Isolation of human multipotent mesenchymal stem cells from second‐trimester amniotic fluid
Culture of MSC from amniotic fluid
1. Twenty
amniotic fluid samples (20 ml) were obtained by amniocentesis performed
between 16 and 20 weeks of gestation for fetal karyotyping.
2. A novel two‐stage culture protocol for isolating MSCs was from amniotic fluid.
3. For
culturing amniocytes (first stage), four primary in situ cultures were
set up in 35 mm tissue culture‐grade dishes using Chang medium (Irvine
Scientific, Santa Ana, CA).
4. Microscopic analysis of
Giemsa‐stained chromosome banding was performed, and the rules for
metaphase selection and colony definition were based on the basic
requirements for prenatal cytogenetic diagnosis in amniocytes.
5. For
culturing MSCs (second stage), non‐adhering amniotic fluid cells in the
supernatant medium were collected on the fifth day after the primary
amniocytes culture and kept until completion of fetal chromosome
analysis.
6. The cells then were centrifuged and plated in 5 ml of
α‐modified minimum essential medium supplemented with 20% fetal bovine
serum (FBS) and 4 ng/ml basic fibroblast growth factor (bFGF) in a 25 cm2 flask and incubated at 37°C with 5% humidified CO2 for MSC culture.
Differentiation assay for MSCs
1. Amniotic fluid‐derived mesenchymal
stem cells (AFMSCs) were cultured to confluence and shifted to
osteogenic medium (α‐MEM supplemented with 10% FBS, 0.1 µmol/l
dexamethason, 10 mmol/l β‐glycerol phosphate, 50 µmol/l ascorbate) and
adipogenic medium (α‐MEM supplemented with 10% FBS, 1 µmol/l
dexamethasone, 5 µg/ml insulin, 0.5 mmol/l isobutylmethylxanthine and 60
µmol/l indomethacin) for 3 weeks.
2. The differentiation potential
for osteogenesis was assessed by the mineralization of calcium
accumulation by von Kossa staining.
3. For adipogenic
differentiation, intracellular lipid droplets could be observed under
the microscope and confirmed by Oil Red O staining.
4. For
differentiation of neural cells, AFMSCs were incubated with α‐MEM
supplemented with 20% FBS, 1 mmol/l β‐mercaptoethanol, 5 ng/ml bFGF for
24 h, and then treated with serum depletion for 5 h.
5. Immunocytochemical
stain with neuron‐specific class III β‐tubulin (TuJ‐1) was used to
assess the capacity of neuronal differentiation.
Reference
Moertel CA, Stupca PJ and
Dewald GW (1992) Pseudomosaicism, true mosaicism, and maternal cell
contamination in amniotic fluid processed with in situ culture and
robotic harvesting. Prenat Diagn 12,671–683.