High glucose is necessary for complete maturation of Pdx1-VP16-expressing hepatic cells into functional insulin-producing cells

Abstract

Pdx1 has been shown to convert hepatocytes into both exocrine and endocrine pancreatic cells in mice, but it fails to selectively convert hepatocytes into pure insulin-producing cells (IPCs). The molecular mechanisms underlying the transdifferentiation remain unclear. In this study, we generated a stably transfected rat hepatic cell line named WB-1 that expresses an active form of Pdx1 along with a reporter gene, RIP-eGFP. Our results demonstrate that Pdx1 induces the expression of multiple genes related to endocrine pancreas development and islet function in these liver cells. We do not however find any expression of the late-stage genes (Pax4, Pax6, Isl-1, and MafA) related to beta-cell development, and the cells do not secrete insulin upon the glucose challenge. Yet when WB-1 cells are transplanted into diabetic NOD-scid mice, these genes become activated and hyperglycemia is completely reversed. Detailed comparison of gene expression profiles between pre- and posttransplanted WB-1 cells demonstrates that the WB-1 cells have similar properties as that seen in pancreatic beta-cells. In addition, in vitro culture in high-glucose medium is sufficient to induce complete maturation of WB-1 cells into functional IPCs. In summary, we find that Pdx1-VP16 is able to selectively convert hepatic cells into pancreatic endocrine precursor cells. However, complete transdifferentiation into functional IPCs requires additional external factors, including high glucose or hyperglycemia. Thus, transdifferentiation of hepatocytes into functional IPCs may serve as a viable therapeutic option for patients with type 1 diabetes.

FIG. 1

Generation and characterization of Pdx1-VP16–expressing WB cell lines. A: Generation of WB-1 cells. WB cells were transfected with Pdx1-VP16 and RIP-eGFP and double-positive single cell–derived GFP-positive clones were selected (a) and expanded (b) as described in RESEARCH DESIGN AND METHODS. Representative cloned cells (c) expressed insulin gene reflected by eGFP as green cells (d). B: Detection of Pdx1 protein by immunocytochemistry. Cytospin slides were made from WB-1 and WB cells and subjected to immunostaining with anti-Pdx1 antibody. Brown nuclear staining represents the Pdx1 protein located in WB-1 cells but not present in WB cells. C: Confirmation of Pdx1 protein by Western blot. Cell lysates extracted from WB, WB-1, and INS-1 cells were separated by SDS-PAGE, and the Pdx1 protein was detected by blotting with Pdx1 antibody. Two forms of Pdx1 proteins are indicated by arrows: activated 46-kDa form (upper band) and inactivated 31-kDa form (lower band). D: Identification of Pdx1-VP16 fusion protein in WB-1 cells. The cellular proteins from WB and WB-1 cells were blotted with anti-VP16 antibody (1:200; BD Biosciences) (left) and anti-Pdx1 antibody (right). Top arrows indicate the position of the exogenous Pdx1-VP16 fusion protein.

FIG. 2

Comparison of gene expression among WB-1, WB, INS-1, and rat pancreas. Total RNA was extracted from the above cells, and RT-PCR was performed. All primers were designed across intron(s). Details regarding primers are presented in Table 1. WB-1 cells are newly generated cells.

FIG. 3

Cell transplantation. 1 × 10⁶/mouse of WB (control, n = 3), WB-1 (n = 6), or INS-1 (n = 4) cells were implanted under the left renal subcapsule after the blood glucose levels reached 400 mg/dl (arrow, transplantation [Tx]) in repeatedly low-dose STZ-induced diabetic NOD-scid mice. The blood glucose levels were monitored regularly under nonfasting conditions through tapping the tail vein. The left kidney of three mice from the WB-1 group was removed at 40 days post-implantation (arrow, explanted [Ex]). The remaining WB-1– and INS-1–transplanted mice were terminated by the end of the observation (4 months).

FIG. 4

Comparison of gene expression profiles among various stages of WB-1 cells (A and B). Total RNA was extracted from the cells, and RT-PCR was performed. All primers were designed cross intron(s). Details regarding primers are presented in Table 1. Gene expression profiles in pre-and posttransplanted (Tx) (40 days and 4 months) WB-1 cells were compared. INS-1 and WB cells transfected with an empty plasmid vector served as positive and negative controls, respectively. Arrows indicate exogenous (mPdx1) and endogenous (rPdx1) gene expression of Pdx1. Arrowheads indicate newly activated genes (Pax4, Pax6, Isl-1, MafA, and PP in A) as well as β-cell function–related genes (SUR1, Kir6.2, SNAP25, and IAPP in B) in 40 days and 4 months posttransplanted WB-1 cells.

FIG. 5

Histology and immunostaining of the explanted tissues. Paraffin sections of explanted tissues from 40 days (A) and 4 months (B) posttransplantation were stained with H&E (upper panels of A and B). The sections were immunostained with antibodies to insulin (1:500), amylase (1:200), and albumin (1:200) and incubated with matched secondary antibodies. The labeled proteins were visualized with DAB reagent. Lower panels of A represent insulin staining (c, pancreatic islet; d, negative control; e, insulin in the explanted tissue) in 40-day explanted WB-1 cells. Lower panels of B represent insulin staining on the explanted tissues containing WB-1 (left) cells, INS-1 (middle), and mouse pancreas tissue (right) as positive controls, as indicated in the figures. Each figure in B contains an internal negative control. C shows immunostaining of amylase and albumin in WB-1, INS-1, and positive controls (liver and pancreas), as indicated in the figure. Tx, transplanted.

FIG. 6

Gene expression of pancreatic exocrine (A) and hepatic markers (B) by RT-PCR. Gene expression studies were performed on WB cells, WB-1 (newly generated), high-glucose–cultured WB-1 cells (WB-1 + HG), and 40-day explanted WB-1 cells (WB-1 Ex), as well as rat liver and pancreas as positive controls. Pancreatic exocrine genes include p48, amylase, carboxypeptidase, and elastase. The liver genes include three transcription factors (TGF-α, GATA-4, and HNF-4) and two genes of liver functional proteins (albumin [Alb] and transthyretin [TTR]).

FIG. 7

Detection of insulin and insulin secretory granules. A: Mature insulin was detected in the differentiated WB-1 cells (lane 2) by Western blot with anti-insulin antibody (1:500; Santa Cruz). All lanes were loaded with 50 μg protein except for INS-1 (loading only one-tenth protein [5 μg] to the well). B: Insulin secretory granules were detected by electron microscopy combined with immunogold labeling using anti-insulin antibody in an in vitro–differentiated WB-1 cell (B, left). Rat islet β-cells serve as the positive control (right). N represents the nucleus, and arrows indicate immunogold-labeled insulin particles.