The Temporal and Hierarchical Control of Transcription Factors-Induced Liver to Pancreas Transdifferentiation

Abstract

Lineage-specific transcription factors (TFs) display instructive roles in directly reprogramming adult cells into alternate developmental fates, in a process known as transdifferentiation. The present study analyses the hypothesis that despite being fast, transdifferentiation does not occur in one step but is rather a consecutive and hierarchical process. Using ectopic expression of Pdx1 in human liver cells, we demonstrate that while glugacon and somatostatin expression initiates within a day, insulin gene expression becomes evident only 2–3 days later. To both increase transdifferentiation efficiency and analyze whether the process indeed display consecutive and hierarchical characteristics, adult human liver cells were treated by three pancreatic transcription factors, Pdx1, Pax4 and Mafa (3pTFs) that control distinct hierarchical stages of pancreatic development in the embryo. Ectopic expression of the 3pTFs in human liver cells, increased the transdifferentiation yield, manifested by 300% increase in the number of insulin positive cells, compared to each of the ectopic factors alone. However, only when the 3pTFs were sequentially supplemented one day apart from each other in a direct hierarchical manner, the transdifferentiated cells displayed increased mature β-cell-like characteristics. Ectopic expression of Pdx1 followed by Pax4 on the 2nd day and concluded by Mafa on the 3rd day resulted in increased yield of transdifferentiation that was associated by increased glucose regulated c-peptide secretion. By contrast, concerted or sequential administration of the ectopic 3pTFs in an indirect hierarchical mode resulted in the generation of insulin and somatostatin co-producing cells and diminished glucose regulated processed insulin secretion. In conclusion transcription factors induced liver to pancreas transdifferentiation is a progressive and hierarchical process. It is reasonable to assume that this characteristic is general to wide ranges of tissues. Therefore, our findings could facilitate the development of cell replacement therapy modalities for many degenerative diseases including diabetes.

Reprogramming of liver cells into insulin-producing cells

“…In most cases NGN-3, NEUROD-1, PAX-4 or MAFA which are instrumental in controlling distinct stages of endocrine pancreas differentiation did not display individual roles in activating the β cell function in liver. However, these pTFs significantly promoted PDX-1 effect on the transdifferetiation process (35, 39,43,50,56,57). In an earlier study, NEUROD-1 expression alone displayed only a moderate effect on the activation of the pancreatic repertoire and functioning liver, in vivo.…”

“…Although no direct proof that PDX-1 functions as a pioneer factor for pancreas organogenesis and in transdifferentiation of adult extra-pancreatic tissues has been provided, numerous studies support its crucial and obligatory roles in M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6 both processes. PDX-1 is used in most transdifferentiation protocols from extrapancreatic tissues (4,(33)(34)(35)(36)(37)(38)(39)(40).…”

“…The capacity of inducing functional transdifferentiation of mature human liver cells carries a substantial therapeutic significance, since it may allow autologous cell replacement therapy for diabetes patients. The potential use of primary cultures of adult human liver cells as pancreatic progenitors has been extensively analyzed in our laboratory using liver samples derived from >80 donors (37)(38)(39)(40). PDX-1 modulation by VP-16 increases the transdifferentiation capacity of the nuclear factor, suggesting that PDX-1 cooperation with other TFs may be synergistic in activating the pancreatic lineage (41,44).…”

“…However, this may not be the case, since despite being fast, transdifferentiation is a sequential and temporally controlled process (39). A recent study suggests that transdifferentiation of human liver cells along the pancreatic lineage is a gradual and consecutive process (39).…”

“…However, this may not be the case, since despite being fast, transdifferentiation is a sequential and temporally controlled process (39). A recent study suggests that transdifferentiation of human liver cells along the pancreatic lineage is a gradual and consecutive process (39). Concomitantly, only sequential ectopic expression of three pTFs initiated by PDX-1 and concluded by MAFA 3 days later, in a hierarchical manner resulted in an increased transdifferentiation efficiency and generation of mature…”