试析全新化学重编程人多能干细胞技术对类器官实验技术应用的推动作用

活病毒培养/核酸提取

动物感染

未灭活样本检测

已灭活材料操作

重组病毒

腺病毒相关病毒

(Adenovirus-associated virus, AAV)

第三类

BSL-2

ABSL-2

BSL-2

BSL-1

BSL-2

逆转录病毒科慢病毒

(Lentivirinae, Lvis)

第三类

BSL-2

ABSL-2

BSL-2

BSL-1

/

仙台病毒(Sendai virus)

第三类

BSL-2

ABSL-2

BSL-2

BSL-1

BSL-2

DMEM

(Gibco, C11965500BT)

DMEM高糖培养基【DMEM为基础培养基, 可支持多种哺乳动物细胞生长。用DMEM已成功培养出包括原代成纤维细胞、神经元、胶质细胞、HUVEC、平滑肌细胞及 HeLa、293、Cos-7 和 PC-12 等多种细胞系】

Mesenchymal Stem Cell Growth Medium 2

(Promo Cell, C-28009)

间充质干细胞生长培养基2 (MSCGM2)【用于人间充质干细胞大规模培养, 不诱导细胞分化】

KnockOut DMEM

 (Gibco, 10829018)

KnockOut D-MEM【一种基础培养基, 经优化用于未分化胚胎干细胞及诱导型多能干细胞生长】

GlutaMAX

(Gibco, 35050-061)

Gibco GlutaMAX 补充剂【是L-谷氨酰胺的替代品, 包含在多种培养基配方中。GlutaMAX补充剂减少了有毒氨气积累,改善细胞活力和生长, 可改善细胞健康状况,适用于哺乳动物细胞的贴壁和悬浮培养】

MEM Non-Essential Amino Acids Solution (NEAA)

(Gibco, 11140050)

MEM非必需氨基酸溶液【用作细胞培养基添加剂, 可以提高细胞生长和活性。】

Penicillin-Streptomycin

(Gibco, 15140-122)

青霉素-链霉素混合溶液【内含10000单位/mL青霉素和10000 μg/mL链霉素, 对革兰阳性细菌和革兰阴性细菌具有有效的联合抗菌作用】

Fetal Bovine Serum (FBS)

(Vistech, VIS93526487)

胎牛血清

Knockout Serum Replacement (KSR)

(Gibco,  10828028)

Knockout 血清替代物【适用于多个物种胚胎干细胞 (ESC) 和诱导多能干细胞 (iPSC) 进行无血清的滋养细胞依赖性培养, 可直接替代现有实验方案中的 FBS。与添加FBS 的培养基相比, 在添加 KnockOut SR 的培养基中培育时, ES和iPS细胞出现的分化显著更少, 种系传递能力应该不会受到影响】

B27 supplement

(Gibco, 17504-044)

B27添加剂【无血清添加剂，由抗氧化剂、蛋白、维生素和脂肪酸以最优比例混合而成，可支持神经细胞的培养】

AlbuMAX-II

(Gibco, 11021-045)

AlbuMAX   II 富含脂质BSA【用于细胞培养的富含脂质牛血清白蛋白】

mTeSR   Plus Medium

(STEMCELL, 100-0276)

多能干细胞培养基【专为人类胚胎干细胞（ES）和诱导多能干细胞（iPS）设计的无血清、无饲养层维持培养基】

Collagenase IV

(Gibco, 1963347)

胶原酶IV

0.25% Trypsin-EDTA

(Gibco, 25200-056)

胰蛋白酶-EDTA

Accutase

(Millipore, SCR005)

Accutase细胞解离溶液【Accutase含细胞解离的蛋白水解酶和胶原蛋白水解酶，可从标准组织培养塑料器具和粘附涂层塑料器具中常规解离细胞。用于诱导性多能干细胞(IPSC)解离或酶解成单个细胞】

ReLeSR

(STEMCELL, 05872)

干细胞集落解离液【一种无酶试剂用于将ES或iPS细胞集落解离为细胞聚集体，且无需对已发生分化的集落进行手工挑选】

Matrigel

(Corning, 354248)

Matrigel基底膜基质【富含细胞外基质蛋白，包括层粘连蛋白 (主要成分)、IV型胶原、硫酸乙酰肝素蛋白聚糖、巢蛋白和多种生长因子】

CellAdhere Laminin-521

(STEMCELL, 77004)

层粘连蛋白521【可与细胞表面的整合素、多聚糖等多种受体结合，其中整合素α6β1与Laminin高亲和力结合，是Laminin的主要细胞粘附受体，直接决定Laminin对细胞贴壁能力的支持。LN521与细胞表面受体结合激活细胞信号通路，从而产生更多功能性细胞】

PBS (Corning, 05418005)

PBS缓冲液

DMSO(Dimethyl sulfoxide)

(Sigma-Aldrich,   D2650)

二甲基亚砜【可在在细胞冷冻培养基中保护细胞免受冰晶引起的机械性损伤，常与BSA或FBS混合使用】

CHIR99021 (CHIR, C)

(WUXI   APPTEC)

糖原合成酶激酶3β抑制剂【可诱导人胚胎干细胞向内胚层分化】

616452 (6)

(WUXI APPTEC)

616452【一种转化生长因子-β抑制剂】

TTNPB (N)

(WUXI APPTEC)

TTNPB/Arotinoid Acid，即芳香维甲酸【视黄酸受体(retinoic acid receptor, RAR)是一种转录转录调节因子，控制特定基因亚群的表达，在细胞生长、分化和器官发生中起重要作用的激动剂，在小鼠肢芽细胞培养物中，可抑制软骨生成】

SAG (WUXI APPTEC)

SAG   (Smoothened Agonist)【一种细胞渗透性Smoothened (Smo)激动剂】

EPZ5676 (MCE, HY-15593)

EPZ-5676即Pinometostat【一种有效 MV4-11 增殖抑制剂，诱导协同和持久的抗增殖作用，增加分化标志物的表达和细胞凋亡】

Ruxolitinib   (Ruxo)

(Selleckchem, S1378)

Ruxolitinib即鲁索利替尼【选择性JAK1/2抑制剂，可诱导自噬并增强细胞凋亡。JAK/STAT信号通路是众多细胞因子信号转导的共同途径，广泛参与细胞增殖、分化、凋亡以及炎症等过程】

3-deazaneplanocin A (DZNep)

(WUXI APPTEC)

DZNep即3-去氮腺嘌呤A【一种有效的组蛋白甲基转移酶 (histone methyltransferase，EZH2) 抑制剂。通过抑制EZH2的表达，激活被抑制的肿瘤抑制基因，诱导细胞凋亡】

Y-27632 (WUXI APPTEC)

小分子抑制剂Y27632【Rho相关蛋白激酶小分子抑制剂，作用于Rho介导的应力纤维的形成，抑制G1-S期细胞周期进程和胞质分裂。还能通过调节上皮-间充质过渡样诱导人多能干细胞选择性地分化为间胚层谱系】

5-Azacytidine (5azaC, 5)

(Sigma-Aldrich, A2385)

5-氮杂胞苷【是DNA甲基转移酶抑制剂（DNMT抑制剂），可以嵌入DNA/RNA上并作用DNA甲基转移酶，使细胞中DNA甲基转移酶活性的缺失和DNA去甲基化】

JNK-IN-8  (J)

(Selleckchem, S4901)

JNK-IN-8【不可逆的JNK抑制剂，作用于JNK1，JNK2和JNK3激酶】

BIRB796 (BIRB)

(Selleckchem, S1574)

即Doramapimod【BIRB 796) 通常与炎症有关，是一种是一种抗炎化合物，通过抑制p38 MAPK而发挥生物活性】

SGC-CBP30 (CBP30)

(Selleckchem, S7256)

SGC-CBP30【一种有效且高度选择性的CBP/p300溴结构域抑制剂，减少Th17细胞中IL-17A的分泌，并具有抗炎作用】

Dorsomorphin (DM)

(MCE, HY-13418A)

Dorsomorphin【一种选择性ATP 竞争性的AMPK 抑制剂，可降低细胞中AMPK 磷酸化水平】

VTP50469

(Selleckchem, S8934)

VTP50469【一种高选择性Menin-MLL相互作用抑制剂，将Menin从蛋白质复合物中置换出来并抑制MLL的染色质占据基因。MLL结合的丧失导致基因表达、分化和细胞凋亡发生变化】

Valproic acid sodium salt (VPA)

(Sigma-Aldrich, P4543)

丙戊酸【抗癫痫药，可影响信号传导通路，如Wnt/β-连环蛋白和ERK通路，干扰肌醇和花生四烯酸代谢，在细胞存活、转录调控、离子稳态、信号转导和细胞骨架修饰的基因表达中发挥作用】

Tranylcypromine (Tranyl, T)

(Enzo,   BML-EI217-0005)

反苯环丙胺【一种抑制单胺氧化酶的抗抑郁药），可抑制组蛋白去甲基化】

PD0325901

(WUXI APPTEC)

即Mirdametinib【对ERK1和ERK2磷酸化抑制作用并诱导细胞凋亡】

IWP-2

(Selleckchem, S7085)

IWP-2【Wnt 加工和分泌的抑制剂，阻止关键的 Wnt 配体棕榈糖基化，在数μM范围即可抑制细胞的增殖】

SB590885

(Selleckchem, S2220)

SB590885【一种有效的Raf/VEGFR 激酶抑制剂】

L-Ascorbic acid 2-phosphate (Vc2P)

(Sigma-Aldrich, A8960)

维生素C磷酸酯【即-抗坏血酸-2-磷酸酯，可促进胶原蛋白的合成】

LiCl (Sigma-Aldrich, L4408)

氯化锂【是糖原合成酶激酶 3β的高度选择性抑制剂，通过抑制GSK-3β的活性，激活 Wnt/β-catenin 信号途径。促进大鼠骨髓间充质干细胞的增殖及向成骨细胞分化】

Nicotinamide (NAM)

 (Sigma-Aldrich, 72340)

烟酰胺【组织培养基的营养成分】

Recombinant Human FGF2

 (oriGene, TP750002)

重组人成纤维细胞生长因子2【参与骨的愈合、软骨修复、骨修复和神经再生。也是一种有丝分裂促进剂，能加速细胞增殖】

Recombinant Human Heregulinβ-1 (HRG)

(PEPROTECH,  100-03)

重组人 Heregulinβ-1【属神经调节蛋白（NRG）中I 型 NRG1，参与组织细胞的发育和成熟】

BMP4

(Stemimmune, HST-B4-0100)

骨形态发生蛋白4（BMP-4）【是转化生长因子 β 家族成员，从早期胚胎发生到胚胎发育期广泛表达，在软骨和骨骼形成、中胚层诱导、牙齿发育、肢体形成和骨折修复中起重要作用】

AKT Kinase Inhibitor (AKTi)

(MCE, HY-10249A)

蛋白激酶B（PKB或Akt）【丝氨酸/苏氨酸特异性蛋白激酶，在葡萄糖代谢，细胞凋亡，细胞增殖，转录和细胞迁移多种细胞过程中起关键作用。AKTi一种细胞渗透化合物，能选择性抑制Akt1，Akt2和Akt3的活性】

SETD2-IN-1 (SET)

(MCE, HY-136328)

EZM0414   (SETD2-IN-1)【对SETD2 甲基转移酶活性具有靶向抑制作用，具有抗增殖作用】

5-Iodotubercidin (5ITU)

(MCE,  HY-15424)

5-碘代杀结核菌素【腺苷激酶抑制剂，可通过激活磷酸化酶和糖原合成酶。也抑制 CK1、胰岛素受体酪氨酸激酶、磷酸化酶激酶】

CX4945

(MCE, HY-50855)

即Silmitasertib【选择性的CK2抑制剂，下调CK2 表达,抑制CK2介导的 PI3K/Akt/mTOR 信号通路的激活，诱导血液肿瘤细胞毒性和凋亡】

Triton X-100

 (Sigma-Aldrich, T8787)

Triton X-100

Donkey serum

(Jackson Immuno Research, 017-000-121)

驴血清

Rabbit monoclonal anti-OCT4

(Invitrogen,   MA5-14845; RRID: AB_10979606)

兔抗OCT4单克隆抗体（一抗）

Alexa Fluor 488-AffiniPure Donkey Anti-Rabbit IgG (H+L)

(Invitrogen, 711-545-152; RRID:AB_2313584)

驴抗兔IgG二抗

Cell incubator (37℃, 21% O2 and 5% CO2)

CO2培养箱

Cell incubator (37℃, hypoxia with 5% O2 and 5% CO2)

三气细胞培养箱

Water bath (37℃)

恒温水浴槽

Scissor

组织用剪刀

Centrifuge

带水平转头的离心机

Cell   counter

细胞计数器

Electric pipettor and pipette tips

电动移液器及移液器吸头

Pipettor and pipette tips

手动移液器及移液吸头

Inverted microscope

倒置相差显微镜

Inverted fluorescence microscope

倒置相差显微镜

12-well tissue culture plates

12孔组织培养板

6-well tissue culture plates

6孔组织培养板

100-mm tissue culture dish

10cm组织培养皿

15ml polystyrene conical tubes

15mL锥形离心管

50ml polypropylene conical tubes

50mL锥形离心管

Adult human adipose derived stromal cells (hADSCs) were isolated from donated adult  adipose tissue that obtained with informed written consent.

成人脂肪来源的基质细胞 (hADSC) ，是从经知情书面同意的捐赠者采集的脂肪组织分离的。

1)   The tissues (2-4 cm3) were washed twice with PBS containing 2%   penicillin-streptomycin, minced as much as possible with scissors to 1-2 mm3.

用含有 2% 青霉素-链霉素的 PBS 洗涤组织 (2-4 cm³)两次, 用剪刀尽可能切碎至 1-2 mm³大小颗粒。

2)   Dissociated with 5-10 ml 2 mg/ml collagenase IV solution in a 100-mm dish at   37℃ for 1-1.5 hour.

在 37℃的 100 mm 培养皿中用 5-10mL浓度2 mg/mL的胶原酶 IV溶液解离 1-1.5小时。

3)   10-20 ml 15% FBS-DMEM medium was added and cells were pipetted up and down   several times for dissociation.

加入 10-20 mL 15% FBS-DMEM 培养基, 用移液器吹打数次将细胞解离。

4)   The suspension was collected to two 50-ml tubes and diluted to 30-40 ml each with 15% FBS-DMEM medium, followed by shaking for 1-2 min to release cells.

将细胞悬浮液收集到两个50mL离心管中, 以 15% FBS-DMEM 培养基稀释至30-40 mL, 然后振荡处理1-2 分钟以释放细胞。

5)   The suspension was centrifuged at 400 g for 5 min and cells were resuspended   in Mesenchymal Stem Cell Growth Medium 2 (Promo Cell) after removing the   supernatant.

将细胞悬浮液400×g离心5分钟, 去除上清, 将细胞重悬于MSCG培养基 2中。

6)   Generally, 1-3 x 106 cells can be obtained from 2-4 cm3 adipose tissue and   are plated in a 100-mm dish (P0) followed by incubation in 37℃ with 5% CO2.

通常,从 2-4 cm³ 脂肪组织中可获得 1-3×10⁶ 个细胞, 接种于100mm 培养皿(作为P0代细胞),在 37℃ - 5% CO2培养箱中孵育。

7)   The next day, fresh Mesenchymal Stem Cell Growth Medium 2 was changed to   remove the non-adherent cells.

第二天, 更换新鲜的MSCG2培养基，以去除非贴壁细胞。

8)   Primary hADSCs usually become confluent in 3-5 days and were ready to passage   for reprogramming) The 0.25% Trypsin-EDTA was used to dissociate primary   hADSCs.

原代hADSCs通常在 3-5 天内融合, 并准备传代以进行重编程。0.25% 胰蛋白酶-EDTA 用于解离原代 hADSC。

9)   For hCiPSCs induction, hADSCs were seeded at a density of 1 x 104 cells per   well of a 12-well plate or 5 x 103 cells per well of a 24-well plate with 15%   FBS-DMEM medium.

对于 hCiPSC 诱导, hADSCs 以 12 孔板每孔 1×10⁴ 个细胞或24 孔板每孔5×10³ 个细胞的密度接种于15% FBS-DMEM 培养基上。

10)   For culture and expansion, hADSCs were seeded at a density of 1.5 x 106 cells   per 100-mm dish with Mesenchymal Stem Cell Growth Medium 2) We recommend to   use the primary hADSCs for the induction of CiPS cells between passages 2 to   4.

对于培养和扩增, 用MSCGM2培养基以每 100 mm培养皿1.5×10⁶个细胞的密度接种 hADSC。我们建议在第2至第4次传代之间使用原代 hADSCs 诱导 CiPS 细胞。

The   15% FBS-DMEM medium: high glucose DMEM (Gibco) supplemented with 15% Fetal   Bovine Serum (FBS) (Vistech), 1% GlutaMAX (Gibco), 1% MEM Non-Essential Amino   Acids Solution (NEAA) (Gibco), 1% Penicillin-Streptomycin.

15% FBS-DMEM 培养基组成为：高葡萄糖 DMEM, 补充有 15% 胎牛血清(FBS)、1% GlutaMAX、1% MEM 非必需氨基酸溶液 (NEAA)、1% 青霉素-链霉素。

Human   adult skin fibroblasts (hASFs) were isolated from donated adult dermis   tissues that obtained with informed written consent.

人成人皮肤成纤维细胞 (hASF) ，是从经知情书面同意的捐赠者采集的脂肪组织分离的。

1)   The tissues (0.5-1 cm2) were washed twice with PBS containing 2%   penicillin-streptomycin, minced by scissors to 0.5-1 mm2 pieces.

用含有 2% 青霉素-链霉素的 PBS 洗涤组织 (0.5-1 cm²)两次, 用剪刀切成 0.5-1 mm² 的块。

2)   The pieces were placed in the 100-mm cell culture dish and 1 drop of 15%   FBS-DMEM medium was put onto each piece of tissue.

将碎片放入 100 mm 细胞培养皿中, 并在每块组织上滴入 1 滴 15% FBS-DMEM 培养基。

3)   The pieces were incubated in 37℃ with 5% CO2 for 4-12   hours (do not allow the pieces go to dry out).

将块状物在 37 ℃和 5% CO2 中孵育 4-12 小时 (不要让块状物变干)。

4)   3-5 ml of Mesenchymal Stem Cell Growth Medium 2 was mildly added to the dish   (do not allow the pieces detached from the dish).

向培养皿中温和加入 3-5 mL MSCGM2 (不要让碎片从培养皿中脱落)。

5)   Fresh Mesenchymal Stem Cell Growth Medium 2 was replaced every 2-3 days.

新鲜MSCGM2培养基每 2-3 天更换一次。

6)   Within 4-7 days, outgrowths of fibroblasts would generate.

在 4-7 天内, 会产生成纤维细胞的生长物。

7)   The primary hASFs usually become confluent in 10-14 days and were ready to   passage for reprogramming.

原代 hASF 通常在 10-14 天内融合, 并准备好传代以进行重编程。

8)   We passaged the hASFs both for reprogramming and expansion in the same way to   hADSCs mentioned above.

以与上述hADSC相同的方式将 hASF 传代以进行重编程和扩增到。

Commercial   human adult dermal fibroblasts (Lonza-CC2511) and ADSCs (Lonza-PT5006) were   also cultured in Mesenchymal Stem Cell Growth Medium 2 and passaged both for   reprogramming and expansion in the same way to hADSCs mentioned above.

商业人成人真皮成纤维细胞 (Lonza-CC2511)和 ADSC (Lonza-PT5006)也在MSCGM2中培养, 并以与上述hADSC相同的方式传代以进行重编程和扩增。

We   recommended to use the primary isolated hADSCs (passages 2-4) for the   induction of hCiPSCs. If the primary hASFs were used, the cells passages   between 2 to 4 were recommended. For these commercially available hADSCs or   hASFs, cultivating two passages in Mesenchymal Stem Cell Growth Medium 2 will   help to improve the state of these cells and reprogramming efficiency. The   longer passaged cells can be also used for the induction of hCiPSCs, but with   quite reduced induction efficiency. Long term passaged commercial cell lines   (IMR90 and BJ, etc.) are not recommended for the induction of hCiPSCs.

建议使用原代第2-4代分离的hADSC来诱导 hCiPSC。如果使用原代 hASF,细胞传代 2 - 4 次（对于市售 hADSC 或 hASF, MSCGM2中培养两次传代将有助于改善细胞的状态和重编程效率）。较长传代的细胞也可用于诱导 hCiPSC, 但诱导效率低。故不建议将长期传代的商业细胞系 (IMR90 和BJ等)用于诱导 hCiPSC。

Stage   I induction medium:

第一阶段的诱导培养基组分

KnockOutTM   DMEM supplemented with 1% KSR, 2% B27 1% GlutaMAXTM, 1% NEAA, 1%   Penicillin-Streptomycin, 50 μg/ml Vc2p, 5mM LiCl, 1 mM NAM, 20 ng/mL BMP4 and   the small molecules CHIR999021 (5 μM), 616452 (10 μM), TTNPB (2 μM), SAG (0.5   μM), EPZ5676 (2 μM), DZNep (0.05 μM), Ruxolitinib (1 μM), VTP50469 (0.5 μM), AKT Kinase Inhibitor   (1 μM), JNKIN8 (0.2 μM), and SETD2-IN-1(0.2 μM)

补充有 1% KSR、2% B27、1% GlutaMAX、1% NEAA、1% 青霉素-链霉素、50 μg/mL Vc2p、5 mM LiCl、1 mM NAM、20 ng/mL BMP4 和小分子 CHIR999021 (5 μM)、616452 (10 μM)、TTNPB (2 μM)、SAG (0.5 μM)、EPZ5676 (2 μM)、DZNep (0.05 μM)、Ruxolitinib (1 μM)、VTP50469 (0.5 μM)、AKT 激酶抑制剂 (1 μM)、 JNKIN8 (0.2 μM)和 SETD2-IN-1 (0.2 μM)

Stage   II induction medium:

第二阶段的诱导培养基组分

KnockOutTM   DMEM supplemented with 2% B27, 1% GlutaMAXTM, 1% NEAA, 1%   Penicillin-Streptomycin, 50 μg/ml Vc2p, 200 ng/ml bFGF (Origene) and the   small molecules  CHIR99021 (5 μM), 616452   (10 μM), TTNPB (2 μM), SAG (0.5 μM), JNKIN8 (0.5 μM), EPZ5676 (2 μM), DZNep   (0.2 μM), Ruxolitinib (1 μM), BIRB796 (2 μM), SGC-CBP30 (2 μM), Dorsormorphin (0.5 μM), VTP50469 (0.5 μM), 5-Iodotubercidin (0.5   μM), 5-Azacytidine (2 μM), AKT Kinase Inhibitor   (0.2 μM), and CX-4945 (1 μM).

KnockOut   DMEM 补充有 2% B27、1% GlutaMAX 、1% NEAA、1% 青霉素-链霉素、50 μg/mL Vc2p、200 ng/mL bFGF (Origene)和小分子 CHIR99021 (5 μM)、616452 (10 μM)、TTNPB (2 μM)、SAG (0.5 μM)、JNKIN8 (0.5 μM)、EPZ5676 (2 μM)、DZNep (0.2 μM)、Ruxolitinib (1 μM)、BIRB796 (2 μM)、SGC-CBP30 (2 μM)、Dorsormorphin (0.5 μM)、 VTP50469 (0.5 μM)、5-碘结核菌素 (0.5 μM)、5-氮杂胞苷 (2 μM)、AKT 激酶抑制剂 (0.2 μM)和 CX-4945 (1 μM)。

Stage   III induction medium:

第三阶段的诱导培养基组分

KnockoutTM   DMEM supplemented with 2% B27 supplement, 1% GlutaMAX, 1% NEAA, 1%   Penicillin-Streptomycin, 5% Knockout Serum Replacement (KSR), 50 μg/ml Vc2p,   20 ng/mL Recombinant Human Heregulinβ-1 (HRG) and the small molecules   CHIR99021 (1 μM), Y-27632 (10 μM), PD0325901 (1 μM), SB590885 (0.5 μM).   During the induction process of stage III, VPA (1000 μM), Tranylcypromine (10   μM), DZNep (0.2 μM), and EPZ5676 (2 μM) were included in the medium for the   first 4 days. Subsequently, VPA (500 μM) was included in the next 4 days   while Tranylcypromine, DZNep, and EPZ5676 were removed. The stage III   induction medium without VPA, Tranylcypromine, DZNep, or EPZ5676 could be   applied for additional 2-4 days to allow the primary hCiPSC colonies to grow   larger.

补充有 2% B27 补充剂、1% GlutaMAX、1% NEAA、1% 青霉素-链霉素、5% 敲除血清替代物 (KSR)、50 μg/mL Vc2p、20 ng/mL 重组人 Heregulinβ-1 (HRG)和小分子 CHIR99021 (1 μM)、Y-27632 (10 μM)、PD0325901 (1 μM)、SB590885 (0.5 μM)的敲除 DMEM。在第三阶段的诱导过程中, 前 4 天将 VPA (1000 μM)、反苯环丙胺(10 μM)、DZNep (0.2 μM)和 EPZ5676 (2 μM)包含在培养基中。

随后, 在接下来的 4 天内加入 VPA (500 μM), 同时去除反式环丙胺、DZNep 和 EPZ5676。而不含 VPA、反式环丙胺、DZNep 或 EPZ5676 的 第三阶段诱导培养基可以再施用 2-4 天, 以使原代 hCiPSC集落更大。

Please   prepare each medium freshly and shake well after configuration and before   use. Prepared induction media can be stored at 4℃ for up to two weeks.

备注：请采用新鲜制备每种培养基, 并在配置后和使用前摇匀（制备诱导培养基可4℃储存长达两周）。

1.   hADSCs and hASFs were seeded in high-glucose DMEM supplemented with 15% FBS   at a density of 1 x 104 cells per well of a 12-well plate or 5-6 x 103 cells   per well of a 24-well plate, and would be changed into freshly prepared stage   I induction medium after 12-24h. Please ensure that the cell density is   appropriate and cells are evenly attached to the bottom of each well. An   appropriate cell density seeded at day-1 is important for the reprogramming   efficiency and induction of the epithelial-like cells in stage I. We highly   recommend to optimize the cell density seeded at day-1 when the percentage or   area of epithelial-like cells is too low at the end of stage I.

hADSCs 和 hASFs 接种在补充有 15% FBS 的高葡萄糖 DMEM 中, 密度为 12 孔板每孔 1×10⁴ 个细胞或 24 孔板每孔 5-6×10³ 个细胞, 12-24 小时后更换为新鲜制备的第一阶段诱导培养基。

确保细胞密度适宜,且细胞均匀地附着在每个孔的底部。在第 1 天接种的适当细胞密度对于第一阶段上皮样细胞的重编程效率和诱导很重要。强烈建议在第一阶段结束时上皮样细胞的百分比或面积过低时优化第 1 天接种的细胞密度。

2.   For stage I induction, single layer epithelial-like cells would emerge at day   4-6. When the epithelial-like cells reach ∼100% confluence (usually after 8-10 days’ stage I   induction for hADSCs and 9-16 days for hASFs). Change the medium into freshly   prepared stage II induction medium. Cells at the end of stage I may not be   firmly attached, so be gentle when changing the medium.

对于第一阶段诱导, 单层上皮样细胞将在第 4-6 天出现。当上皮样细胞达到近100%融合度时 (通常在 hADSC 的第一阶段诱导 8-10 天和 hASF 的第一阶段诱导 9-16 天后)，将培养基更换为新鲜制备的第二阶段诱导培养基。第一阶段结束时的细胞可能没有牢固附着, 因此更换培养基时要轻柔。

3.   For stage II induction, multi-layered cell colonies appeared after 4-6 days   treatment and these cell colonies would continually grow larger. In the last   four days of stage II, due to the strong proliferation of cells, the medium   can be changed every two days, or you can add the same volume of stage II   medium to the original well on the penultimate day. It is normal that there   is cell death in late stage II.

对于第二阶段诱导, 处理 4-6 天后出现多层细胞集落, 这些细胞集落会不断变大。在第二阶段的最后 4 天, 由于细胞增殖较强, 可以每两天更换一次培养基, 也可以在倒数第二天向原始孔中加入相同体积的第二阶段培养基（第二阶段晚期有细胞死亡是正常的）。

4.   Change the medium into freshly prepared stage III induction medium after 8-12   days’ treatment of stage II medium when the colonies grow larger. The   induction days of stage II is important for the induction efficiency of   hCiPSCs. You can switch stage II to stage III after 8, 10 or 12 days of stage   II induction, and compare efficiency.

当细胞集落变大时, 在第二阶段培养基处理 8-12 天后, 将培养基更换为新鲜制备的第三阶段诱导培养基。第二阶段的诱导天数对 hCiPSC 的诱导效率很重要。您可以在第二阶段诱导 8、10 或 12 天后将第二阶段切换到第三阶段, 并比较效率。

5.   For stage III induction, VPA (1000 μM), Tranylcypromine (10 μM), DZNep (0.2   μM), and EPZ5676 (2 μM) were included in the induction medium for the first 4   days. Subsequently, VPA (500 μM) was included in the next 4 days while   Tranylcypromine, DZNep, and EPZ5676 were removed. The stage III induction   medium without VPA, Tranylcypromine, DZNep, or EPZ5676 could be applied for   additional 2-4 days to allow the primary hCiPSC colonies to grow larger. In   stage III, it is normal that there will be a large amount of cell death.

对于第三阶段诱导, 前 4 天在诱导培养基中加入 VPA (1000 μM)、反苯环丙胺(10 μM)、DZNep (0.2 μM)和 EPZ5676 (2 μM)。随后, 在接下来的 4 天内加入 VPA (500 μM), 同时去除反式环丙胺、DZNep 和 EPZ5676。不含 VPA、反式环丙胺、DZNep 或 EPZ5676 的第三阶段诱导培养基可以再施用 2-4 天, 以使原代 hCiPSC 集落更大（这一阶段出现大量细胞死亡是正常的）。

6.   Immunofluorescent staining of OCT4 at the end of stage III is recommended and   the OCT4-positive colonies were regarded as primary hCiPSC colonies. Please   leave at least one well of living cells for establishment of hCiPS cell   lines.

建议在第三阶段结束时对 OCT4 进行免疫荧光染色, 并将 OCT4 阳性集落视为原代 hCiPSC 集落。请至少保留一个活细胞孔以建立 hCiPS 细胞系。

1.   Preparation plates coated with Laminin-521. Briefly, thaw the Laminin-521 at   2-8℃ before use. Dilute it in PBS to a final concentration   of 5-10 µg/mL. Gently mix and immediately add 0.5mL diluted Laminin-521 to   12-well plate. Incubate at 2-8 ℃ overnight.

涂有层粘连蛋白-521 的制备板。

简而言之, 使用前在 2-8℃下解冻层粘连蛋白-521。在 PBS 中将其稀释至终浓度为 5-10 μg/mL。轻轻混合并立即将 0.5 mL 稀释的层粘连蛋白-521 添加到 12 孔板中。2-8 ℃下孵育过夜。

2.   Preparation medium for derivation hCiPS cell lines.

用于衍生 hCiPS 细胞系的制备培养基。

3.   After 8-12 days stage III condition treatment, cells were dissociated by   Accutase (Millipore) and replated at a ratio from 1:3 to 1:12 on Laminin-521   (STEMCELL) coated plates in the medium for derivation hCiPS cell lines:   Knockout DMEM supplemented with 2% B27 supplement, 1% GlutaMAX, 1% NEAA, 1%   Penicillin-Streptomycin, 50 μg/ml Vc2p, 2 mg/mL AlbuMAXTM-II (AlbuMAXTM-II   could be replaced by 4% KSR) and the small molecules CHIR99021 (1 μM),   PD0325901 (0.5 μM), Y-27632 (10 μM), HRG (20 ng/mL), and bFGF (100 ng/mL).   Cells were incubated under 21% O2, 5% CO2 at 37℃ and the medium was   changed every day.

第三阶段条件处理 8-12 天后, 用 Accutase解离细胞, 并以 1：3 至 1：12 的比例重新接种在层粘连蛋白-521包被的平板上, 用于衍生 hCiPS 细胞系：补充有 2% B27 补充剂、1% GlutaMAX、1% NEAA、1% 青霉素-链霉素、50 μg/mL Vc2p、2 mg/mL AlbuMAXTM-II (AlbuMAXTM-II 可被 4% KSR 替代)和小分子 CHIR99021 (1 μM)的敲除 DMEM,  PD0325901 (0.5 μM)、Y-27632 (10 μM)、HRG (20 ng/mL)和 bFGF (100 ng/mL)。将细胞在 21% O2、5% CO2 和 37℃下孵育, 每天更换培养基。

4.   After 7-12 days, single compact hCiPSC colony was mechanically picked and   dissociated into small clamps and transferred onto Matrigel coated plates in   mTeSR Plus Medium supplemented with Y-27632 (10 μM).

7-12 天后, 将单个紧凑的 hCiPSC 集落机械挑取并解离成小夹具, 并转移到补充有 Y-27632 (10 μM)的 mTeSR Plus 培养基中的基质胶包被板上。

5.   After 24 hours, the culture was replaced by fresh mTeSR Plus Medium without   Y-27632.

24 小时后, 用不含 Y-27632 的新鲜 mTeSR Plus 培养基替换培养物。

hCiPS   cell lines were maintained in mTeSR Plus Medium on Matrigel coated plates   under 21% O2, 5% CO2 at 37 oC. The medium was changed every day. Cells were   passaged when they reach ~85% confluence. This typically occurred at day 3–7   after passaging with split ratios of around 1:10 to 1:20. For passaging,   hCiPS cell lines were dissociated by ReLeSRTM (STEMCELL), and the detached   cell aggregates were transferred onto Matrigel-coated plates in mTeSR Plus   Medium supplemented with Y-27632 (10 µM). Allow the colonies to attach to the   culture plate for 24 hours before replacing the spent medium with fresh mTeSR   Plus Medium without Y-27632.

将 hCiPS 细胞系维持在 mTeSR Plus 培养基中, 在 21% O2、5% CO2 和 37℃下基质胶包被板中。介质每天都在更换。当细胞达到约85%融合度度时传代。这通常发生在传代后的第 3-7 天, 分流比约为1/10至1/20。对于传代, 通过 ReLeSR解离 hCiPS 细胞系, 并将分离的细胞聚集体转移到补充有 Y-27632 (10 μM)的 mTeSR Plus 培养基中的基质胶包被板上。让菌落附着在培养板上 24 小时, 然后用不含 Y-27632 的新鲜 mTeSR Plus 培养基替换用过的培养基。