1) Human iPSC culture (feeder-free)
The here described induction and differentiation conditions are based on the feeder-free attachment (2D) culture of human induced pluripotent stem cells (iPSCs) 201B77 and 1231A38 on laminin-coated (iMatrix-511 silk, Nippi) sterile cell culture dishes with StemFit®AK02N (Reprocell) being the iPSC-culture and maintenance medium of choice. Stable culture of fully reprogramed high-quality human iPSCs is necessary for subsequent successful differentiation steps and efficient induction of mesodermal cell types described in this protocol exchange. The here defined culture and differentiation conditions are compatible with and can be applied to other high-quality human iPSC-lines (e.g. 428C2)9. Please note that for human embryonic stem cells (ESCs) and pluripotent stem cells of other species culture conditions may need to be optimized.
The here exemplified culture and differentiation conditions are for single-cell dissociation and culture of human iPSCs in a laminin-coated 6 well culture plate. Please adjust initial seeding densities and volumes of culture (maintenance) media according to the sterile cell culture plates and dishes utilized for initial feeder-free iPSC culture (see Table 1 for details).
1.1 Coating of cell culture dishes & plates for feeder-free iPSC culture
1. Described conditions are for a sterile 6 well culture plate but can be adjusted to other culture plates and dishes accordingly. (see Table 1 for details)
2. Add 1.5 ml of PBS containing iMatrix-511 silk (0.5 µg/cm2) into each well of a 6 well sterile culture plate. Other Laminin-511 fragment sources/types such as iMatrix-511 (Nippi, Cat# 892012) can be also used.
3. Incubate the plate with coating agent either for >1 hour at 37°C, for >3 hours at room temperature or overnight at 4°C. Usually coating is performed overnight at 4°C.
4. Add 750 µl (or half the volume of initial iMatrix-511 solution) of StemFit®AK02N and mix thoroughly.
5. Aspirate the medium and add 1.5 ml of StemFit®AK02N containing Y-27632 (10 µM). Place plate into humidified CO2 incubator at 37°C. Do not let the plate dry out when changing solutions.
1.2 Passaging of human feeder-free iPSCs
1. Prepare ~ 80% confluent culture of human feeder-free iPSCs.
2. Aspirate the culture medium.
3. Wash the iPS cell colonies with PBS.
4. Add 300 µl of 0.5x TrypLETM Select enzyme solution (diluted with 0.5 mmol/l-EDTA/PBS); distribute it evenly, and put the plate into a humidified CO2 incubator set to 37°C.
5. After 1 minute, take out the plate and re-distribute TrypLETM Select enzyme solution and put it back into the incubator (for another 3 minutes).
6. Take out the plate and observe the cells under the microscope whether they have started separating/detaching; if not, incubate for another 30 seconds to 1 minute.
7. Aspirate the solution and wash with PBS gently.
8. Aspirate PBS and add 1 ml of StemFit®AK02N with Y-27632 (10 µM).
9. Harvest cells with cell scraper into a 1.5 ml microtube.
10. Dissociate the collected cells gently by pipetting up and down ~ 10 times (using 1,000 µl tip) and measure cell density.
11. Seed 1.3x104 cells/well onto iMatrix-511 silk-coated 6 well culture plate containing pre-warmed medium (for other plates or dishes see Table 1).
12. Distribute cells evenly by gentle shaking and incubate plate with cells in humidified CO2incubator at 37°C.
13. On the next day, change the medium to StemFit®AK02N without Y-27632.
14. Change medium every other day unless the medium color turns yellow or iPSCs start to prematurely differentiate; if so, change the medium every day.
15. Passage once every week.
1.3 Preparing freezing stocks of feeder-free iPSCs
Initial steps are same with the procedure for passaging until step 10.
1. Transfer the required number of cells collected from the culture plate or dish into a 1.5 ml microtube and centrifuge (800 rpm, 22°C, 5 minutes).
2. Remove the supernatant and resuspend the cells with Stem-cellbanker (Takara) (1.0x106 cells/ml).
3. Add 200 µl of cell suspension (2.0x105 cells) into cryogenic tube (Thermo Fisher Scientific).
4. Freeze vials in pre-cooled freezing container at -80°C for overnight.
5. On the next day, transfer frozen vials into a liquid nitrogen tank.
1.4 Thawing and culture of frozen feeder-free iPSC stocks
1. Set water bath to 37°C.
2. Add 5 ml of StemFit®AK02N with Y-27632 (10 µM) into a 15 ml tube.
3. Thaw a frozen vial of iPSCs in the water bath until only a few small ice particles remain.
4. Transfer the cells into a 15 ml tube and centrifuge (800 rpm, 22°C, 5 minutes).
5. Aspirate the supernatant and add 500 µl of StemFit®AK02N with Y-27632.
6. Dissociate the cells gently by pipetting (about 10 times) and measure the cell density using hemocytometer or automated cell counter.
7. Seed 1.3x104 single cells/well onto laminin-coated 6 well culture plate. Make sure that viability of cells is high and seed appropriate number of living cells into utilized culture plates or dishes.
2) Stepwise induction of human somitic mesoderm and its major derivatives
Feeder-free cultured human iPSCs are utilized for stepwise somitic mesoderm induction at day 5 post single-cell dissociation and culture. Human iPSC colonies should be clearly visible and the condition of cells should be excellent without presence of prematurely differentiating cells in the culture. For all here described differentiation and induction conditions of human somitic mesoderm and its derivatives a chemically defined medium (CDM)10 is used as base medium. Composition of the utilized CDM is described in Table 2. All growth factors and small molecules necessary at each induction step should be added to CDM just before use of media. A master mix of the required induction media can be made and distributed into each well/dish as needed. Exemplified induction protocols are based on culture of human iPSCs in a 6 well sterile culture plate. Necessary minimal media volumes for different plates or dishes are described in Table 3. Our stepwise somitic mesoderm induction protocol is similar to a recent published protocol, albeit with some differences4.
2.1 Preparation of Chemically Defined Medium (CDM)
1. Perform all described steps under a clean-bench; use sterile tips and bottles (see Table 2 for composition of CDM).
2. Dissolve bovine serum albumin (BSA) (10 mg/ml) in Ham’s F-12 Nutrient Mix (F12) and add Penicillin-Streptomycin (P/S) (100 unit/100 µg/ml).
3. Pass dissolved mixture through filter bottle (0.22 µm).
4. Mix filtered BSA and P/S containing F12 medium with IMDM (1:1).
5. Add sterile reconstituted Apo-transferrin (15 µg/ml), 1-Thioglycerol (450 µM), CD Lipid (1%) and rh-Insulin (7 µg/ml) and store at 4°C until use. Concentrations given in brackets are the final concentrations at the time of adding components. CDM should be used up within two weeks.
2.2 Primitive Streak (PS) induction
PS cells are induced via activation of FGF, WNT and TGFβ signaling pathways in human feeder-free cultured iPSCs. Adjust induction media volumes to utilized type of dishes or plates (Table 3).
1. Prepare PS induction medium by adding CHIR99021 (10 µM), rh-Activin A (50 ng/ml) and rh-bFGF (20 ng/ml) to CDM. See Table 4 for details. Usually a master mix of the required induction medium is made and distributed into each well or dish as needed to reduce pipetting errors and well-to-well variability.
2. Prior to induction of PS cells, iPSCs are cultured in humidified CO2 incubator on laminin-coated plates for 5 days from initial seeding of iPSCs at single cell level. At time of induction clear epithelial colonies of iPSCs have formed and are visible under the microscope. If iPSC conditions are not good (e.g. many spontaneously differentiated cells are present or colony sizes/shapes are unusual) do not use cells for induction.
3. Aspirate the maintenance medium (StemFit®AK02N).
4. Add 2 ml of PS induction medium into each well (for volume of induction medium to be added when using other plates or dishes see Table 3).
5. Culture iPSCs in PS induction medium at 37°C for 24 hours in humidified CO2 incubator.
6. Use induced PS cells for subsequent differentiation into human presomitic mesoderm (PSM) cells; use PS cells for characterization and quality control (i.e. RNA and protein isolation, immunocytochemistry, FACS and quantitative PCR (qRT-PCR) etc.).
2.3 Presomitic Mesoderm (PSM) induction
PSM cells are induced from human iPSC-derived primitive streak (PS) cells (for details on how to induce PS cells see 2.2). Our PSM induction protocol is similar to a recent published protocol, albeit with some differences4. Other protocols aiming to induce presomitic mesoderm from human pluripotent stem cells (hESCs or hiPSCs) have also been reported2,3,5,6. Following the 24 hours of initial PS induction, induce PSM cells by simultaneous activation of WNT and FGF signaling and inhibition of TGFβ and BMP signaling pathways.
1. Prepare PSM induction medium by adding CHIR99021 (3 µM), LDN193189 (250 nM), rh-bFGF (20 ng/ml) and SB431542 (10 µM) to CDM. See Table 5 for details on components of the PSM induction medium. Usually a master mix of the required induction medium is made and distributed into each well or dish as needed to reduce pipetting errors and well-to-well variability.
2. Aspirate previous (PS) induction medium.
3. Add 2 ml of freshly made PSM induction medium.
4. Culture cells in humidified CO2 incubator at 37°C for 24 hours.
5. Use induced PSM cells for subsequent differentiation into human somitic mesoderm (SM) cells or analysis of in vitro features of induced PSM cells including e.g. oscillatory expression of segmentation clock genes. For details on measurement and visualization of oscillatory expression of segmentation clock genes in in vitro induced human PSM cells see our concurrent paper in Nature and related recent papers by other groups2. In case of bioluminescent measurement of oscillatory gene activity, in vitro induced PSM cells derived from a luciferase-reporter line of e.g. human HES7 are cultured in CDM-based PSM medium containing D-luciferin (BMS). The presence of bioluminescent activity is measured using a luminometer set-up which allows extended culture of PSM cells in vitro (Atto). The here described PSM induction approach can also be used to measure and visualize oscillatory activity in other human and non-human pluripotent stem cells, but may require further adjustments and optimization of the culture conditions e.g. adjustment of the level of WNT activity during in vitro culture. Other human in vitro PSM induction protocols can also be used to characterize aspects of the in vitro human segmentation clock2,11. Already described in our concurrent paper1, stepwise in vitro induced PSM cells can be utilized for further molecular characterization and functional analysis as well as quality control e.g. RNA and protein isolation, immunocytochemistry, flow cytometric (FACS) analysis and quantitative PCR (qRT-PCR). Brief descriptions of these methods making use of the induced cells are given below (see 3.1 – 3.5).
2.4 Somitic Mesoderm (SM) induction
SM cells are induced from human in vitro derived presomitic mesoderm (PSM) cells via inhibition of FGF and WNT signaling pathways for 24 hours (for details on how to induce human PSM cells from iPSCs see 2.3).
1. Prepare SM induction medium by adding PD173074 (100 nM) and XAV939 (1 µM) to CDM. See Table 6 for details on how to prepare the SM induction medium. Usually a master mix of the required induction medium is made from higher concentrated stocks and distributed into each well or dish as needed to reduce pipetting errors and well-to-well variability.
2. Aspirate previous (PSM) induction medium.
3. Add 2 ml of freshly made SM induction medium.
4. Culture cells in humidified CO2 incubator at 37°C for 24 hours.
5. Use induced SM cells for subsequent differentiation into human sclerotome (SCL) or dermomyotome (DM) cells; utilize SM cells for further characterization and quality control (e.g. RNA and protein isolation, immunocytochemistry, FACS and quantitative PCR (qRT-PCR)).
2.5 Dermomyotome (DM) induction
DM cells are induced from human iPSC-derived somitic mesoderm (SM) cells (for details on how to induce SM cells see 2.4). DM cells are induced by activating WNT and BMP signaling pathways and inhibiting hedgehog (HH) signaling for 48 hours4.
1. Prepare DM induction medium by adding CHIR99021 (3 µM), GDC-0449 (150 nM) and rh-BMP4 (50 ng/ml) to CDM. See Table 7 for details on how to prepare the DM induction medium. Usually a master mix of the required induction medium is made from higher concentrated stocks and distributed into each well or dish as needed to reduce pipetting errors and well-to-well variability.
2. Aspirate previous (SM) induction medium.
3. Add 2 ml of freshly made 2 ml of DM induction medium.
4. Culture cells in humidified CO2 incubator at 37°C for 48 hours.
5. Use induced DM cells for subsequent differentiation into human skeletal muscle cells in vitro or in vivo; utilize DM cells for further characterization and quality control (e.g. RNA and protein isolation, immunocytochemistry, FACS and quantitative PCR (qRT-PCR)).
2.6 Sclerotome (SCL) induction
SCL cells are induced from stepwise in vitro derived human somitic mesoderm (SM) cells (for details on how to induce SM cells see 2.5). Human SCL cells are induced by simultaneous activation of the hedgehog (HH) signaling pathway and inhibition of BMP signaling for 72 hours as previously reported in the context of murine ESC-based in vitro chondrogenic induction12.
1. Prepare SCL induction medium by adding LDN193189 (600 nM) and SAG (100 nM) to CDM. See Table 8 for details on how to prepare human SCL induction medium. Usually a master mix of the required induction medium is made from higher concentrated stocks and distributed into each well or dish as needed to reduce pipetting errors and well-to-well variability.
2. Aspirate previous (SM) induction medium.
3. Add 2 ml of freshly made SCL induction medium.
4. Culture cells in humidified CO2 incubator at 37°C for 72 hours.
5. Use induced SCL cells for subsequent differentiation into 2D chondrocytes, for 3D in vitro human cartilage induction or in vivo xeno-transplantation (described briefly below); utilize SCL cells for further molecular and functional characterization and quality control (e.g. RNA and protein isolation, immunocytochemistry, FACS and quantitative PCR (qRT-PCR)).
3) Functional analysis and further differentiation of induced human somitic mesoderm derivatives
3.1 In vitro skeletal muscle induction from human DM cells
Human skeletal muscle cells are derived in vitro from stepwise-induced dermomyotome (DM) cells. Protocols for the induction of human skeletal muscle cells from iPSCs have been reported previously3,13,14. The protocol described here for skeletal muscle induction from stepwise in vitro induced human DM cells is similar to the protocol published by Chal et al.13. For further molecular and functional characterization of the in vitro DM-derived human skeletal muscles cells see our concurrent paper in Nature1.
3.1.1 Plate coating for in vitro skeletal muscle induction from human DM cells
1. Dilute Matrigel® (Corning) with DMEM (Nacalai, Cat#: 08456-65) (1:50).
2. Add 500 µl of the Matrigel® solution to each well (12 well sterile culture plate).
3. Incubate for >1h at 37°C, for >3h at room temperature or overnight at 4°C. Usually coating is performed overnight at 4°C. Aspirate the coating solution and add 1 ml of the skeletal muscle induction medium and pre-warm in incubator prior to use.
3.1.2 In vitro skeletal muscle induction protocol
The here-exemplified protocol utilizes stepwise in vitro derived human dermomyotome (DM) cells induced in 10 cm sterile culture dishes.
1. Wash the DM cells with PBS.
2. Add 1 ml of AccutaseTM and distribute it evenly.
3. Incubate for 2 minutes.
4. Take out the plate, re-distribute the AccutaseTM and incubate it for another 2 minutes.
5. Observe the cells under the microscope whether they are starting to detach or not; if not, incubate for another 30 seconds to 1 minute.
6. Add 4 ml of the induction medium with Y-27632 (10 µM) for neutralization.
7. Harvest cells with cell scraper into a 15 ml tube and centrifuge (800 rpm, 22°C, 4 minutes).
8. Aspirate the supernatant and add 3 ml of the induction medium with Y-27632 (10 µM).
9. Dissociate the cells gently by pipetting and measure the cell concentration.
10. Seed 2.5x105 DM cells/well onto Matrigel® coated 12 well culture plate.
11. On the next day, change to induction medium without Y-27632.
12. Cells are cultured in a humidified CO2 incubator set at 37°C for 3 weeks. Change skeletal muscle induction medium every 3 days (see Table 9). Spontaneously contracting colonies of induced human skeletal muscle cells will appear within two weeks of culture. In vitro induced human skeletal muscle cells can be used for further molecular and functional characterization including immunocytochemistry or imaging of spontaneous calcium activity using a dye-based calcium reporter system or a GCaMP reporter line (Gen1C)15.
3.2 In vitro 3D chondrogenic induction (3D-CI) of human SCL cells
Human 3D cartilage can be induced from stepwise in vitro derived human sclerotome cells. Our human SCL-based 3D chondrogenic induction (3D-CI) approach is utilizing a previously described chondrogenic induction medium16.
3.2.1 In vitro 3D chondrogenic induction (3D-CI) protocol
Human SCL derived 3D cartilage induction is performed in floating culture using 60 mm low attachment dishes (Sumitomo Bakelite). Floating human sclerotome-derived 3D cartilage is emerging within the first 3-4 weeks and increasing in size and maturation state during further culture. The here-exemplified protocol is for stepwise-induced human sclerotome (SCL) cells derived in 10 cm sterile culture dishes.
1. Wash the SCL cells with PBS.
2. Add 1 ml of AccutaseTM and distribute it evenly.
3. Incubate for 2 minutes.
4. Take out the plate, re-distribute the AccutaseTM and incubate for another 2 minutes.
5. Observe the cells under the microscope whether they are detached or not; if not, incubate for another 30 seconds to 1 minute.
6. Add 4 ml of CDM with Y-27632 (10 µM) for neutralization.
7. Harvest cells with cell scraper into a 15 ml tube and centrifuge (800 rpm, 22°C, 4 minutes).
8. Aspirate the supernatant and add 3 ml of the SCL induction medium with Y-27632 (10 µM).
9. Dissociate the cells gently by pipetting and measure cell concentration.
10. Prepare cell suspension (2.0x106 cells/ml) in SCL induction medium with Y-27632 (10 µM).
11. Transfer 100 µl/well of the cell suspension (2.0x105 cells) in 96 well low attachment plate to make 3D-SCL spheres and incubate for overnight.
12. Take out 3D-SCL spheres into a 35 mm dish and wash them with base medium twice.
13. Add supplemental growth factors just before using.
14. Add 7 to 10 ml of 3D-CI medium into the low attachment dish (Sumitomo Bakelite). (add rh-bFGF from 3 days of culture)
15. Distribute all spheres evenly to not let them fuse to each other.
16. Change the medium every 3 days (see Table 10 for composition of medium).
17. Culture 3D-SCL spheres in humidified CO2 incubator set at 37°C for 30 days or longer depending on experimental schedule. 3D cartilage should appear in culture within 2 to 3 weeks. Induced human 3D cartilage can be used for in vivo transplantation (described briefly below) and in vitro characterization e.g. RNA isolation and sectioning. For immunostaining and sectioning, in vitro derived human 3D cartilage should be fixed with 4% PFA for overnight at 4°C and then embedded into paraffin.
3.2.2 In vivo xeno-transplantation of in vitro induced human SCL cells
In vitro induced human SCL cells can be transplanted into immunodeficient (SCID) mice for in vivo formation of iPSC-derived cartilage and endochondral bone. The cartilage and bone forming ability of stepwise induced human SCL cells are described in our concurrent paper in Nature. The here described protocol is briefly summarizing the in vivo xeno-transplantation of in vitro induced human SCL cells. SCL cells should be treated as described for 3D chondrogenic induction until step 9 (see 3.2.1).
1. Prepare SCL cell suspension (5.0x105-1.2x106 cells/100 µl) in SCL induction medium with Y-27632 (10 µM).
2. Mix 100 µl of cell suspension with the same amount of Matrigel®.
3. Inject mixture of cells and Matrigel® into NOD/ShiJic-scidJcl mice subcutaneously using 1ml syringe with 26 G needle.
4. Evaluate after two months (or longer) the formation of hard cartilage/bone tissue from the transplanted human sclerotome cells in the recipient mice using e.g. µCT analysis or excision and histological analysis of formed tissues.
5. In case of xeno-transplantation of 3D cartilage, make an incision into the back of NOD/ShiJic-scidJcl mice, transplant ~20-30 of in vitro induced human 3D cartilage and suture the incision. Evaluate the formation of endochondral bone from transplanted 3D cartilage two months post-transplantation using e.g. µCT analysis or excision and histological analysis of emerged tissues.
3.3 Immunocytochemistry of stepwise in vitro induced cells
The here-exemplified protocol is for stepwise induced cells (PS, PSM, SM, DM, SCL) and iPSCs grown in 12 well sterile culture plates.
1. Aspirate the induction or maintenance medium.
2. Wash the samples with PBS (1 ml).
3. Immerse in 2% PFA for 30 minutes at room temperature.
4. Aspirate added PFA and wash the samples twice with PBS (1 ml).
5. Permeabilize with 0.2% TritonTM X-100 in PBS for 10 minutes at room temperature and then wash with PBST (1% TWEEN®20 in PBS).
6. Samples are blocked in 5% skim milk in distilled water (DW) for 1 hour at room temperature.
7. Aspirate the blocking solution.
8. Stain samples with primary antibodies diluted in 10% of blocking solution (5% skim milk in DW) for overnight at 4°C.
9. Aspirate the primary antibodies and wash the samples with PBST three times and stain with secondary antibodies for 1 hour at room temperature.
10. Aspirate the secondary antibodies and wash the samples with PBST twice and stain with DAPI (2 µg/ml) for 10 minutes at room temperature.
11. Wash the samples with PBST.
12. Store the samples at 4°C shaded from light until microscopic evaluation.
3.4 FACS analysis of stepwise in vitro induced cells
The here-exemplified protocol is for stepwise induced cells (PS, PSM, SM, SCL, DM) and iPSCs grown in 6 well sterile culture plates.
1. Aspirate the medium.
2. Wash the cells with PBS.
3. Add AccutaseTM and incubate for 4 minutes at 37°C.
4. Neutralize the cells with CDM with Y-27632 (10 µM) and collect with cell scraper.
5. Measure the concentration of cell suspension and centrifuge (800 rpm, 4 minutes, 4°C)
6. Aspirate the supernatant.
7. Resuspend the cells in FACS buffer (0.1% BSA in PBS) with Y-27632 (10 µM) (1.0x107 cells/ml).
8. Stain the cells with primary antibody or conjugated antibody (or isotype control) for 30 minutes at 4°C protected from light.
9. Wash the cells with PBS and centrifuge (800 rpm, 4 minutes, 4°C).
10. Aspirate the supernatant.
11. Resuspend the cells in FACS buffer with Y-27632 (10 µM) (5.0x106 cells/ml) and stain the cells with DAPI (500 ng/ml).
12. Filtrate the cells through a fine mesh.
13. Analyze with cell analyzer.
14. In case of co-staining of intracellular molecules, fix the cells with 4% PFA for 20 minutes at 4°C after initial staining of surface markers. Permeabilize and stain the fixed cells with primary antibodies against intracellular molecules of interest.
3.5 RNA isolation of stepwise in vitro induced cells
The here-exemplified protocol is for induced cells (PS, PSM, SM, SCL, DM) and iPSCs grown in 6 well sterile culture plates.
1. Add 2-Mercaptoethanol (2-ME) to RLT Buffer (10 µl 2-ME/ml RLT Buffer).
2. Aspirate culture medium and wash the cells with PBS (2 ml).
3. Aspirate the PBS and add 350 µl of RLT Buffer + 2-ME.
4. Collect the cells with scraper into a 1.5 ml microtube.
5. Mix the sample thoroughly by vortexing (30 seconds).
6. Store the sample until isolation of RNA at -30°C.
7. Isolate RNA with RNeasy Mini kit (Qiagen) or other RNA isolation kit.
8. RNA can be used for e.g. cDNA generation and qRT-PCR or library preparation and RNA-sequencing analysis.
9. In case of isolation of RNA from in vitro derived human 3D cartilage, collect and freeze in vitro derived 3D cartilage in liquid nitrogen and then crash with multi beads shocker (Yasui Kikai) before mixing with 350 µl of RLT Buffer + 2-ME. RNA can then be isolated according to the manufacturer’s instruction with RNeasy Mini kit (Qiagen) or other RNA isolation kit.