Abstract
Abstract 2622
Numerous somatic cells can be reprogrammed by ectopic expression of defined transcription factors including OCT4, SOX2, MYC, KLF4 (Yamanaka's factors), NANOG, and LIN28 to pluripotent cells, referred to as induced pluripotent stem (iPS) cells which can then be differentiated into a variety of somatic cell types. Production efficiency of iPS cells by these transcription factors is extremely low, and therefore a large portion of the cells remain unprogrammed or incompletely reprogrammed. Thus, identification of additional factors required for enhancing iPS cells production efficiency has been an intensive research subject. We generated iPS cell from CD34+ cells of human umbilical cord blood that had been frozen in an unseparated state for twenty plus years using lentiviruses expressing Yamanaka's factors. During the iPS cell production processes, we monitored cell surface expression of TRA1-60, a marker for human embryonic stem (hES) cells, within colonies using a live cell staining method. Three to four weeks after gene transduction, TRA1-60 positive cells emerged in about 5% of the colonies. So, we mechanically separated TRA1-60 negative cells from TRA1-60 positive cells in colonies. Further culture did not allow TRA1-60 negative cells to convert to TRA1-60 positive cells even after 10 passages, indicating that TRA1-60 negative cells were stable at an incompletely reprogrammed state. TRA1-60 negative cells were similar to hES cells in morphology and still demonstrated expression of exogenous Yamanaka's factors. TRA1-60 negative cells were distinct from TRA1-60 positive cells with regards to methylation pattern of OCT4 promoter regions and differentiation potential. In contrast to TRA1-60 positive cells, incompletely reprogrammed TRA1-60 negative cells lacked hESC-specific miRNAs (miR-302 and 371 clusters), which are known to be involved in controlling self-renewal and pluripotency of hES cells. We hypothesized that these miRNAs can promote a transition from incompletely to fully reprogrammed iPS cells. To test this hypothesis, we introduced the miRNA clusters of miR-302 and 371 using lentiviruses to TRA1-60 negative incompletely reprogrammed cell to determine whether these miRNAs could convert TRA-60 negative cells to TRA1-60 positive completely reprogrammed cells. Our results showed that these miRNAs were able to convert more than 10% of TRA1-60 negative incompletely reprogrammed cells to TRA1-60 positive iPS cells with characteristics of completely reprogrammed iPS cells, such as differentiation of three germ layers and acquisition of typical hES cell-specific cell cycling patterns with an unrestricted G1 to S phase transition. These results indicate that hES-specific miRNAs have a strong potential to promote partially reprogrammed cord blood CD34+ cells to iPS cells with extensive self-renewal capacity. Our study suggests that current techniques with low iPS cell production efficiency can be improved by ectopic overexpression of hES-specific miRNAs (miR-302 and371 clusters).
Disclosures:
Broxmeyer: Corduse: Honoraria, Membership on an entity's Board of Directors or advisory committees.
SOX2 Is Regulated Differently from NANOG and OCT4 in Human Embryonic Stem Cells during Early Differentiation Initiated with Sodium Butyrate