Side population cells in the human decidua of early pregnancy exhibit stem/progenitor cell-like characteristics  Chun Guo, Huili Zhu, Wei Huang, Shengfu Li, Wenwen Qu, Yaofang Liu, Aixiang Tan  Reproductive BioMedicine Online  Volume 21, Issue 6, Pages 783-793 (December 2010) DOI: 10.1016/j.rbmo.2010.07.010 Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Surface markers of decidual cells of human first-trimester pregnancy for (A–D) primary decidual cells and (E–H) cells of first passage. (A) Control; (B and F) E-cadherin expression; (C and G) vimentin expression; and (D and H) PRL expression. Bars=100μm. Reproductive BioMedicine Online 2010 21, 783-793DOI: (10.1016/j.rbmo.2010.07.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Sorting of SP cells from decidual cells. Decidual cells were digested and labelled with Hoechst 33342 in the presence or absence of verapamil and at least 1×106 cells were analysed by flow cytometry. Populations of decidual cells were separated using a two-window discrimination of red and blue fluorescence emissions along the abscissa and ordinate axis respectively. (A and B) On the fluorescence profile, whole decidual cells could be divided into two distinct major populations (annotated P3 and P4). The SP cells exhibited low Hoechst staining correlating with the fluorescence feature of an active Hoechst efflux (A; 0.6% P3) and disappeared in the presence of verapamil (B; 0.2% P3), therefore the percentage of real SP cells is 0.4%. (C) Phycoerythrin-CD34 (anti-human) and (D) Allophycocyanin-CD45 (anti-human) were added to decidual cells after Hoechst staining, their prevalence in SP cells (C; P2) were analysed (D) Q1 and Q4 represent the percentage of CD34 and CD45 positive cells respectively, and Q2 represents the percentage of both CD34 and CD45 positive cells, while Q3 represents both CD34 and CD45 negative cells. SP cells were mainly detected in Q3 and were negative for both CD34 and CD45. Reproductive BioMedicine Online 2010 21, 783-793DOI: (10.1016/j.rbmo.2010.07.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Phycoerythrin-CD13 staining of SP cells and NSP cells after Hoechst sorting. (A) After 48h of culture in vitro, primary decidual cells were stained with Hoechst 33342 and subsequently PE-CD13 (anti-human); SP-gated and NSP-gated cells are indicated as P2 and P3. (B and C) Percentage of CD13-positive cells from P2 and P3 are indicated as P4 and P5. (D) 8.8%±4.13% and 95.46%±3.61% of CD13-positive cells were detected in SP cells and NSP cells, respectively. Results are from experiments performed in triplicate. Reproductive BioMedicine Online 2010 21, 783-793DOI: (10.1016/j.rbmo.2010.07.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Immunophenotypes in human decidua of first-trimester pregnancy of (A–D) NSP cells and (E–H) SP cells. (A and E) Phase-contrast microscopy; (B and F) phycoerythrin (PE)-CD13 staining; (C and G) vimentin staining; and (D and H) NSP cells were positive for prolactin (PRL) staining. Bars=100μm (A–D), 50μm (E), 20μm (F–H). Reproductive BioMedicine Online 2010 21, 783-793DOI: (10.1016/j.rbmo.2010.07.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Clonogenicity of decidual SP cells in in-vitro culture. (A and B) Clones (arrows) were formed after 21days of culture in CFM. (C) Formation of clones (arrow) 28days after digestion and reseeding in CFM. (D) No clones were formed after 21days of culture in media without IL-6, SCF and TPO. Bars=100μm (A), 50μm (B), 50μm (C), 100μm (D). Reproductive BioMedicine Online 2010 21, 783-793DOI: (10.1016/j.rbmo.2010.07.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Growth of decidual SP cells in different conditioned medium (A) at 1week, (B) at 3weeks and with (C) prolactin (PRL) staining. PRL-positive cells were found in group II (NSP cells cultured in DMEM/F12 and 10% FBS) and groups III–VI (DMEM/F12 and 10% FBS plus: 50% CCM (group III), 50% DCM (group IV), 10nmol/l oestradiol and 10μmol/l progesterone (group V) or 50% (CCM+DCM) (group VI)); stain intensity varied among different groups. Bars=100μm (1 and 3weeks), 50μm (PRL staining). Reproductive BioMedicine Online 2010 21, 783-793DOI: (10.1016/j.rbmo.2010.07.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 7 (A) Expression of CD13 in differentiated cells from groups II–VI by fluorescent immunophenotyping. CD13 was expressed in each group. (B) Group IV, from medium with DCM, forming a multilayered structure, which looked like a meshwork and was consistent with characteristics of decidual stromal cells. Prolactin (PRL) was positively expressed. (C and D) When decidual SP cells formed clones after 21 days of culture, they were digested and reseeded in CFM for another 28 days until colonies formed again, then the cells were digested and the medium was replaced by DMEM/F12 and 10% FBS for 20 days: stroma-like tissues were found and CD13 was expressed again. Bars=20μm (A, C, D), 50μm (B). Reproductive BioMedicine Online 2010 21, 783-793DOI: (10.1016/j.rbmo.2010.07.010) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions