Mouse studies showed that gene silencing in iPSCs did not impact ability to develop into differentiated cells.

Researchers have demonstrated that induced pluripotent stem cells (iPSCs) from mice have the same capacity as mouse embryonic stem cells (ESCs) to differentiate into endodermal progenitors. They believe that these results could allay concerns that differences between the two cell types could impact on the ability of iPSCs to generate tissues such as lung, liver, thyroid, and pancreas.

The work, by Boston University School of Medicine-led scientists, found that with few exceptions, the two cell types displayed similar surges in gene expression of specific master transcriptional regulators and global transcriptomes that define the developmental milestones of definitive endoderm (DE) differentiation. They report their findings in The Journal of Clinical Investigation in a paper titled “Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes.”

The team compared gene expression in iPSC and ESC lines triggered to differentiate into DE in defined culture media over five days; it has previously been shown that it takes about this much time for the majority of cells in culture to change to DE but not yet undergo lineage specification to liver, thyroid, or lung.

The investigators found that the four iPSC lines and two ESC lines evaluated all responded to the culture protocol by downregulating the expression of pluripotent transcriptional regulators and upregulating the set of essential endodermal master transcriptional regulators. While there was some clone-to-clone variability in the magnitude of endodermal response across the cell lines tested, the two iPSC and one ESC lines allowed to further differentiate over the complete 15-day endoderm differentiation protocol all finished the process.

“All three cell lines showed the capacity to robustly proliferate in these conditions, although growth kinetics were slightly better for the parental ES cell line,” the authors note.

The researchers then compared the capacity of both ESC- and iPSC-derived putative multipotent DE progenitors to undergo further lineage specification in response to inductive signals. When each cell line was exposed to an established three-stage culture protocol designed to favor pancreatic lineage specification, all three clones displayed similar early pancreatic lineage specification.

To demonstrate the in vivo functional potential to form endoderm, unsorted day-five iPSC-derived putative endodermal progenitors were transplanted beneath the kidney capsules of immunosuppressed mice. “These cells displayed robust capacity to form endodermal epithelia expressing nuclear Foxa2 protein, confirming the in vivo functional potential of iPS cell-derivatives following in vitro directed differentiation,” the team states.

“Taken together, these waves of gene expression during differentiation to endoderm-derived lineages further supported the definitive endodermal capacity of the day-five cells derived from each cell line in vitro.”

When studying the directed differentiation of pluripotent stem cells, an important issue is determining how closely a putative lineage generated in vitro mimics the phenotype of its authentic counterpart during normal development in the embryo. The researchers therefore compared the expression profiles of 1,000 genes in differentiating ESCs and iPSCs with those involved in DE development in the mouse embryo in vivo.

Although there were significant differences, the native embryonic DE kinetic signature overlapped with more than 50% of the in vitro 1,000-gene endoderm signature. “These results indicate that ES and iPS cell-derived DE resembles but is not identical to embryonic DE,” they note.

Interestingly, there were significant differences between the gene expression profiles of iPSCs and ESCs in both the fully differentiated and five-day state, with the iPSC lines demonstrating aberrant silencing of imprinted genes known to participate in endoderm differentiation. However, aberrant gene silencing appeared to have no detrimental effect on the iPSCs’ capacity to undergo directed differentiation to DE or early hepatic lineage specification in vitro.

“Our results indicate that iPS and ES cells undergo directed differentiation to DE with induction of remarkably similar global gene expression programs,” the authors conclude. “The key pioneer factors and transcriptional regulators known to be important in DE development, such as Foxa2, Gata4/6, and Sox17, are all similarly upregulated during endodermal directed differentiation of ES and iPS cells, and the waves of marker genes expressed during subsequent lineage specification of ES and iPS cell-derived endoderm also follow a sequence that has been described in the developing embryo.

“Overall our results have considerable implications for those wishing to develop cell-based therapies to reconstitute diseased endoderm-derived tissues. Regardless of imprinted status, iPS cells can be differentiated efficiently into DE precursors using the same serum-free culture protocols developed to derive endoderm from ES cells.”

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