*Purpose: Epigenetic states such as histone modifications and DNA methylation that influence transcription are well-characterized regulators of stem cell pluripotency and differentiation to specific lineages. Despite the importance of alternative splicing (the process of intron removal and exon ligation in which an RNA-binding protein (RBP; trans-acting factor) directly interacts with a cis-element in the RNA molecule to influence more than one outcome on the mature mRNA, and thus protein, product) in regulating disease, pluripotency, and tissue-specific gene expression, how this additional epigenetic form of regulation is involved during early cell fate decisions is not known. Here we identify specific splicing patterns do exist during differentiation and they can be useful biomarkers for lineage-specificity.

*Methods: We identify lineage-specific splicing patterns by comparing splicing changes (using VASTtools to analyze high throughput RNA sequencing (RNA-seq) data) between undifferentiated human embryonic stem cells (hESCs), 3 different samples from 3 day differentiated hESCs (spontaneous, endoderm, and cardiac mesoderm), and 2 different samples from 7 day differentiated hESCs (spontaneous and hepatic progenitors). We have validated a number of these changes using RT-PCR.

*Results: We find evidence of lineage-specific splicing patterns at the global level. One particularly interesting example is a previously reported developmentally-regulated alternative exon in the FOXP1 transcription factor that produces protein isoforms with opposing functions. When exon 18a is included FOXP1 promotes ESC differentiation, but with it is skipped promotes self-renewal (Gabut et al 2011). We find here FOXP1 splicing actually shows lineage-specificity in partial skipping in day 3 endoderm and spontaneously differentiated cells, but is almost completely included in day 3 cardiac mesoderm (see figure).

*Conclusions: As evidenced by our RNA-seq datasets and individual examples a distinctive splicing pattern is associated with not only stem cell differentiation but also lineage progression. These patterns can serve as exon-specific biomarkers for a high-resolution, systems-level analysis of specific early lineages. Further studies are warranted to determine if these are actual drivers of cell fate and lineage progression along the way to becoming hepatocytes or other mature cell types.

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