The mean EL of offsprings over 4-day training periods in the Morris water maze test.

<div><p><b>A</b>: The learning curves for the offspring rats from different groups over the course of 4-day training periods in the Morris water maze test were showed. </p> <p><b>B</b>: The mean EL of the offspring rats from different groups (control group, n= 12; 5 min HIE group, n= 13; 10 min HIE group, n= 12; 15 min HIE group, n= 10) were expressed as mean ± S.D.. <i>a</i>: compared with the control group; <i>b</i>: compared with the 5 min HIE group; <i>c</i>: compared with the 10 min HIE group. The P values were showed in the bracket.</p></div

Survival curves of neonatal offspring rats from different groups.

<p>The survival curves of offspring rats were displayed at PND 35 (control group, n=12; 5 min HIE group, n= 14; 10 min HIE group, n= 21; 15 min HIE group, n= 24).</p

Expression of BDNF in the hippocampal of offspring rats from different groups.

<div><p><b>A</b>: A representative Western blot of BDNF protein in the hippocampal of offspring rats from different groups was showed. </p> <p><b>B</b>: Relative levels of BDNF in the hippocampal of offspring rats from different groups (control group, n= 12; 5 min HIE group, n= 13; 10 min HIE group, n= 12; 15 min HIE group, n= 10) at PND 35 were expressed as mean ± S.D.. <i>a</i>: compared with the control group; <i>b</i>: compared with the 5 min HIE group; <i>c</i>: compared with the 10 min HIE group. The P values were showed in the bracket.</p></div

TUNEL staining of apoptotic cells in the brains of neonatal offspring rats from different groups.

<div><p><b>A</b>: Apoptotic cells were detected by TUNEL staining (dark brown, indicated by arrows) in the brains of offspring rats at PND 1 after ischemic injury. Scale bar: 400μm. </p> <p><b>B</b>: Total TUNEL-positive cells were calculated in six randomly chosen views (three in hippocampus and three in cerebral cortex). Total TUNEL-positive cells per view from different groups (n=12 in each group) at PND 1 were showed. The results were expressed as mean ± S.D.. <i>a</i>: compared with the control group; <i>b</i>: compared with the 5 min HIE group; <i>c</i>: compared with the 10 min HIE group. The P values were showed in the bracket.</p></div

Levels of cleaved caspase-3 to procaspase-3 in the brain of neonatal offspring rats from different groups.

<div><p><b>A</b>: Representative Westernblots of cleaved caspase-3 and procaspase-3 from brain tissues of offspring rats with or without ischemic injuries were showed. </p> <p><b>B</b>: Levels of cleaved caspase-3 to procaspase-3 in the brain of neonatal offspring rats from different groups (n= 12 in each group) at PND 1 were calculated relative to that of β-actin. The results were expressed as mean ± S.D.. <i>a</i>: compared with the control group; <i>b</i>: compared with the 5 min HIE group; <i>c</i>: compared with the 10 min HIE group. All <i>P</i> values were <0.001.</p></div

Nissl staining showed neuronal cells in the hippocampus of offspring rats from different groups.

<div><p><b>A</b>: Representative histological sections of Nissl staining of neurons in the hippocampus (CA1) of neonatal rat brains from different groups were showed (scale bar: 400μm).</p> <p><b>B</b>: Total Nissl-positive neuronal cells were calculated in six randomly chosen views. Total Nissl-positive neuronal cells per view in the hippocampus (CA1) of offspring rats from different groups (n= 12 in each group) at PND 35 were showed. The results were expressed as mean ± S.D.. <i>a</i>: compared with the control group; <i>b</i>: compared with the 5 min HIE group; <i>c</i>: compared with the 10 min HIE group. All <i>P</i> values were <0.001.</p></div

Nissl staining of neuronal cells in the brain of neonatal offspring rats from different groups.

<div><p><b>A</b>: Representative photographs of Nissl staining of neurons in the cortex and hippocampus (CA1) of neonatal rat brains from different groups were showed (scale bar: 400μm).</p> <p><b>B</b>: Nissl-positive neuronal cells were counted in six randomly chosen views. Total Nissl-positive cells per view in the cortex and hippocampus (CA1) of offspring rats from different groups (n= 12 in each group) at PND 1 were showed. The results were expressed as mean ± S.D.. <i>a</i>: compared with the control group; <i>b</i>: compared with the 5 min HIE group; <i>c</i>: compared with the 10 min HIE group. All <i>P</i> values were <0.001.</p></div

Karyotype analysis.

<p>(A) HUMSCs exhibited a normal Karyotype (46, XX). (B) A representative karyotype of tHUMSCs exhibited triploid and hypotriploid with 69 chromosomes. (×1000).</p

In vitro life span of donor HUMSCs.

<p>The life span of cells was defined as the number of culture days before observation of senescence. Donor HUMSCs were treated with or without 3-MCA. HUMSCs treated with 3-MCA from donors 1 and 7, while still growing, were terminated at 318 days to allow for data analysis.</p

Analysis of telomerase activity.

<p>(A) The relative telomere length of HUMSCs was measure by qPCR, which was decreased as the cell passage number increased. *P<0.05 (B) There was no statistical difference in the relative telomere length of tHUMSCs among passages as analyzed by qPCR. (C) RT-PCR revealed the expression of hTERT in tHUMSCs at P50 (lane 4), while HUMSCs showed no detectable expression of hTERT at P3 (lane 5). The transcripts of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from tHUMSCs (lane 2) and HUMSCs (lane 1) were used as positive controls.</p