Improved Efficiency and Pace of Generating Induced Pluripotent Stem Cells from Human Adult and Fetal Fibroblasts

Abstract

It was reported recently that human fibroblasts can be reprogrammed into a pluripotent state that resembles that of human embryonic stem (hES) cells. This was achieved by ectopic expression of four genes followed by culture on mouse embryonic fibroblast (MEF) feeders under a condition favoring hES cell growth. However, the efficiency of generating human induced pluripotent stem (iPS) cells is low, especially for postnatal human fibroblasts. We started supplementing with an additional gene or bioactive molecules to increase the efficiency of generating iPS cells from human adult as well as fetal fibroblasts. We report here that adding SV40 large T antigen (T) to either set of the four reprogramming genes previously used enhanced the efficiency by 23-70-fold from both human adult and fetal fibroblasts. Discernible hES-like colonies also emerged 1-2 weeks earlier if T was added. With the improved efficiency, we succeeded in replacing MEFs with immortalized human feeder cells that we previously established for optimal hES cell growth. We further characterized individually picked hES-like colonies after expansion (up to 24 passages). The majority of them expressed various undifferentiated hES markers. Some but not all the hESlike clones can be induced to differentiate into the derivatives of the three embryonic germ layers in both teratoma formation and embryoid body (EB) formation assays. These pluripotent clones also differentiated into trophoblasts after EB formation or bone morphogenetic protein 4 induction as classic hES cells. Using this improved approach, we also generated hES-like cells from homozygous fibroblasts containing the sickle cell anemia mutation Hemoglobin Sickle.

Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors

“…The SV40 large T antigen (T) and the catalytic subunit of human telomerase, hTERT, have been reported to significantly improve the efficiency of generating iPS cells [23,24]. Therefore, we attempted to establish ovine iPS cell lines by adding SV40 large T and hTERT.…”

The emerging role of p53 in stem cells

“…The first evidence for a role of p53 in the control of this process came from studies on human fibroblasts in which either SV40 large T antigen (a repressor of p53 and pRb pathways) [24] or p53 small interfering RNA (siRNA) [25] were used together with the Yamanaka factors to improve the efficiency of iPSC reprogramming. Subsequently, it was demonstrated that inhibition or loss of p53 increases reprogramming efficiency by 3-to 10-fold, depending on the cell system or the manner of deletion or inactivation of p53 [22,23,[26][27][28].…”

Mechanism and methods to induce pluripotency

“…Suppression of the p53 pathway may compromise a cell's genome stability. For instance, adding SV40 large T into the reprogramming mix accelerated the speed of iPSC formation; however, many of the iPSCs gained chromosomal abnormality (Mali et al, 2008). Thus, during reprogramming, one must take extra care to maintain the intricate balance between safeguarding genome integrity and change of cell fate.…”