에서 따버릴 후, 모낭은 (B) 머리카락의 길이를 둘러싼 상피의 소매와 머리카락의 바닥을 둘러싼 세포의 공을 telogen 클럽 머리카락 (A), 또는 anagen의 머리카락 중으로 나타납니다. anagen의 팽창이 덜 뚜렷한 반면 telogen의 볼록한 지방은 마이크로 해부하다하는 독특한 morphologic 지역을 형성하고 있습니다. anagen 전구는 격리 및 교양 수있는 대중 교통 - 확장 전지를 나타내는 매트릭스 keratinocytes을 포함하고 있습니다. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="그림 2" /> 그림 2. 줄기 세포 문화. (A) 전지 (F 지정)의 피더 레이어와 KCM의 교양 피부 양식 꽉 상피 식민지 (E 지정)에서 상피 줄기 세포. (B) 헤어 뿌리의 explants는 상피 outgrowths을 일으키다. telogen의 팽창 지역은 (T 지정) 접시에 붙어 피더 세포와 함께 상피 세포 콜로니로 둘러싸여 있습니다. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="그림 3" /> 그림 3. 전지 차별 줄기. (A) 줄기 세포의 식민지는 cytokeratin 15 상피 마커의 표현을 유지합니다. (B) 피부 상피 줄기 세포는 같은 K6Hf 표현식에 의해 결정 머리 뿌리의 가계 따라 차별화된 수 있습니다. (C) 세포는 공기 - 액체 인터페이스에서 드 epidermalized 진피 또는 다른 매트릭스에 교양 수 있으며 cornified 계층, 세분화된 레이어와 spinous 레이어와 층상 표피를 형성합니다. (D) 줄기 세포는 오일 레드 긍정 globules과 피지선 혈통을 따라 유도하실 수 있습니다.

<ol>
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M., Wagne, J. E., Blaza, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+epidermolysis+bullosa+by+transfer+of+wild-type+bone+marrow+cells.">Amelioration of epidermolysis bullosa by transfer of wild-type bone marrow cells.</a> Blood. 113, 1167-1174 (2009).</li><li>Wagne, J. E., Ishida-Yamamot, A., McGrat, J. A., Hordinsk, M., Keen, D. R., Riddl, M. J., Osbor, M. J., Lun, T., Dola, M., Blaza, B. R., Tolar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+transplantation+for+recessive+dystrophic+epidermolysis+bullosa.">Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.</a> N Engl J Med. 363, 629-639 (2010).</li><li>Muraue, E. M., Gach, Y., Grat, I. K., Klausegge, A., Mus, W., Grube, C., Meneguzz, G., Hintne, H., Baue, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Correction+of+Type+VII+Collagen+Expression+in+Dystrophic+Epidermolysis+Bullosa.">Functional Correction of Type VII Collagen Expression in Dystrophic Epidermolysis Bullosa.</a> J Invest Dermatol. , (2010).</li><li>Y, H., Kuma, S. M., Kossenko, A. V., Show, L., X, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+with+neural+crest+characteristics+derived+from+the+bulge+region+of+cultured+human+hair+follicles.">Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.</a> J Invest Dermatol. 130, 1227-1236 (2010).</li><li>Nishimur, E. K., Grante, S. R., Fishe, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+hair+graying:+incomplete+melanocyte+stem+cell+maintenance+in+the+niche.">Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche.</a> Science. 307, 720-724 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

설명 세포 추출과 문화 방법은 놀라울 정도로 손쉬운하고 재현할 수 있습니다. 우리는 상속 피부 결함 18 환자를 포함하여 넓은 연령 범위에 걸쳐 개인의 수십에서 상피 줄기 세포 문화를 생성합니다. 조직 수확 당일에 과정을 시작하는 것이 좋습니다이지만 전지는 필요한 경우 야간 선적을 촉진시켜, 며칠 동안 얼음 미디어 가능한 상태로 유지됩니다. 폐기 facelift의 피부가 단세포 suspensio...

Keratinocyte 미디어 (KCM) [DMEM과 햄의 F12 (GIBCO, 3시 1분), 아데닌 (시그마, 180 ㎜) 10 % 태아 소 혈청 (GIBCO), 콜레라 독소 (ICN 0.1 NM), 페니실린 / 스트렙토 마이신 (GIBCO, 100 U / ML 각각 100 MG / ML), 하이드로코티손 (시그마, 0.4 MG / ML, 1.1 ㎜), T/T3 (트랜스페린, GIBCO, 5 μg / ML, 649 NM 및 triiodo - L - thyronine, 시그마, 2 NM) , 인슐린 (시그마, 5 MG / ML, 862 NM), 그리고 EGF (시그마, 10 NG / ML, 1.6 NM), 산도 7.2]

isolation and culture of adult epithelial stem cells from human skin

모든 자기 갱신 조직의 항상성은 성인 줄기 세포에 따라 좌우됩니다. undifferentiated 줄기 세포는 비대칭 분열을 받다, 그들은 줄기 세포 표현형, 그리고 줄기 세포 틈새에서 마이 그 레이션 급속한 확산을 받다하고 말기 조직을 repopulate하기 위해 차별화 교통 - 확장 세포를 (TA 세포) 유지 딸 세포를 생성합니다. 상피 줄기 세포는 체외 proliferative 가능성이 높은과 같은 느린 - 자전거 생체내 1-3에서 세포를 레이블 - 유지와 세포로 표피, 머리 뿌리하고, 소장에서 발견되었습니다. 성인, 조직 고유의 줄기 세포들이 정상적인 physiologic 매출 동안뿐만 아니라 스트레스의 4-5 시간 동안 거주하고있는 조직의 재생에 대한 책임이 있습니다. 또한, 줄기 세포는 일반적으로 조직 내에서 여러 셀에 6 타입 야기할 수있는 능력을 보유하고 다중 강력한 것으로 간주됩니다. 예를 들어, 쥐 머리 뿌리의 줄기 세포는 표피, 피지선 땀샘과 모낭 7-9를 생성할 수 있습니다. 우리는 인간의 머리 뿌리의 팽창 지역에서 줄기 세포가 여러 가능성 10 전시 것으로 나타났습니다. 줄기 세포가 발달 생물학, 분화, tumorigenesis과 가능한 치료 유틸리티에 대한 공부를 위해 체외 시스템에 해당하는 유틸리티로 인해 생명 의학 연구에 가치있는 도구가되었습니다. 그것은 성인 상피 줄기 세포는 같은 ectodermal dysplasias, monilethrix, 네더턴 증후군, Menkes 질환, 유전 epidermolysis bullosa 및 alopecias 11-13으로 질병의 치료에 유용하게 사용될 가능성이 높습니다. 또한, 같은 화상 상처, 만성 상처와 궤양 등의 피부 문제 요법 14,15 관련 줄기 세포에서 도움이 될 것입니다. pluripotent 상태 (IPS 세포) 16,17로 성인 세포의 프로그래밍에 대한 잠재력을 감안할 때, 인간의 피부에 쉽게 접근 및 확장 성인 줄기 세포를 포함한 질환의 광범위한 유도 및 다운 스트림 치료를위한 세포의 중요한 원천을 제공할 수 있습니다 당뇨병과 파킨슨병.

Watch this Scientific Journal Video about Isolation and Culture of Adult Epithelial Stem Cells from Human Skin at JoVE.com

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

인간의 피부에서 가능한 성인 상피 줄기 세포를 분리의 신속하고 강력한 방법이 설명되어 있습니다. 방법은 모낭과 세포 배양에 대한 단세포 suspensions 또는 조직의 파편의 격리에 따버릴 다음 피부 콜라겐 매트릭스의 효소 소화를 활용합니다.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

isolation-and-culture-of-adult-epithelial-stem-cells-from-human-skin

Research

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Biology

28.1K 조회수.  University of Massachusetts Medical School. 인간의 피부에서 가능한 성인 상피 줄기 세포를 분리의 신속하고 강력한 방법이 설명되어 있습니다. 방법은 모낭과 세포 배양에 대한 단세포 suspensions 또는 조직의 파편의 격리에 따버릴 다음 피부 콜라겐 매트릭스의 효소 소화를 활용합니다.

비디오: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

분리 및 성인 인간 내피 콜로니 형성 전구 세포의 대규모 확장

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

연구

생물학

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

인간 췌장암의 Xenografts에서 줄기 세포의 분리

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

인간 Lipoaspirates에서 지방 유래 줄기 세포를 수동으로 분리

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

근육 위성 세포의 분리, 문화, 및 이식

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...