This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C2116) and by Research Fund of Seoul St. Mary's Hospital, The Catholic University of Korea.

이 연구 프로토콜은 NIH 지침에 따라 가톨릭 대학교 (KC16EAME0162)의 기관 검토위원회의 승인을 수행 하였다. 모든 조직은 서면 통보 동의를 얻었다. 1. 원심 중력로드 및 펠렛 문화 <ol><li> 세포 배양 및 수확 <ol><li> 배양의 ASC (P2-P3, 자재 목록 참조), 37 ℃에서 10 % 소 태아 혈청 (FBS) 및 1 % 페니실린 / 스트렙토 마이신 (P / S)이 보충 된 둘 베코 변형 이글 배지 낮은 글루코즈 (DMEM-LG)에서 5 % CO 2를 포함하는 가습 인큐베이터있다. </li><li> 세포가 80 % 합류에 도달했을 때, 배지를 버리고 1X 인산 완충 생리 식염수 5 ㎖ (PBS)으로 세포를 세척 하였다. </li><li> PBS 함유 1mM의 EDTA 1 ㎖를 첨가하고, 5 % CO 2를 포함하는 가습 인큐베이터에서 37 ℃에서 2 분간 배양한다. </li><li> 에 세포를 전송, 부드럽게 문화 판을 눌러 새로운 배지 4 mL를 넣고15 ML 원뿔 튜브와 원심 분리기 실온에서 2 분 (RT) 250 × g에서 세포. </li></ol></li><li> 원심 중력 하중 <ol><li> 원심 분리 (단계 1.1.4) 후, 펠렛을 교란시키지 않고 상층 액을 제거하고, 10 % FBS와 DMEM-LG 10ml에 펠렛을 재현 탁. 혈구 세포를 사용하여 계산. </li><li> CG로드를 들어, 15 분 동안 2,400 × g에서 새로운 15 ML 원뿔 튜브와 원심 분리기에 2.5 × 10 5 세포를 전송합니다. </li></ol></li><li> 펠렛의 문화 <ol><li> 즉시 원심 분리 후, 상등액을 흡인하고 정의 연골 분화 배지 500 μL를 추가 (CDM은 고 글루코스 DMEM는 1 % FBS, 1 % ITS + 프리믹스, 100 나노 덱사메타손, 1X MEM 비 필수 아미노산 용액, 50 μg의 보충 / ㎖ L - 프롤린, 1 % 페니실린 / 스트렙토 마이신). 모든 매체 변화에 새로 제조 된 L- 아스 코르 빈산 2- 인산의 50 μg의 / ML을 추가합니다. 양성 대조군으로, 연골 유도 differentiati추가하여 원심 분리하지 않은 세포에 CDM은 10 NG / mL의 TGF-β1을 포함. </li><li> 서 위치 펠렛을 함유하는 튜브를 캡핑 느슨하게 삽입하고, 5 % CO 2를 포함하는 가습 인큐베이터에서 37 ℃에서 배양한다. </li><li> 삼주 위해 매일 매체를 변경합니다. </li></ol></li><li> Micromass 문화 <ol><li> 즉시 원심 분리 (단계 1.2.2 15 분 동안 2,400 ×의 g) 후, 뜨는을 대기음 및 CDM의 10 μL에 펠렛을 재현 탁. </li><li> micromass을 형성하기 위해, 24- 웰 플레이트의 웰의 중앙에 재현 탁하고 세포를 배치했다. </li><li> 2 시간 후, 조심스럽게 micromass을 방해하지 않도록 접시의 벽에 잘 피펫에 CDM 1 mL를 넣어. 양성 대조군으로, 10 NG / ㎖ TGF-β1을 포함하는 CDM을 첨가하여 원심 분리하지 않은 세포와 micromass 배양을 수행한다. </li><li> 5 % CO 2를 포함하는 가습 인큐베이터에서 37 ° C에서 배양 한 micromass. </li><li> 매체 EV 변경삼주에 대한 다른 일 ERY. </li></ol></li></ol> 2. 역전사 중합 효소 연쇄 반응 (RT-PCR)을 연골 세포 분화 마커 전사 상향 조절을 검출하도록 <ol><li> 14 일에, 1X PBS에서 그들을 새로운 1.5 ML의 튜브에 회전 타원체 펠렛을 전송하고 씻어 피펫을 사용합니다. </li><li> 구 아니 디늄 티오 시아 네이트 페놀 - 클로로포름 추출 방법 (9)를 사용하여 펠렛에서 총 RNA를 추출합니다. </li><li> 제조사의 프로토콜에 따라 역전사 효소를 이용하여 총 RNA로부터 2 μg의 cDNA의 합성 (1 시간 동안 42 ° C에서 부화 한 다음 5 분 동안 70 ℃에서 효소를 실활). </li><li> 연골 분화 마커 (10)에 대한 특정 프라이머로 PCR을 수행합니다. </li></ol> 3. 염색은 연골 분화 마커 단백질의 과발현을 감지하는 <ol><li> 파라핀 포함 세포 펠렛 <ol><li> 21 일에,회전 타원체 펠렛을 수확하고 1X PBS로 씻어 피펫을 사용합니다. </li><li> 24 시간 동안 4 % 파라 포름 알데히드의 침수에 의한 회전 타원체의 알약을 수정합니다. 주의 : 파라 포름 알데히드는 매우 독성이다. 눈, 피부 또는 점막과의 접촉을 피하십시오. 노출을 최소화하고 준비하는 동안 흡입을 피하십시오. 적절한 개인 보호 장비를 착용하십시오. </li><li> 정착액을 취소하고 탈 이온수 (DW)와 세포 펠렛을 씻어. </li><li> 카세트에 거즈의 한 층을 삽입하고 피펫을 사용하여 고정 된 펠렛을 전송합니다. 거즈를 접어 펠렛을 덮고 카세트 뚜껑을 닫습니다. </li><li> 펠렛을 탈수. <ol><li> RT에서 5 분 동안 70 % 에탄올 (EtOH 중)의 펠릿을 담근다. </li><li> RT에서 5 분 동안 80 % EtOH로의 알약을 담가. </li><li> RT에서 5 분 동안 95 % EtOH로의 알약을 담가. </li><li> RT에서 5 분 동안 100 %의 EtOH의 알약을 담가. 두 번 반복합니다. </li><li> 실온에서 15 분 동안 100 % 크실렌의 알약을 담가. 반복 트위어가전. </li></ol></li><li> 금형에 56 ° C에서 파라핀에 고정 된 세포 펠렛을 포함; 펠릿 전문 자동화 조직 처리 시스템을 사용하여 파라핀에 매립 될 수있다. </li><li> 회전 마이크로톰을 이용하여 파라핀 세포 펠릿으로부터 두께 5㎛의 부분을 잘라. </li><li> 50 %의 EtOH의 섹션 플로트 다음 슬라이드를 사용하여 50 ℃의 수조로 옮긴다. </li><li> 슬라이드에 떠있는 섹션을 배치합니다. </li></ol></li><li> Safranin O 및 알 시안 블루 염색 <ol><li> 파라핀 섹션 재수. <ol><li> 15 분 동안 크실렌의 슬라이드를 담가. 두 번 반복합니다. </li><li> 5 분 동안 100 % EtOH 중 슬라이드를 담근다. 두 번 반복합니다. </li><li> 5 분 동안 90 %의 EtOH에 슬라이드를 담근다. </li><li> 5 분 동안 80 %의 EtOH에 슬라이드를 담근다. </li><li> 5 분 동안 70 % EtOH로의 슬라이드를 담가하고 1X PBS에서 그들을 씻는다. </li></ol></li><li> 1 % Safranin O 솔루션 3 % 알 시안 블루 용으로 재수 섹션을 얼룩30 분. </li><li> 염색 솔루션을 취소하고 DW와 섹션을 씻어. </li><li> 1 분 동안 헤 마톡 실린 / 에오신 용액 (Counterstain과)와 섹션을 얼룩. </li><li> 염색 솔루션을 취소하고 DW와 섹션을 씻어. </li><li> 잔여 물을 제거 한 후 커버 슬립에 장착 매체의 드롭을 놓습니다. 조심스럽게 장착 매체를 포함하는 커버 슬립에 슬라이드 (아래 셀 측)을 배치합니다. </li><li> 슬라이드는 실온에서 2 ~ 3 시간 동안 건조하도록 허용합니다. </li><li> 밝은 필드 현미경 (자동화 직립 현미경, 50X 및 200X)를 사용하여 이미지를 캡쳐. </li></ol></li><li> 면역 형광 분석 <ol><li> 섹션 3.2.1에 설명 된대로 섹션을 재수. </li><li> 시트르산 나트륨 완충액에서 슬라이드 (10 mM의 시트르산 나트륨, 0.05 % 트윈 20, pH를 6.0)에 담가하고, 마이크로파를 이용하여 10 분 동안 서브 끓는 온도들을 유지한다. </li><li> 실온에서 20 분 동안 슬라이드를 냉각 한 후 수돗물로 씻어. </li><li> 3 %에서 슬라이드를 부화H 2 O 다음 15 분 동안 (1X PBS)에 2, 15 분 11 수돗물로 씻는다. </li><li> 1 시간 동안 완충액 (PBS 중 10 % 정상 염소 혈청)을 차단와 슬라이드 블록. </li><li> 블로킹 용액을 흡인하고 슬라이드 상 5 % 정상 염소 혈청으로 희석 차 항체를 적용한다. </li><li> 4 ℃에서 밤새 슬라이드를 품어. </li><li> 5 분마다 1X PBS에서 슬라이드를 세 번 씻으십시오. </li><li> 어둠 속에서 실온에서 1 시간 동안 5 % 정상 염소 혈청으로 희석하고, 형광 접합 된 이차 항체의 슬라이드를 부화. </li><li> 어둠 속에서 5 분마다 1X PBS에서 슬라이드를 세 번 씻으십시오. </li><li> 10 분 동안 DAPI (1 μg의 / mL)으로 슬라이드를 품어하고 5 분마다 1X PBS에서 그들에게 두 번 씻는다. </li><li> 잔여 물을 제거 한 후 커버 슬립에 장착 매체의 드롭을 놓습니다. 조심스럽게 설치 매체에 슬라이드 (아래 셀 측)을 배치합니다. </li><li> 슬라이드 2-3에 대한 건조하도록 허용실온에서 어둠 속에서 시간. </li><li> 형광 현미경을 사용하여 슬라이드를 시각화 (594 nm의 DAPI : 루비로드 340 nm의, 60X 및 200X 배율) 12. </li></ol></li></ol>

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N., de Crombrugghe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX9+is+a+potent+activator+of+the+chondrocyte-specific+enhancer+of+the+pro+alpha1(II)+collagen+gene.">SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.</a> Mol Cell Biol. 17 (4), 2336-2346 (1997).</li><li>Jang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adipose+tissue-derived+stromal+vascular+fraction+for+clinical+application+to+cartilage+regeneration.">Characterization of adipose tissue-derived stromal vascular fraction for clinical application to cartilage regeneration.</a> In Vitro Cell Dev Biol Anim. 51 (2), 142-150 (2015).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+regeneration+of+articular+cartilage+and+subchondral+bone+in+vivo+using+MSCs+induced+by+a+spatially+controlled+gene+delivery+system+in+bilayered+integrated+scaffolds.">Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds.</a> Biomaterials. 32 (21), 4793-4805 (2011).</li><li>Janzen, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-cell+ageing+modified+by+the+cyclin-dependent+kinase+inhibitor+p16INK4a.">Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.</a> Nature. 443 (7110), 421-426 (2006).</li><li>Muraglia, A., Cancedda, R., Quarto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+mesenchymal+progenitors+from+human+bone+marrow+differentiate+in+vitro+according+to+a+hierarchical+model.">Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model.</a> J Cell Sci. 113, 1161-1166 (2000).</li></ol>

We declare that we have no conflicts of interest associated with this work.

세포의 stemness 상태는 SOX9의 CG에 의한 과잉 발현에 매우 중요합니다. 우리의 연구에서, SOX9 표현은 아니지만 나중에-통로의 ASC에서, 초기 통로의 ASC (2-3)에서 CG에 의해 유도 될 수있다. 배양 동안의 ASC 3 통로 (16)까지 CD34 + 세포를 포함하는 것으로보고되었다. 의 ASC는 세포가 CG로 낮은 반응의 결과로 계대 CD34의 발현을 상실하는 경향이있다. 원심 중력으로, 수...

관절 연골의 결함은 자연적으로 치유되지 않습니다. 따라서, 세포 이식이 손상 연골의 수리를 위해 유망한 방법으로 제안 된 줄기. 그러나,이 방법은 줄기 세포의 충분한 수의 취득 및 연골 분화를 받아야하는 이러한 세포의 유도를 모두 요구한다. 골수 (BM)은 널리 줄기 세포의 공급원으로서 사용되었지만, BM 세포의 분리는 두 가지 단점이있다 : 침입 불충분 수율. 때문에 취득 용이성, 지방 조직에서 줄기 세포의 바람직한 소스이다. 이전의 연구는 지방 조직으로부터 줄기 세포를 분리 및 TGF-β1 (1, 2)과 같은 사이토 카인을 사용하여 이들 세포의 연골 분화를 유도하는 가능성을 입증 하였다. 이러한 방법은 효과가 있지만 비싸다. 사이토킨에 대한 저비용 대안으로서, 기계적 응력에 사용될 수있다연골 분화를 유도한다. 기계적 부하는 관절 연골 (3)의 상태를 유지하는데 중요한 역할을하고, 여러 가지 세포의 연골 세포 표현형을 유도 할 수있다. 예를 들어, 수압은 MAP 키나아제 / JNK 경로 4를 통해 활막 유래 전구 세포의 연골 세포 표현형을 유도하고, 기계 압축비는 연골 세포의 유전자 5 상승 조절하여 인간 중간 엽 줄기 세포 (MSC들)에서 연골을 유도한다. 또한, 전단 응력은 인간 중간 엽 줄기 세포의 연골 6 관련된 세포 외 기질 (ECM)의 발현에 기여한다. 원심 중력 (CG)를 원심 분리에 의해 용이하게 생성 된인가 제어 기계적 응력은 셀 7에서 차등 유전자 발현을 유도 할 수있다. 예를 들어, 폐 상피 암종 세포에서 인터루킨 (IL) -1b의 발현을 원심 분리 (8)에 의해 상향 조절된다. 티herefore, 실험적으로 유도 기계적 응력으로서, CG는 줄기 세포의 연골 세포에서 유전자 발현을 유도 할 수있다. 그러나, CG는 줄기 세포의 연골 분화를 유도 할 수 있는지 불분명. 본 연구에서는 CG가 연골 세포 유전자의 과발현의 결과로, 인간의 ASC에서, SOX9의 상향 조절, 연골의 마스터 레귤레이터를 유도 한 것으로 나타났습니다. 또, TGF-β1의 것과 연골에 CG의 효과, 성장 인자는 가장 일반적으로 줄기 세포에서 시험 관내 연골을 유도하는 데 비해.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Plasticware&lt;/strong&gt;</td></tr><tr><td>100 mm Dish</td><td>TPP</td><td>93100</td><td></td></tr><tr><td>60 mm Dish</td><td>TPP</td><td>93060</td><td></td></tr><tr><td>50 mL Cornical Tube</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>15 mL Cornical Tube</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>10 mL Disposable Pipette</td><td>Falcon</td><td>7551</td><td></td></tr><tr><td>5 mL Disposable Pipette</td><td>Falcon</td><td>7543</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;ASC Culture Media Materials&lt;/strong&gt;</td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>DMEM Low glucose</td><td>Life Technologies</td><td>11885-084</td><td>growth base media</td></tr><tr><td>Penicilin Streptomycin</td><td>Sigma Aldrich</td><td>P4333</td><td>1%</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>16000-044</td><td>10%</td></tr><tr><td>PBS/1 mM EDTA</td><td>Life Technologies</td><td>12604-039</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Chondrogenic Differentiation Media Materials&lt;/strong&gt;</td></tr><tr><td>DMEM High glucose</td><td>Life Technologies</td><td>11995</td><td>chondrogenic differentiation base media</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma Aldrich</td><td>D2915</td><td>100 nM</td></tr><tr><td>Penicilin Streptomycin</td><td>Life Technologies</td><td>P4333</td><td>1%</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>16000-044</td><td>1%</td></tr><tr><td>Ascorbate-2-phosphate</td><td>Sigma Aldrich</td><td>A8960</td><td>50 &amp;#956;g/mL</td></tr><tr><td>L-proline</td><td>Sigma Aldrich</td><td>P5607</td><td>50 &amp;#956;g/mL</td></tr><tr><td>ITS</td><td>BD</td><td>354352</td><td>1%</td></tr><tr><td>Human TGF&amp;beta;1</td><td>Peprotech</td><td>100-21</td><td>10 ng/mL</td></tr><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>18 mm Cover Glass</td><td>Superior</td><td>HSU-0111580</td><td></td></tr><tr><td>4% Paraformaldyhyde</td><td>Tech &amp;amp; Innovation</td><td>BPP-9004</td><td></td></tr><tr><td>Tween 20</td><td>BIOSESANG</td><td>T1027</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Vector Lab</td><td>SP-5050</td><td></td></tr><tr><td>Anti-Collagen II antibody</td><td>abcam&amp;nbsp;</td><td>ab34712</td><td>1:100</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td><td>Molecular Probe</td><td>A-11037</td><td>1:200</td></tr><tr><td>DAPI</td><td>Molecular Probe</td><td>D1306</td><td></td></tr><tr><td>Prolong gold antifade reagent</td><td>Invitrogen</td><td>P36934</td><td></td></tr><tr><td>Slide Glass, Coated</td><td>Hyun Il Lab-Mate</td><td>HMA-S9914</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-018</td><td></td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>366919</td><td></td></tr><tr><td>Isoprypylalcohol</td><td>Millipore</td><td>109634</td><td></td></tr><tr><td>Ethanol</td><td>Duksan</td><td>64-17-5</td><td></td></tr><tr><td>RevertAid First Strand cDNA Synthesis kit</td><td>Thermo Scientfic</td><td>K1622</td><td></td></tr><tr><td>i-Taq DNA Polymerase</td><td>iNtRON BIOTECH</td><td>25021</td><td></td></tr><tr><td>UltraPure 10x TBE Buffer</td><td>Life Technologies</td><td>15581-044</td><td></td></tr><tr><td>loading star</td><td>Dyne Bio</td><td>A750</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>9012-36-6</td><td></td></tr><tr><td>1 kb (+) DNA ladder marker</td><td>Enzynomics</td><td>DM003</td><td></td></tr><tr><td>Human adipose-derived stem cells (ASCs)</td><td>Catholic MASTER Cells</td><td></td><td></td></tr></tbody></table>

chondrogenic differentiation induction of adipose-derived stem cells by centrifugal gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

원심 중력 지방 유래 줄기 세포의 연골 세포 분화 마커의 과다 발현을 유도한다. 연골 분화를 유도하기에 적합한 원심 중력의 정도를 결정하기 위해,의 ASC는 15 분 동안 다른 CG의도 (0, 300, 600, 1,200, 2,400 XG)로 자극 하였다. 자극 후, ASC를 배양 플레이트에 다시 접종하고 24 시간 동안 배양 하였다. 도 1a에 도시 된 바?...

Watch this Scientific Journal Video about Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity at JoVE.com

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

원심 중력에 의해 지방이 유래 줄기 세포의 연골 분화 유도

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

chondrogenic-differentiation-induction-adipose-derived-stem-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.4K 조회수.  The Catholic University of Korea. Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

비디오: Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

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. ...

연구

발생학

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

인간 제대혈 및 태아 태반에서 중간 엽 기질 세포의 분리 및 특성

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Lipoaspirate에서 수확 지방 - 파생 Stromal 셀을 사용하여 임계 크기 Calvarial 결함 모델의 수리

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

의학

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

말초 혈액 유래 인간 유래 다 능성 줄기 세포에서 연골 세포 분화

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

제대혈 유래 다 능성 줄기 세포로부터 연골 형성 펠렛 형성

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

신경 문장 원래 지방 유래 줄기 세포의 격리, 문화 및 지방 유도는 periaortic 지방 조직에서

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

로컬 컨트롤 표면 점착을 고르지 Nanopatterns Dendrimer 기반: Chondrogenic 차별화 하는 방법

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

생체공학

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

인간의 대동맥 혈관 주위 지방 조상 세포의 분화 능력

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

생물학

지방 조직 유래 줄기세포 분리 및 특성 분석

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

저자 스포트라이트: Sprague Dawley Rats의 지방 조직에서 유래한 중간엽 줄기 세포의 분리 및 식별  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Sprague Dawley 쥐의 지방 조직에서 유래한 중간엽 줄기 세포의 분리 및 식별

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...