Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells - Fig 4

<p>A and D) total population of cells are presented and highlighted population are transferred and tested for different markers, B) CD90-EP positive cells, C) CD44-FITC positive cells, and E) CD45 negative cells and F) C31 negative cells population.</p

Human amniotic fluid stem cells (hAFSCs) expressing p21 and cyclin D1 genes retain excellent viability after freezing with (dimethyl sulfoxide) DMSO

Human amniotic fluid stem cells (hAFSCs) have features intermediate between embryonic and adult SCs, can differentiate into lineages of all three germ layers, and do not develop into tumors in vivo. Moreover, hAFSCs can be easily obtained in routine procedures and there is no ethical or legal limitations regarding their use for clinical and experimental applications. The aim of this study was to assess the effect of slow freezing/thawing and two different concentrations of DMSO (10% DMSO + 90% fetal bovine serum [FBS] and 5% DMSO + 95% FBS) on the survival of hAFSCs. hAFSCs were obtained from 5 pregnant women during amniocentesis at 16–22 weeks of gestation. The expression of pluripotency markers (Octamer-binding transcription factor 4 [Oct4] and NANOG) by reverse transcription polymerase chain reaction and cell surface markers (cluster of differentiation [CD31], CD44, CD45, and CD90) by flow cytometry was analyzed before and after the slow-freezing. Cell viability was assessed by trypan blue exclusion or MTT assay. Quantitative mRNA expression of Oct4, NANOG, cyclin D1 and p21 was determined by real-time PCR before and after the slow-freezing. Pluripotency of hAFSCs was confirmed by NANOG and POU5F1 (Oct4) gene expression before and after slow-freezing. All hAFSC cultures were positive for CD44 and CD90. A higher viability of hAFSCs was observed after freezing with 90% FBS + 10% DMSO. There was increased expression of NANOG and decreased expression of POU5F1 gene after freezing, compared to control cells (before freezing). DMSO and the process of freezing did not significantly change the expression of p21 and cyclin D1 genes in hAFSCs. Overall, our results indicate the applicability of slow-freezing and DMSO in cryopreservation of SCs

Human amniotic fluid stem cells (hAFSCs) expressing p21 and cyclin D1 genes retain excellent viability after freezing with (dimethyl sulfoxide) DMSO

Human amniotic fluid stem cells (hAFSCs) have features intermediate between embryonic and adult SCs, can differentiate into lineages of all three germ layers, and do not develop into tumors in vivo. Moreover, hAFSCs can be easily obtained in routine procedures and there is no ethical or legal limitations regarding their use for clinical and experimental applications. The aim of this study was to assess the effect of slow freezing/thawing and two different concentrations of DMSO (10% DMSO + 90% fetal bovine serum [FBS] and 5% DMSO + 95% FBS) on the survival of hAFSCs. hAFSCs were obtained from 5 pregnant women during amniocentesis at 16–22 weeks of gestation. The expression of pluripotency markers (Octamer-binding transcription factor 4 [Oct4] and NANOG) by reverse transcription polymerase chain reaction and cell surface markers (cluster of differentiation [CD31], CD44, CD45, and CD90) by flow cytometry was analyzed before and after the slow-freezing. Cell viability was assessed by trypan blue exclusion or MTT assay. Quantitative mRNA expression of Oct4, NANOG, cyclin D1 and p21 was determined by real-time PCR before and after the slow-freezing. Pluripotency of hAFSCs was confirmed by NANOG and POU5F1 (Oct4) gene expression before and after slow-freezing. All hAFSC cultures were positive for CD44 and CD90. A higher viability of hAFSCs was observed after freezing with 90% FBS + 10% DMSO. There was increased expression of NANOG and decreased expression of POU5F1 gene after freezing, compared to control cells (before freezing). DMSO and the process of freezing did not significantly change the expression of p21 and cyclin D1 genes in hAFSCs. Overall, our results indicate the applicability of slow-freezing and DMSO in cryopreservation of SCs

Regulators which can regulate both NANOG, Oct-4, adipogenesis and osteogenesis.

<p>The network was constructed based on Common Regulators algorithm and the relationships were extracted by text mining using Pathway Studio 11.0.5 (Elsevier).</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

The growth curve of HAFCs in different passages.

<p>As shown here, by increasing the time of cell culture, the cell number increased. The growth rate of P4 is faster than P7 and P10. Except the p-value of 0 h vs 24 h, other p-vales were significant.</p

Relative expression of Oct4 and NANOG at early and late passages.

<p>Expression level of Oct4 and NANOG were high at P5 (early passage) compared with P7 (late passage) (P< 0.05).</p

Two lineage differentiation of AFSCs.

<p><b>A.1, 2</b> Before and after osteogenic differentiation and cell aggregates (were stained with alizarin red staining).<b>B1, 2</b> Before and after differentiation into adipose cells. Arrows show lipid vacuoles generated after adipose differentiation and oil red staining. (40x magnification was used)</p

The doubling time of HAFSCs in different passages.

<p>As shown in this figure, by increasing the number of passages, the time of doubling time were increased. So, the doubling time related to P10 is significantly more than P7 and P4. All p-value were significant and p-value for P10 vs P7, P10 vs P4 and P7 vs P4 were .000.</p

Expression analysis of MIR302A, MIRLet7G, MIR 21, and MR 137 in Human Amniotic Fluid Stem Cells.

<p>SC1: Human Amniotic Fluid Stem Cell, Control: Human fibroblasts.</p