hAMSCs were obtained from normal post-partum placenta. The amnion and villus layer were bluntly separated and repeatedly washed with D-Hank’s solution, including double resistant (100 U/ml penicillin and 100 μg/ml streptomycin). After rinsing, the amnion was cut into several 1×1-mm sections with ophthalmic scissors and digested at 37°C in a water bath for ~30 min with the action of 2.5 g/l trypsin (Gibco-BRL, Carlsbad, CA, USA). The digestion of the amnion was terminated with Dulbecco’s modified Eagle’s medium (DMEM) containing 5% calf serum and filtered through a 200 mesh cell sieve. The filtered amnion products were digested again in a 37°C water bath for ~0.5 h with the addition of 1.0 g/l collagenase II (Sigma-Aldrich, St. Louis, MO, USA). Subsequent termination and filtrations were performed as described above. Finally, the harvested cell suspensions were centrifuged at 1,000 rpm for 5 min and the cell pellet was resuspended in low-glucose DMEM (L-DMEM; Hyclone Laboratories, Inc., South Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin-streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA). The cells were then plated in 25-cm² culture flasks at a density of 1×10⁶ cells/ml and incubated at 37°C with 5% carbon dioxide. The medium was changed every 2 days. When the established adherent cell colonies reached 70% confluence, they were detached with 2.5 g/l trypsin and replated at a ratio of 1:2 in 25-cm² flasks.

Second or third generation hAMSCs were plated onto 6-well plates. When 60% confluence was achieved, the harvested cells were washed with phosphate-buffered saline (PBS). To induce neural differentiation, the hAMSCs were incubated with serum-free medium containing DMSO (2%) and butylated hydroxyanisole (BHA) (100 μM). The media were changed every 3 days and culturing was continued for 14–21 days. The neural induced cells were then confirmed by neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) immunofluorescence staining.

Immunofluorescence was performed on hAMSCs cultured for 24 h. The cells were grown to 60% confluence on 6-well plates, washed with PBS three times, fixed in 4% paraformaldehyde for 30 min, washed as previously described, permeabilized in 0.3% Triton X-100 for 20 min and then rinsed with PBS three times. The cells were then blocked with goat serum for 20 min, incubated with the appropriate primary antibody in PBS for 2 h at 37°C, washed with PBS three times, incubated with secondary antibodies in PBS for 30 min at 37°C (in the dark) and then viewed under a fluorescence microscope. The following primary antibodies were used: rabbit anti-human NSE (1:500) and rabbit anti-human GFAP (1:500), both from Boster Biological Technology, Ltd). The secondary antibody for immunofluorescence was goat anti-rabbit IgG (1:500; Sigma).

Third generation cells from the AMSCs were collected and plated in 25-cm² culture flasks and 6-well plates at a density of 1×10⁵ cells/ml. The cell pellet was resuspended in L-DMEM supplemented with 10% FBS, penicillin-streptomycin (100 μg/ml), 5 ng/ml bFGF and 10 μg/ml BrdU, and then incubated at 37°C with 5% carbon dioxide for 48 h. BrdU-labeled AMSCs were centrifuged at 1,000 rpm for 10 min and the cell pellet was resuspended in PBS at 1×10⁶ cells/μl. Finally, 5-μl cell suspensions were used for cell transplantation.

Healthy male Wistar rats, aged 3–4 months and weighing 250–300 g, were obtained from the Schistosomiasis Prevention and Control Center of Jiangsu Province, China. Briefly, the rats were placed in a supine position on an operating table following the intraperitoneal injection of 10% chloral hydrate (4 m1/kg) anesthetic. A blunt dissection of the sternocleidomastoid was made through the middle line neck incision, and the carotid artery (CCA) was isolated and then separated into the right external carotid artery (ECA), internal carotid artery (ICA) and the wing jaw artery. A slipknot was left under the ECA and the wing jaw artery, after threading deeply into all the arteries. The CCA was clamped, a small incision was made in the proximal sidewall of the ECA and a nylon suture filament (0.24 mm) was inserted and advanced to a depth of ~18.5±0.5 mm away from the CCA bifurcation. The suture was removed following a 2-h right MCAO, the ECA was ligated and the skin was sutured.

Out of 45 rats subjected to focal cerebral ischemia, 13 died and 8 did not exhibit paralysis of the limbs. The remaining 24 rats were randomly divided into two groups (n=12 per group).

Two weeks after MCAO, the rats were placed in a stereotactic apparatus and the bregma was exposed through a median head scalp incision. Coordinates were marked to enable targeting of the striatum (1 mm anterior to the skull, left margin 2.5 mm; depth 4.5–5.5 mm), and a 5 μl BrdU-labeled AMSC suspension or PBS control was then injected into the striatum with a Hamilton syringe for 10 min.

Neurological deficit evaluations were carried out prior to the transplantation and 1, 3, 6 and 8 weeks after MCAO using the neurological severity score (NSS; Table I), beam balance test (BBT; Table II) and elevated body swing test (EBST). For the EBST, observers were instructed to record data only when the rat head moved >10° from the vertical axis within 30 sec of the rat being raised by the tail. This procedure was followed by 1 min of rest, and the test was then repeated 20 times. For the three tests, recordings were taken on every last day of weeks 1, 3, 6 and 8, and all rats were tested three times at different time-points for each test, and the average result was determined.

TTC staining was used to show the ischemic area of the brain tissue following 2 h of perfusion. A coronal cut was then made in the brain tissue, 2 mm behind the optic chiasm, the latter part of the brain was immersed in 1% TTC (Sigma-Aldrich) in PBS at 37°C for 30 min and then into 10% neutralized formalin overnight.

Eight weeks after MCAO, the rats were anesthetized intraperitoneally with 400 mg/kg chloral hydrate, perfused transcardially with 4% paraformaldehyde in PBS, and their brains were quickly extracted. An ~2 cm ischemic area of brain tissue, including the lateral ventricles and basal ganglia, striatum and hippocampus, was excised and post-fixed in 4% paraformaldehyde. Coronal brain slices (5-μm) were then consecutively sampled using paraffin sections or frozen sections.

Sections were transferred to distilled water with xylene and ethanol, placed into the working solution (an equal parts mixture of ferrocyanide and hydrochloric acid) for 15 min, rinsed with distilled water and then with tap water. Sections were then stained with neutral red for 1 min, rinsed well with tap water, dehydrated with ethanol and finally cleared with xylene. Out of 400 slices, every 20th slice was stained using this method to confirm the needle placement and injected sites.

Data were presented as the mean ± standard deviation. Comparisons of neurological scores were carried out by ANOVA (F test, q test), using SPSS 10.0. The paired t-test was used for the cell count. In the analysis, a value of P<0.05 indicated a statistically significant result.

Adherent cells were observed 4 h after the cells were plated, and clone-like growth was observed 48 h later. The morphology of these cells was similar to that of BMSCs: spindle-shaped with fibroblast-like colonies adhering to the plastic surface. Flow analysis (Fig. 1) showed that the AMSCs expressed the typical MSC markers (CD73, CD105 and CD90), but were negative for hematopoietic markers (CD34 and CD45), the monocytic marker (CD14) and HLA-DR. A large number of BrdU-positive cells were observed using fluorescence microscopy, suggesting successful transplantation of the AMSCs.

Morphological changes, including condensed cell bodies with outgrowth in a few sites, were detected in some of the cells 2 h after incubation (Fig. 2A), with more cells showing these neural cell-like changes 1 h later (Fig. 2B). In addition to the morphological changes, differentiated cells expressed NSE, a marker for neural progenitor cells, and GFAP, a marker for astrocytes (Fig. 2C and D, respectively).

The rats were tested for neurological function at different time-points using the NSS, BBT and EBST tests (Fig. 3A-C). In each test, the neurological behaviors were markedly improved, and there was a significant difference between the AMSC-transplanted and the PBS-injected groups.

TTC staining is a standard for the measurement of infarct size and has previously been used for assessment of infarct size resulting from apoptosis and necrosis (6). Normal brain tissue was shown in gray, while the ischemic area was white in TTC staining (Fig. 4D). The ischemic region was observed in the dorsolateral striatum and was lateral to the ischemic hemisphere, which is consistent with the blood supply area of the middle cerebral artery (Fig. 4A). In the ischemic hemisphere, H&E staining indicated a large necrotic area in the 8th week after transplantation. Within this region, there was a significant loss of neurons, with only a few remaining astrocytes, and a marked interstitial edema. The surviving neurons had varying degrees of morphological changes, with the most significant changes occurring in pyramidal cells, which showed a shrunken cell body, retracted processes and loss of Nissl bodies. Furthermore, the chromatin became cloudy and the nuclear membrane decreased in size (Fig. 4B). Fewer degenerated cells were observed in the AMSC-transplanted area (Fig. 4C).

The injection sites and needle tracks were identified in each of the 24 rats using specific hemosiderin staining (Fig. 5A) and showed that 17 rats were injected into the striatum and the other 7 rats in the cerebral cortex. Small amounts of lymphocytic infiltration and glial cell proliferation were observed around the needle tracks (Fig. 5B and C). In the experimental group, BrdU-positive AMSCs near the ischemic lesion were found to be distributed around the needle passages (Fig. 5D), with some cells residing at a distance of 2 mm away. No BrdU-positive cells were observed around the needle passages in the control group.