Real-time PCR experiments were performed using a standard process and as the reference gene. 4.7. Four hESC and 11 hiPSC lines were analyzed in this study (Table 1). Human PSCs were assayed after an average of 33 passages and differentiated into hematopoietic progenitors from EBs, using established hematopoietic permissive culture conditions. Their hematopoietic potential was evaluated by flow cytometry, colony formation, and whole transcriptome analysis in day-16 EBs. Two sub-groups of hPSCs were thereby identified according to their hematopoietic competence. Table 1 Human pluripotent stem cell (hPSC) lines used in this work. or master transcription factors such as were found down-regulated in hematopoietic-deficient iPSC-derived EBs. The same samples were also tested for their capability to differentiate into endoderm, mesoderm or ectoderm (Figure S2). In this context, several genes involved in mesoderm (was previously described to be down-regulated during hematopoietic development, with its expression inversely correlated to the hematopoietic potential of PSCs [17]. However, we found no significant change in expression level between hematopoietic-competent and -deficient AQ-13 dihydrochloride hPSC lines in our study. 2.3. Gene Expression Analysis of the NODAL/ACTIVIN Signaling Pathway This pathway belongs to the TGF-beta signaling pathway and is involved in many AQ-13 dihydrochloride developmental processes, including hematopoiesis (Figure S3A). The mRNA levels of several genes from the NODAL/ACTIVIN/BMP pathways were evaluated by microarray analysis in day-16 EBs from H1, PB6, PB6.1, PB7, and PB12.1 hPSCs, and by quantitative RT-PCR in all 15 hPSC lines at the pluripotent undifferentiated stage (Table S2 AQ-13 dihydrochloride and Figure S3B). None of these genes were differentially altered either in EBs or at the pluripotent stage. Rabbit polyclonal to KCTD1 Hence, they did not enable us to discriminate hematopoietic-deficient from -competent hPSCs solely based on their expression (Figure S3C,D). 2.4. Hematopoiesis-Related miRNA Expression during Hematopoietic Differentiation The role of miRNAs has been extensively explored in adult tissues including hematopoietic compartment, with functions in stem cell self-renewal, differentiation and in hematological disorders such as acute myeloid leukemia. Aside from their putative function, the role of miRNAs in early hematopoietic development has yet to be explored. As cell reprogramming and differentiation may be altered by miRNA expression, we have investigated the kinetics of hematopoiesis-related miRNA expression in hESC and hiPSC during hematopoietic commitment (Table S3). The expression kinetics of five miRNAs with recognized role in hematopoiesis (hsa-miR-125b-5p, hsa-miR-142-3p, hsa-miR-150-5p, hsa-miR-155-5p, hsa-miR-223-3p) and those of the PSC-specific hsa-miR-302-3p (used as control) were analyzed in hematopoietic-deficient (PB6, PB9) and -proficient hPSCs (PB 6.1, PB7, SA01, H1, H9), in the pluripotent undifferentiated stage (day time 0) and in day time-3 and day time-16 EBs (Number 2). As expected, miR-302 manifestation decreased upon hPSC differentiation into EBs. Open in a separate window Number 2 Hematopoiesis-related miRNA manifestation during EB tradition. Five hematopoietic-competent PSCs (PB 6.1, PB7, SA01, H1, H9) and two hematopoieticCdeficient ones (PB6, PB9) were analyzed at 0, 3 and 16 days in the course of hematopoietic differentiation (day time 0 representing the undifferentiated stage) by qRT-PCR. Graphs symbolize the manifestation kinetics of hsa-miR-125b-5p, hsa-miR-142-3p, hsa-miR-150-5p, hsa-miR-155-5p, hsa-miR-223-3p, and the hPSC-specific miR-302-3p, estimated by a CCt calculation (with Ct = Ct miRNA C Ct RNU48). Hematopoietic-competent and hematopoietic-deficient PSCs are displayed by green and reddish lines, respectively. Interestingly, miR-302 manifestation level remained elevated in hematopoietic-deficient PB6 and PB9 iPSCs, as compared to most hematopoietic-competent cells. Manifestation of miR-125b, related to multipotent HSC, was improved early in day time-3 EBs and partially reduced in day time-16 EBs. Blood-specific miR-223 was mainly up-regulated in day time-16 EBs, whereas the relative manifestation of miR-142 appeared to AQ-13 dihydrochloride be somewhat stable. Notably, the hematopoietic-deficient PB9 iPSC collection displayed a reduced manifestation level of miR-223 and miR-142 in both day time-3 and day time-16 EBs. We also mentioned substantial variations among the PSC lines concerning the manifestation of miR-155 and miR-150 (Number 2). 2.5. Global microRNA Manifestation Profiling in Human being PSCs To demonstrate a.
