Name: epigenetic conversion as a safe and simple method to obtain insulin-secreting cells from adult skin fibroblasts
Uploaded: 2016-03-18T15:00:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts at JoVE.com

이 작품은 Carraresi 재단과 당뇨병 연구를위한 유럽 재단 (EFSD)에 의해 투자되었다. GP는 밀라노 대학의 후 문서의 교제에 의해 지원됩니다. 후성 유전학과 Periconception 환경과 BM1308 공유 큰 동물 모델에서 진행 비용 조치 (살람) : 저자는 비용 작업 FA1201 Epiconcept의 구성원입니다. TALB은 비용 조치 CM1406 성적인 화학 생물학 (EPICHEM)의 회원입니다.

주의 : 아래에 기재된 모든 절차를 무균 상태에서 층류 후드에서 수행되어야한다. 그들의 처리에 걸쳐 37 ° C에서 모든 문화 절차가 온도 조절 단계에서 수행하는 세포가 유지되어 있는지 확인합니다. 1. 피부 섬유 아세포의 분리 <ol><li> 문화 접시 코팅액을 제조 <ol><li> 물 (최종 농도 0.1 %) 100 ㎖에 돼지 젤라틴 0.1 g을 녹인다. 오토 클레이브와 솔루션을 소독. </li><li> 35mm 배양 접시에 무균 0.1 % 돼지 젤라틴의 1.5 ML을 추가합니다. 실온에서 그들을 유지, 코트에 2 시간을 기다립니다. 주 : 인간의 피부 생검 전완의 앞쪽 양태의 무 혈관 영역에서 국소 마취 절제하여 수거하고, 사용하기 전에 4 ℃에서 2 % 항생제 항진균제 용액이 보충 된 둘 베코 인산염 완충 식염수 (PBS)에 저장된다. </li></ol></li><li> 새로운 PBS로 세척 생검은 2 % antibi 보충귀 안티 곰팡이 솔루션입니다. </li><li> 장소 a를 100mm 페트리 접시에 조직 검사와 멸균 메스와 약 2mm 3 조각으로 잘라. </li><li> 단편 도금 직전에 도포 액의 과도한 제거. </li><li> 사전 코팅 35mm 페트리 접시에 5-6 피부 조각을 놓습니다. </li><li> 섬유 아세포 배양 배지를 준비 : 77 % 둘 베코 변형 이글 중간 (DMEM) 고 글루코스, 20 % 우 태아 혈청 (FBS), 1 % L- 글루타민 용액 및 2 % 항생제 항진균제 용액. </li><li> 5 % CO 2 37 ° C에서 각각의 조각 (조각 당 보통 100 μL), 문화 그들을 섬유 아세포 매체의 방울을 추가합니다. </li><li> 24 시간 후, 5 % CO 2에서 37 ℃에서 습윤을 유지하기 위해 조각 위에 섬유 아세포 배양 배지 500 μl를 추가한다. </li><li> 적어도 한 번 피펫마다 48 시간과 매체를 변경합니다. </li><li> 배양 6 일 후, 조심스럽게 조직 조각을 제거하고 폐기합니다. 참고 : 6 일 후, 섬유 아세포S는 조직 절편에서 성장하고 세포 단층을 형성하기 시작한다. </li><li> 매체 고침 섬유 아세포 배양액 2 ㎖를 추가 한 5 % CO2 인큐베이터에서 37 ℃에서 세포 단층 배양을 계속한다. </li></ol> 2. 섬유 아세포 문화 <ol><li> 37 ° C에서 배양 섬유 아세포 5 % CO 2에서 80 %의 합류까지 </li><li> 조직 배양 접시에서 계대, 흡인 섬유 아세포 배양 배지하십시오. PBS의 4 ㎖로 세포를 세 번 씻으 1 % 항생제 항진균제 용액으로 보충. </li><li> 트립신 EDTA 수용액 (0.5 g / 리터 돼지 트립신 및 0.2 g / L EDTA)의 얇은 층 (배양 배지 용적의 10 %)를 첨가하고, 세포 단층을 조직의 바닥으로부터 분리되기 시작할 때까지 37 ° C 부화 배양 접시에 세포를 떼어 놓다. </li><li> 트립신 작용을 중화 섬유 아세포 배지 9 부를 세포 현탁액을 희석. 원심 분리는 필요하지 않습니다. </li><li> 새로운 배양 접시에서 플레이트 세포 (와젤라틴 아웃)과 문화 5 % CO 2 배양기에서 37 ° C에서. 성장 속도에 따라, 4 : 2, 1 : 1 사이의 통로 비율을 유지한다. </li><li> 세포 (주당 보통 회) 약 80 %의 합류 통로를 도달하면. </li></ol> 성적인 변환 3. 섬유 아세포 도금 <ol><li> 세포 배양 접시에 0.26 ㎖ / 0.1 % 돼지 젤라틴 cm 2 (1.1에 설명 된대로 준비) 추가합니다. 코트에 2 시간을 기다립니다. </li><li> 섬유 아 세포를 도금하기 전에 도포 액을 초과하는 10 ~ 30 분을 제거합니다. </li><li> 배양 접시에서 섬유 아세포 배양 배지를 제거합니다. PBS로 세포를 세 번 씻으 1 % 항생제 항진균제 용액으로 보충. </li><li> 트립신 EDTA 수용액 (0.5 g / 리터 돼지 트립신 및 0.2 g / L EDTA)의 얇은 층 (배양 배지 용적의 10 %)를 첨가하고, 세포 단층을 조직의 바닥으로부터 분리되기 시작할 때까지 37 ° C 부화 배양 접시에 세포를 떼어 놓다. </li><li> fibrobl 9 부분으로 세포 현탁액을 희석AST 배지는 트립신 중화 작용. </li><li> 실온에서 현미경 카운팅 챔버를 이용하여 세포 수를 계산. / ㎠ 7.8 × 104 섬유 아세포의 세포 농도를 얻기 위해, 세포를 재현 탁하는 섬유 아세포 배양액의 필요한 부피를 계산한다. 이것은 사용 챔버의 특정 유형에 의존 할 것이다. 셀 / μL = 작은 그리드 X 90 (곱셈) × 희석 당 세포의 평균 수 </li><li> 실온에서 5 분 동안 150g로 원심 분리 세포 현탁액. 섬유 아세포 배지 이전에 계산 된 양으로 재현 탁하고 세포 상등액을 제거한다. </li><li> 0.1 % 젤라틴 미리 코팅 요리와 5 % CO 2 배양기에서 37 ° C에서 24 시간 동안 문화를 접시에 세포. </li></ol> 탈 메틸화 에이전트 5 아자 - CR을 사용하여 4. 증가 셀 소성 <ol><li> 날 0 <ol><li> DMEM 고 글루코스 메디칼 10ml에 5- 아자 CR 2.44 mg을 용해시켜 5- 아자 - CR 원액을 준비음. 여과 소독. 바로 사용하기 전에 5 아자 - CR 주식을 준비합니다. </li><li> 섬유 아세포 배양 배지 (최종 농도 1 μM) 1 ㎖에 5 아자 CR 원액 1 μl를 희석. </li><li> 세포의 가소성, 세포 도금 (소호 3.8) 후 24 시간을 늘리려면 시드 섬유 아 세포에서 배지를 제거하고 CO 2 배양기 5 %에서 37 ° C에서 18 시간 동안 1 μM 5 아자 - CR 원액과 문화를 추가 할 수 있습니다. </li></ol></li><li> 1 일 <ol><li> 표 1에 기재된 바와 같이 신선한 인간 다 능성 (HP) 배지를 준비한다. </li><li> 1 μM 5 아자 CR 함께 배양 한 결과, 5- 아자 - CR 멀리 세정되도록 PBS로 3 회 배지를 제거하고 세포를 세척한다. </li><li> 부화 -5- 아자 - CR 5 % CO 2에서 37 ℃에서 3 시간 (회복 기간)에 대한 HP 매체 처리 아세포. </li><li> 회복 기간 후, 중간 제거 PBS로 3 회 세척한다. </li><li> 5- 아자 - CR 처리의 효율을 모니터링하기 위해,이 단계 f 체크인또는 형태 학적 변화의 존재는 (결과 섹션에 설명). 세포는 섬유 아세포의 전형적인 긴 형태를 잃고 확대 핵으로, 크기가 작아지고, 원형 또는 타원형 모양을 취득. </li><li> 췌장 분화를 진행합니다. </li></ol></li></ol> 5. 췌장 분화 프로토콜 <ol><li> 일 1-6 <ol><li> 표 2에 기술 된 바와 같이 췌장 기초 배지를 준비합니다. </li><li> 원액을 티빈 준비 : 멸균의 166.6 μL에서 재조합 인간 단백질을 티빈의 5 μg의 용해. </li><li> 배양 5 아자 CR 5 % CO 2 인큐베이터에서 37 ° C에서 6일 위해 (5.1.2 참조) 1 μL / ㎖ 액 티빈 원액 보충 0.26 ㎖ / cm 췌장 기초 배지 2에서 섬유 아세포를 처리 하였다. 매체 매일을 변경합니다. </li></ol></li><li> 일 7-8 <ol><li> 레티노 ACI 50 mg의 디메틸 설폭 사이드 (DMSO) 16.6 ml에 첨가하여 레티노 산 원액을 준비디. </li><li> 췌장 기초 배지 0.26 ㎖ / cm 2 배양 세포는 1 μL / ㎖ 37 ℃에서 원액 (5.1.2 참조) 2 일간 1 μL / ㎖ 레티노 산 원액을 (5.2.1 참조) 티빈 C 보충 5 %의 CO 2. 매체 매일을 변경합니다. </li></ol></li><li> 일 9-36 <ol><li> 췌장 기초 배지 0.26 ㎖ / cm 2 배양 세포 (v / v)의 인슐린 트랜스페린 - 셀레늄 (ITS), 2 % (v / v)의 B27 0.1 % (v / v)의 재조합 인간 FGF 기본 1 %로 보충 5 % CO 2 인큐베이터에서 37 ℃에서 (bFGF의) 원액 (표 1 참조). 처음 15 일 동안 매일 매체를 변경합니다. </li><li> 이후 하루에 16에서, 배양 접시의 바닥에서 분리 할 수​​ 있습니다 집계 세포를 형성하기 때문에, 현미경, 매일 매체를 새로 고칩니다. </li></ol></li></ol>

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저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

본 논문은 조직 특이 적, 유도 프로토콜이어서 5- 아자 CR에 과도 짧은 노광, 인슐린 생산 세포에 인간의 피부 섬유 아세포의 변환을 허용하는 방법을 설명한다. 이 방법은 전사 인자 또는 마이크로 RNA의 강제 표현이나 세포가 더 불안정하고 실수 (14)하는 경향이 있습니다 안정적인 만능 상태의 취득없이, 중배엽에서 내배엽과 관련된 세포로 전환 할 수 있습니다. 첫 ?...

재생 의료의 기본적인 목적은 수리되거나 손상된 대체 할 수있는 새로운 기능성 세포의 세대, 조직 변성. 다른 하나의 세포 유형에서 그들을 변환하여, 새로운에 쉽게 사용할 성인 세포 개조하기 필요한 세포 집단 액세스가 풍부하거나 어렵지 않다 특히, 특히 매력적인 방법이다. 그러나, 성인 세포는 매우 안정하다. 그들은 성숙한 터미널 전문화에 도달하면, 그들은 안정적는 1을 유지, 자신의 선택에 점진적으로 제한을 통해 차별화 된 상태를 취득합니다. 체세포 (IPS)의 다 능성을 재 프로그래밍을 가능하게하는 프로토콜 번호가 개발되었으며 최근 년에 전사 세트 2,3- 요인의 강제 발현을 통해 달성. 또한, 셀 변환 (4) 하나를 소개, 직접 혈통의 분화에 의해 얻을 수있다 </sup는&gt; 또는 전사의 조합은 5-7 요인. 이 전략은 역 분화 상태를 통해 전환을 포함하지만, 특정 전사가 8 요인의 고 발현을 필요로하지 않습니다. 우리는 최근 시티 딘 유사체 5- 아자 시티 딘 (5 아자 - CR), 잘 특징 DNA의 메틸화 억제제의 탈 메틸화 특성 성체 세포의 짧은 노출에 기초하여 변환 프로토콜을 개발했다. 탈 메틸화 단계는 즉시 필요한 단말 표현형을 얻을 수있는 특정 분화 프로토콜 9-11 따른다. 이 방법은 다른 혈통의 세포로 성숙, 분화 세포로 변환 할 수 있으며, 바이러스 벡터의 사용 및 외인성 전사 인자의 형질을 모두 회피하는 상당한 이점을 갖는다. 안정적인 만능 상태의 취득 및 불안정성을 셀에 관련 증가 감수성도 피할 수있다. <p class=&quot;jove_content&quot;&gt; 여기에 제시되는 모든 기능 인슐린 분비 세포로 성인 인간의 피부 섬유 아세포의 전환을 할 수 있습니다 자세한 프로토콜입니다. 그러나, 본 기술은 상이한 유형의 세포에 적용된 다양한 분화 세포를 향해 경로를 다루는 경우, 긍정적 인 결과를 생성 한 것을 주목할 가치가있다. 또한, 후생 유전 학적 변환이 성공적으로 인간 및 돼지 9-13 종뿐만 아니라 개에서 사용 된 방법의 다양한 효능과 안정성을 제안 (원고 제출).

<table><tbody><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma</td><td>D5652</td><td>PBS; for cell wash and solution preparation</td></tr><tr><td>Antibiotic Antimycotic Solution</td><td>Sigma</td><td>A5955</td><td>Component of Fibroblast, HP and Pancreatic media</td></tr><tr><td>100 mm Petri dish</td><td>Sarstedt</td><td>83.3902</td><td>For Fibroblast isolation</td></tr><tr><td>Porcine Gelatin</td><td>Sigma</td><td>G1890</td><td>For dish coating</td></tr><tr><td>Water</td><td>Sigma</td><td>W3500</td><td>For solution preparation</td></tr><tr><td>35 mm Petri dishes</td><td>Sarstedt</td><td>83.39</td><td>For Fibroblast isolation</td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life Technologies</td><td>41966052</td><td>For Fibroblast culture medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10500064</td><td>FBS; Component of Fibroblast and HP media</td></tr><tr><td>L-Glutamine solution</td><td>Sigma</td><td>G7513</td><td>Component of Fibroblast, HP and Pancreatic media</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma</td><td>T3924</td><td>For Fibroblast dissociation</td></tr><tr><td>KOVA GLASSTIC SLIDE 10 WITH GRIDS</td><td>Hycor Biomedical</td><td>87144</td><td>Cell counting</td></tr><tr><td>5-Azacytidine</td><td>Sigma</td><td>A2385</td><td>5-aza-CR, for increrase cell plasticity in fibroblasts</td></tr><tr><td>Ham&#039;s F-10 Nutrient Mix</td><td>Life Technologies</td><td>31550031</td><td>For HP medium</td></tr><tr><td>DMEM, low glucose, pyruvate</td><td>Life Technologies</td><td>31885023</td><td>For HP medium</td></tr><tr><td>KnockOut Serum Replacement</td><td>Life Technologies</td><td>10828028</td><td>Component of HP medium</td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Life Technologies</td><td>11140035</td><td>Component of HP and Pancreatic Basal media</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M7522</td><td>Component of HP and Pancreatic Basal media</td></tr><tr><td>Guanosine</td><td>Sigma</td><td>G6264</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Adenosine</td><td>Sigma</td><td>A4036</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Cytidine</td><td>Sigma</td><td>C4654</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Uridine</td><td>Sigma</td><td>U3003</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Thymidine</td><td>Sigma</td><td>T1895</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Millex-GS 0,22 &amp;micro;m</td><td>Millipore</td><td>SLGS033SB</td><td>For sterilizing of solution</td></tr><tr><td>FGF-Basic (AA 1-155) Recombinant Human Protein</td><td>Life Technologies</td><td>PHG0261</td><td>bFGF; Component of HP and Pancreatic Basal medium</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3311</td><td>BSA; Component of Pancreatic Basal medium</td></tr><tr><td>DMEM/F-12</td><td>Life Technologies</td><td>11320074</td><td>For Pancreatic Basal medium</td></tr><tr><td>B-27 Supplement Minus Vitamin A</td><td>Life Technologies</td><td>12587010</td><td>Component of Pancreatic medium</td></tr><tr><td>N-2 Supplement</td><td>Life Technologies</td><td>17502048</td><td>Component of Pancreatic Basal medium</td></tr><tr><td>Activin A Recombinant Human Protein</td><td>Life Technologies</td><td>PHG9014</td><td>For Pancreatic medium</td></tr><tr><td>Retinoic Acid</td><td>Sigma</td><td>R2625</td><td>For Pancreatic medium</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td>DMSO; for Retinoic Acid stock preparation</td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Life Technologies</td><td>41400045</td><td>ITS; for Pancreatic Final medium</td></tr><tr><td>Anti-Vimentin antibody&amp;nbsp;</td><td>Abcam</td><td>ab8069</td><td>For immunocytochemical analisys. Working dilution 1:100</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride</td><td>Sigma</td><td>32670</td><td>DAPI. For immunocytochemical analisys. Working dilution&amp;nbsp; 1 &amp;micro;g/ml</td></tr><tr><td>5-Methylcytidine</td><td>Eurogentec</td><td>MMS-900P-B</td><td>For immunocytochemical analisys. Working dilution 1:500</td></tr><tr><td>Anti-C Peptide antibody&amp;nbsp;</td><td>Abcam</td><td>ab14181</td><td>For immunocytochemical analisys. Working dilution 1:100</td></tr><tr><td>Anti-PDX1 antibody&amp;nbsp;</td><td>Abcam</td><td>ab47267</td><td>For immunocytochemical analisys. Working dilution 1:500</td></tr><tr><td>Mercodia Insulin ELISA</td><td>Mercodia</td><td>10-1113-10</td><td>For insulin release detection</td></tr></tbody></table>

epigenetic conversion as a safe and simple method to obtain insulin-secreting cells from adult skin fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

피부 생검에서 차 문화의 설립 피부 생검은 작은 조각으로 잘라 젤라틴 미리 코팅 요리에 넣었다. 육일 후, 섬유 아세포는 조직 조각에서 성장하기 시작하고 세포 단층 (그림 1A)을 형성했다. 세포는 전형적인 긴 형태를 보여, 예상대로 섬유 아세포 특정 마커 멘틴 (빔, 그림 1B)의 균일 한 면역 양성을 표시. <p class="jove_content" fo:keep-together.wit...

Watch this Scientific Journal Video about Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts at JoVE.com

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

성인 피부 섬유 아 세포에서 인슐린 분비 세포를 얻을 수있는 안전하고 간단한 방법으로 성적인 변환

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

epigenetic-conversion-as-safe-simple-method-to-obtain-insulin

Research

JoVE Journal

Developmental Biology

6.7K 조회수.  Università degli Studi di Milano. Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

비디오: Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

체외 식민지 분석 실험에서 트라이 강력한 전구 세포를 특성화를위한 성인 쥐 췌장에서 분리

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

연구

발생학

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

능성의 효율적인 분화 NKX6-1에 줄기 세포<sup&gt; +</sup&gt; 췌장 전구 세포

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

인간의 태아 췌장 세포 클러스터의 분리, 문화, 및 이미징

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

의학

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

비 계, 3 차원 인슐린 표현 Pancreatoids 마우스 췌 장 창시자에 체 외에서 의 세대

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

성인 인간 섬유 아세포의 유도 및 확장 신경 전구 세포에 자신의 직접 변환

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

신경과학

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

인간 만능 줄기세포를 인슐린 생산 섬 클러스터로 분화(Differentiation of Human Pluripotent Stem Cells into Insulin-producing Islet Clusters)

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

높은 콘텐츠<em&gt; 체외</em&gt; 췌장 작은 섬의 β 세포 복제 디스커버리 플랫폼

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

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

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

성인 인간 피부 섬유 아 세포에서 유도 된 신경의 고수익 문화의 간단한 세대

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

후성유전학적 변형과 생체역학적 단서를 결합하여 포유류 다능성 세포를 생성하는 두 단계 전략

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

성인 피부 섬유 아 세포에서 인슐린 분비 세포를 얻을 수있는 안전하고 간단한 방법으로 성적인 변환

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