AF samples were obtained from 17 patients undergoing amniocentesis for routine prenatal diagnosis at 15 - 22 weeks of pregnancy approved by the New York University School of Medicine Institutional Review Board. Fresh amniotic fluid (4 - 6 ml) samples were centrifuged at 300 g (4˚C) for 5 minutes to harvest cells and cultured in a-MEM medium (Gibco, Invitrogen) containing 15% ES-FBS, 1% glutamine and 1% penicillin/streptomycin (Gibco), supplemented with 18% Chang B and 2% Chang C (Irvine Scientific). Cells from the same patient were also used, after they had been maintained in culture for 2 - 3 weeks in the cytogenetics lab in 11 patients. The cell culture were incubated under 5% CO2 at 37˚C and sub-cultured at 70% - 80% confluence.

At passage 2 or 3, after assessing that an adequate number of cells were available, amniotic fluid cells were sorted using magnetic bead technique to obtain a CD117 + AFSC initial isolate. These CD117 positive cells were divided into 2 parts: one half was used to isolate the mRNA noted as early samples, the other was maintained in culture and subcultured for 4 - 5 passages. At that time, these cells were sorted again and a high concentration of CD117 positive cells was obtained and noted as late samples.

Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, #74134) according to the manufacturer’s instructions. After DNase digestion, RNA samples were quantified, aliquoted and stored at –80˚C until use. For quality control, RNA purity and integrity were evaluated by denaturing gel electrophoresis, OD 260/280 ratio, and analysis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA was amplified and purified using the standard Affymetrix 3’IVT express Kit to yield biotin-labeled cDNA according to the manufacturer’s instructions.

The NYU Genome Technology Center was utilized for both microarray and Q-PCR technologies (Affymetrix and ABI 7900 HT SDS, respectively). The labeled cDNA hybridization to the Affymetrix U133 2.0 arrays, array fluidics processing and scanning were performed under standardized conditions as recommended by Affymetrix. Analysis of the microarray expression profile was performed using Genespring Multiomics software suite under the NYU institutional license in consultation with NYU Center for Health Informatics and Bioinformatics.

Cellular senescence PCR Array plates (Qiagen, PHAS- 050) were used to assay the 84 genes related to cellular senescence expression. Following the manual we purified RNA from each sample using the RNeasy Plus Mini Kit (Qiagen 74134), prepared cDNA from purified RNA using RT2 Strand Kit (Qiagen 330401) and added cDNA to RT2 SYBR Green Mastermix (Qiagen330513), aliquoting the mix into RT2 Profile PCR Arrays plates. PCR for each plate consisted of the following sequence: 95˚C, 10 sec for initial denaturation, and 40 cycles of 95˚C for 10 sec and 60˚C for 45 sec. To determine primer specificity, three stages (95˚C for 15 s, 60˚C for 20 s, and 95˚C for 15 s, with a ramping time of 20 min) were added at the end of the PCR to obtain melt curves for each gene. All realtime PCR reactions were performed using a Bio-Rad iCycler optical module system. Analysis data were obtained by PCR Array analysis software provided by SABioscience. The housekeeping genes used in PCR array were average of 5 genes including ACTB, B2M, GAPDH, HPRT1 and RPLP according to SABioscience guide.

Genomic DNA of cells was prepared using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). Average telomere length was measured from total genomic DNA using a real-time PCR assay, as described [7] . PCR reactions were performed on the iCycler iQ real-time PCR detection system (Bio-Rad, Hercules, CA, USA), using telomeric primers, and primers for the reference control gene (human 36B4 single copy gene). For each PCR reaction, a standard curve was made by serial dilutions of known amounts of DNA. The telomere signal was normalized to the signal from the single copy gene to generate a T/S ratio indicative of relative telomere length. Equal amounts of DNA (35 ng) were used for each reaction, with at least three replicates for each specimen.

Genespring Muti-omics Software Suite was used for microarray analysis and processed to normalized intensive values for comparison of early and late passages. Principal component analysis was done with 3 components for comparison of early and late passages of expression of senescence genes. Data mining analysis using the NIH DAVID base was performed in the 1685 gene probes of interest using functional annotation analysis. Calculation of p-values from a large dataset with a low 1.33 fold threshold increases the chance of random false discovery. Therefore Benhamini-Hochberg multiple comparison testing was used to eliminate false discovery. It is used extensively for microarray analysis. Where applicable, student t-test was employed.

Sixteen samples from 15 patients at 15 - 22 weeks were assayed for population doubling (Table 1). In early (1 - 4) passage of fresh samples and (1 - 3) passage of cytogenetics samples, the generation doubling time could be determined as 30 to 40 hours, but in the late passages the doubling time would prolong gradually. Every sample could be passaged more than 6 times and yield more than 10 doublings. The range of population doublings was from 10 to 81, the average doubling of AF cells was 24. However one sample from a 15 week patient had 81 population doublings and was maintained in culture for 150 days. By the late passage these cells stopped proliferating at which time their morphology was flat and large, which is one of the characteristics of cellular senescence observed after at least 10 passages. This is distinct from the morphology of the early passage human AF derived cells.

To identify gene expression shift in culture, we performed microarray chip assays using AF derived cells sorted by CD117 conjugated microbeads at early passage and late passage from 5 different patients. Differentially expressed genes were grouped according to their expression profiles. Of the 54675 genes on the chip, at fold change threshold of 1.33 fold and p value of <0.05, a total of 1685 gene probes showed differential expression from early to late passage. This represents 3.08% of the

Table 1. Life span of human amniotic stem cells.

^aNPD = Number of Population Doublings.

total gene probes showing statistically significant change from early to late passage. The 1.33 fold threshold was chosen to maximize detection of small changes in gene expression which might be missed at the usual 2 fold level. Using more than one appropriate statistical analysis was relied on to correct for potential false positives.

Functional annotation analysis of 1685 genes of interest revealed that most of the genes belonged to several biological processes (Table 2). The genes with molecular function in binding, cellular organization, and catalytic activity were the most differentially expressed.

To investigate senescence of AF derived cells, the differentially expressed genes were compared with gene set known to influence cellular senescence. Expression was analyzed before the cells became obviously senescent in an attempt to identify the markers that would be useful as predictors of the onset of senescence. Out of 1685 investigated, only 8 known to influence cellular senescence were differentially expressed genes (Table 3). This represents 0.47% of differentially expressed genes and 0.02% of all genes investigated in the study. Of the 8 differen-

Table 2. Gene annotation summary of differentially expressed genes in AFSC culture.

Table 3. Differentially expressed senescence related genes in AFSC during in-vitro culture.

tially expressed genes, only THBS1 and ATM genes were up-regulated, while the majority of the differentially regulated genes were down-regulated in early passage compare to late passage. The results suggest effective down regulation of apoptotic genes in the early cells, which would be consistent with their potential for longevity and regeneration. It is important to note that the majority of senescence related genes did not show a statistically significant change from passage 2 - 3 to passage 4 - 5.

In 5 patients, 84 genes whose expression is known to be related to cellular senescence were measured between cultures early (P1-3) and onset of senescence (P11-29) as shown by the change in morphology as well as prolongation of doubling time and/or arrest of growth. Both early and senescent cultures express the telomerase gene at a level below detection in the Cellular Senescence PCR Array. Early passage cells (n = 5) had higher expression of AKT1, E2F1, IRF5 and RBL1 compared to senescent cells, and less expression in CDKN1C if the P value was set at <0.1 (Figure 1 and Table 4). There is significantly higher expression in early passage for AKT1 and RBL1 at P ≤ 0.05. Both of them belong to the senescence initiators in p53/pRB signaling pathway.

One cell line (11 - 18), with 81 doublings over 150 days, was established by AF derived cells. These cells revealed homogenous small spindle-shape morphology and clone formation. They continued growing and not in islands or in cell groups, as found in the other AF cell lines. The 11 - 18 cells were maintained with 81 population doublingsTable 4. Gene expression of AF cells at early passage (n = 5) compare to late passage (n = 5).

Red dots are the genes whose expression increase more than 2 fold while green dots are decrease more than 2 fold and the dots upward the blue horizontal line are the genes whose p-value < 0.1. Genes whose expression changes and p-values beyond the selected boundaries are listed in the right table. The arrows indicate more than 2 fold change and p < 0.10.

Figure 1. Volcano plot of AF cells at early passage (group 1, n = 5) compare to late passage (control group, n = 5).

far more than the other cell lines whose average doubling was 20.

The longevity cell line 11 - 18 compared with short lived cell lines showed a greater gene expression difference than that seen when senescent and early passage cells were compared for gene expression. 11 - 18 cells had significant up-regulation in 6 genes and down-regulation in 3 genes compared to short lived lines (n = 6) when cellular-senescence related gene expression was tested in early passage (P1-6) cell lines (Figure 2 & Table 5). All the AF cells were compared at early passages (P1-3) to see if longevity as predictable. Significant up-regulated genes include CDC25C (Cell division cycle 25 homolog C S. pombe), CDKN2C (Cyclin-dependent kinase inhibitor 2C, p18, inhibits CDK4), CDKN2C (Cyclin-dependent kinase inhibitor 2D, p19, inhibits CDK4), COL3 A1 (Collagen, type III, alpha 1), TBX2 (T-box 2) and TWIST (Twist homolog 1 Drosophila). Significant downregulated genes include IGFBP7 (Insulin-like growth factor binding protein 7), CREG1 (Cellular repressor of E1A-stimulated genes 1) and IRF3 (Interferon regulatory factor 3).

There was minimal telomerase gene expression when tested by microarray and cellular senescence PCR Array as the signal was not detected. Using relative telomere length assay by T/S Ratio by quantitative PCR, following the method of Cawthon [7] , the results showed that short telomere length was present, consistent with the minimal telomerase mRNA expression by PCR data and by its absence on the microarray data. Longevity cell line 11-18 did not have longer telomere length than the short lived lines and all were shorter than tumor cells (Figure 3). Single cell telomere length measurement also showed 11 - 18 had shorter telomere length than the tumor cell. The other 2 cell lines were in the normal life span rangeTable 5. Gene expression of longevity cell line versus shortlifespan cells.

Red dots are the genes whose expression increased more than 2 fold while green dots decreased more than 2 fold. The dots above the blue horizontal line are the genes with p < 0.05. Genes whose fold expression changes and p-values exceeded the boundaries are seen in the table.

Figure 2. Volcano plot of senescence related genes expression in longevity cell line (group1) versus short-lifespan cells (control group).

The first four poles obtained from different cell populations, showed no significant difference between long-lived cells 1118 and short-lived cells 1122,1113, but they are all have shorter telomeres than the tumor cell line 1301. SC1-6 are single cell telomere length assays, confirming that 1118 telomeres are significantly shorter than 1301 telomeres.

Figure 3. Telomere length measurement of longevity cell and short lifespan cell.

11 - 22 with 40 doublings, and 11 - 13 with 13.