Expression levels of miRNAs in PSC derived hematopoietic progenitors were compared to bone marrow and cord blood CD34+ cells. Several of the miRNAs (miR-142, miR-223) are not expressed in the ‘negative’ non-hematopoietic populations, but highly expressed in all of the hematopoietic populations, which supports the literature saying they are hematopoietic specific. miR-146 on the other hand was shown to be expressed at equivalent or higher levels than bone marrow and cord blood in all the differentiated populations, and miR-181 is expressed at high levels
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in the negative population at day 12, so these miRNAs are probably less useful as markers for hematopoiesis in PSC derived cultures (Figure 4.4a).
The ‘flat-up’ group miRNAs are mostly hematopoietic specific miRNAs and were shown by the microarray data to be expressed at too low a level in P-HPCs. The results of the qPCR analysis show a different pattern; in definitive hematopoietic progenitors (which are the ones that this project is most interested in) the miRNAs are either not expressed at significantly different levels to bone marrow and cord blood or in some cases are expressed at significantly higher levels than either bone marrow or cord blood progenitors (Figure 4.4a). Although these results contradict the microarray data the high expression of hematopoietic-specific miRNAs is encouraging as it suggests that the differentiation protocol which was used for the qPCR analysis promotes more effective
hematopoiesis than the earlier protocol used for the microarray data (see methods section 2.1.2). The fact that the expression of some of these miRNAs is higher than that in cord blood or bone marrow reflects the more-differentiated nature of the sorted cell population, as these miRNAs are expressed in CD34+ progenitors but increase in expression as the cells differentiate towards specific blood lineages. All of these miRNAs can be ruled out as targets for manipulation of expression as they seem to be expressed correctly in the definitive population.
qPCR analysis for flat-down miRNAs: The results from the flat-down group can be divided into
two broad categories as shown in Figure 4.4b; group A, which has miRNAs that eventually down- regulate to a level not statistically significantly different from cord blood or bone marrow, and group B, in which the miRNAs maintain high expression throughout development in all of the sorted populations.
The group A results include all of the miRNAs from the miR-302 cluster as well as miR-18b. This data shows that the cells rapidly lose expression of the miR-302 cluster miRNAs upon exit form pluripotency, which is what would be expected based on the literature. Where miR-302a and miR- 18b are different from bone marrow and cord blood they are significantly lower in expression, indicating that the cells have fully exited the pluripotent state. miR-302a* is expressed at
significantly higher levels in the definitive population than in bone marrow in both H9 and SB-Ad3 populations, but it is not significantly different from the expression in cord blood, which may be due to the more foetal nature of cord blood. miR-302a* is also the inactive strand, and is present in quantities which are at the limits of detection, which reduces the accuracy of the results.
It is not clear why so many of the miR-302 cluster fell into the flat-down group in the microarray data, it is possible that the early stage that the cells were tested as well as the lesser effectiveness of the protocol that was used meant that the cells were still expressing some molecular markers for
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pluripotency. Group A miRNAs can be ruled out as candidates for inhibition, as they appear to be expressed at the correct level in definitive hematopoietic cells when the Glasgow protocol is used.
The group B miRNAs (miR-9, miR-134, miR-148a, miR-150, miR-150*, miR-200b, miR-200c, miR-205, miR-424) are consistently highly expressed throughout development from pluripotent stem cells, in all the sorted populations, both hematopoietic and non-hematopoietic. Although for the definitive population the expression of some miRNAs such as miR-200b and miR-205 the difference is not statistically significant this is due to the large amount of variation and is a technical problem caused by the low numbers of this population reducing the accuracy of the results. The qPCR data for this group is consistent with the microarray data. Failure to down-regulate all of these 9 miRNAs could impair the function of hematopoietic cells. Their expression indicates problems in the regulation of the cell cycle, adhesion/motility and hematopoiesis specifically. All these miRNAs are therefore possible candidates for inhibition.
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Figure 4.4: qPCR data for expression of miRNAs calculated using ΔΔCT method using RNU44 and RNU48 as endogenous controls; all values were
calculated with respect to a reference value of expression of RNU44 and RNU48 in bone marrow. Data is presented as average +/- SEM. n=3. See methods section 2.3.1 for experimental details.
P values were calculated using unpaired two tailed t-tests comparing the three replicates for each of the cell types for each miRNA against both bone marrow and cord blood.
Figure 4.4a: Flat-up miRNAs Figure 4.4b: Group A micro-RNAs (down regulated by day 12), Group B micro-RNAs (maintained high expression
throughout differentiation).
Different time-points are shown in different colours:
Columns are labelled according to their cell types as sorted in section 4.3
Hemogenic endothelium= CD31+ CD34+ KDR+ CD45- Primitive= CD34low/-CD41a+CD43+CD235a+CD45- Definitive= CD34low/-CD41a-CD43+CD235a-CD45+ Negative= negative for all markers used in the sort
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