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

Universidad de Cartagena UDEC

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

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

에피 솜 벡터를 이용하여 인간 말초 혈액 단핵 세포로부터의 통합없이 유도 다 능성 줄기 세포의 생성

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

연구

발생학

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

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

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 ...

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 ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

섬유아세포에서 신경 전구세포로의 직접 변환을 이용한 신경질환 시험관 모델링 및 성상세포로의 분화

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

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 ...

생물학

피부 생검을 사용하여 환자 유래 족세포(Patient-derived Podocyte) 생성

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

저자 스포트라이트: Generation of Patient-Derived Podocytes from Skin Biopsyes  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

피부 생검에서 환자 유래 Podocytes의 생성

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...