MicroRNA Profiling Reveals Distinct Mechanisms Governing Cardiac and Neural Lineage-Specification of Pluripotent Human Embryonic Stem Cells

Abstract

Realizing the potential of human embryonic stem cells (hESCs) has been hindered by the inefficiency and instability of generating desired cell types from pluripotent cells through multi-lineage differentiation. We recently reported that pluripotent hESCs maintained under a defined platform can be uniformly converted into a cardiac or neural lineage by small molecule induction, which enables lineage-specific differentiation direct from the pluripotent state of hESCs and opens the door to investigate human embryonic development using in vitro cellular model systems. To identify mechanisms of small molecule induced lineage-specification of pluripotent hESCs, in this study, we compared the expression and intracellular distribution patterns of a set of cardinal chromatin modifiers in pluripotent hESCs, nicotinamide (NAM)-induced cardiomesodermal cells, and retinoic acid (RA)-induced neuroectodermal cells. Further, genome-scale profiling of microRNA (miRNA) differential expression patterns was used to monitor the regulatory networks of the entire genome and identify the development-initiating miRNAs in hESC cardiac and neural lineage-specification. We found that NAM induced nuclear translocation of NAD-dependent histone deacetylase SIRT1 and global chromatin silencing, while RA induced silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family to high levels. Genome-scale miRNA profiling indentified that a unique set of pluripotence-associated miRNAs was down-regulated, while novel sets of distinct cardiac- and neural-driving miRNAs were up-regulated upon the induction of lineage-specification direct from the pluripotent state of hESCs. These findings suggest that a predominant epigenetic mechanism via SIRT1-mediated global chromatin silencing governs NAM-induced hESC cardiac fate determination, while a predominant genetic mechanism via silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family governs RA-induced hESC neural fate determination. This study provides critical insight into the earliest events in human embryogenesis as well as offers means for small molecule-mediated direct control and modulation of hESC pluripotent fate when deriving clinically-relevant lineages for regenerative therapies.

Figure 1. NAM-induced cardiomesodermal cells have acquired a silenced chromatin while RA-induced neuroectodermal cells retain an embryonic acetylated chromatin

(A) NAM (cardiac) or RA (neural) treatment induced differentiation under the defined culture system, as indicated by the appearance of Oct-4 (red) negative cells within the colony. Untreated cells were used as the control. (B) The strong expression and nuclear localization of a set of active chromatin modifiers in untreated pluripotent hESCs (control), including acetylated histones H3 (acH3, green), acetylated histones H4 (acH4, green), ATP-dependent active chromatin-remodeling factors Brg-1 (red) and hSNF2H (green), and HDAC1 (red), and cytoplasmic localization of histone methyltransferase SUV39H1 (red). NAM-induced cardiomesodermal cells (cardiac) displayed significantly decreased levels of histone H3 and H4 acetylation, and Brg-1 and HDAC1 expression, while RA-induced neuroectodermal cells (neural) retained the similar expression levels and localization patterns. All cells are indicated by DAPI staining of their nuclei (blue) in the insets. (C) The weak or undetectable expression of repressive chromatin remodeling factor Brm (green) and K9 methylated histone H3 (red). (D) NAM induces nuclear translocation of NAD-dependent HDAC SIRT1 (red). All cells are shown by DAPI staining (blue) of their nuclei. Scale bars: 10 μm.

Figure 2. Genome-scale miRNA profiling of hESC cardiac and neural specification by small molecule induction

(A) Hierarchal clustering of differentially expressed miRNAs in undifferentiated hESCs (hESC), cardiac-induced hESCs by NAM (cardiac), and neural-induced hESCs by RA (neural). Statistically significant p values and clustering analysis were provided by LC Sciences as part of miRNA microarray service. (B) Pie charts showing decreased contributions of a set of pluripotence-associated miRNAs (purple) and increased contributions of distinct sets of cardiac- (green) and neural- (blue) driving miRNAs to the entire miRNA populations upon cardiac and neural induction of pluripotent hESCs by small molecules.

Figure 3. Down-regulation of a unique set of human pluripotence-associated miRNAs upon lineage-specific induction by small molecules

(A) The expression of two most prominent clusters of pluripotence-associated miRNAs hsa-miR-302 and hsa-miR-371/372/373 was significantly suppressed upon lineage-induction of hESCs by small molecules. (B) A novel group of miRNA clusters was found to be significantly down-regulated upon small-molecule-induced lineage differentiation, albeit to less extents. (C) Log2 ratios of down-regulation of the human pluripotence-associated miRNAs. The levels of down-regulation: *: 5–10 fold, **: 10–200 fold, and ***: 200–1000 fold (green lines: cardiac induction by NAM, blue lines: neural induction by RA).

Figure 4. Up-regulation of a novel set of human embryonic cardiac-driving miRNAs upon cardiac induction by NAM

(A) Hierarchal clustering of differentially expressed miRNAs in cardiac-induced hESCs by NAM, compared with pluripotent hESCs and neural-induced hESCs by RA. Statistically significant p values and clustering analysis were provided by LC Sciences as part of miRNA microarray service. (B) A group of cardiac-driving miRNAs displayed an expression pattern of up-regulation upon cardiac induction and down-regulation upon neural induction. (C) A group of cardiac-driving miRNAs had an expression pattern of up-regulation upon cardiac induction but was not significantly affected upon neural induction.

Figure 5. Up-regulation of a novel set of human embryonic neural-driving miRNAs upon neural induction by RA

(A) Hierarchal clustering of differentially expressed miRNAs in neural-induced hESCs by RA, compared with pluripotent hESCs. Statistically significant p values and clustering analysis were provided by LC Sciences as part of miRNA microarray service. (B) A group of neural-driving miRNAs displayed an expression pattern of up-regulation upon neural induction and down-regulation upon cardiac induction. (C) A group of neural-driving miRNAs had an expression pattern of up-regulation upon neural induction but was not significantly affected upon cardiac induction. (D) Log2 ratios of up-regulation of the human embryonic neural-driving miRNAs. The levels of up-regulation: *: 5–10 fold, **: 10–50 fold, and ***: 50–200 fold.