mRNA expression and promoter DNA methylation status of CDKi p21 and p57 genes in ex vivo expanded CD34⁺ cells following co-culture with mesenchymal stromal cells and growth factors

Figures & data

Table 1. Sequence of primers used for real-time RT-PCR and MSP analysis

Primers	Sense primer 5′ → 3′	Antisense primer 5′ → 3′	Size (bp)
P21-RT	TTGTACCCTTGTGCCTCGCTCA	AGATCAGCCGGCGTTTGGAGT	128
P57-RT	AGATCAGCGCCTGAGAAGTCGTC	GGCTCTAAATTGGCTCACCGCAG	125
β 2M	TCTTTCTGGCCTGGAGGCTATC	CGGATGGATGAAACCCAGACAC	119
P21-M	TGTAGTACGCGAGGTTTCG	TCAACTAACGCAACTCAACG	202
P21-U	TTTTTGTAGTATGTGAGGTTTTGG	AACACAACTCAACACAACCCTA	200
P57-M	CGCGGTCGTTAATTAGTCGC	ACACAACGCACTTAACCTATAA	263
P57-U	TTTGTTTTGTGGTTGTTAATTAGTTGT	ACACAACACACTTAACCTATAA	270

Figure 1. Flow cytometric analysis of fresh CB CD34⁺ enriched cells (A) and ex vivo expanded cells in cytokine liquid culture (B), co-culture system with cytokine (C) and co-culture system without any cytokine (D). Specific staining was performed with anti-CD34-FITC and anti-CD38-PE antibodies. (A) CD34: 94.25%; CD38: 54.63%. (B) CD34: 71.63%; CD38: 67.38%. (C) CD34: 79.48%; CD38: 65.07%. (D) CD34: 77.22%; CD38: 64.42%.

Figure 2. Mean percentage of CD34⁺/CD38⁻ cells among fresh CB CD34⁺ isolated cells and expanded cells at day 10 in three culture conditions (n = 5).

Figure 3. Mean fold change of TNC (A) and CD34⁺ cells (B) in cytokine liquid culture and in co-culture system with and without cytokine.

Figure 4. Mean fold increase of CD34⁺/CD38⁻ cells in cytokine supplemented liquid culture and in co-culture system with and without cytokine on day 10 of expansion in three culture conditions.

Figure 5. CFU assay of ex vivo expanded hematopoietic cells at day 10 of culture.

Figure 6. mRNA expression fold changes of p21 (A) and p57 (B) in expanded CD34⁺ cells relative to fresh CB CD34⁺ cells in cytokine liquid culture and in co-culture system with cytokine. Data are plotted as mean ± SD of 2^−ΔΔCT, which is directly proportional to the relative gene expression.

Figure 7. Methylation-specific PCR of bisulfite-modified DNA using primer pairs specific for methylated and unmethylated p21 and p57 genes' promoter sequence. (A, B) representing p21 MSP results and (C, D) representing p57 MSP results for expanded cells in cytokine liquid culture and in co-culture system with cytokine, respectively. M, primers specific for methylated DNA; U, primers specific for unmethylated DNA. 1, DNA ladder; 2, unmethylated DNA control; 3, methylated DNA control; 4, 6, 8, 10, and 12 illustrate DNA amplification at days 0, 3, 7, 10, and 14 of culture, respectively, with unmethylated primers; 5, 7, 9, 11, and 13 had no band indicating lack of DNA amplification for expanded cells at days 0, 3, 7, 10, and 14 of culture, respectively, with methylated primers.