REAGENT SETUP
Matrigel-coated culture plates Refer to manufacturer’s instructions for reagent handling and thawing. !CAUTION It is important to keep Matrigel and all equipment used to prepare and aliquot stocks chilled. Thaw Matrigel on ice overnight. When thawed, dilute Matrigel 1:2 using ice-cold KO-DMEM and immediately aliquot into 15 ml chilled centrifuge tubes (1 ml/tube). Aliquots can be stored at -80°C until use. To prepare Matrigel-coated plates, thaw 1 ml stock aliquot overnight at 4°C and dilute 1:15 using ice cold KO-DMEM. Using chilled pipette tips, aliquot 1 ml/well for a 6-well plate. Swirl to coat plate surface evenly. Plates can be used after incubation at 37°C for 1 hour, or at 4°C overnight, and can be kept at 4°C for up to 2 weeks wrapped in Parafilm. Plates kept at 4°C should be transferred to 37°C for at least 30 minutes before use. To use Matrigel-coated plates for cell culture, aspirate the Matrigel solution, replace with 3ml of pre-warmed Nutristem media and place back into incubator to equilibrate. Passage cells onto the prepared plates after a minimum of 10 minutes.
Collagenase Type IV solution Final concentration is 200U/ml. Dissolve 20,000 U of collagenase Type IV in 100 ml KO-DMEM. Add all components to a 250 ml filter unit and filter. Aliquot into 15 ml sterile tubes and store at -20°C until use. Thawed solution can be stored at 4°C for up to 1 week.
Isolation medium The isolation medium consists of DMEM supplemented with 2 mM L-Glutamine, 100 U/ml Penicillin/Streptomycin and 10% FBS. Solutions of L-Glutamine and Penicillin/Streptomycin are aliquoted in 5 ml tubes and stored at -20°C until use. FBS is filtered (optional) and aliquoted in 50 ml falcon tubes and stored at -20°C until use. To make 500 ml isolation medium, take one DMEM bottle and remove 60 ml of liquid. Thaw one aliquot of L-Glutamine, Penicillin/Streptomycin and FBS in a 37°C waterbath. Add solutions to the DMEM bottle. !CAUTION gently swirl the bottle to mix avoiding the liquid touching the ridges of the bottle. The isolation medium should be kept at 4°C for up to 2 weeks and a small aliquot should be pre-warmed in a 37°C waterbath before use.
Expansion medium The expansion medium consist of Nutristem supplemented with 100 U/ml Penicillin/Streptomycin. To prepare one 500 ml bottle of expansion medium, remove 5 ml of solution from a 500 ml Nutristem bottle and replace with 5ml of thawed Penicillin/Streptomycin. !CAUTION gently swirl the bottle to mix avoiding the liquid touching the ridges of the bottle. The expansion medium should be aliquoted into 50 ml aliquots in falcon tubes and stored at -20°C until use. When needed, thaw one aliquot in a 37°C waterbath and at keep at 4°C for up to 1 week. Prior to use for cell culture, a small aliquot should be pre-warmed in a 37°C waterbath before use.
Reprogramming medium Reprogramming medium consists of expansion medium supplemented with 1 mM VPA. Make 50 mM VPA stock by diluting 100 mg of powder VPA into 12 ml expansion medium and sterilize by filtering using a 0.2 μm syringe filter. Take 1 ml from this solution and add it to 50 ml expansion medium to make up a final VPA concentration of 1mM. CRITICAL STEP The 50 mM VPA stock and the reprogramming medium should be made fresh before use.
Freezing medium The freezing medium consists of 60 % (vol/vol) DMEM, 30 % FBS and 10 % DMSO. To make 10 ml of freezing medium, add 6 ml of DMEM, 3 ml of FBS and 1 ml of DMSO. CRITICAL STEP Add the DMSO last and keep the freezing medium at 4°C until use.
Solution for Flow cytometry and cell separation The solution for flow cytometry and cell separation consists of 1% BSA in DPBS. First, make a stock solution of 10 % BSA by dissolving 5 g of BSA into 50 ml of DPBS and sterile filter using a 0.2 μm syringe filter. !CAUTION BSA does not readily dissolve in DPBS. To fully dissolve, put a little bit of DPBS in a 50 ml falcon tube and add the BSA powder carefully. Then, affix the tube horizontally on a shaking plate, shaking slowly. When dissolved, complete the volume up to 50 ml with DPBS and sterile filter using a 0.2 μm syringe filter. To make the 1 % BSA solution, add 5 ml of the filtered solution into 45 ml of DPBS. CRITICAL STEP 1% BSA in DPBS should be stored at -20°C and thawed before use. The solution can be kept at 4°C for 24 h.
PROCEDURE
Isolation of AFSC from human amniotic fluid TIMING ∼10 min
1| Collect human amniotic fluid after amniocentesis (usually 1-2 ml) in a sterile syringe.
2| Transfer to 15 ml centrifuge tube and centrifuge at 300g for 5 minutes.
3| Resuspend the resulting pellet in 1 ml of pre-warmed isolation medium and transfer to one well of a 6-well plate, add 2 more ml of pre-warmed medium, working in aseptic conditions.
!CAUTION any study involving use of human tissue must conform to national and institutional ethics regulations.
Expansion of isolated AFSC cells TIMING ∼ 2 weeks
4| Check cells daily for attachment without changing the medium for the first week.
5| When the first colonies are forming (∼ 10-15 cells each), replace the medium. Aspirate the medium from the well carefully without touching the bottom of the well. Wash the well carefully with 2 ml of pre-warmed DPBS. Replace with 3 ml of pre-warmed isolation medium. !CAUTION place the pipette end on the side of the well and release the solution very slowly to avoid disturbance of the colonies.
6| Allow the colonies to grow until 70% confluence has been reached, changing the medium every Monday, Wednesday and Friday.
7| When the culture has reached 70% confluence, passage the cells.
8| Remove the medium and wash with DPBS as mentioned above.
9| Add 1 ml of Trypsin solution, swirl the plate gently to cover plate surface evenly and place back in the incubator for 2-3 minutes, until cells detach.
10| Add 1 ml of isolation medium to neutralize the trypsin, and collect in a 15 ml tube.
11| Centrifuge at 300g for 5 minutes.
12| Carefully remove the supernatant and resuspend the pellet slowly in 200 μl of isolation medium to obtain a single cell suspension
13| Add a further 800μl of isolation medium, resuspending slowly
14| Add a further 2 ml of isolation medium and resuspend carefully, avoiding the formation of air bubbles
15| Transfer 1 ml of cell suspension into each of 3 wells of a 6 well plate
16| Add 2ml of pre-warmed isolation medium to each well containing cells
17| Place the plate in the incubator
Selection of c-KIT+ cells. TIMING 1h
When the cells have been expanded to ∼10 x106 cells, proceed with selection of c-KIT+ cells
18| Repeat steps 8-11
19| Carefully remove the supernatant and resuspend the pellet slowly in 200 μl of solution for cell separation to obtain a single cell suspension
20| Add a further 800 μl of solution for cell separation, resuspending slowly
21| Count the cells using a haemocytometer or cell counter, using methylene blue to determine cell viability
22| Wash the cells by adding 4 ml to the cell suspension and resuspending gently
23| Centrifuge at 300g for 5 minutes
24| Discard the supernatant and resuspend the pellet as before
25| Repeat wash 2 more times, centrifuging after each wash
26| Resuspend the cell pellet in 300 μl of AutoMACS running buffer, in a FACS round bottom tube
27| Add 100 μl of CD117 microbeads
28| Mix well and incubate at 4°C for 15 minutes
29| Wash cells by adding 4 ml of running buffer, resuspend well
30| Centrifuge at 300g for 10 minutes
31| Remove supernatant completely and resuspend in 500 μl of running buffer
32| Place an MS column in the magnetic field of the MACS separator
33| Prime column by rinsing with 500 μl of running buffer
34| Apply cell suspension to the column, and collect the flow through that contains the unlabelled cells in a 15 ml falcon tube
35| Wash column 3 times with 500 μl of running buffer, collecting the flow through in the same 15 ml tube
36| After the washes, remove the separation column and place it in a 15ml falcon tube
37| Add 1 ml of isolation medium onto the column and immediately flush out the CD117+ magnetically labelled cells by firmly pushing the plunger into the column.
? TROUBLESHOOTING
PAUSE POINT If needed, cells can be frozen and kept in liquid nitrogen long-term
38| Plate the cells in 6-well plates at a cell density of 2 x 105 cells/well, topping up with isolation medium to 3 ml per well
39| Place the plate in the incubator
Adaptation of cells to medium sustaining pluripotency TIMING ∼ 2 weeks
To transfer the cells from isolation medium to expansion medium:
40| First prepare Matrigel-coated plates and place the plates in the incubator with 2 ml per well of pre-warmed 1:1 isolation medium:expansion medium to equilibrate for a minimum of 10 minutes.
41| When the cells reach 70% confluence in isolation medium, detach the cells with trypsin as describe in steps 8-11.
42| Resuspend the cells in 1 ml of a solution made of pre-warmed 1:1 isolation medium:expansion medium.
43| Count the cells and resuspend at a cell density of 2 x 105 cells/ml
44| Add 1 ml of single cell suspension per well.
? TROUBLESHOOTING
45| The next day, check for cell viability (should be 100%) and replace the media with 3 ml of pre-warmed expansion medium per well
46| The pre-warmed expansion medium is then replaced daily
47| When the cells reach 70 % confluence, passage the cells
48| Prepare Matrigel plates and place the plates in the incubator with 2 ml per well of pre-warmed expansion medium to equilibrate for a minimum of 10 minutes.
49| Pre-warm the collagenase in a 37°C waterbath for 15 minutes
50| Aspirate carefully the medium from the wells
51| Add carefully 1 ml of collagenase on the side of the well without touching the cells
52| Place the plate back in the incubator for 7 minutes !CAUTION after 5 minutes of incubation, check the cells under light microscope. The cells must not detach completely but the sides of the cytoplasm should slightly detach while the nucleus remain attached. This might take 5 to 7 minutes depending on cell type.
53| Carefully aspirate the collagenase from the side of the well without touching the cells
54| Wash the well twice by gently pipetting 2 ml of room temperature DPBS on the side of the well, paying attention not to detach the cells
55| Add 1 ml of pre-warmed expansion medium per well
56| Gently scrape cells with a 1 ml pipette tip until cells are uniformly dispersed into small clumps (50 to 500 cells) !CAUTION do not triturate the cells to a single cell suspension
57| Add 2 ml of expansion medium
58| Add 1 ml of cell suspension into one well of a 6 well plate (1:3 split ratio)
59| Place the plate back into the incubator. The cells should now start growing as compact spherical colonies of small cells, which are difficult to disaggregate and with time increase in size on top of large fibroblastic cells arranged as flat colonies.
PAUSE POINT If needed, cells can be frozen and kept in liquid nitrogen long-term
Derivation of pluripotent cells TIMING ∼ 5-14 days
60| Prepare the reprogramming medium and pre-warm in a 37°C waterbath for 15 minutes
61| Remove the expansion medium from the cells and replace with 3 ml of pre-warmed reprogramming medium per well
62| Change the medium daily, for 5 days in total. !CAUTION At this stage, the cells will stop growing
? TROUBLESHOOTING
63| After 5 days, extract RNA from one well and synthesize cDNA. Using qRT-PCR, verify that the levels of expression of OCT4A, SOX2, c-MYC, KLF4 and FBX015 are upregulated
Stabilization of pluripotent lines TIMING ∼ 3 weeks
To stabilize the cells after reprogramming:
64| Remove reprogramming medium and rinse cells carefully with 2 ml DPBS
65| Replace the medium with 3 ml of pre-warmed expansion medium per well
66| When the cells reach 70% confluence, you can either passage the cells by following steps 48-59 or freeze the cells.
Characterisation of pluripotent lines
67| The pluripotency status of stabilised reprogrammed cells can be characterised using flow cytometry for cell surface markers (option A), flow cytometry for nuclear markers (option B), EB formation (option C), immunofluorescence (option D), quantitative real-time PCR (option E), teratoma formation (option F), or in vitro differentiation assays (option G).
REAGENT SETUP
EB differentiation medium: 80% (vol/vol) KO-DMEM supplemented with 1 mM L-glutamine, 0.1 mM b-mercaptoethanol, 1% non-essential amino acids stock and 20% FBS.
Immunofluorescence blocking solution: DPBS supplemented with 1 % (vol/vol) BSA, 0.2 % (vol/vol) fish skin gelatin and 0.1 % (vol/vol) casein (pH 7.6).
Hepatic differentiation medium: High glucose DMEM supplemented with 15% (vol/vol) FBS, 1 % (vol/vol) Penicillin/Streptomycin, 2 mM L-Glutamine, 300 μM Monothioglycerol, 20 ng/ml Hepatocyte Growth Factor, 10 ng/ml Oncostatin M, 10-7 Dexamethasone, 100 ng/ml Fibroblast Growth Factor 4 and 1X ITS (Insulin, Transferrin, selenium).
Ectoderm differentiation medium: DMEM/F12 (1:1) supplemented with 1 % (vol/vol) Penicillin/Streptomycin, 2 mM L-Glutamine, 0.6 % (vol/vol) glucose, 3 mM sodium bicarbonate, 5 mM HEPES buffer, 25 mg/ml insulin, 100mg/ml transferrin, 20nM progesterone, 60 mM putrescine, 30 nM selenium chloride, 20 ng/ml Epidermal Growth Factor, 10 ng/ml basic Fibroblast Growth Factor and 10 ng/ml Leukemia Inhibitory Factor.
Neuronal differentiation medium: High glucose DMEM supplemented with 0.5 % (vol/vol) FBS, 1 % (vol/vol) Penicillin/Streptomycin, 2 mM L-Glutamine and 0.1% (vol/vol) Baicalin.
CRITICAL STEP Neuronal medium must be made fresh and used immediately, as Baicalin becomes toxic for the cells when left at high concentration.
Oligodendrocyte differentiation medium: high glucose DMEM supplemented with 1% (vol/vol) Penicillin/Streptomycin, 2mM L-Glutamine, 1X N1 supplement, 1μg/ml biotin, 5ng/ml basic Fibroblast Growth Factor, 1ng/ml Platelet Derived Growth Factor and 30% B104 conditioned medium.
(A) Flow cytometry for cell surface markers: CD105, CD24, CD29, CD90, SSEA3, SSEA4, TRA-1-60, TRA-1-81 TIMING 3 hours
(i) Detach the cells as described in steps 49-56.
(ii) Collect the cells in a 15 ml falcon tube and centrifuge at 300g for 5 minutes.
(iii) Resuspend the cells in 1 ml of flow cytometry buffer and count using a haemocytometer.
(iv) Wash the cells 3 times by resuspending in 4 ml of flow cytometry buffer and centrifuging at 300g for 5 minutes after each wash.
(v) For cell surface staining, stain cells with antibodies for 1 h at 4°C.
(vi) When using unconjugated primary antibodies, wash cells twice in 4 ml of flow cytometry buffer, centrifuging at 300g for 5 minutes after each wash.
(vii) Add secondary fluorochrome-conjugated antibody and incubate for 30 min at 4°C.
(viii) Wash the cells 3 times by resuspending in 4 ml of flow cytometry buffer and centrifuging at 300g for 5 minutes after each wash.
(B) Flow cytometry for nuclear markers: OCT4A, SOX2, C-MYC, NANOG TIMING 3 hours
(i) Detach the cells as described in steps 49-56.
(ii) Collect the cells in a 15 ml falcon tube and centrifuge at 300g for 5 minutes.
(iii) Resuspend the cells in 1 ml of flow cytometry buffer and count using a haemocytometer.
(iv) Wash the cells 3 times by resuspending in 4 ml of flow cytometry buffer and centrifuging at 300g for 5 minutes after each wash.
(v) Fix cells in 2 ml of 0.01% PFA for 10 minutes at room temperature
(vi) Wash twice with 4 ml of DPBS, centrifuging at 300g for 5 minutes after each wash
(vii) Resuspend the cells in DPBS with 1% (vol/vol) Triton and centrifuge at 300g for 5 minutes
(viii) Stain cells with antibodies for 1 h at 4°C.
(ix) When using unconjugated primary antibodies, wash cells twice in 4 ml of flow cytometry buffer, centrifuging at 300g for 5 minutes after each wash.
(x) Add secondary fluorochrome-conjugated antibody and incubate for 30 min at 4°C.
(xi) Wash the cells 3 times by resuspending in 4 ml of flow cytometry buffer and centrifuging at 300g for 5 minutes after each wash.
(C) EB formation TIMING ∼ 25 days
(i) Detach the cells from 5-6 confluent wells of a six-well plate as described in steps 49-55.
(ii) Scrape the cells using an 18 cm cell scraper, taking care to detach the cells in small clumps. CRITICAL STEP Do not overdo it to avoid breaking up clumps of cells too much into single cells. Check under the microscope after scraping to ensure all cells have detached from surface of the well.
(iii) Plate the cells in a 100mm square low-attachment petri dish in 15 ml of EB differentiation medium, using a 5 ml plastic disposable pipette to transfer the cell solution.
(iv) To change medium twice per week transfer cells to a 50 ml falcon tube and centrifuge at 300g for 5 minutes.
(v) Carefully remove supernatant without disturbing the cell pellet, and resuspend pellet in 5 ml of EB differentiation medium using a 5 ml plastic disposable pipette
(vi) Transfer cell solution to a new low-attachment petri dish, and add 10 ml of EB differentiation medium.
(vii) Allow the cells to develop into 15 day old EBs.
(viii) Transfer EBs suspensions to gelatin-coated plates for another 7-10 days before fixation in 4% PFA and immunostaining for markers representative of the three embryonic germ layers, i.e. NESTIN and PAX6 (ectoderm), BMP4 (primitive endoderm), CK3, CK19 (endoderm), GATA6 (mesoderm), or SYCP1 (testis), with concomitant down-regulation of OCT4A. !CAUTION PFA is a toxic material. Avoid inhalation, ingestion and skin contact.
(D) Immunofluorescence TIMING 3 days
(i) Fix cells on coverslips in 1 ml of 4% PFA in 125 mM HEPES (pH 7.6) per well for 10 minutes at 4°C. !CAUTION PFA is a toxic material. Avoid inhalation, ingestion and skin contact.
(ii) Remove 4% PFA and replace by 1 ml of 8% PFA in the same buffer for 50 minutes at 4°C.
(iii) Remove 8% PFA and wash cells twice with 2 ml DPBS per well.
(iv) Transfer coverslips into a 24-well plate (1 coverslip per well).
(v) Permeabilize cells in 1 ml of 0.5% Triton in DPBS for 30 min at room temperature.
(vi) Remove Triton solution and wash cells 6 times with DPBS.
(vii) Incubate cells with 1 ml 20mM glycine in DPBS for 30 minutes at room temperature.
(viii) Remove glycine solution and wash cells 3 times with DPBS.
(ix) Incubate cells with 1 ml of immunofluorescence blocking solution for 1 hour at room temperature.
(x) Incubate cells with primary antibodies diluted in immunofluorescence blocking solution for 2 hours at room temperature.
(xi) Wash cells every 20 minutes with 1 ml of immunofluorescence blocking solution for a total of 5 times.
(xii) Incubate the cells with secondary antibodies diluted in immunofluorescence blocking solution for 2 hours at room temperature.
(xiii) Wash the cells in 1 ml of immunofluorescence blocking solution overnight at 4°C.
(xiv) Wash the cells 3 times with 1 ml DPBS
(xv) Mount the coverslips using 1 drop of VectaShield containing DAPI
(E) Quantitative real time PCR TIMING ∼ 8 hours
(i) Detach the cells as described in steps 49-56.
(ii) Collect the cells in a 15 ml falcon tube and centrifuge at 300g for 5 minutes.
(iii) Remove the supernatant and extract RNA from the pellet using the RNeasy minikit according to manufacturer’s instructions.
(iv) Synthesise cDNA using the Reverse transcription system kit, according to manufacturer’s instructions.
(v) Prepare one mastermix per gene of interest containing SYBR green PCR mastermix, custom oligonucleotide pairs and water, and aliquot into each well of a MicroAmp fast 96-well reaction plate, running each gene in triplicate for each sample.
(vi) Add cDNA (10-20 ng) to each well, mixing the cDNA into the mastermix.
(F) Teratoma formation assay TIMING ∼ 13 weeks
(i) Detach 2x106 cells as described in steps 49-56.
(ii) Collect the cells in a 15 ml falcon tube and centrifuge at 300g for 5 minutes.
(iii) Remove the supernatant and resuspend the cells in 30% (vol/vol) Matrigel on ice.
(iv) Inject the cells subcutaneously into the dorsal flank of 8-12 week old common γ chain -/-, RAG2-/-,C5-/- immunodeficient mice without preconditioning.
(v) Observe mice for the growth of solid tumours up to 12 weeks or up to 10mm3 tumour volume.
(vi) Dissect the formed teratomas, and proceed with histopathological and immunohistochemical analysis to confirm the presence of cell and tissue derivatives from all three embryonic germ layers.
(G) In vitro differentiation protocols
Hepatic differentiation TIMING ∼ 25 days
(i) Detach 2x106 cells as described in steps 49-56.
(ii) Collect the cells in a 15 ml falcon tube and centrifuge at 300g for 5 minutes.
(iii) Seed cells at a concentration of 5000 cells/cm2 on tissue culture plastic plates and coverslips coated with Matrigel in 3 ml isolation medium per well.
(iv) 3 days later, remove the isolation medium and wash each well carefully with 2 ml DPBS.
(v) Add 3 ml of pre-warmed hepatic differentiation medium per well.
(vi) Replace the medium every 3 days for 21 days in total. Collect the supernatant after each change and store at -80°C for analysis of urea secretion.
(vii) After 21 days, fix the cells with 4% (vol/vol) PFA and proceed with immunofluorescence analysis for the expression of Alpha Fetoprotein (AFP) and ALBUMIN. !CAUTION PFA is a toxic material. Avoid inhalation, ingestion and skin contact.
(viii) Measure urea in the supernatant using the Urea/Ammonia determination kit according to the manufacturer’s instructions.
Ectoderm differentiation TIMING ∼ 25 days
(i) Detach 2x106 cells as described in steps 49-56.
(ii) Collect the cells in a 15 ml falcon tube and centrifuge at 300g for 5 minutes.
(iii) Seed cells at a concentration of 3000 cells/cm2 on tissue culture plastic plates and coverslips coated with Matrigel in 3 ml of pre-warmed ectoderm differentiation medium.
(iv) Replace the medium every 3 days for 21 days in total.
(v) After 21 days, fix the cells with 4% (vol/vol) PFA and proceed with immunofluorescence analysis for the expression of NESTIN and VIMENTIN. !CAUTION PFA is a toxic material. Avoid inhalation, ingestion and skin contact.
Neuronal differentiation TIMING 9 days
(i) Detach 2x106 cells as described in steps 49-56.
(ii) Collect the cells in a 15 ml falcon tube and centrifuge at 300g for 5 minutes.
(iii) Seed cells at a concentration of 5000 cells/cm2 on tissue culture plastic plates and coverslips coated with Matrigel in 3 ml of isolation medium for 1 day.
(iv) Remove the isolation medium from the wells and wash with 2 ml of DPBS.
(v) Replace with 3 ml of pre-warmed, freshly made neuronal differentiation medium per well.
(vi) Add one co-culture membrane insert per well, and plate C17.2 mouse neural progenitor cells at a concentration of 10,000 cells/ cm2 into the insert.
(vii) Allow the cells to differentiate for 5 days without changing the medium.
(viii) After 5 days, fix the cells with 4 % (vol/vol) PFA and proceed with immunofluorescence analysis for the expression of the neuronal markers β-TUBULIN and MAP2 and for the NMDA receptor NR1. !CAUTION PFA is a toxic material. Avoid inhalation, ingestion and skin contact.
Oligodendrocyte differentiation TIMING 9 days
(i) Detach 2x106 cells as described in steps 49-56.
(ii) Collect the cells in a 15 ml falcon tube and centrifuge at 300g for 5 minutes.
(iii) Seed cells at a concentration of 5000 cells/cm2 on tissue culture plastic plates and coverslips coated with Matrigel in 3 ml of oligodendrocyte differentiation medium for 1 day.
(iv) The following day add one co-culture membrane insert per well, and plate CG4 rat oligodendrocyte progenitor cells at a concentration of 10,000 cells/ cm2 into the insert.
(v) Replace the medium every 2 days with 3 ml of pre-warmed oligodendrocyte differentiation medium per well, for 5 days in total.
(vi) After 5 days, fix the cells with 4% (vol/vol) PFA and proceed with immunofluorescence analysis for the expression of the oligodendrocyte markers O2 and NG2. !CAUTION PFA is a toxic material. Avoid inhalation, ingestion and skin contact.