Understanding the much more complex human embryonic development has been hindered by the restriction on human embryonic and fetal materials as well as the limited availability of human cell types and tissues for study. In particular, there is a fundamental gap in our knowledge regarding the molecular networks and pathways underlying human embryonic development. The enormous diversity of human somatic cell types and the highest order of complexity of human genomes, cells, tissues, and organs among all the eukaryotes pose a big challenge for characterizing, identifying, and validating functional elements in human embryonic development in a comprehensive manner. Many of the biological pathways and mechanisms of lower-organism or animal model systems do not reflect the complexity of humans and have little implications for the prevention and cure of human diseases in the clinical setting. As a result of lacking a readily available human embryonic model system, the mainstream of biomedical sciences is becoming increasingly detached from its ultimate goal of improving human health.
Pluripotent human embryonic stem cells (hESCs) have the unconstrained capacity for long-term stable undifferentiated growth in culture and the intrinsic potential for differentiation into all somatic cell types in the human body, holding tremendous potential for restoring tissue and organ function. Derivation of hESCs, essentially the in vitro representation of the pluripotent inner cell mass (ICM) or epiblast of the blastocyst, provides not only a powerful in vitro model system for understanding the human embryonic development, but also a pluripotent reservoir for derivation of a large supply of disease-targeted human somatic cells that are restricted to the lineage in need of repair. However, realizing the developmental and therapeutic potential of hESCs has been hindered by the current state of the art for generating functional cells through multi-lineage differentiation of pluripotent cells in a 2-dimentional (2D) culture, which is uncontrollable, inefficient, highly variable, difficult to reproduce and scale-up, and often causes phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity following transplantation. Development of novel strategies for well-controlled efficient differentiation of hESCs into functional lineages is crucial not only for unveiling the molecular and cellular cues that direct human embryogenesis, but also to harnessing the power of hESC biology for cell-based therapies.
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. MicroRNA (miRNA) expression profiling using microarrays is a powerful high-throughput tool capable of monitoring the regulatory networks of the entire genome and identifying functional elements in development and disorders. Recently advances in human miRNA expression microarrays have provided powerful genome-wide, high-throughput, and high resolution approaches that will lead to great advances in our understanding of the global phenomena of developmental processes. 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 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. This original research article of Dr. Parsons supported by the National Institutes of Health was published in The International Open Access Journal of Stem Cell Research & Therapy.
