Better Induction and Differentiation Strategy for Rat Pancreatic Stem Cells: Transplant in Liver Niche

Better Induction and Differentiation Strategy for Rat Pancreatic Stem Cells: Transplant in Liver Niche B. Chen, L. Zhou, L. Wang, S. Hu, and R. Wang ABSTRACT Current in vitro induction protocols cannot generate mature islet cells from stem cells. Transplantation into the liver niche may greatly contribute to the maturity of pancreatic stem cells (PSCs) due to its similarity to the pancreas. To determine the effect of the liver niche on the differentiation of PSCs, we used neonatal Wistar rat pancreata for cultivation of PSCs, which were transplanted into diabetic Wistar rats via the portal vein or beneath the renal capsule. After transplantation, we measured random blood glucose, weight, and serum insulin and performed an intraperitoneal glucose tolerance test. Specimens were examined by immunofluorescence. As a result, highly proliferating harvested cells showed the characteristics of stem cells. The PSCs could be induced to form large islet-like structures (150 –200 ␮m diameter) in the liver, which resulted in better therapeutic efficacy. In contrast, there were smaller islet-like structures (about 50 ␮m diameter) when PSCs were transplanted beneath the renal capsule. These findings demonstrated that the liver niche benefits the in vivo differentiation of PSCs into endocrine and exocrine cells that may contribute to the generation of insulin producing cells. ANCREATIC STEM CELLS (PSCs) have been shown to differentiate into functional endocrine cells under suitable in vitro conditions.1 At present, however, cultivation and induction strategies of PSCs mostly include application of limited growth factors and cytokines. It is known that complex in vivo processes determine differentiation from duodenal endoderm to an insulin-secreting ␤-cell as the result of evolutionary selection during several million years, driven by environmental pressures rather than by conscious design. Therefore in vitro induction strategies have resulted in so-called insulin-secreting cells with the characteristics of low insulin content and release, high apoptotic rate, and failure to normalize blood glucose levels in diabetic animals.2– 4 Thus the current in vitro differentiation protocols do not generate real mature islet cells.5 Another superior induction protocol transplants PSCs into the liver niche based on the following evidence. First, some authors have observed that unpurified islet auto transplantations via the portal vein are more efficacious than purified islets (no PSCs). The former grafts showed therapeutic effect at 6-years, while the latter only survived 1 year.6 Second, the assessment of 83 human islet grafts transplanted using the Edmonton protocol since 19997 showed significant positive correlation between the number of pancreatic ductal epithelial cells transplanted and long-

P

term metabolic success as assessed by intravenous glucose tolerance tests at approximately 2 years posttransplantation. Thus PSCs transplanted via the portal vein together with islet cells showed satisfying results. Therefore we designed this experiment to examine whether the liver niche was better for the differentiation of PSCs than other sites such as beneath the renal capsule, one of the most common transplantation sites.

MATERIALS AND METHODS Animals Neonatal Wistar rats were used for cell culture and male Wistar rats weighing 180 to 200 g as recipients, all of which were obtained from the Shandong University Animal Center. They were handled in accordance with the guidelines approved by our Animal Care and Use Committee.

From the Department of General Surgery, Qilu Hospital of Shandong University, Jinan, Shandong, China. This work was supported by Research Fund for Doctoral Program of Shandong Province, China (2005BS03011). Address reprint requests to Prof. Lei Wang, Qilu Hospital of Shandong University, 107 Wenhua Xi Road, Jinan, Shandong, China. E-mail: [email protected]

0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.06.208

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Transplantation Proceedings, 41, 3898 –3904 (2009)

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Cultivation and Induction of Pancreatic Stem Cells The neonatal Wistar rats were sacrificed, and the pancreata, removed and dissected into 2 to 3 mm segments. Ductal epithelium was isolated by the collagenase digestion method, as reported previously by us.8 The pancreata tissue, which was suspended in modified RPMI1640 medium (33.3 mmol/L Glu) (GIBCO, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA), 100 U/mL penicillin, 100 ␮g/mL streptomycin, 10 mmol/L HEPES buffer, 20 ng/mL epidermal growth factor (EGF), and 20 ng/mL basis fibroblast growth factors (bFGF) (Sigma, USA), was transferred into 75 cm2 flasks (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) coated with concanavalin A (ConA). Pancreata segments were primarily cultured for 48 hours at 37 °C with 95% air and 5% CO2. In these conditions, most islets remained in suspension (floated). After a 48-hour incubation, the medium containing the suspended islets was removed and new modified RPMI1640 medium was changed every 3 days for continuous primary cultures. Stem cells were induced with RPMI1640 medium (11.1 mmol/L Glu) without EGF, bFGF, and FBS but containing 10 mmol/L nicotinamide and 71.5 umol/L ␤-mercaptoethanol (Sigma, USA).

Identification of Pancreatic Stem Cells Transmission Microscope Electron. The stem cells were collected in Eppendorf tubes fixed with 2.5% glutaraldehyde at 4 °C for 2 hours and then with 1% osmium tetrachloride at 4 °C for 30 minutes. The JEM-1010 (JEOL, Japan) was used to observe stem cell ultrastructure. Dithizon Staining. Freshly prepared dithizon (DTZ) solution (0.56 mg/m) was mixed with media (1:3 v/v) with a final concentration of DTZ of 0.14 mg/mL. We calculated the positive growth rate of cells in high and low glucose medium. Radioimmunoassay. We cultured 1 ⫻ 105 cells in every well of 24-well plates, which contained 0.5 mL phosphate-buffered saline (PBS) with different glucose concentrations. Three parallel wells were designated for every glucose concentration. Insulin concentrations in culture medium were determined using ultrasensitive radioimmunoassay kits purchased from China Atomic Energy Institute; the procedure followed the kit instructions. Immunocytochemistry. Immunostaining was performed for CK19, PDX-1, Ngn3, insulin, and glucagon (Sigma, USA; dilution 1:100). Antibodies for PDX-1 and Ngn3 were monoclonal rabbit antirat IgG1 type, while the other antibodies were mouse anti-rat.

Fig 1.

3899 Monolayers were fixed with 4% paraformaldehyde at 4 °C. Plates were stained with multiple primary antibodies using greased 8-mm cloning cylinders (Bellco Biotechnology, Vineland, NJ), which were attached to the areas of interest after the plates were washed with PBS and blocked with 5% lamb serum (Gibco Brl, Burlingto, Ontario, Canada). Primary antibodies (50 –100 ␮L/cylinder) in appropriate dilutions were added for overnight incubation at 4 °C. Normal IgG1 isotype antibodies (same dilution) were used as negative controls to exclude nonspecific staining. Slides were then rinsed with PBS and cover-slipped with fluorescent mounting medium (Peking Zhongshan Bio, China). Fluorescence images were obtained using a Zeiss Epifluorescence microscope equipped with an Optronics TEC470 CCD camera (Optronics Engineering, Goleta, Calif, USA) interfaced with a PowerMac 7100 installed with IP Lab Spectrum analysis software (Signal Analytics, Vienna, Vir, USA). Reverse Transcription and Polymerase Chain Reaction. Total cellular RNA prepared from cultured cells was reverse transcribed and amplified by polymerase chain reactions (PCR) for 35 cycles. Oligonucleotides used as amplimers for the PCR were: Rat nestin: forward, 5=-GCGGGCGGTGCGTGACTAC-3=; reverse, 5=-AGGCAAGGGGGAAGAGAAGGATGT-3=. Rat PDX-1: forward, 5=-GGCTTAACCTAAACGCCACA-3=; reverse, 5=-GGGACCGCTCAAGTTTGTAA3=. Rat CK-19: forward, 5=-CTGGGTGGCAATGAGAAGAT-3=; reverse, 5=-TCAAACTTGGTCCGGAAGTC-3=. Rat Ngn3: forward, 5=-GGCGCCTCATCCCTTGGATG-3=; reverse, 5=-CAGTCACCCACTTCTGCTTCG-3=. Rat ␤-actin: forward, 5=-TGTCACCAACTGGGACGATA-3=; reverse, 5=-ACCCTCATAGATGGGCACAG3=. Primers selected from two different exons encompassed at least one intronic sequence. In addition, a reverse transcription (RT) minus control was run for most samples. PCR cycling was at 94 °C for 1 minute followed by 94 °C for 10 seconds, 58/56/54 °C for 10 seconds 72 °C for 1 minute (35 cycles), and 72 °C for 2 minutes. The annealing temperature was 58 °C for rat nestin, 45 °C for human insulin, and 56/54 °C for the remaining primer pairs.

Establishment and Grouping of T1DM Models Intravenous administration of streptozotocin (STZ) at a dose of 60 mg/kg body weight to male Wistar rats was performed as recommended by a previous study.9 The random blood glucose (RBG) was monitored at the same time everyday from the second day. According to Korec et al,10 the diagnostic criteria for a successful T1DM were an RBG continuously more than 16.7 mmol/L for at

(A) The epithelial cells grew as a monolayer; (B): the islet-like structure formed after induction via low-glucose medium.

3900 least 1 week. Once the T1DM models were successfully established, the diabetic rats were randomly divided into three groups: A (n ⫽ 7), PBS control injected via the portal vein; B (n ⫽ 10), PSCs transplanted via the portal vein; and C (n ⫽ 10), PSCs transplanted beneath the renal capsule.

Cells Labeled by BrdU PSCs were cultured in 24-well plastic tissue culture plates coated with collagen. BrdU (10 ␮mol/L) was added 48 hours before cell confluence. Then the cover slip was removed and washed thrice with PBS. Cells fixed in 4% formaldehyde were treated with 0.3% Triton plus PBS. After 1 N HCl was used for degeneration and 0.5% bovine

Fig 2. Immunofluorescence, DTZ staining, and TME: (A) PDX-1; (B) CK-19; (C) Ngn3; (D) glucagon; (E) insulin. A, B, and C were also stained by DAPI (blue); (F) DTZ staining; (G) secretory granules; (H) rough (surfaced) endoplasmic reticulum.

CHEN, ZHOU, WANG ET AL serum albumin for blockage, the cells stained with anti-BrdU (1:75) were incubated at 4 °C overnight. After 0.3% Triton plus PBS was used again; the cells were incubated with the FITC-labeled secondary goat antisera (Peking Zhongshan Bio, China) for 1 hour at room temperature. The cover slip was washed again with 0.3% Triton plus PBS, and then DAPI (50 ␮g/mL) was added as a counterstain. Then we used the following formula to calculate the BrdU-labeled cell positive rate: BrdU labeled cell positive rate ⫽ (BrdU labeled cells/DAPI labeled cells ⫻ 100%)

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Fig 3. RT-PCR for mRNA of CK19, PDX-1, Ngn3, and nestin. Two days after BrdU treatment cell viability was determined by a trypan blue exclusion assay in which cell survival (%) was calculated as (living cell numbers/living ⫹ the dead cell numbers) ⫻ 100

embedding in paraffin. To detect cellular grafts in the liver, we sampled all liver lobes. Sections were double stained with antiinsulin (1:50) and anti-BrdU. The detailed steps were similar to the above processes.

Pancreatic Stem Cell Transplantation Procedure

Statistical Analysis

We administered 10% chloral hydrate to diabetic rats at a dose of 0.3 mL/100 g BW. BrdU-labeled cell suspensions (0.3 mL containing 5 ⫻ 106 cells) were transplanted via the portal vein in group B and beneath the renal capsule in group C. After the operation, a regular diet was given to all rats. Intraperitoneal injection of cyclosporine (5 mg/kg BW) was performed on the first day after operation and every 3 days thereafter.

Data are presented as mean values ⫾ standard deviation. Results were analyzed using analysis of variance. Statistical significance was set at P ⬍ .05.

Postoperative Monitoring of Random Blood Glucose Weight, and Insulin The RBG and weights of the three groups were monitored immediately before transplantation, as well as on days 1, 2, 4, 6, 8, 10, 14, 18, 22, 26, and 30 after transplantation. Blood was collected from the tail tip of the rats to determine glucose levels as measured with a portable glucometer. According to the change in RBG, we measured the insulin level using radioimmunoassay every week. The detailed steps were identical to those previously described in the Radioimmunoassay section.

Intraperitoneal Glucose Tolerance Test Intraperitoneal glucose tolerance test (IPGTT) was performed by intraperitoneal injection of a 40% glucose solution (2 g/kg BW) in a fasting state. Blood samples were collected from the tail tip using heparinized microhematocrit tubes immediately before (zero time) as well as 30, 60, and 120 minutes after glucose loading. Blood glucose level measured with a standard glucose/triglyceride/cholesterol meter (Accutrend GCT, Roche, Germany) was expressed as milligrams per deciliter.

Observation of Cell Grafts by Hematoxylin-Eosin and Immunohistochemical Examination Livers and kidneys of recipient rats at 14 and 30 days, respectively, after cell transplantation were fixed in 4% formaldehyde for

RESULTS Isolation, Proliferation, and Identification of Stem Cells

Collagenase digestion in combination with modified RPMI 1640 medium containing high glucose allowed us to isolate and purify pancreatic ductal epithelial cells on ConAcoated dishes. The islets do not adhere to the plates under these culture conditions; they were easily removed by the change of high glucose medium. The epithelial cells attached to the plates at 24 to 48 hours after incubation grew as a monolayer within 2 weeks (Fig 1A). After the monolayer was induced by changing high into low glucose medium, the small elliptical cells that emerged formed isletlike structures (ILSs), which consisted of 10 to 15 elliptical cells ranging from 100 to 150 ␮m in diameter (Fig 1B). The cultured cells had the ability of self-replication, which showed the characteristics of undifferentiated cells, expressing the markers of PSCs: CK-19, PDX-1, and Ngn3. After induction, some of these cells expressed insulin and glucagon (Fig 2A, B, C, D, E). To confirm the immunocytochemical identification of CK-19, PDX-1, and Ngn3 expression by the cells, we performed RT-PCR for PDX-1 and Ngn3 mRNA using total RNA prepared from cells before induction. The RT-PCR– generated products showed the correct size (Fig 3). Both DTZ staining and insulin secretion tests did not show insulin-generated cells before induction. Once the cells were induced, they displayed an increased insulin response to glucose over the

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operated rats did not display a significant change at 4 weeks after the operation. However, the body weights of groups B and C increased 55.5% and 36.9%, respectively, at 4 weeks after implantation of PSCs (Fig 6B). The three groups showed similar tendency of the plasma insulin curves as those of RBG (Fig 6C). Intraperitoneal Glucose Tolerance Test

Fig 4. The cells’ ability to secret insulin in response to the glucose solutions of different concentrations; all data are represented as mean values ⫾ SD.

range of 10 to 30 mmol/L (Fig 4), with the ratio of DTZpositive cells increased to 25 ⫾ 6% (Fig 2F). Concerning TME observation, abundant rough endoplasmic reticulum was observed in cells. Many secretory vacuoles containing granules were close to the cell surface (Fig 2G, H). BrdU Labeling

The nuclei of cells labeled with BrdU were stained green, while those of all other cells were stained blue by DAPI. The positive rate of BrdU-labeled cells was more than 80% (Fig 5A, B, C), and the cell viability was 88% ⫾ 5.6%. Monitoring of Random Blood Glucose, Weight and Plasma Insulin

The 27 rats that received STZ through the caudal vein all became diabetic as evidenced by continuous monitoring of RBG. Group A included seven rats and the other two groups of 10 rats each received 5 ⫻ 106 cells. Stem cell transplantation led to a decrease of RBG in both groups B and C compared to the sham-operated diabetic rats (group A). Group B displayed better control of RBG than group C shown by longer control of hyperglycemia and a transiently normalized RBG (Fig 6A). The body weight of sham-

To assess the ability of the transplanted rats to dispose of a glucose load, an IPGTT was performed on days 13 and 28. As noted in Fig 6D, PSC-implanted rats (via the portal vein) showed significantly lower plasma glucose levels at 30 minutes than did control diabetic rats. Among diabetic rats recovery of RBG was faster in B group. Similar results were obtained during the day 28 glucose tolerance test, (Fig 6E). Morphological Observation of Liver and Kidney Samples

Many ILS grew in the sinus hepaticus among the B group on day 14 after transplantation that resembled real islets, that is, with clear edges, cells of different morphologies, rich extracellular matrix, and new vessels. Lymphocytes surrounded the sinus hepaticus with some in the center of the grafts. Many BrdU- and insulin-double-positive cells were present in B group sections, wherein insulin in cells stained red surrounding a green nucleus labeled with BrdU. Most ILS were 150 to 200 ␮m in diameter (Fig 7A). The number of insulin positive cells was 4.73 ⫾ 0.74. ILS in group C were mostly 50 ␮m in diameter (Fig 7B). New vessels were observed in group C, but the extracellular matrix was not abundant. Compared to B group, the cell staining among group C was weak with sparse positive cells (2.30 ⫾ 0.69 per visual field; P ⬍ .01). On day 30, BrdU- and insulin-doublepositive cells were fewer, and insulin-positive cells, 1.00 ⫾ 0.55 per visual field. Insulin-positive cells were hardly observed in group C. DISCUSSION

According to the criteria of an ideal islet transplant site, such as a simple procedure, normal insulin release pathways, and avoidance of rejection,11 we concluded that they should also include the niche contribution to the matura-

Fig 5. BrdU staining: (A) the nuclei of cells labeled by BrdU (green); (B) the nuclei of cells labeled by DAPI (blue); (C) the dual-stained cells (blue-green).

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Fig 6. (A) The RBG of three groups after operation; (B) the weight of three groups after operation; (C) the insulin of three groups after operation; (D) IPGTT on day 13 postoperatively; (E) IPGTT on day 28 postoperatively. All data are represented as the mean values ⫾ SD. Values in the same row not sharing the same letters are significantly different from each other at P ⬍ .05 by ANOVA.

tion of stem cells. PSC transplantation can be divided into orthotopic and heterotopic sites: the former includes the portal vein regions such as pancreas, liver, spleen, and

greater omentum, and the latter subcutaneous, beneath the renal capsule, and the brain. Some authors have recently judged stem cell infusions based on their ability to localize

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Fig 7. The morphous of stem cell grafts in liver and beneath renal capsule day 14 after transplantation. (A) Larger ILS in liver (bar ⫽ 50 ␮m); (B) smaller ILS in liver (bar ⫽ 50 ␮m).

in and repair injury sites.12 Unfortunately, due to etiological disease and special autoimmune attack, the pancreas cannot offer a microenvironment for PSC growth.13 In this study, we compared the contribution to maturation of transplanted PSCs of the liver niche to that beneath the renal capsule. The liver (via portal vein) is currently the most common clinical transplant site for islets. Some clinicians have observed the ductal epithelium that serve for PSC differentiation into islet cells and for generation of appropriate extracellular matrix greatly improving the clinical effects of islet transplantation.14 In the laboratory the most common transplant site is under the renal capsule which is not within the portal vein region, but it has the advantage of immune priveledged. In this study, high RBG was transiently reversed in group B. The niche beneath the renal capsule showed a less important role to differentiate PSCs. The reasons why the two niches show disparate effects on differentiation are due to the special anatomic and physiological microenvironments of the liver, which are similar to the pancreas.15 It has been reported that hepatic oval cells can be induced in vitro to arrange like ILSs, expressing islet cell markers and controlling the blood glucose when transplanted into mice,16 which illustrates the intimate relationship between the liver and the pancreas. In this study we did not use in vitro differentiated cells but selected undifferentiated PSCs for transplantation based on the following theories. First, bone marrow derived stroma cells have been directly transplanted into the circulation of diabetic rats because workers have believed that stem cells have the ability to repair the injury region.17 Secondly, Li et al observed that transplantation of pancreatic ductal epithelium or PSCs in vivo controlled diabetes.18 Compared to group C, rats in group B showed RBG to be reduced to normal, with insulin secretion levels higher and more durable than those in group C. The findings in pathological sections supported these data. ILSs induced from PSCs in vivo in Group B ranged from 150 to 200 ␮m, while group C included many 50 ␮m small ILSs beside 50% of them at 150 to 200 ␮m ILSs. The 150 to 200 ␮m ILSs showed a more mature phenotype.19 In addition, there was more extracellular matrix in group B than C. An abundant blood supply and extracellular matrix not only provide nutrient sub-

stances for the maturation of stem cells, but also the signals to induce differentiation. We propose that the doublepositive stem cells of PDX-1 and CK-19 represent undifferentiated PSCs, which can theoretically differentiate into all kinds of pancreatic cells, including endocrine and exocrine cells. The exocrine cells can serve as the inducers of endocrine islet cell differentiation, as illustrated by Street et al.9 If the undifferentiated PSCs are induced in vitro, there stem cells can differentiate into endocrine precursor cells or even into ILSs. Thus, the reason why transplantation of differentiated cells is not as good as that of undifferentiated PSCs is that purified islet cells are transplanted, which are short-lived.

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