|Pluripotency Factors Worm their Way towards Mammals|
Original article from STEM CELLS
Planarians are free-living freshwater flatworms well-known for their extreme regenerative abilities linked to the existence of a large population of pluripotent adult stem cells (ASCs) (Wagner et al 2011) that comprise approximately 10–20% of the cells in the animal (Eisenhoffer et al 2008). While the existence of stem cells is ancestral to multicellular animals (Watanabe et al 2009 and Bosch 2009) it is unknown whether stem cells from diverse species share conserved transcriptional networks associated with stem cells, pluripotency and self-renewal. Now in a study from the laboratory of Bret J. Pearson, the transcriptomes of planarian adult stem cells and those of human (hESCs) and mouse embryonic stem cells (mESCs) have been compared, which has uncovered conserved factors that can affect the stem cell-associated functions in planarians (Labbé et al 2012).
To define the planarian stem cell transcriptome, RNA-Seq analysis was performed on three distinct cellular compartments: stem cells, stem cell progeny, and differentiated tissues and then differential expression (DE) between the three compartments analysed. A previously described fluorescence-activated cell sorting (FACS) protocol was used to sort highly purified planarian stem cells (Hayashi et al 2006), and differentiated tissue cells were collected after a lethal dose of irradiation which leaves planarians devoid of stem cells and their lineage progeny (Eisenhoffer et al 2008). This demonstrated that 2,177 planarian transcripts showed over 5-fold DE in stem cells compared with differentiated tissues and, interestingly, only 539 of those transcripts were also over 5-fold DE over stem cell progeny, which suggested that 1,638 transcripts are shared between stem cells and stem cell progeny. Finally, 219 transcripts were found to be specific to the stem cell progeny, being over 5-fold higher than both stem cells and differentiated tissues. Gene orthologs were then mapped between planarian, human and mouse, giving a total of 4,432 of planarian transcripts that were assigned as orthologs to both mouse and human genes.
Gene set enrichment analysis (GSEA), a powerful method to analyze genetic pathways enriched in a particular dataset, was then performed for several comparisons: (1) stem cells versus stem cell progeny, (2) stem cells versus differentiated tissues and (3) stem cell progeny versus differentiated tissues. Overall, this found that the stem cell compartment was enriched for genes associated with the cell cycle, DNA damage and repair (including significant enrichment of p53, Rb, and Fanconi pathways) and RNA processing/transport pathways. Stem cell progeny were enriched for pathways associated with development and differentiation, spliceosome and chromatin modification pathways while differentiated tissue was shown to be enriched for processes excluded from stem cells, such as genes involved in maintenance of the nervous system and pathways implicated in cell adhesion, muscular maintenance and osmoregulation.
Using RNA-seq data for hESCs (H1 and H9) and mESCs (V6.5 cells), DE in mammalian ESCs was next determined in order to compare this against planarian ASC data. Only orthologous genes were used for comparison and, of the 4,432 orthologous genes, 538 in mESCs, 421 in hESCs and 605 in planarian ASCs were found to have over 5-fold DE in the stem cell compartment over their respective differentiated tissues, with a similar gene overlap for planarian ASCs with human (46%) and mouse (36%). Overall, 123 genes were found to be specifically over 5-fold DE in the stem cells of all three species. These genes were then assayed by RNAi-mediated knockdown in planarian and effects were measured against three criteria: 1) defects in tissue homeostasis, 2) the ability to regenerate missing head structures - a process that depends on proper stem cell function, and 3) the expression of the stem cell marker piwi-1. Of 100 genes assayed, the knockdown of 26 gave multiple common stem cell defects, such as head regression (e.g. NHP2-like, TDRD9, PHB, RPA, CSTF3, CBX3, and PLK) and dorsal lesions (e.g. PSD12, TTC27, RUVB2, NOP58, NUP93, TBL3, TDRD9, SMD1, FBRL, NOP2 and RACK1). All 26 knockdowns caused the loss of stem cells (as measured by piwi-1 staining), except for RACK1 which gave minimal effects, while several knockdowns caused a spatial-specific loss in stem cells; NOP2 (lateral), TDRD9, RPA2, SMD1 (posterior), C1orf107, PSMC4 (anterior) and TBL3, PHB (complete loss).
In conclusion, genes that were found to be differentially expressed in planarian, human and mouse stem cell populations were shown to be regulators of planarian stem cells which may make up an ancestral network which controls pluripotency and self-renewal. Further, as the genes found are conserved, their role in mammalian systems can now be examined. One potential caveat to the study is the non-neutral effect of irradiation on differentiated cells (Eisenhoffer et al 2008 and Solana et al 2012), however FACS purification, RNAi studies and careful array analysis suggest that the findings in this study are relevant.
Eisenhoffer GT et al.
Hayashi T et al.
Labbé RM et al.
Solana J et al.
Wagner DE et al.
Watanabe H et al.
STEM CELLS correspondent Stuart Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.