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

JoVE Journal

Biology

28.3K 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

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

등쪽 마우스 피부에서 헤어 난포 줄기 세포와 표피 각질 형성 세포를 분리

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

연구

발생학

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

인간의 모공에서 신경 크레스트 줄기 세포의 분리와 문화

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

신경과학

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

생성 및 기본 인간 Keratinocytes 성인 피부에서의 경작

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

성인 피부 조직에서 기본 인간의 Keratinocytes를 분리 하는 단순 하 고 효율적인 방법

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

각질 줄기 세포 가소성을 모델링하기 위해 높은 처리량 표피 스페로이드 배양 시스템 구축

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

비통합 방법을 사용하여 복부 피부에서 성인 인간 진피 섬유 아세포의 분리 및 유도 만능 줄기 세포의 생성

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

생체공학

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&#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 ...

생물학

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

인간 포피로부터의 일차 슈완 세포, 각질세포 및 섬유아세포의 분리, 배양 및 특성화

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

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 ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

성인 마우스 입천장에서 1 차 구강 각막 세포의 격리 및 문화

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...