活病毒培養/核酸提取

動物感染

未滅活樣本檢測

已滅活材料操作

重組病毒

腺病毒相關病毒

(Adenovirus-associated virus, AAV)

第三類

BSL-2

ABSL-2

BSL-2

BSL-1

BSL-2

逆轉錄病毒科慢病毒

(Lentivirinae, Lvis)

第三類

BSL-2

ABSL-2

BSL-2

BSL-1

/

仙臺病毒(Sendai virus)

第三類

BSL-2

ABSL-2

BSL-2

BSL-1

BSL-2

DMEM

(Gibco, C11965500BT)

DMEM高糖培養基【DMEM為基礎培養基, 可支持多種哺乳動物細胞生長。用DMEM已成功培養出包括原代成纖維細胞、神經元、膠質細胞、HUVEC、平滑肌細胞及 HeLa、293、Cos-7 和 PC-12 等多種細胞系】

Mesenchymal Stem Cell Growth Medium 2

(Promo Cell, C-28009)

間充質干細胞生長培養基2 (MSCGM2)【用于人間充質干細胞大規模培養, 不誘導細胞分化】

KnockOut DMEM

 (Gibco, 10829018)

KnockOut D-MEM【一種基礎培養基, 經優化用于未分化胚胎干細胞及誘導型多能干細胞生長】

GlutaMAX

(Gibco, 35050-061)

Gibco GlutaMAX 補充劑【是L-谷氨酰胺的替代品, 包含在多種培養基配方中。GlutaMAX補充劑減少了有毒氨氣積累,改善細胞活力和生長, 可改善細胞健康狀況,適用于哺乳動物細胞的貼壁和懸浮培養】

MEM Non-Essential Amino Acids Solution (NEAA)

(Gibco, 11140050)

MEM非必需氨基酸溶液【用作細胞培養基添加劑, 可以提高細胞生長和活性?！?/span>

Penicillin-Streptomycin

(Gibco, 15140-122)

青霉素-鏈霉素混合溶液【內含10000單位/mL青霉素和10000 μg/mL鏈霉素, 對革蘭陽性細菌和革蘭陰性細菌具有有效的聯合抗菌作用】

Fetal Bovine Serum (FBS)

(Vistech, VIS93526487)

胎牛血清

Knockout Serum Replacement (KSR)

(Gibco,  10828028)

Knockout 血清替代物【適用于多個物種胚胎干細胞 (ESC) 和誘導多能干細胞 (iPSC) 進行無血清的滋養細胞依賴性培養, 可直接替代現有實驗方案中的 FBS。與添加FBS 的培養基相比, 在添加 KnockOut SR 的培養基中培育時, ES和iPS細胞出現的分化顯著更少, 種系傳遞能力應該不會受到影響】

B27 supplement

(Gibco, 17504-044)

B27添加劑【無血清添加劑，由抗氧化劑、蛋白、維生素和脂肪酸以最優比例混合而成，可支持神經細胞的培養】

AlbuMAX-II

(Gibco, 11021-045)

AlbuMAX   II 富含脂質BSA【用于細胞培養的富含脂質牛血清白蛋白】

mTeSR   Plus Medium

(STEMCELL, 100-0276)

多能干細胞培養基【專為人類胚胎干細胞（ES）和誘導多能干細胞（iPS）設計的無血清、無飼養層維持培養基】

Collagenase IV

(Gibco, 1963347)

膠原酶IV

0.25% Trypsin-EDTA

(Gibco, 25200-056)

胰蛋白酶-EDTA

Accutase

(Millipore, SCR005)

Accutase細胞解離溶液【Accutase含細胞解離的蛋白水解酶和膠原蛋白水解酶，可從標準組織培養塑料器具和粘附涂層塑料器具中常規解離細胞。用于誘導性多能干細胞(IPSC)解離或酶解成單個細胞】

ReLeSR

(STEMCELL, 05872)

干細胞集落解離液【一種無酶試劑用于將ES或iPS細胞集落解離為細胞聚集體，且無需對已發生分化的集落進行手工挑選】

Matrigel

(Corning, 354248)

Matrigel基底膜基質【富含細胞外基質蛋白，包括層粘連蛋白 (主要成分)、IV型膠原、硫酸乙酰肝素蛋白聚糖、巢蛋白和多種生長因子】

CellAdhere Laminin-521

(STEMCELL, 77004)

層粘連蛋白521【可與細胞表面的整合素、多聚糖等多種受體結合，其中整合素α6β1與Laminin高親和力結合，是Laminin的主要細胞粘附受體，直接決定Laminin對細胞貼壁能力的支持。LN521與細胞表面受體結合激活細胞信號通路，從而產生更多功能性細胞】

PBS (Corning, 05418005)

PBS緩沖液

DMSO(Dimethyl sulfoxide)

(Sigma-Aldrich,   D2650)

二甲基亞砜【可在在細胞冷凍培養基中保護細胞免受冰晶引起的機械性損傷，常與BSA或FBS混合使用】

CHIR99021 (CHIR, C)

(WUXI   APPTEC)

糖原合成酶激酶3β抑制劑【可誘導人胚胎干細胞向內胚層分化】

616452 (6)

(WUXI APPTEC)

616452【一種轉化生長因子-β抑制劑】

TTNPB (N)

(WUXI APPTEC)

TTNPB/Arotinoid Acid，即芳香維甲酸【視黃酸受體(retinoic acid receptor, RAR)是一種轉錄轉錄調節因子，控制特定基因亞群的表達，在細胞生長、分化和器官發生中起重要作用的激動劑，在小鼠肢芽細胞培養物中，可抑制軟骨生成】

SAG (WUXI APPTEC)

SAG   (Smoothened Agonist)【一種細胞滲透性Smoothened (Smo)激動劑】

EPZ5676 (MCE, HY-15593)

EPZ-5676即Pinometostat【一種有效 MV4-11 增殖抑制劑，誘導協同和持久的抗增殖作用，增加分化標志物的表達和細胞凋亡】

Ruxolitinib   (Ruxo)

(Selleckchem, S1378)

Ruxolitinib即魯索利替尼【選擇性JAK1/2抑制劑，可誘導自噬并增強細胞凋亡。JAK/STAT信號通路是眾多細胞因子信號轉導的共同途徑，廣泛參與細胞增殖、分化、凋亡以及炎癥等過程】

3-deazaneplanocin A (DZNep)

(WUXI APPTEC)

DZNep即3-去氮腺嘌呤A【一種有效的組蛋白甲基轉移酶 (histone methyltransferase，EZH2) 抑制劑。通過抑制EZH2的表達，激活被抑制的腫瘤抑制基因，誘導細胞凋亡】

Y-27632 (WUXI APPTEC)

小分子抑制劑Y27632【Rho相關蛋白激酶小分子抑制劑，作用于Rho介導的應力纖維的形成，抑制G1-S期細胞周期進程和胞質分裂。還能通過調節上皮-間充質過渡樣誘導人多能干細胞選擇性地分化為間胚層譜系】

5-Azacytidine (5azaC, 5)

(Sigma-Aldrich, A2385)

5-氮雜胞苷【是DNA甲基轉移酶抑制劑（DNMT抑制劑），可以嵌入DNA/RNA上并作用DNA甲基轉移酶，使細胞中DNA甲基轉移酶活性的缺失和DNA去甲基化】

JNK-IN-8  (J)

(Selleckchem, S4901)

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

BIRB796 (BIRB)

(Selleckchem, S1574)

即Doramapimod【BIRB 796) 通常與炎癥有關，是一種是一種抗炎化合物，通過抑制p38 MAPK而發揮生物活性】

SGC-CBP30 (CBP30)

(Selleckchem, S7256)

SGC-CBP30【一種有效且高度選擇性的CBP/p300溴結構域抑制劑，減少Th17細胞中IL-17A的分泌，并具有抗炎作用】

Dorsomorphin (DM)

(MCE, HY-13418A)

Dorsomorphin【一種選擇性ATP 競爭性的AMPK 抑制劑，可降低細胞中AMPK 磷酸化水平】

VTP50469

(Selleckchem, S8934)

VTP50469【一種高選擇性Menin-MLL相互作用抑制劑，將Menin從蛋白質復合物中置換出來并抑制MLL的染色質占據基因。MLL結合的喪失導致基因表達、分化和細胞凋亡發生變化】

Valproic acid sodium salt (VPA)

(Sigma-Aldrich, P4543)

丙戊酸【抗癲癇藥，可影響信號傳導通路，如Wnt/β-連環蛋白和ERK通路，干擾肌醇和花生四烯酸代謝，在細胞存活、轉錄調控、離子穩態、信號轉導和細胞骨架修飾的基因表達中發揮作用】

Tranylcypromine (Tranyl, T)

(Enzo,   BML-EI217-0005)

反苯環丙胺【一種抑制單胺氧化酶的抗抑郁藥），可抑制組蛋白去甲基化】

PD0325901

(WUXI APPTEC)

即Mirdametinib【對ERK1和ERK2磷酸化抑制作用并誘導細胞凋亡】

IWP-2

(Selleckchem, S7085)

IWP-2【Wnt 加工和分泌的抑制劑，阻止關鍵的 Wnt 配體棕櫚糖基化，在數μM范圍即可抑制細胞的增殖】

SB590885

(Selleckchem, S2220)

SB590885【一種有效的Raf/VEGFR 激酶抑制劑】

L-Ascorbic acid 2-phosphate (Vc2P)

(Sigma-Aldrich, A8960)

維生素C磷酸酯【即-抗壞血酸-2-磷酸酯，可促進膠原蛋白的合成】

LiCl (Sigma-Aldrich, L4408)

氯化鋰【是糖原合成酶激酶 3β的高度選擇性抑制劑，通過抑制GSK-3β的活性，激活 Wnt/β-catenin 信號途徑。促進大鼠骨髓間充質干細胞的增殖及向成骨細胞分化】

Nicotinamide (NAM)

 (Sigma-Aldrich, 72340)

煙酰胺【組織培養基的營養成分】

Recombinant Human FGF2

 (oriGene, TP750002)

重組人成纖維細胞生長因子2【參與骨的愈合、軟骨修復、骨修復和神經再生。也是一種有絲分裂促進劑，能加速細胞增殖】

Recombinant Human Heregulinβ-1 (HRG)

(PEPROTECH,  100-03)

重組人 Heregulinβ-1【屬神經調節蛋白（NRG）中I 型 NRG1，參與組織細胞的發育和成熟】

BMP4

(Stemimmune, HST-B4-0100)

骨形態發生蛋白4（BMP-4）【是轉化生長因子 β 家族成員，從早期胚胎發生到胚胎發育期廣泛表達，在軟骨和骨骼形成、中胚層誘導、牙齒發育、肢體形成和骨折修復中起重要作用】

AKT Kinase Inhibitor (AKTi)

(MCE, HY-10249A)

蛋白激酶B（PKB或Akt）【絲氨酸/蘇氨酸特異性蛋白激酶，在葡萄糖代謝，細胞凋亡，細胞增殖，轉錄和細胞遷移多種細胞過程中起關鍵作用。AKTi一種細胞滲透化合物，能選擇性抑制Akt1，Akt2和Akt3的活性】

SETD2-IN-1 (SET)

(MCE, HY-136328)

EZM0414   (SETD2-IN-1)【對SETD2 甲基轉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-碘代殺結核菌素【腺苷激酶抑制劑，可通過激活磷酸化酶和糖原合成酶。也抑制 CK1、胰島素受體酪氨酸激酶、磷酸化酶激酶】

CX4945

(MCE, HY-50855)

即Silmitasertib【選擇性的CK2抑制劑，下調CK2 表達,抑制CK2介導的 PI3K/Akt/mTOR 信號通路的激活，誘導血液腫瘤細胞毒性和凋亡】

Triton X-100

 (Sigma-Aldrich, T8787)

Triton X-100

Donkey serum

(Jackson Immuno Research, 017-000-121)

驢血清

Rabbit monoclonal anti-OCT4

(Invitrogen,   MA5-14845; RRID: AB_10979606)

兔抗OCT4單克隆抗體（一抗）

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

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

驢抗兔IgG二抗

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

CO2培養箱

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

三氣細胞培養箱

Water bath (37℃)

恒溫水浴槽

Scissor

組織用剪刀

Centrifuge

帶水平轉頭的離心機

Cell   counter

細胞計數器

Electric pipettor and pipette tips

電動移液器及移液器吸頭

Pipettor and pipette tips

手動移液器及移液吸頭

Inverted microscope

倒置相差顯微鏡

Inverted fluorescence microscope

倒置相差顯微鏡

12-well tissue culture plates

12孔組織培養板

6-well tissue culture plates

6孔組織培養板

100-mm tissue culture dish

10cm組織培養皿

15ml polystyrene conical tubes

15mL錐形離心管

50ml polypropylene conical tubes

50mL錐形離心管

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

成人脂肪來源的基質細胞 (hADSC) ，是從經知情書面同意的捐贈者采集的脂肪組織分離的。

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-鏈霉素的 PBS 洗滌組織 (2-4 cm³)兩次, 用剪刀盡可能切碎至 1-2 mm³大小顆粒。

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 培養皿中用 5-10mL濃度2 mg/mL的膠原酶 IV溶液解離 1-1.5小時。

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 培養基, 用移液器吹打數次將細胞解離。

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.

將細胞懸浮液收集到兩個50mL離心管中, 以 15% FBS-DMEM 培養基稀釋至30-40 mL, 然后振蕩處理1-2 分鐘以釋放細胞。

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.

將細胞懸浮液400×g離心5分鐘, 去除上清, 將細胞重懸于MSCG培養基 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,從 2-4 cm³ 脂肪組織中可獲得 1-3×10⁶ 個細胞, 接種于100mm 培養皿(作為P0代細胞),在 37℃ - 5% CO2培養箱中孵育。

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

第二天, 更換新鮮的MSCG2培養基，以去除非貼壁細胞。

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 天內融合, 并準備傳代以進行重編程。0.25% 胰蛋白酶-EDTA 用于解離原代 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.

對于 hCiPSC 誘導, hADSCs 以 12 孔板每孔 1×10⁴ 個細胞或24 孔板每孔5×10³ 個細胞的密度接種于15% FBS-DMEM 培養基上。

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.

對于培養和擴增, 用MSCGM2培養基以每 100 mm培養皿1.5×10⁶個細胞的密度接種 hADSC。我們建議在第2至第4次傳代之間使用原代 hADSCs 誘導 CiPS 細胞。

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 培養基組成為：高葡萄糖 DMEM, 補充有 15% 胎牛血清(FBS)、1% GlutaMAX、1% MEM 非必需氨基酸溶液 (NEAA)、1% 青霉素-鏈霉素。

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

人成人皮膚成纖維細胞 (hASF) ，是從經知情書面同意的捐贈者采集的脂肪組織分離的。

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-鏈霉素的 PBS 洗滌組織 (0.5-1 cm²)兩次, 用剪刀切成 0.5-1 mm² 的塊。

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.

將碎片放入 100 mm 細胞培養皿中, 并在每塊組織上滴入 1 滴 15% FBS-DMEM 培養基。

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

將塊狀物在 37 ℃和 5% CO2 中孵育 4-12 小時 (不要讓塊狀物變干)。

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).

向培養皿中溫和加入 3-5 mL MSCGM2 (不要讓碎片從培養皿中脫落)。

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

新鮮MSCGM2培養基每 2-3 天更換一次。

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

在 4-7 天內, 會產生成纖維細胞的生長物。

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 天內融合, 并準備好傳代以進行重編程。

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

以與上述hADSC相同的方式將 hASF 傳代以進行重編程和擴增到。

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.

商業人成人真皮成纖維細胞 (Lonza-CC2511)和 ADSC (Lonza-PT5006)也在MSCGM2中培養, 并以與上述hADSC相同的方式傳代以進行重編程和擴增。

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.

建議使用原代第2-4代分離的hADSC來誘導 hCiPSC。如果使用原代 hASF,細胞傳代 2 - 4 次（對于市售 hADSC 或 hASF, MSCGM2中培養兩次傳代將有助于改善細胞的狀態和重編程效率）。較長傳代的細胞也可用于誘導 hCiPSC, 但誘導效率低。故不建議將長期傳代的商業細胞系 (IMR90 和BJ等)用于誘導 hCiPSC。

Stage   I induction medium:

第一階段的誘導培養基組分

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)

補充有 1% KSR、2% B27、1% GlutaMAX、1% NEAA、1% 青霉素-鏈霉素、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 激酶抑制劑 (1 μM)、 JNKIN8 (0.2 μM)和 SETD2-IN-1 (0.2 μM)

Stage   II induction medium:

第二階段的誘導培養基組分

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 補充有 2% B27、1% GlutaMAX 、1% NEAA、1% 青霉素-鏈霉素、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-碘結核菌素 (0.5 μM)、5-氮雜胞苷 (2 μM)、AKT 激酶抑制劑 (0.2 μM)和 CX-4945 (1 μM)。

Stage   III induction medium:

第三階段的誘導培養基組分

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.

補充有 2% B27 補充劑、1% GlutaMAX、1% NEAA、1% 青霉素-鏈霉素、5% 敲除血清替代物 (KSR)、50 μg/mL Vc2p、20 ng/mL 重組人 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。在第三階段的誘導過程中, 前 4 天將 VPA (1000 μM)、反苯環丙胺(10 μM)、DZNep (0.2 μM)和 EPZ5676 (2 μM)包含在培養基中。

隨后, 在接下來的 4 天內加入 VPA (500 μM), 同時去除反式環丙胺、DZNep 和 EPZ5676。而不含 VPA、反式環丙胺、DZNep 或 EPZ5676 的 第三階段誘導培養基可以再施用 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.

備注：請采用新鮮制備每種培養基, 并在配置后和使用前搖勻（制備誘導培養基可4℃儲存長達兩周）。

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 接種在補充有 15% FBS 的高葡萄糖 DMEM 中, 密度為 12 孔板每孔 1×10⁴ 個細胞或 24 孔板每孔 5-6×10³ 個細胞, 12-24 小時后更換為新鮮制備的第一階段誘導培養基。

確保細胞密度適宜,且細胞均勻地附著在每個孔的底部。在第 1 天接種的適當細胞密度對于第一階段上皮樣細胞的重編程效率和誘導很重要。強烈建議在第一階段結束時上皮樣細胞的百分比或面積過低時優化第 1 天接種的細胞密度。

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.

對于第一階段誘導, 單層上皮樣細胞將在第 4-6 天出現。當上皮樣細胞達到近100%融合度時 (通常在 hADSC 的第一階段誘導 8-10 天和 hASF 的第一階段誘導 9-16 天后)，將培養基更換為新鮮制備的第二階段誘導培養基。第一階段結束時的細胞可能沒有牢固附著, 因此更換培養基時要輕柔。

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.

對于第二階段誘導, 處理 4-6 天后出現多層細胞集落, 這些細胞集落會不斷變大。在第二階段的最后 4 天, 由于細胞增殖較強, 可以每兩天更換一次培養基, 也可以在倒數第二天向原始孔中加入相同體積的第二階段培養基（第二階段晚期有細胞死亡是正常的）。

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.

當細胞集落變大時, 在第二階段培養基處理 8-12 天后, 將培養基更換為新鮮制備的第三階段誘導培養基。第二階段的誘導天數對 hCiPSC 的誘導效率很重要。您可以在第二階段誘導 8、10 或 12 天后將第二階段切換到第三階段, 并比較效率。

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.

對于第三階段誘導, 前 4 天在誘導培養基中加入 VPA (1000 μM)、反苯環丙胺(10 μM)、DZNep (0.2 μM)和 EPZ5676 (2 μM)。隨后, 在接下來的 4 天內加入 VPA (500 μM), 同時去除反式環丙胺、DZNep 和 EPZ5676。不含 VPA、反式環丙胺、DZNep 或 EPZ5676 的第三階段誘導培養基可以再施用 2-4 天, 以使原代 hCiPSC 集落更大（這一階段出現大量細胞死亡是正常的）。

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.

建議在第三階段結束時對 OCT4 進行免疫熒光染色, 并將 OCT4 陽性集落視為原代 hCiPSC 集落。請至少保留一個活細胞孔以建立 hCiPS 細胞系。

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.

涂有層粘連蛋白-521 的制備板。

簡而言之, 使用前在 2-8℃下解凍層粘連蛋白-521。在 PBS 中將其稀釋至終濃度為 5-10 μg/mL。輕輕混合并立即將 0.5 mL 稀釋的層粘連蛋白-521 添加到 12 孔板中。2-8 ℃下孵育過夜。

2.   Preparation medium for derivation hCiPS cell lines.

用于衍生 hCiPS 細胞系的制備培養基。

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.

第三階段條件處理 8-12 天后, 用 Accutase解離細胞, 并以 1：3 至 1：12 的比例重新接種在層粘連蛋白-521包被的平板上, 用于衍生 hCiPS 細胞系：補充有 2% B27 補充劑、1% GlutaMAX、1% NEAA、1% 青霉素-鏈霉素、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)。將細胞在 21% O2、5% CO2 和 37℃下孵育, 每天更換培養基。

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, 將單個緊湊的 hCiPSC 集落機械挑取并解離成小夾具, 并轉移到補充有 Y-27632 (10 μM)的 mTeSR Plus 培養基中的基質膠包被板上。

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

24 小時后, 用不含 Y-27632 的新鮮 mTeSR Plus 培養基替換培養物。

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.

將 hCiPS 細胞系維持在 mTeSR Plus 培養基中, 在 21% O2、5% CO2 和 37℃下基質膠包被板中。介質每天都在更換。當細胞達到約85%融合度度時傳代。這通常發生在傳代后的第 3-7 天, 分流比約為1/10至1/20。對于傳代, 通過 ReLeSR解離 hCiPS 細胞系, 并將分離的細胞聚集體轉移到補充有 Y-27632 (10 μM)的 mTeSR Plus 培養基中的基質膠包被板上。讓菌落附著在培養板上 24 小時, 然后用不含 Y-27632 的新鮮 mTeSR Plus 培養基替換用過的培養基。