Generation and characterization of the WB-1 cell line
To establish an in vitro system to study the molecular mechanisms of the transdifferentiation of hepatic cells into insulin-producing cells (IPCs), rat WB cells were cotransfected with CMV-Pdx1-VP16/neor and RIP-eGFP/zeor plasmids. Five single cell– derived green fluorescence protein–positive clones (, part a) were isolated and named WB-1 to -5. These clones were further expanded (), and the cells were found to exhibit an intense cytoplasmic green fluorescence, indicating activation of the insulin promoter by Pdx1-VP16 (). To confirm the nuclear localization of the Pdx1 protein, WB-1 cells were subjected to immunocytochemistry with anti-Pdx1 antibodies. As expected, Pdx1 protein was mainly distributed in the nuclei, as shown by dark brown nuclear staining in WB-1 cells (, left), and there was no staining in WB cells (, right). Cells stained with control antibody showed no detectable staining (data not shown). Western blot analysis of whole-cell lysates from WB, WB-1, and INS-1 cells demonstrated abundant expression of activated 46-kDa Pdx1 phosphorylated protein in WB-1 and INS-1 cells, but not in parental WB cells (, WB-1). Similar expression of the Pdx1 protein was also observed in other WB-derived clones 2–5 (data not shown). Because all five Pdx1-VP16 – expressing WB clones expressed similar amounts of Pdx1-VP16 protein and RIP-eGFP, the remaining studies were performed with the WB-1 clone.
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 (more ...)
To confirm that the WB-1 cells indeed expressed the Pdx1-VP16 fusion protein, we performed a Western blot using anti-VP16 antibody. A single band at ~58 kDa of the Pdx1-VP16 fusion protein was detected in WB-1 cells, but not in the parental WB cells (, left). When a Pdx1 antibody was used to examine the Pdx1 expression, we were able to not only detect the Pdx1-VP16 fusion protein, but also a phosphorylated/active (46-kDa) and an unphosphorylated/inactive (31-kDa) endogenous Pdx1 protein that did not normally express in WB cells (, right). These results indicate that Pdx1-VP16 activates the expression of the endogenous Pdx1 during the transdifferentiation to pancreatic endocrine cells. In conclusion, we have successfully established an in vitro system consisting of a single hepatic cell– derived clone expressing Pdx1-VP16 with a built-in reporter RIP-eGFP gene to reflect the activity of the insulin promoter.
To characterize the gene expression profile of the newly generated WB-1 cells, we examined the expression of various genes related to pancreatic development and β-cell function by RT-PCR and compared the results with parental WB cells, rat insulinoma cells (INS-1), and normal pancreas (). Expression of Pdx1-VP16 in WB cells resulted in the transcription of multiple genes related to endocrine pancreas development and β-cell function. These include HNF1
, endogenous Pdx1
, insulin I
, and glucagon
. This gene expression profile is similar to that seen in the INS-1 cells and rat pancreas. However, there was no detectable expression of the genes Pax4
, or MafA
, which are involved in the late stages of differentiation of pancreatic endocrine cells (15
). To determine whether the newly generated WB-1 cells are capable of glucose-responsive insulin release, they were challenged with 20 mmol/l glucose for 2 h, and insulin secretion was determined by ELISA. We found that although the WB-1 cells express multiple pancreatic endocrine genes, including insulin, they do not respond to glucose stimulation by releasing insulin (data not shown). Moreover, there was no detectable mature insulin by Western blot with an anti-insulin antibody (data not shown). Taken together, these results indicate that Pdx1-VP16 – expressing WB-1 cells are precursors of pancreatic endocrine cells that do not exhibit mature β-cell function in the absence of further differentiation.
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 . WB-1 cells are (more ...)
Reversal of diabetes in WB-1 cell–transplanted diabetic mice
To determine whether the WB-1 cells possess the ability to further differentiate into mature functional pancreatic endocrine β-like cells, they were transplanted into the left renal subcapsular space of STZ-induced diabetic NOD-scid mice. As demonstrated in , WB-1 cells are capable of reducing blood glucose levels from ~400 to ~200 mg/dl in the diabetic mice within 2–3 weeks after cell transplantation, and by day 60, blood glucose levels were normalized (70 –100 mg/dl). In contrast, mice implanted with WB cells did not show any reduction in blood glucose levels and remained hyperglycemic during the entire observation period. As expected, mice receiving INS-1 cell transplantation showed a sharp reduction in blood glucose levels within 7–9 days and maintained blood glucose levels near 150 –200 mg/dl for a long time, but eventually became hypoglycemic (~30 – 40 mg/dl). Furthermore, removal of implanted WB-1 cells by left nephrectomy induced a rebound persistent hyperglycemia, confirming that the implanted WB-1 cells are indeed responsible for the reduction of blood glucose levels. To evaluate the long-term effects of the implanted WB-1 and INS-1 cells, the remaining mice from the two groups were continuously observed for 4 months. WB-1–transplanted mice displayed a consistent euglycemia (70 –100 mg/dl), whereas INS-1–transplanted mice had persistent hypoglycemia (30 – 40 mg/dl). All mice maintained normal body weight comparable to the aged-matched normal mice without diabetes (data not shown). These results demonstrate that although WB-1 cells do not appear to be mature endocrine cells in vitro, they are able to further differentiate and mature in vivo, as well as function like β-cells and rescue diabetic mice.
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 (more ...)
Gene expression profiles of pre- and posttransplanted WB-1 cells
To explore the molecular mechanism responsible for the functional shift of WB-1 cells from being glucose insensitive in vitro to being functional in vivo, we compared the gene expression profiles of posttransplanted WB-1 cells at 40 days and 4 months to that of the functional rat insulinoma INS-1 cells and to pretransplanted WB-1 cells as well as to their parental WB cells. We observed several noticeable changes in the expression of some genes related to β-cell development and function (). First, after 40 days in vivo, the WB-1 cells now express the genes Pax4
, and Isl-1
, which were not expressed before transplantation. Second, Ngn3
, a key transcription factor that is transiently expressed in the pancreatic endocrine precursors but not in mature pancreatic endocrine cells (15
), was now undetectable. Third, we found increased expression of NKx2.2
, and insulin 2
genes in the day 40 posttransplanted WB-1 cells. Fourth, MafA
genes became activated in WB-1 cells 4 months after transplantation. Last, we confirmed that the exogenous Pdx1-VP16 fusion gene was persistently expressed in the explanted WB-1 cells throughout the entire observation period. (see supplemental figure
[available at http://diabetes.diabetesjournals.org
]). The profile of gene expression at 4 months posttransplantation is similar to that of INS-1 cells. Taken together, the changes in gene expression profiles of WB-1 cells suggest a correlation of the sequence of gene activation, β-like cell differentiation and maturation, and the ability of glucose-regulating function in the WB-1 cells.
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 . Gene (more ...)
To characterize the molecular components of the glucose sensing, insulin secretion– coupling machinery, and β-cell function, we investigated the expression profiles of genes known to be involved in β-cell function: SUR1, Kir6.2, Snare 25, PC1/3, PC2, IAPP, and chromagranin A (Chrom A). SUR1 and Kir6.2 are ATP-sensitive K+ channel proteins, Snare 25 is involved in coupling and fusing vesicles to the cell membrane, IAPP is colocalized with insulin in secretory granules, and Chrom A, an abundant protein, is present in all islet cells. We also examined the gene expression of hexokinase (HK) to compare the levels to glucokinase (GK) during various stages of WB-1 cell maturation. Several interesting findings are demonstrated in ) The gene expression of GLP-1R and PC2 is weak in WB-1 cells (pretransplantation) but becomes strong in mature (posttransplantation) WB-1 cells at 40 days and 40 months posttransplantation. 2) Several genes including SUR1, Kir6.2, Snare 25, and IAPP are not expressed in immature WB-1 cells but become highly expressed in mature WB-1 cells, indicating that these proteins are related to mature β-cell functions. 3) HK gene expression appears to gradually decrease as the cells become mature. In contrast, GK gene expression increases as the WB-1 cells become mature during their in vivo differentiation. These results suggest that the estimated GK/HK ratio increases as the WB-1 cells undergo maturation. 4) Chrom A, a widely distributed protein in all islet cells, is expressed at all stages of WB-1 cell maturation, but is not detected in the parental liver epithelial WB cells. As expected, the rat insulinoma cell line INS-1 (823/13) expresses all genes with the exception of HK, whereas the parental WB cells express GK, HK, PC1/3, and PC2. These results demonstrate that upon maturation, the WB-1 cells indeed express many of the molecular components involved in regulated insulin secretion in mature β-cells.
Histology and pancreatic hormone production in the explanted tissues
To further characterize the identity of the transplanted WB-1 cells, we examined their histological appearance and insulin protein expression after in vivo differentiation at 40 and 120 days posttransplantation. The explanted WB-1 cells formed glandular or islet-like clusters with a rich network of microvasculature (). The cytology of the posttransplanted WB-1 cells at 40 days revealed large nuclei and relatively scant cytoplasm, which is morphologically consistent with less mature cells (). In contrast, at 120 days, the cells showed a decreased nucleus/cytoplasm ratio with “salt and pepper” chromatin and abundant cytoplasm, which is characteristic of mature pancreatic endocrine cells (). Immunostaining for insulin protein showed that >95% of the implanted cells expressed insulin () with no detectable glucagon-positive cells present (data not shown). Neither amylase nor albumin was detectable in WB-1 and INS-1 cells in comparison to positive controls (). These results demonstrate that in vivo, Pdx1-VP16 – expressing WB-1 cells selectively differentiate into mature functional IPCs with no detectable pancreatic exocrine or liver proteins.
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 (more ...)
Effects of high-glucose culture on WB-1 cell maturation
Based on the results of cell transplantation, as well as our previous studies on hepatic oval cells (3
) and bone marrow– derived stem cells (14
), we hypothesize that high-glucose condition is a critical factor to promote further differentiation of precursor WB-1 cells. To determine whether WB-1 cells could be induced to mature in vitro into functional IPCs, we cultured them in high-glucose media for 4 weeks. summarizes the insulin content and release in WB, WB-1, and INS-1 cells upon stimulation with 20 mmol/l glucose. We found a 1.8-fold increase in insulin release in WB-1 cells in response to glucose stimulation when compared with unstimulated WB-1 cells. A similar ratio is seen with INS-1 cells under our experimental conditions, demonstrating that high glucose can indeed promote the maturation of nonfunctional WB-1 cells into functional IPCs. These results support our hypothesis that Pdx1-VP16 – expressing hepatic cells are endocrine precursor cells that selectively differentiate into mature functional IPCs only when placed in the proper microenvironment, such as in high-glucose culture or in a hyperglycemic diabetic mouse.
Insulin content and insulin release after 2 h of glucose stimulation
To examine whether the functional WB-1 cells can maintain their differentiated state when glucose levels are switched back to basic levels, we continuously followed the WB-1 cells in the basic medium (11.1 mmol/l glucose) and measured static insulin levels in the culture medium. We observed that the static insulin secretion in the medium was maintained at a high level (>1.7 ng/ml culture medium) for five passages, decreased to 0.8 ng/ml for the next two passages, and then remained at 0.3 ng/ml for six additional passages before it became undetectable. This phenomenon of changing sensitivity to glucose is commonly observed and has been well documented, even in true β-cell lines or genetically engineered β-cell lines (18
) when they were cultured in vitro for a long time. These results indicate that functional WB-1 cells can indeed maintain their differentiated state during in vitro culture conditions.
Selective transdifferentiation of WB-1 into IPCs without evidence of exocrine differentiation
Previously, we showed that amylase protein is not detected in the explanted WB-1 cells. To confirm that transdifferentiation is selective toward an endocrine cell fate, we examined the expression of both early (p48) and late exocrine (amylase, elastase, and carboxypeptidase) genes in various stages of WB-1 cells by RT-PCR. In agreement with our hypothesis that WB-1 cells are pancreatic endocrine precursors, we found no detectable expression of exocrine markers (). In contrast, we do find expression of hepatic mRNA, though we do not detect protein expression (). These results indicate that expression of Pdx1-VP16 selectively transdifferentiates hepatic cells into pancreatic endocrine IPCs cells and the gene expression profile that we examined in WB-1 cells become identical to that of mature β-cell line INS-1 cells after transplantation into diabetic mice.
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), (more ...)
Analysis of insulin protein and insulin secretory granules
To determine if the in vitro– differentiated WB-1 cells process proinsulin to insulin and if there are any insulin secretory granules present in these cells, we performed Western blotting to detect mature insulin and electron microscopy studies combined with immunogold labeling with anti-insulin antibody to detect insulin secretory granules. WB-1 cells were continuously cultured in high-glucose medium for further differentiation and maturation. The presence of mature insulin in high-glucose–cultured WB-1 cells was evaluated after the cells became glucose responsive to release insulin, as detected by ELISA. As indicated in , WB-1 cells in high glucose culture (lane 2) produce mature insulin (arrow), compared with the positive control INS-1 (823/13) cells (lane 4). No insulin was detected in immature WB-1 (lane 3) or WB (lane 1) cells. At the ultrastructural level, these cultured mature WB-1 cells show scattered cytoplasmic globular structures containing insulin molecules, which were confirmed by immunogold-labeled anti-insulin antibody. As shown in , insulin-containing electron dense granules were present in mature WB-1 cells (left) and were similar to that seen in β-cells (right). These results indicate that cultured mature WB-1 cells can indeed process insulin and form insulin secretory granules.
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 (more ...)