Experimental differentiation of embryonic stem cells to cardiomyocytes
Experimental differentiation of embryonic stem cells to cardiomyocytes
Describes several methods used to generate and differentiate cardiomyocytes differentiated from hESC, which are as effective as the methods used to isolate cardiomyocytes into single-cell suspensions, the preparation of which can be used for antibody labeling to differentiate the cells and for electrophysiological detection of the cells, and which are also used in the preparation of cells implanted in the (animal) heart. Author: Stacey et al, Translated by Jingbo Zhang, from "Human Stem Cell Culturing
Operation method
Differentiation of human embryonic stem cells into cardiomyocytes Move I. Preparation of medium and reagents 1. Feeder cell culture medium I M E F medium The "cut-and-paste" approach has been reported to be the most effective for preserving karyotypic stability. However, most hESC cell lines can be adapted to high-volume culture methods (trypsin digestion, collagenase digestion), and they can be cultured on top of mouse embryonic fibroblasts or human feeder cells (e.g., foreskin fibroblasts). In our experiments, routine passaging of hES2 and hES3 cells, performed once a week, was cultured on top of M EFs. During passaging, the colony diameter is usually 0.5 ~ £ mm, but no confluence occurs. Reagents and Materials Sterile or aseptic preparation □ h E S 2 or h E S 3 cells cultured in organ culture dishes □ M E F feeder layer cultured in organ culture dishes, freshly prepared Standard hESC culture medium □ Dulbecco's P B S (D -P B S ) containing C a 2+ and M g 2 . □ Neutral protease solution, 10 m g /m l in standard h E S C medium, freshly prepared, filter sterilized. □ 2 Petri dishes, 3.5 cm, add D-PBS and incubate at 37°C in an incubator. □ Glass needles were heated over a flame, elongated and broken into two pieces. Sterilize at high pressure before passaging. □ sterile filter, 0.22um □ Yellow colored pipette tip (10~200ul) Non-sterile Micro Pipette, 100ul □ 4 X magnification stereo dissecting microscope (with heated stage if possible) Steps (a) Examine the culture and select a petri dish containing undifferentiated hESC colonies. (b) Cut the colony using a glass needle (similar to cutting a pizza) and select the undifferentiated pieces, and pass them on to a new dish (Figure 4.1) Removing the differentiated portion is important for long-term culture of undifferentiated cells. This is most conveniently done under a stereo dissecting microscope with a 4 X magnified field of view. (c) Remove the medium and add 0.5 m I neutral protease solution. (d) Place the petri dish in an incubator for about 2 min or, if possible, on a heated microscope stage. (e) Remove the preheated petri dish containing D-PB S from the incubator. (f) Pick out undifferentiated colonies from the neutral protease-treated dishes; use an I00ul pipette with a yellow tip to transfer the colonies to the first dish containing D-PB S, and then to the second dish, which results in two washes with D-PBS. (g) Distribute these colonies evenly into a freshly prepared organ culture dish containing the M EF feeder layer (see Scheme 4. 5), which contains I m l of standard hESC culture medium (see section 4. 3. 2), and contains approximately 9 colonies per dish. Colonies should not be inoculated too close to the edge of the dish or one next to the other in order to provide enough space for colony growth and to facilitate the next passaging. (h) Carefully place the organoids in a 5 % C02, 37°C incubator. (i) Change the medium daily with standard hESC medium. Reagents and Materials Sterile or aseptic preparation The starting material for two 12-well hESC-END2 co-culture plates is: 12 organoids, each containing 9 to 10 colonies (hES2 or hES3) (see Scheme 4.1). 12-well plates containing mitomycin C-treated END2 cells (see Scheme 4. 6). □ serum-free hESC medium (see section 4. 3. 2. 2). Standard hESC medium (see section 3.2.1). □ D-PBS □ Neutral protease digest, 10 mg/m l dissolved in standard hESC medium, freshly prepared, filtered for sterilization. □ Blue pipette tip (200~lOOOpl). Non-sterile □ I m l Pipette Steps (a) At least I.5 h prior to inoculation of hESC cell clusters, exchange END2 cells with F B S-free hESC cultured with a _realistic mitomycin C-treated END2 cell. (b) Prepare petri dishes for washing undifferentiated hESC colonies: i) Add D- P B S solution to six 3. 5 cm diameter flat dishes. ii) Add D- P B S solution to six 3. 5 cm diameter flat dishes. iii) Add D- P B S solution to six 3. 5 cm diameter flat dishes. ii) Add I ml of standard hESC medium (i.e., containing FBS) to both organoids. iii) Place all dishes in an incubator at 37°C, 5 % C0 2 . (c) Add 0.5 m l of neutral protease to the organoid petri dishes and place them in the incubator for 7 m in, to isolate the hESC colonies from the M E F s . (d) Collect all undifferentiated hESC colonies from 1 2 organoids using an Im l pipette with a blue tip and wash them in 3 flat dishes pre-filled with D- P B S. The hESC colonies will be separated from the M E F s by adding 0.5 m l of neutral protease to the organoids and placing them in an incubator for 7 m in. (e) Transfer the colonies to 3 new petri dishes containing D-P B S (remove M E F s cells attached to the colonies). (f) Transfer these colonies to 2 organotypic dishes containing Iml standard hESC medium. (g) Blow vigorously up and down (2-3 times depending on the size of the colonies) with an Im l pipette against the bottom of the Petri dish to disperse the colonies into small pieces (see Scheme 4.1). (h) Transfer the hESC microcolony to two 12-well dishes (containing pooled mitomycin C-treated END2 cells). (j) One or two days after inoculation, the pulsatile areas are evaluated by microscopic examination. The pulsation usually begins on days 5 to 7 and is strongest on day 12. Immunostaining with antibodies against actin (diluted 1:800) and pro-myosin (diluted 1:400) shows the number of cardiomyocytes in the pulsatile zone, which is usually 20% to 25%. As an alternative, protein tracing can also be used for quantification (e.g., with troponin I antibody; 1:100 dilution). Reagents and Materials aseptic □ co-culture dish (see Option 4.2) Standard hE SC medium (see Section 4.3.2.1) Separation buffer (see Section 4.3.3 and Table 4.I) Buffer 2, Enzyme Buffer Buffer 3, K B Petri dish, 3.5 c m □ 0. 1 % gelatin-coated coverslips or petri dishes □ Blue colored pipette tip (200~IOOOul) □ Fine surgical scissors (e.g., iridectomy scissors) Non-sterile Paraffin Sealing Film □ Im l pipette Chapter 4 Differentiation of Human Embryonic Stem Cells into Cardiomyocytes Steps (a) Use scissors to cut and separate the pulsatile region from within the co-culture plate and collect the cut tissue in standard h E S C medium containing F B S . (b) Three buffers are required for the isolation process (see section 4.3.3). To begin the separation, use a pipette with a blue tip to transfer small pieces of cut tissue to a flat dish containing Buffer 1 and keep it at room temperature for 30 m i n . (c) Transfer the cell pellet from Buffer 1 to Buffer 2 at 37 . (Incubate for about 45 m i n (seal the mouth of the dish with sealing film before transferring to the CO2 incubator). (d) Transfer the cell pellet from Buffer 2 to Buffer 3 and incubate gently on a nonpivoting shaker at 100 rpm for 1 h at room temperature. (e) Transfer the cell pellet from Buffer 3 to standard hESC medium containing 20% FBS to promote adhesion and survival. Disperse the cell pellet by blowing up and down (2 to 4 times) against the bottom of the dish with an I m l pipette. The degree of dispersion required depends on the different experiments performed with cardiomyocytes: i) transplantation into animals or immunofluorescence staining, obtaining a mixture of cell clusters and single cells is sufficient for the experiment. ii) Electrophysiological tests require dispersed single cells. Reagents and Materials aseptic Pregnant mice of gestational age were recorded by examination of vaginal plugs (= 0 days). □ MEF medium (see section 4.3.1) □ Freezing medium: MEF medium with 20 % DMSO □ PBSA: D-PBS without Ca2+ and Mg2+. □ Trypsin/EDTA : 0.5 % trypsin, 0.5 mmol/L EDTA dissolved in Ca2+ and Mg2+ free D-PBS. 0.5 % trypsin, 0.5 mmol/L ED T A in Ca2+ and Mg2+ free Hank's B S S Centrifuge tubes, 15 ml □ Freezing tubes (cryopreservation tubes) □ Separation equipment □ injectors, 18G and 21G needles Steps (a) Painless execution of female rats at E 13.5 days of pregnancy. (b) The embryos were isolated and washed once with P B SA: i) Remove the uterus from the mouse and isolate each embryo [Freshney, 2005]. ii) Remove the head and internal organs (liver, heart, lungs, etc.). iii) Wash the embryos twice with PBSA. (c) Transfer the embryos into 3 cm diameter bacterial flat dishes with two embryos per dish. (d) Add a small amount of PBSA and cut the two embryos into tiny pieces with two crossed surgical blades. (e) Add I m l trypsin/ED TA to each embryo. (f) Leave at room temperature for IOmin, while gently shaking the dish. Alternatively, the embryos were placed at 4°C for 18 h and incubated at 37°C for 100-20 min; this method, which improves yield and viability, has been described (see Freshney [2005]). (g) The digested tissue was separated into a single cell suspension using a syringe with a pink 18 G needle followed by a green 21 G needle. The cell suspension was transferred to test tubes and 5 ml of M EF medium was added to each embryo (see section 4.3.1). (h) Sediment the large tissue fragments and transfer the supernatant to a clean centrifuge tube and centrifuge at 250 g for 5 min. (i) Resuspend the cell sediment with M E F medium and inoculate the suspension in 175 cm2 tissue culture flasks at approximately one embryo per flask. (j) Incubate the cells for 24 to 48 h and trypsinize them. (k) Resuspend cells in M E F medium. (l) Mix with freezing liquid I : 1 and dispense into freezing tubes (cryotubes), two tubes per embryo. (m) Freeze according to standardized steps (see Freshney [2005], Chapter 20) □ mitomycin C storage solution, 2 mg/ml □ PBSA □ Trypsin/EDTA □ Tissue culture flask, 25 cm2 , coated with 0.1 % gelatin (see section 4.3.4). □ Tissue culture flasks, 175 cm2 , coated with 0.1 % gelatin (see section 4.3.4). □ Multiwell plate, 12 wells, coated with 0.1 % gelatin (see section 4.3.4). 1 % gelatin (see section 4.3.4). □ Coverslips coated with 0.1 % gelatin in 12-well plates (see section 4.3.4). Steps (a) On day 1 (preferably Monday), inoculate END2 cells with END2 medium in a 25 cm2 tissue culture flask coated with 0.1% gelatin (see section 4.3.1.2). END2 cells are transferred from the confluent culture flasks at a ratio of 1 : 8. (b) On day 5, all END2 cells from the previous 25 cm2 culture flask are inoculated into a 175 cm2 culture flask coated with 0.1% gelatin. (C) On day 8, cells in the 175 cm2 culture flask should be grown to 100 % confluence and ready for mitomycin c treatment as described for M E F s (see Scheme 4. 5). (d) Add mitomycin C at a final concentration of lOug/ml by adding 5 ul of 2 mg/ml stock solution per m l of medium. (e) Incubate the culture flasks at 37°C for at least 2% of the incubation time and up to 3 hours. (f) Aspirate the medium; wash the wells once with END2 medium and twice with PBSA. Note: Waste solution containing mitomycin C is highly toxic. (g) Trypsin digestion and cell counting. (h) Cells were resuspended with END2. (i) When further experiments are required, inoculate cells at a cell concentration of 1.75X l05 m l in a 12-well plate coated with 0.1% gelatin or a 1.5-cm diameter coverslip coated with gelatin. 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(h) Transfer of the hESC cell clusters into two 1 2-well dishes (containing pooled mitomycin C-treated END2 cells).
(i) Change the medium on days 5, 9 and 12.
Buffer 1, Low C a 2+
END2 medium (see section 4.3.1.2)