Adding Rarg and Lrh-1 to transcription factor mix enabled efficient production of exogenous factor-independent iPSCs from adult fibroblasts.

Scientists claim to have improved the efficiency of the four transcription factor approach to generating human induced pluripotent stem cells (iPSCs) from somatic cells. A team led by Wellcome Trust Sanger Institute researchers says that somatic cells transfected with vectors delivering the RAR receptor Rarg (retinoic acid receptor gamma) and Lrh-1 (liver receptor homolog 1) in addition to the known iPSC-promoting factors Oct4, Sox2, Klf4, and c-Myc resulted in the generation of human iPSCs from both neonatal and adult human fibroblasts.

Using this six factor mix of reprogramming genes, resulting IPSCs proliferated independently without the need for ectopic expression of any of the exogenous factors and closely resembled ground-state mouse embryonic stem cells (ESCs) in terms of growth properties, gene expression, and signaling dependency.

Pentao Liu, PhD., Wei Wang, Ph.D., and colleagues, report their research in PNAS in a paper titled “Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1.”

To try and identify factors that could increase the efficiency of reprogramming somatic cells to iPSCs, the authors looked at developmentally important signaling pathways, in particular retinoic acid (RA) signaling. They cloned cDNAs of the four transcription factors Oct4, Sox2, Klf4, and cMyc, as well as Rara (RA receptor-α), Rarg, and Rara-DN (dominant-negative form of Rara) into piggyback (PB)-murine stem cell virus (MSCV) expression vectors.

Individual vectors were transfected in different combinations into mouse embryonic fibroblasts (MEFs). Transfection of the 4F vector was used as a control and compared with transfection using 4F plus either Rarg (4F+Rarg), Rara (4F+Rara), or Rara-DN (4F+Rara-DN).

By day 30, it was evident that one and two orders of magnitude more ESC-like colonies had developed as a result of expressing the 4F+Rara and 4F+Rarg vectors, respectively, compared with expression of 4F. In contrast, expression of the Rara-DN vector almost completely blocked reprogramming.

The researchers then investigated the specificity and temporal requirement of RA signaling in reprogramming by using a synthetic RA agonist specific to Rarg (CD437) or to Rara (AM580). After transfecting MEFs with 4F, addition of CD437 in the medium for up to eight days substantially increased the number of iPSCs. This indicated a temporal requirement for RA signaling in reprogramming. Similar results were observed when the Rara agonist AM580 was added instead of CD437.

Interestingly, studies using cell lines in which activation of the pluripotency marker gene Oct4 was accompanied by GFP fluorescence suggested that the requirement for RA signaling occurs early in reprogramming. However, while Rarg overexpression increased the numbers of cells undergoing reprogramming, it didn’t markedly improve the overall iPSC quality nor did it alter the reprogramming kinetics: transfection with  either 4F or with 4F+ Rarg led to the formation of pre-iPSC clones with incomplete Oct4 activation and lower expression levels of key pluripotency genes than wild-type ES cells.

The next step was to see whether additional genetic factors could cooperate with Rarg in reprogramming somatic cells. Based on data from previous studies, the team focused on Lrh-1 (liver receptor homolog 1), a nuclear receptor that has previously been shown to play an essential role in the maintenance of Oct4 expression in ESCs.

When Lrh-1 and Rarg were co-expressed alongside 4F (i.e., 6F expression) in MEF, up to 20 time more iPSC colonies developed compared with iPSC colony development after expression of 4F alone. And, remarkably, the researchers report, over 80% of these expressed high levels of endogenous Oct4 as evidenced by GFP fluorescence.

The 6F combination also markedly accelerated the reprogramming process, they continue. Microscopic ESC-like GFP+ colonies appeared as early as four days after transfection of 6F into the reporter MEFs. In contrast, when cells were transfected using 4F, GFP+ cells weren’t found until day seven after transfection.

And while 10–16 days of continuous expression of 4F in MEFs is generally needed before some of the reprogrammed cells will become independent of exogenous factor expression, transfection of MEFs with doxycycline (dox)-inducible 6F demonstrated that some colonies expressed SSEA-1 and Nanog after just four days and could subsequently generate large iPSC colonies even when doxycycline was withdrawn. “This is consistent with a recent report that activation of Nanog coincides with irreversible commitment to reprogramming, or point of no return,” the team remarks.

Rex1 is expressed in mouse ES cells but not in epiblast stem cells (EpiSCs) and thus represents a better marker for ground state pluripotency than Oct4. The researchers generated a Rex1-GFP mouse cell line and used this to evaluate the effects of 6F co-expression. Again, it took only four days before dox-independent iPSC colonies were generated, while expression of 4F was required for at least 12 days.

To confirm the pluripotency of iPSCs generated following 6F expression, 20 dox-indepenent cell lines were characterized in vitro and in vivo. Analyses confirmed that iPSCs expressed expected levels of pluripotency genes. The promotor regions of Oct4 and Nanog showed nearly complete DNA demethylation. 6F iPSCs also proliferated well and maintained pluripotency in N2B27/leukemia inhibitory factor (LIF) medium supplemented with PD0325901 (MAPK inhibitor) and CHIR99021 (glycogen synthase kinase 3 inhibitor), without feeders.

The iPSCs were able to differentiate to somatic cell types representing all three germ layers both in vitro and in vivo and efficiently contributed to the germline in chimeras. “Importantly, because most iPSC lines did not express detectable levels of the exogenous factors, the chimeras did not develop tumors,” the authors stress.

The team then turned their attention to testing the effects of 6F expression (compared with 4F) on reprogramming human somatic cells, in this case human neonatal foreskin dermal fibroblast (HDFn) cells. Primary 4F colonies in M15/LIF medium were morphologically distinct from the 6F ones and no stable lines could be established.

In contrast, colonies formed from human HDFn cells in M15/LIF mouse ESC medium were morphologically similar to mouse ESC colonies. Representative colonies were picked on day 12 to 14 and cultured using standard mouse ESC protocols on STO feeders.

Interestingly, the team notes, iPSC 6F colonies (but not 4F colonies) in M15/LIF expressed key pluripotency genes and human ESC markers and could also be maintained in N2B27/2i/LIF medium. Even after extensive in vitro culture, these iPSCs retained a normal karyotype, demonstrating that they were genetically stable, and formed teratomas in immune-compromised NSG mice, with cell types representing all three germ layers.

To establish human iPSC lines that were independent of exogenous reprogramming factor expression, the researchers then used the Tet-On system to express 6F. Colonies consisting of ES-like cells appeared after 8 to 10 days of dox induction, and these expressed endogenous NANOG. It was also possible to withdraw dox from the medium from as early as day eight after transfection.

Resulting cells could subsequently be cultured in DMEM/F12 medium supplemented with KSR/2i/LIF. Calculations suggested that from 1 million human neonatal dermal fibroblast cells, it was possible to obtain typically 800–1,000 colonies, most of which were dox-independent.

The dox-independent human iPSCs expressed endogenous Oct4, Sox2, and Nanog at levels comparable to those of human ESCs and were also positive for human ESC surface markers. “Critically, expression of the exogenous factors in these cells was undetectable, which confirmed that pluripotency was self-sustainable in the KSR/2i/LIF medium,” the authors note.

“Even after extensive in vitro culture, these iPSCs retained the normal karyotype demonstrating that these cells were genetically stable.” Human iPSCs could in addition differentiate to cells of three germ layers in vitro and formed mature teratomas when injected into NSG mice.

Encouragingly, the same strategy also led to the successful generation of iPSCs from primary human adult dermal fibroblasts (HDFa). iPSCs derived from HDFn and HDFa displayed similar gene-expression patterns including stable expression of key endogenous plurpiotency genes.

The researchers called their fibroblast growth factor (FGF)-indpendent human iPSCs Sanger human iPSCs (SH-iPSCs) to distinguish them from those produced in conventional human ESC medium that does contain FGF. Further studies showed that similar to mouse ESCs , SH-iPSCs were easily expanded, and their Oct4 and Nanog promoter regions were almost completely demethylated. Cell proliferation analysis suggested that roughly seven times more SH-iPSCs underwent cell division than human ESCs cultured in KSR/FGF medium.

Indeed, rather than requiring FGF, the SH-iPSCs grew well and kept pluripotency gene expression in KSR/2i/LIF medium even in the presence of a FGF receptor inhibitor. SH-iPSCs also expressed lower levels of lineage-specific genes such as PAX6, GATA6, and SOX17 and proliferated well in the presence of BMP4.

As a final test the researchers cultured SH-iPSCs in KSR/FGF medium to see whether they could could become human ESC-like cells. After three passages, the FGF-cultured human iPSCs became morphologically similar to human ESCs.

While they still expressed comparable levels of pluripotency genes including Oct4, Nanog, and REX1, KLF4 expression dropped four-fold. They also downregulated STELLA and NODAL expression but retained human ESC markers such as SSEA3. When the culture medium had been switched back to KSR/2i/LIF, these FGF-adapted iPSCs appeared differentiated and expressed high levels of Sox1 with concomitant loss of Oct4 and Nanog expression, “resembling the behavior of human ES cells,” the Sanger team notes.

Comparative analysis of human ESCs with SH-iPSCs that were subsequently cultured in KSR/FGF medium indicated that the cells exhibited similar expression profiles that were, importantly, distinct from SH-iPSCs cultured in 2i/LIF medium.

Further analysis of the SH-iPSCs was carried out in terms of telomerase activity. Stem cells express telomerase, and human ESCs have high telomerase activity, the team explains. They found that human iPSCs growing in the 2i/LIF medium had about 10-fold higher telomerase activity than human ESCs or the FGF-cultured iPSCs. “These results thus demonstrated that SH-iPSCs growing in KSR/2i/LIF medium had the potential to covert to cells similar to human ES cells but not vice versa,” they state.

“Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency,” the authors conclude. SH-iPSCs are similar in many aspects to the envisioned ground-state human pluripotent stem cells and should facilitate functional dissection of the human genome and for modeling human diseases.”

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