胚胎干细胞分化到间充质

HuES9 human ESCs are maintained on mouse embryonic
fibroblast feeder as previously described (11).
2. To generate MSCs, a starting confluent 6-cm plate of HuES9
hESCs is recommended.
3. Aspirate the culture medium and rinse cells with 5 ml of PBS.
4. Add 1 ml of trypsin/EDTA and return the plate to the 37°C
CO 2 incubator for 8 min (see Note 3).
5. After incubation, remove the plate and tilt the plate from side
to side to dislodge and disperse the cells into a cell suspension.
6. Neutralize trypsin with 5 ml of hES-MSC medium. Pipet cell
suspension up and down gently (~5 times) to break up any
cell clumps.
7. Centrifuge the cell suspension for 5 min at 800 × g, 4°C.
8. Discard the supernatant and loosen the cell pellet by flicking
the side of tube with your finger.
9. Add 5 ml of hES-MSC medium to resuspend the cells (see
Note 4).
10. Plate cell suspension on a 6-cm gelatinized plate (see Note 5).
No feeder is required from this point.
11. After plating, return the cell suspension to the CO 2 incubator
for 48 h to allow cells to adhere to the plate.
12. After 48 h, remove culture medium and cell debris, wash the
cell culture with 5 ml of PBS, and replenish cells with fresh
hES-MSC medium (see Note 6).
13. Repeat trypsinization when the culture is 90~100% confluent.
14. Seeding density is recommended at 500 cells per cm 2 .
15. After the third or fourth trypsinization, the characteristic
fingerprint whorl of confluent MSC cultures starts to become
evident (see Notes 7, 8). Figure 1 shows differentiated hES-
MSCs, HuES9.E1.
2.4. Differentiation
Assays
3. Methods
3.1. Derivation
of MSCs from hESCs
(hES-MSCs)
3.1.1. hES-MSC Derivation:
Protocol 1 (see Note 2)
145 Derivation and Characterization of Human ESC-Derived Mesenchymal Stem Cells
16. When the fingerprint whorl of confluent MSC cultures
forms, trypsinize the cell culture and plate cells on a gelati-
nized 10 cm plate. MSC cultures should not be expanded
at more than a 1:3–1:4 split. When cells have reached a
population size of 10 7 the culture is designated P1 (see
Note 9).
1. After Subheading 3.1.1 step 3, add 2 ml hES-MSC medium
and mechanically dissociate hESC into clumps using a pipette
tip.
2. Detach the cells with a cell scrapper and transfer the clumps
into two wells of a six-well low adhesion culture dish contain-
ing an additional 3 ml hES-MSC medium per well to allow
EB formation.
3. Culture the EBs in suspension for 7 days with medium change
every alternate day. Collect the EBs in a 15-ml conical centri-
fuge tube and centrifuged at 500 × g, 4°C for 3 min. The
supernatant is discarded and 3 ml of fresh hES-MSC medium
(AC: typically we allow the EBs to settle in a 15-ml tube,
remove most of the media – leaving ~0.5 ml and resuspend in
fresh media. Rationale for this is that it is easier to resuspend
the EBs when compared with centrifugation).
4. After 7 days, transfer the EBs into two gelatinized wells of a
six-well tissue culture dish containing hES-MSC medium and
allow the EBs to adhere to the dish.
5. Differentiate the attached EBs for an additional 7 days with
medium change every alternate day.
6. Resume from step 13 of Subheading 3.1.1.
3.1.2. hES-MSC Derivation:
Protocol 2
Fig. 1. hES-MSCs. Cellular morphology of huES9.E1 (hES-MSCs) under phase contrast.
146 Lai, Choo, and Lim
1. Trypsinize the differentiating hESCs with 1 ml of trypsin for
8 min at 37°C. Tilt plate from side to side to ensure that all
cells are lifted off the plates and there are no visible cell clumps
(see Notes 10, 11).
2. Neutralize the trypsin with an equal volume of hES-MSC
medium. Gently pipet the cells up and down about 5 times to
break up any cell clumps.
3. Centrifuge the cell suspension for 5 min at 800 × g, 4°C.
4. Discard the supernatant and resuspend the cell pellet in 10 ml
culture medium.
5. Plate the cell suspension on 10 cm bacterial culture dish and
place the plate on an orbital shaker with gentle shaking for
2 h in a CO 2 incubator at 37°C.
6. After 2 h shaking, harvest the cells and centrifuge the cell
suspension for 5 min at 800 × g, 4°C.
7. Wash cells twice with PBS by resuspending the cell pellet in
10 ml PBS each time followed by centrifuging the cell
suspension for 5 min at 800 × g, 4°C.
8. Resuspend 1× 10 6 cells in 0.5 ml 2% FBS or KSR in PBS (v/v).
9. Add 10 ml of FITC-conjugated anti-human CD105 and 10 ml
of PE-conjugated anti-human CD24 or the corresponding
FITC- or PE-conjugated mouse IgG1 isotype controls to the
cell samples.
10. Incubate with gentle shaking for 40 min at room temperature
in the dark.
11. Centrifuge the cell suspension for 5 min at 800 × g, 4°C.
12. Repeat step 7.
13. Resuspend cells in 500 ml medium and sort for CD105+,
CD24− cells on a FACS Aria using FACS Diva software.
14. After sorting, plate cells at a density of about 500 cells per cm 2
on a 15-cm gelatinized tissue culture plate with hES-MSC
medium. Incubate the cellsat 37°C in a CO 2 incubator.
1. Source expanded hES-MSCs from the CO 2 incubator (see
Note 12).
2. Aspirate medium and rinse cells with PBS.
3. For a 15-cm plate, add 5 ml trypsin to cells and place in the
incubator for 8 min.
4. Remove plates from incubator, tilt plate from side to side to
ensure that all cells are lifted off the plates and there are no
visible cell clumps.
5. Add 5 ml hES-MSC medium to the plate to neutralize trypsin.
6. Gently pipette the cells up and down about 5 times to break
up any remaining cell clumps.
3.2. hES-MSC Cell
Sorting
3.3. Passaging
hES-MSCs
147 Derivation and Characterization of Human ESC-Derived Mesenchymal Stem Cells
7. Transfer cell suspension into a 50-ml falcon tube.
8. Wash the plate with another 10 ml culture medium. Transfer
wash into the same 50 ml Falcon tube.
9. Centrifuge at 800 × g for 3 min, 4°C.
10. Aspirate supernatant, dislodge cell pellet by tapping on the
outside of the tube and resuspend cells in 40 ml hES-MSC
medium. Pipette up and down until cells are evenly dispersed.
11. Distribute 10 ml of the cell suspension onto new gelatinized
15-cm plates.
12. Add an additional 10 ml of hES-MSC medium to each plate.
13. Feed cells every 48 h with fresh medium until the culture is
~60–75% confluent and thereafter every 24 h. Cells reach
90–100% confluency in about 7–9 days. The cells should ide-
ally be split before they are 100% confluent, preferably at 80%
confluent.
1. Freezing Medium (2×), contains 80% (v/v) FBS and 20%
(v/v) DMSO . Mix well. Keep medium at 4°C until use. Store
complete freezing medium for £24 h.
2. Trypsinize a 15-cm MSC plate (~90% confluence) with 5 ml
trypsin for 8 min at 37°C.
3. Remove plate from incubator, tilt plate from side to side to
ensure that all cells are lifted off the plates and there are no
visible cell clumps.
4. Neutralize the trypsin with an equal volume of hES-MSC
medium. Gently pipette the cells up and down (~5 times) to
break up any cell clumps.
5. Centrifuge the cell suspension for 5 min at 800 × g, 4°C.
6. Discard the supernatant and resuspend the cell pellet in 2 ml
hES-MSC medium.
7. Add 2 ml of 2× Freezing Medium slowly while gently shaking
the tube to mix the freezing medium and the cell suspension.
8. When addition is complete, pipette the cell suspension up and
down a few times to ensure complete and even distribution of
the freezing medium.
9. Quickly aliquot 1 ml of the cell suspension into prelabeled
cyrovials.
10. Transfer the cryovials to a Cryo 1°C Freezing Container and
place at −80°C for 48 h before transferring to long-term stor-
age in liquid nitrogen or at −150°C.