Isolation and culture of HEFs
Human embryonic fibroblasts (HEFs) were isolated from12- to 18-week-old embryos that obtained from elective termination of pregnancy with informed written consent. Briefly, the donated embryonic dermis tissues (0.5-1 cm2) were washed twice with PBS (Corning) containing 2% Penicillin-Streptomycin (Gibco), minced by scissors to 1-2 mm2 and dissociated with 5-10 ml 2 mg/ml collagenase IV solution (Gibco) in a 100-mm dish at 37 oC for 1 hour. Next, 10-20 ml 15% FBS-DMEM medium was added and cells were pipetted up and down several times for dissociation. The suspension was collected to 50-ml tube and shook for 1-2 min to release cells. Then the suspension was centrifuged at 400 g for 5 min and cells were resuspended in 15% FBS-DMEM medium after remove the supernatant. Generally, 1-3 x 106 cells can be obtained from 0.5-1 cm2 dermis and are plated in a 100-mm dish (P0) followed by incubation in 37 oC with 5% CO2. The next day, fresh 15% FBS-DMEM medium was changed to remove the non-adherent cells. Primary HEFs usually become confluent in 3-4 days and were ready to passage for reprogramming. 0.25% Trypsin-EDTA (Gibco) was used to dissociate primary HEFs. For hCiPSCs induction, HEFs were seeded at a density of 1.5 x 104 cells per well of 12-well plate with 15% FBS-DMEM medium. For culture and expansion, HEFs were seeded at a density of 1.5 x 106 cells per 100-mm dish. We recommend to use the primary HEFs for the induction of CiPS cells within 7 passages.
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 and 0.055 mM 2-mercaptoethanol (Gibco).
Isolation and culture of hADSCs
Adult human adipose derived mesenchymal stromal cells (hADSCs) were isolated from donated adult adipose tissue that obtained with informed written consent. Briefly, the tissues (2-4 cm3) were washed twice with PBS containing 2% penicillin-streptomycin, minced by scissors to 1-2 mm3 and dissociated with 5-10 ml 2 mg/ml collagenase IV solution in a 100-mm dish at 37 oC for 1 hour. Next, 10-20 ml 15% FBS-DMEM medium was added and cells were pipetted up and down several times for dissociation. 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. Then 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. 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 oC with 5% CO2. The next day, fresh Mesenchymal Stem Cell Growth Medium 2 was changed to remove the non-adherent cells. 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 HEFs. For hCiPSCs induction, hADSCs were seeded at a density of 1 x 104 cells per well of a 12-well plate with 15% FBS-DMEM medium. 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 within 4 passages.
Isolation and culture of hASFs
Human adult skin fibroblasts (hASFs) were isolated from donated adult dermis tissues that obtained with informed written consent. Briefly, the tissues (0.5-1 cm2) were washed twice with PBS containing 2% penicillin-streptomycin, minced by scissors to 0.5-1 mm2 pieces. Then 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. Next, the pieces were incubated in 37 oC with 5% CO2 for 4-12 hours (do not allow the pieces go to dry out). Then 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). Fresh Mesenchymal Stem Cell Growth Medium 2 was replaced every 2-3 days. Within 4-7 days, outgrowths of fibroblasts would generate. The primary hASFs usually become confluent in 10-14 days and were ready to passage for reprogramming. We passaged the hASFs both for reprogramming and expansion in the same way to hADSCs mentioned above.
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.
Generation of hCiPSCs from HEFs
Medium preparation for hCiPSCs induction
Stage I induction medium:
KnockOutTM DMEM (Gibco) supplemented with 10% Knockout Serum Replacement (KSR) (Gibco), 10% FBS, 1% GlutaMAXTM, 1% NEAA, 1% Penicillin-Streptomycin, 0.055 mM 2-mercaptoethanol, 50 μg/ml L-Ascorbic acid 2-phosphate (Vc2P) (Sigma-Aldrich), 5 mM LiCl (Sigma-Aldrich), 1 mM Nicotinamide (NAM) (Sigma-Aldrich), 2 mg/mL AlbuMAXTM-II (Gibco) and the small molecules CHIR999021 (10 μM), 616452 (10 μM), TTNPB (2 μM), SAG (0.5 μM), ABT-869 (1 μM), Rock inhibitor (Y-27632 (2 μM) or Tzv (2 μM)).
Stage II induction medium:
KnockOutTM DMEM supplemented with 10% KSR, 10% FBS, 1% GlutaMAXTM, 1% NEAA, 1% Penicillin-Streptomycin, 0.055 mM 2-mercaptoethanol, 50 μg/ml Vc2p, 5 mM LiCl, 1 mM NAM, 40 ng/ml bFGF (Origene) and the small molecules CHIR99021 (10-12 μM), 616452 (10 μM), TTNPB (2 μM), SAG (0.5 μM), ABT-869 (1 μM), Y27632 (10 μM), JNKIN8 (1 μM), Tranylcypromine (10 μM), 5-Azacytidine (10 μM).
Note: To enhance the reprogramming efficiency, the small molecules UNC0224 (1 μM), Ruxolitinib (1 μM) and SGC-CBP30 (2 μM) were recommended in stage II induction medium.
Stage III induction medium:
KnockoutTM DMEM supplemented with 1% N2 supplement (Gibco), 2% B27 supplement (Gibco), 1% GlutaMAXTM, 1% NEAA, 1% Penicillin-Streptomycin, 0.055 mM 2-mercaptoethanol, 50 μg/ml Vc2p, 5 mg/mL AlbuMAXTM-II, 20 ng/mL Recombinant Human Heregulinβ-1 (HRG) (PEPROTECH) and the small molecules CHIR99021 (1 μM), 616452 (10 μM), Y-27632 (10 μM), PD0325901 (1 μM), Tranylcypromine (10 μM), VPA (500 μM), DZNep (0.2 μM), EPZ004777 (5 μM), UNC0379 (1 μM).
Stage IV induction medium:
KnockoutTM DMEM supplemented with 1% N2 supplement, 2% B27 supplement, 1% GlutaMAXTM, 1% NEAA, 1% Penicillin-Streptomycin, 0.055 mM 2-mercaptoethanol, 50 μg/ml Vc2p, 20 ng/mL HRG and the small molecules CHIR99021 (1 μM), Y-27632 (10 μM), PD0325901 (1 μM), IWP-2 (2 μM), SB590885 (0.5 μM). The VPA (500 μM) was included in the first 4 days.
Induction process of hCiPSCs from HEFs
Cells were maintained at 37 ℃ with 21% O2 and 5% CO2. The induction medium was changed every 3-4 days.
1. HEFs were seeded at a density of 1-1.5 x 104 cells per well of a 12-well plate in 15% FBS-DMEM medium. Change the medium into stage I induction medium on the next day.
2. For stage I induction, single layer epithelial-like cells would emerge at day 4-6 and approach 80% confluence at day 8-12, then change the medium into stage II induction medium.
3. For stage II induction, multi-layered colonies appeared after 8-12 days treatment and these cell colonies would continually grow larger. After total 16-20 days’ stage II treatment, change the medium into stage III condition.
4. For stage III induction, 12 day’s treatment of stage III medium was recommended and then change the medium into stage IV condition.
5. For stage IV induction, VPA (500 μM) was included in the first 4 days. Primary hCiPSC colonies would emerge after 6-8 days’ treatment.
Note: At the end of stage IV, immunofluorescent staining of co-expression of OCT4 and NANOG was recommended to confirm the generation of primary hCiPSC colonies. Primary hCiPSC colony number was calculated as the number of the compact OCT4 positive colonies. Reprogramming efficiency was calculated as the number of primary hCiPSC colonies divided by the number of input HEFs.
Generation of hCiPSCs from hADSCs and hASFs
Medium preparation for hCiPSCs induction
Stage I induction medium:
KnockOutTM DMEM supplemented with 10% KSR, 10% FBS, 1% GlutaMAXTM, 1% NEAA, 0.055 mM 2-mercaptoethanol, 1% Penicillin-Streptomycin, 50 μg/ml Vc2p, 5mM LiCl, 1 mM NAM, 2 mg/mL AlbuMAXTM-II and the small molecules CHIR999021 (10 μM), 616452 (10 μM), TTNPB (2 μM), SAG (0.5 μM), ABT-869 (1 μM), Rock inhibitor (Y-27632 (2 μM) or Tzv (2 μM)), Dot1L inhibitor (EPZ004777 (2 μM) or EPZ5676 (2 μM)), DZNep (0.02 μM), Ruxolitinib (1 μM). For hCiPSCs induction from hASF, 0.5 μM VTP50469 was recommended to enhance the induction efficiency.
Stage II induction medium:
KnockOutTM DMEM supplemented with 10% KSR, 10% FBS, 1% GlutaMAXTM, 1% NEAA, 0.055 mM 2-mercaptoethanol, 1% Penicillin-Streptomycin, 50 μg/ml Vc2p, 5 mM LiCl, 1 mM NAM, 100 ng/ml bFGF (Origene) and the small molecules CHIR99021 (12 μM), 616452 (10 μM), TTNPB (2 μM), SAG (0.5 μM), ABT-869 (1 μM), Y-27632 (10 μM), JNKIN8 (1 μM), Tranylcypromine (2 μM), 5-Azacytidine (10 μM), UNC0224 (1 μM), Ruxolitinib (1 μM), BIRB796 (2 μM), Dorsormorphin (0.5 μM), SGC-CBP30 (2 μM). For hCiPSCs induction from hASFs or hADSCs, 0.5 μM VTP50469 was recommended to enhance the induction efficiency in stage II.
Stage III induction medium:
KnockoutTM DMEM supplemented with 1% N2 supplement, 2% B27 supplement, 1% GlutaMAXTM, 1% NEAA, 0.055 mM 2-mercaptoethanol, 1% Penicillin-Streptomycin, 50 μg/ml Vc2p, 5 mg/mL AlbuMAXTM-II, 20 ng/mL HRG and the small molecules CHIR99021 (1 μM), 616452 (10 μM), Y-27632 (10 μM), PD0325901 (1 μM), Tranylcypromine (10 μM), VPA (500 μM), DZNep (0.2 μM), EPZ004777 (5 μM), UNC0379 (1 μM). For hCiPSCs induction from hASFs, 0.5 μM DMH1 was recommended to enhance the induction efficiency.
Stage IV induction medium:
KnockoutTM DMEM supplemented with 1% N2 supplement, 2% B27 supplement, 1% GlutaMAXTM, 1% NEAA, 0.055 mM 2-mercaptoethanol, 1% Penicillin-Streptomycin, 50 μg/ml Vc2p, 20 ng/mL HRG and the small molecules CHIR99021 (1 μM), Y-27632 (10 μM), PD0325901 (1 μM), IWP-2 (2 μM), SB590885 (0.5 μM). VPA (500 μM), Tranylcypromine (10 μM), DZNep (0.05 μM), and EPZ004777 (5 μM) were included in the first 4 days.
Induction process of hCiPSCs from hADSCs and hASFs
Hypoxia with 5% O2 was used in stage I induction. After stage I induction, cells were cultured in 21% O2 condition. The induction medium was changed every 3-4 days.
1. ADSCs and hASFs were seeded at a density of 1 x 104 cells per well of a 12-well plate in 15% FBS DMEM medium. Change the medium into stage I induction medium on the next day.
2. For stage I induction, single layer epithelial-like cells induced from hADSCs would emerge at day 4-6 and approach 80% confluence at day 8-12. For ASFs, epithelial-like cells would approach 80% confluence at day 12-20. Then change the medium into stage II induction medium.
3. For stage II induction, multi-layered cell colonies appeared after 8-12 days treatment and these cell colonies would continually grow larger. After total 20 days’ treatment of stage II medium, change the medium into stage III induction medium.
4. For stage III induction, 12-day’s treatment of stage III induction medium was recommended. Then change the medium into stage IV condition.
5. For stage IV induction, VPA (500 μM), Tranylcypromine (10 μM), DZNep (0.05 μM), and EPZ004777 (5 μM) were included in the first 4 days of stage IV induction medium. Primary hCiPSC colonies would emerge after 6-8 days’ treatment.
Derivation and culture of human CiPS cell lines
After 8-12 days stage IV condition treatment, cells were dissociated by Accutase (Millipore) and replated at a ratio from 1:3 to 1:12 on feeder layers of mitomycin C (Sigma-Aldrich)-treated MEFs (2-3 x 104 per cm2) in the modified stage IV condition: Knockout DMEM supplemented with 1% N2 supplement, 2% B27 supplement, 1% GlutaMAXTM, 1% NEAA, 1% Penicillin-Streptomycin, 0.055 mM 2-mercaptoethanol, 50 μg/ml Vc2p, 2 mg/mL AlbuMAXTM-II and the small molecules CHIR99021 (1 µM), PD0325901 (0.5 µM), IWP-2 (2 µM), Y-27632 (10 µM), HRG (20 ng/mL), and bFGF (100 ng/mL, Origene). Cells were incubated under 21% O2, 5% CO2 at 37 oC and the medium was changed every day. After 3-7 days, compact CiPS cell colonies appeared. After 10-12 days, these colonies were manually picked up and mechanically dissociated into small clamps and transferred onto Matrigel (Corning) coated plates in mTeSR™ Plus Medium (STEMCELL) 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.
Human CiPSCs 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, human CiPS cells 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.