Stepwise in vitro induction of human somitic mesoderm and its derivatives

Our understanding of human somitogenesis is limited and largely based on insights gained from model organisms. Pluripotent stem cell-based in vitro approaches aiming to recapitulate distinct aspects of this core developmental process have recently been reported, including our recent paper on the in vitro recapitulation of the human segmentation clock 1 . Here we describe in detail our stepwise induction protocol of presomitic mesoderm (PSM), somitic mesoderm (SM), and its two major derivatives, sclerotome (SCL) and dermomyotome (DM) from human induced pluripotent stem cells (iPSCs). We further brie�y address the subsequent molecular and functional analysis of these in vitro induced human mesodermal lineages and cell-types.


Introduction
Mesoderm, one of the three principal germ layers is formed and speci ed in the primitive streak (PS) during early embryonic development.The primitive streak is the anatomical correlate of gastrulation, a process during which the pluripotent cells of the epiblast undergo epithelial to mesenchymal transition (EMT) and give rise to mesoderm and de nitive endoderm.Paraxial or presomitic mesoderm is one the mesodermal lineages which are formed and speci ed along the anterior-posterior axis of the primitive streak.
During somitogenesis these presomitic mesoderm (PSM) cells then give rise to somites, transiently epithelialized balls of somitic mesoderm (SM), which further differentiate into the two major somite compartments, sclerotome (SCL) and dermomyotome (DM) respectively.Somitic mesoderm and its derivatives contribute to the formation and patterning of the musculoskeletal system, giving rise to vertebrae, ribs and skeletal muscle.This entire process has been extensively studied using model organisms such as chick, mouse or zebra sh but remains largely elusive in the human context.This also applies to studies focusing on the segmentation clock, a species-speci c molecular oscillator believed to control the process of somitogenesis.
In order to gain insights into human somitogenesis, we and others have recently established induction protocols and approaches for the reproducible in vitro derivation of presomitic and somitic mesoderm-like cells from human pluripotent stem cells [1][2][3][4][5][6] .Molecular and functional analysis of these in vitro derived human presomitic and somitic mesoderm-like cells and their derivatives have increased our understanding of this core developmental process in human.This Nature Protocol Exchange is an addition to our recent paper on the in vitro recapitulation of the human segmentation clock from pluripotent stem cells, and describes in detail the stepwise induction of presomitic and somitic mesoderm and its derivatives from human iPSCs.Reagents 0.5%-Trypan blue stain solution (Nacalai, Cat#: 29853-34) 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 exempli ed 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/cm 2 ) 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 humidi ed CO 2 incubator at 37°C.Do not let the plate dry out when changing solutions.
3. Wash the iPS cell colonies with PBS.
4. Add 300 µl of 0.5x TrypLE TM Select enzyme solution (diluted with 0.5 mmol/l-EDTA/PBS); distribute it evenly, and put the plate into a humidi ed CO 2 incubator set to 37°C. 5.After 1 minute, take out the plate and re-distribute TrypLE TM 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.
12. Distribute cells evenly by gentle shaking and incubate plate with cells in humidi ed CO 2 incubator 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.

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).
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.
3. Thaw a frozen vial of iPSCs in the water bath until only a few small ice particles remain.
6. Dissociate the cells gently by pipetting (about 10 times) and measure the cell density using hemocytometer or automated cell counter.
7. Seed 1.3x10 4 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 de ned 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.Exempli ed 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 differences 4 .

Preparation of Chemically De ned Medium (CDM)
1. Perform all described steps under a clean-bench; use sterile tips and bottles (see Table 2 for composition of CDM).

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 humidi ed CO 2 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.
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 humidi ed CO 2 incubator.

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.).

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 differences 4 .Other protocols aiming to induce presomitic mesoderm from human pluripotent stem cells (hESCs or hiPSCs) have also been reported 2,3,5,6 .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.
3. Add 2 ml of freshly made PSM induction medium.
4. Culture cells in humidi ed CO 2 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 groups 2 .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 clock 2,11 .Already described in our concurrent paper 1 , 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, ow 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).

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.
3. Add 2 ml of freshly made SM induction medium.
4. Culture cells in humidi ed CO 2 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)).

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 hours 4 .
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.

Aspirate previous (SM) induction medium.
3. Add 2 ml of freshly made 2 ml of DM induction medium.
4. Culture cells in humidi ed CO 2 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)).

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 induction 12 .
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.

Aspirate previous (SM) induction medium.
3. Add 2 ml of freshly made SCL induction medium.
4. Culture cells in humidi ed CO 2 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 brie y 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

In 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 previously 3,13,14 .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 Nature 1 .
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.

In vivo xeno-transplantation of in vitro induced human SCL cells
In vitro induced human SCL cells can be transplanted into immunode cient (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 brie y 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).
2. Mix 100 µl of cell suspension with the same amount of Matrigel ® .
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.

Immunocytochemistry of stepwise in vitro induced cells
The here-exempli ed 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.
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% Triton TM X-100 in PBS for 10 minutes at room temperature and then wash with PBST (1% TWEEN ® 20 in PBS).
12. Filtrate the cells through a ne mesh.13.Analyze with cell analyzer.
14.In case of co-staining of intracellular molecules, x the cells with 4% PFA for 20 minutes at 4°C after initial staining of surface markers.Permeabilize and stain the xed cells with primary antibodies against intracellular molecules of interest.

RNA isolation of stepwise in vitro induced cells
The here-exempli ed protocol is for induced cells (PS, PSM, SM, SCL, DM) and iPSCs grown in 6 well sterile culture plates.
2. Aspirate culture medium and wash the cells with PBS (2 ml).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.

Troubleshooting
Troubleshooting of stepwise induction Cells do not differentiate properly Ø Use fresh media; add all growth factors and inhibitors just before using.Ø Store stocks of growth factors and small molecules at appropriate temperatures (-30°C/-80°C) Ø Avoid excess freeze and thaw cycles of recombinant protein stocks used in media.
Ø Do not vortex recombinant proteins or media after adding recombinant proteins.Ø Passage a few times after thawing cells until stable iPSC culture is established, especially in cases where thawed cells appear not healthy (i.e.growth speed is low or cells spontaneously differentiate).

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.

Ø
Use low passage number cells.If condition of iPSCs is not optimal, thaw earlier passage number cells and re-start experiment.Ø Maintain iPSCs in undifferentiated state.Check whether iPSCs maintain the pluripotent state.Check the expression of pluripotent markers (i.e.OCT3/4, NANOG, SOX2) by qRT-PCR and immunocytochemistry.Troubleshooting of skeletal muscle induction Cells are detaching Ø Use fresh Matrigel ® and increase dilution rate with DMEM.Ø Change medium gently during in vitroinduction.Ø Shorten the time when the plate is outside the incubator as much as possible.Ø Avoid taking out the plate from the incubator unnecessarily.Contracting colony/cell number is low Ø Check the induction e ciency of the previous stages e.g.DLL1 expression at the PSM stage by FACS.Ø Check proper marker gene expression at DM stage and/or previous steps by immunocytochemistry and/or qRT-PCR.Troubleshooting of 3D cartilage induction 3D-SCL spheroids or forming 3D cartilage fuse to each other Ø Distribute spheroids and forming 3D cartilage evenly and put dish back into the incubator gently.Ø Use lager dish and higher media volume for culture.Spheroids don't form cartilage Ø Prolong the 3D-CI induction period.Ø Check the induction e ciency of the previous stages e.g.DLL1 at the PSM stage by FACS.Ø Check proper marker gene expression at SCL stage and/or previous steps by immunocytochemistry and/or qRT-PCR.Time Taken Passaging: 30-60 minutes Freezing stocks: 30-60 minutes Thawing stocks: 30 minutes Stepwise induction (each step): 15-30 minutes Skeletal muscle induction: 60-90 minutes 3D chondrogenic induction: 90-120 minutes In vivo xeno-transplantation: 240-300 minutes Immunocytochemistry: 2 days Flow cytometric (FACS) analysis: 150-180 minutes RNA isolation: 90-120 minutes Anticipated Results If induction is successful, stage-speci c makers are expressed robustly.Validate developmental stage speci c expression of genes and proteins by qRT-PCR, immunocytochemistry, FACS or RNAsequencing analysis.PS induction