IsolationandDifferentiationofAmnionDerivedStemCells(ADSCs)

ADSC isolation, culture, and cloning

1. Rat amnion membrane is mechanically separated from the chorion of embryonic day 18.5 Sprague–Dawley rat embryos

2. The tissue is washed extensively with phosphate-buffered saline (PBS) and subsequently cut into small pieces

3. Membrane fragments is placed in six-well plastic tissue culture dishes in a minimal volume (0.5 ml) of Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA) to encourage attachment

4. Cells will emerge from the explanted tissues within 24 hr of plating

5. After 1 week, the tissue explants are removed and the remaining adherent cells are trypsinized and replated into 100 mm plastic culture dishes

6. Cultures are passaged at confluency. All cultures are maintained under a humidified atmosphere of 5% CO2 at 37°C

Generation of clonal lines

1. Clonal lines are generated from single cells as described previously. Briefly, ADSCs are plated at a density of 1 cell/cm2 in 150 mm plastic culture dishes and incubated for 2 hr to allow cellattachment.

2. The supernatant is removed and dishes are washed with PBS to eliminate unattached cells. Dishes are examined microscopically and isolated single cells are identified and marked. Colonies arising from these single cells are then expanded into clonal lines.

Neuronal differentiation

1. Cells in neural induction media (NIM) are maintained under a humidified atmosphere of 5% CO2 in air at 30°C. The media are supplemented with 10 ng/ml bFGF at 24 hr postinduction and every 48 hr thereafter.

2. Neural differentiation is determined by morphological changes and immunocytochemical analysis.

Adipogenic induction

1. Before adipogenic differentiation, ADSCs are rinsed extensively with PBS.

2. ADSCs are exposed to adipogenic induction media (AIM) consisting of 10% FBS/DMEM, 500 μM IBMX, 1 μM dexamethasone, 10 μg/ml insulin, and 100 μM indomethacin.

3. After 3 days in the AIM, ADSCs are cultured in adipogenic maintenance media (AMM) for 3 days. AMM consists of 10% FBS/DMEM and 10 μg/ml insulin in a base of DMEM.

4. ADSCs are maintained at 37°C for 3 weeks, with media changed every third day.

5. Cells are fixed with 4% PFA and stored at 4°C until stained. Control cultures are grown in 10% FBS/DMEM. Oil Red O is utilized to visualize fat droplets.

Osteogenic induction

1. ADSCs are maintained in osteogenic induction media (OIM) up to 6 weeks, with fresh media added every third day.

2. Cells are fixed with 4% PFA and stored at 4°C until stained. Alizarin red is utilized to visualize the mineralized matrix.

Hepatogenic induction

1. ADSCs are seeded onto fibronectin-coated dishes at a density of 3,000 cell/cm2 and grown to 70% confluency in serum-containing media (SCM).

2. Cultures are subsequently transferred to hepatocyte induction media (HIM) consisting of 20 ng/ml hepatocyte growth factor (HGF), 10 ng/ml oncostatin M (OSM), 10 ng/ml FGF-4, 10?8 M dexamethasone, and 1% FBS in low-glucose (LG) DMEM and maintained in this media until harvested.

3. Media are changed every 3–4 days.

4. Hepatic differentiation of the ADSCs is determined by Dil-Ac-LDL (low-density lipoprotein) uptake (Biomedical Technologies, Stoughton, MA) and periodic Schiff acid (PAS) staining for glycogen.

References

Marcus AJ, et al. Isolation, characterization, and differentiation of stem cells derived from the rat amniotic membrane. Differentiation. 2007 Jul 2.