Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone

We developed an optimized Dipheylthiocarbazone or Dithizone (DTZ) with improved physical and chemical properties to characterize human islets and insulin-producing cells differentiated from embryonic stem cells. Application of the newly formulated iDTZ (i stands for islet) over a range of temperatures, time intervals and cell and tissue types found it to be robust for identifying these cells. Through high transition zinc binding, the iDTZ compound concentrated in insulin-producing cells and proved effective at delineating zinc levels in vitro.

www.nature.com/scientificreports www.nature.com/scientificreports/ readily complexes with transition metals, including zinc, and can identify zinc-replete cells such as islets 17 , and can distinguish islets from exocrine and ductal cells 18 . For these reasons, DTZ staining is a standard technique for determining the number of islets in pancreatic digest and for assessing islet quality 18 . Traditionally, this process involved manual assessment of DTZ-stained islets and was inherently variable. At the same time, studies employing dithizone analogues are few. One such DTZ analogue, 1,5-Bis(2,5-difluorophenyl)-3-mercaptoformazan, was noted to have greater affinity for Co 2+ and Hg 2+ compared to Zn 2+ and has not been used to identify islets 19 . Interestingly, the analogue [11C]-DTBZ, while not used to stain isolated islets, was used to image islets in vivo 20 .
Automated methods for the quantification of stained islets have been reported with mixed results 9,21-23 . We hypothesized this was secondary to limitations in standard DTZ solution. We tested this hypothesis comparing standard DTZ to iDTZ solution under varied conditions and parameters 9 . Interestingly, over 24 hours, islets stained with iDTZ displayed normal morphology and minimal increases in background staining. In contrast, islets stained with standard DTZ displayed intense background staining preventing automated quantification (Fig. 2a). In this regard, iDTZ solution is distinct in supporting islet quality validation over an extended period of time, an important factor in qualifying islets for clinical transplantation. Six months after preparation, solutions of iDTZ were found to provide islet-specific staining on par with freshly made solutions, an effect that persisted for 48 hours (Fig. 2b). These findings indicate that iDTZ staining creates a stable color signal that will allow for greater sharing of stained biomaterial without signal degradation.
Both the pancreas and prostate have abundant concentrations of zinc compared to other organs 24 , whereas prostate cancer is associated with decreased zinc levels 25 . However, we found that solutions of iDTZ were sufficient for localizing islet-based zinc in pancreatic cryo-sections (Fig. 2c). These findings suggest that this reagent could be used as a simple, rapid means for detecting zinc in tissues. Further, the process of isolating islets from cadaveric pancreata is complex, expensive and lengthy. Given this, employing iDTZ solution to stain pancreatic cryo-sections prior to proceeding with islet isolation would be advantageous, especially when considering islet isolation from pancreata from individuals with high HbA1c, chronic diabetes and extensive insulin usage 26 .
The application of standard DTZ solutions beyond staining of mature islets is very limited. In one report, standard DTZ was used to stain stem cell-derived insulin-producing cells 27 . This suggested that stem cell-derived islet-like cells, on differentiation, may display increased levels of zinc and insulin. To interrogate this idea, we utilized the human embryonic cell line H1 28 . Cells were cultured under appropriate conditions for differentiation into insulin-producing cells using an established 7-stage protocol 28 . Clusters of cells were collected at every differentiation stage and stained with iDTZ. At stage 7, cells displayed a classic islet phenotype and were positive for intra-cellular zinc (Fig. 3a,b), concurrent with the presence of immuno-reactive insulin (Fig. 3c), a finding previously noted in stage 7 cells 28 . Stage 7 clusters, despite staining with iDTZ, were noted to be de-granulated compared to mature adult human islets, indicating that stage 7 cell clusters would need further steps to attain full maturation and development (Fig. 3). Interestingly, we found that approximately 10% of stage 6 cells retained iDTZ, indicating that a portion of such cells are immature insulin-producing cells. Thus, iDTZ can be employed www.nature.com/scientificreports www.nature.com/scientificreports/ as a simple and robust method to identify fully mature insulin-producing β-cells prior to transplantation into recipients. Parenthetically, iDTZ could be used as a rapid and simple agent to detect insulin producing cells transduced by several methods 29,30 .
Currently, characterization of zinc in liquids is done using a double beam flame absorption spectrophotometer or commercially available kits. However, these methods are complex and expensive. To further characterize the sensitivity of iDTZ to measure zinc levels we developed a standard curve and measured dye levels at 570 nm in samples containing known concentrations of zinc chloride (Fig. 4). As these data indicate, iDTZ can be used in microplate and plate readers available in most laboratories. Extrapolating these results, it is likely that solutions of iDTZ could detect zinc in a range of tissue and fluid samples. Additionally, solutions of iDTZ may find application in detecting other metals such as lead, mercury, and cadmium.

Methods
Isolation of human pancreatic islets. Human islets were isolated from cadaveric donor pancreata obtained from a local organ procurement organization following a standard operational procedure 31 . The use of human islets was approved by the Institutional Review Board of City of Hope and informed consent was obtained from the legal next of kin of each donor. All islets were cultured for 24-48 hrs at 37 °C, 5% CO 2 prior to staining with iDTZ or standard DTZ. All experiments were performed in accordance with relevant guidelines and regulations.  www.nature.com/scientificreports www.nature.com/scientificreports/

Assessment of cell staining by standard DTZ and iDTZ solutions.
To evaluate the staining efficacy, solutions were prepared fresh and used for staining isolated islets. Islet morphology was observed and imaged using an automated islet cell counter (ICC) (Biorep Technologies, Miami, USA). Briefly, 100 μL of islet sample was pipetted (Drummond pipette, Fisher Scientific, USA) into an ICC-provided dish 9 . A 250 μL aliquot of standard DTZ or iDTZ solution was then added and the dish agitated gently prior to placement on the imaging stage with the temperature at 22 °C. Data was acquired at time zero (T0), 1 hr (T1), 2hrs (T2) and 24hrs (T24).

Assessment of islet staining with stored iDTZ solution.
Human islets were stained with iDTZ solution that was stored for 6 months at −20 °C prior to use. In control experiments, freshly made iDTZ solution was used to stain islets from the same cell preparation.

Assessment of zinc and insulin status in differentiated stage 7 human embryonic stem cells.
Human embryonic stem cells were differentiated following a previously published method 28   Technologies, Vancouver, Canada, Cat #85850) that was changed daily until cells were confluent. Cultured H1 cells were dissociated into single cells by incubation with Accutase (STEMCELL Technologies, Vancouver, Ca., Cat #07920) for 5-10 minutes at 37 °C. Single cells were counted and 5.5 × 10 6 cells in 5.5 mL mTeSR medium supplemented with 10 µM Y27632 (Stemgent, 04-0012-H-10) were seeded into 6-well low-attachment plates (Costar, Cat #3471), placed on an orbital shaker (New Brunswick Innova 2000, #M1190-0000) set to 100 rpm to promote cluster formation and then cultured at 37 °C, 5% CO 2 . Forty-eight hours later, the culture medium was changed into differentiation medium. Stage-specific differentiation was conducted by the controlled addition of specific small molecules as described 28 . At each stage, representative clusters were transferred into new 6-well culture dishes and washed 3 times with 3 mL of COHSII (Gemini, Sacramento, CA, Cat#900-7500) prior to staining with iDTZ. After 3 minutes, the cells were washed 3 times with 3 mL of COHSII and suspended with COHSII. Cell clusters were examined using a ckx31 Olympus microscope and pictures were acquired using a camera. The iDTZ solution employed was a single batch stored at −20 °C until use. Human islets stained with iDTZ were used as control.

Figure 4.
Zinc levels can be measured using iDTZ. There was a highly significant correlation between zinc concentration and the absorbance readings at 570 nm (R 2 = 0.9985, p < 0.0001).