Peer review of: Long-term culture of human pancreatic slices as a model to study real-time islet (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
This paper is a major breakthrough in pancreatic tissue long term culture of up to a remarkable 10 
days, which is of sufficient duration for numerous applications in pancreatic islet and exocrine 
biology, but here it was applied to understanding neogenesis of beta cells from ductal cells using 
viral mediated lineage tracing in normal pancreas, and to lesser extent also in T1D and T2D human 
pancreas. I strongly believe that this technical capability is extremely important and useful to the 
large community of pancreatic investigators and should be disseminated. I also think that the data 
on BMP induced neogenesis is novel. I however would like some improvements and more rigor in 
some of their data. 
 
Figure 1. The data shown here are very convincing that the pancreatic slices are much more viable 
particularly after Day 5 with the PFC wells than the control trans wells. Along with these functional 
studies, an oxygen-deficient environment could induce increased expression of hypoxia-inducible 
factors which would also be alleviated in PFC wells. 
 
Figure 2. 2j. The purported evidence of islet health at D10 is that the respective cell type 
immunolabeling remains discrete in the PFC and much less (or as they said - lower defined 
boundaries) so with the transwell. Is this because of cell death or anoxic injury, some de-
differentiation. EM on D10 with characteristic features of each cell (granule appearance) and 
organelle injury would have made these conclusions more apparent. 
2m: It is difficult to assess from these images the acinar intactnesss and injury. High power views 
to show more obvious acinar morphology with amylase (in zymogen granules) concentrated in the 
apical poles of cells within an acinus should be shown. Acinar injury is typically shown as 
intracellular vauole formation (from perturbed autophagy), large blebs forming on the basal 
membrane, junctional complex disruption, and necrotic markers. 
 
Figure 3. 3a: in this pancreatic slice perfusion study, the insulin content of the slice should have 
been determined, and customarily the secreted insulin should be expressed as percentage of total 
tissue insulin content. 
3d: Why in control acute 1 mM glucose stimulation secreted more insulin than 16mM? 
3f: Error bars missing 
3h: No legend as to which trace correspond to what. Was this single beta cell or whole islet 
response? Were the beta cells within the whole islet at Day 10 synchronous and oscillatory in 
calcium response like Day 0 or was there disruption in coupling. How about the TW, although little 
response to 18G were the calcium response just weaker, in fewer cells within the islet, or loss of 
synchrony. 
3k: Similar comments as 3h. Calcium responses in acini are observed as sequential light up of all 
acinar cells and oscillatory in pattern- was this pattern observed at Day 10. This is important for 
both islet and acini as this demonstrates that the coupling of beta and acinar cells remain intact at 
Day 10 
3i: no labels for each trace. Author should put the acute sample for control as other figures. 
 
Figure 4 (mouse) and Figure 5 (human) are very impressive in the longitudinal tracking of beta cell 
neogenesis induced by BMP-7 especially after alloxan destruction of the mouse beta cells. The 
techniques employed were likewise clever and impressive. Fig. 4c: They showed that insulin+ cells 
were generated de novo from ductal cells, bud did not use a ductal cell marker, and should do so 
to be truly convincing that these beta cells genuinely differentiated from the ductal cells. 
 
BMP7 induction of beta cell neogenesis in the T1D and T2D human islets in the supplementary 
figures should be shown as a main figure. 
 
Methods. A bit description of the differences in the buffer that protected the pancreatic slices. 

Good that they did mention using the better protease inhibitors - aprotinin and chymotstatin. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This manuscript reports an improvement in culturing pancreatic slices in their native-like 
environment for long-term applications. First, the author designed a novel culture system that 
provides an improved system to deliver oxygen to the pancreatic explants. Then they 
characterized cell viability and function of endocrine and exocrine pancreas in this culture system 
during a 10-day time-period. Finally, they provided data on the potential application of this 
improved system to study beta cell regeneration in mouse and human. The improvement in the 
maintenance of the culture system for longer-time is of great importance to study physiological 
and pathological processes of human pancreas. This definitely highlights the importance of this 
study. However, the study lack of substantial experimental setting to support the scientific claims. 
In particular, the evidence indicating beta cell neogenesis using this model in not sufficient. 
Therefore, several key experiments and further evidence are required to clarify and support the 
finding of this manuscript. 
 
Major points 
-Using a modeling prediction, the author estimated and characterized the required oxygen for each 
type of culture systems. However, there is no direct experimental data indicating that oxygen is 
the only parameter that improved the maintenance of the culture. I was wondering if the authors 
can support this claim experimentally, for instance, through culturing PFC system in different 
levels of oxygen and analyze metabolic and morphological features. 
 
-The characterization of the new system has been done mainly on immunohistochemistry (IHC). 
To provide further supporting evidence, the authors need to check some key markers (such as 
transcription factors) for different types of endocrine and exocrine cells. This can be done using 
IHC, qPCR analysis or western blotting. This analysis are important to better characterize the 
changes between TW and PFC systems that results in improvement of PFC culture. For instance, in 
Fig2c, in TW condition more dead cells are shown, however, it remains unclear, which cell type are 
undergo cell death, as total endocrine cells (Fig2K) and exocrine cells look normal between two 
systems. Therefore, it is important to check some apoptotic markers contained with different types 
of pancreatic cells. Additionally, no data is shown on the maturity of beta cells in tow systems. 
Only showing Insulin or Glucagon does not clearly indicate the presence of mature functional beta 
or alpha cells in the system. Finally, in supporting loss of islet integrity and its improvement in PFC 
vs TW, more analysis needs to be presented. For instance, high-resolution imaging of E-cadherin 
and/or Beta-catenin might indicate cell-cell interaction changes between two systems. 
 
-On the functional analysis of HPSs, regarding the data presented in Fig3, the freshly sectioned 
(acute) samples show lower GSIS. Of course the authors discuss that this might be due to stress 
of sectioning and so on, but I am really wondered why they didn’t rest the samples for a day and 
then perform the experiments. The problem here is the HPSs cultured 10 days in TW perform 
similar GSIS to the control acute samples. For having proper control at least they should compare 
TW and PFC samples at day 1 vs day 10. 
 
-In Figure 3a, in the PFC culture the total secreted insulin in higher even in the KCl. Is this 
indicating that the total insulin levels is higher in this condition, or we have more insulin granules? 
This can be very important as in later figures the authors show increase in insulin+ cells in the PFC 
condition. They should clearly rule out if PFC condition increase total levels if insulin or insulin 
granules that might contribute to increased EGFP signals in later figures. 
 
-In figure 4 the authors claim that PFC culture of mouse slices result in islet neogenesis upon 
treatment with BMP-7 during 10 day culturing. They used slices from InsCre, mTmG mice and 

showed increased yellow cell population as well as EGFP signal upon treatment with BMP-7 
indicating conversion of non-beta to beta cells. One important point here is, there is no evidence 
that the observed process in neogenesis. The term of neogenesis refers to regeneration of beta 
cells from progenitors. There is not supporting data to show the existence of progenitors and their 
differentiation towards beta cells. To claim this, the authors need at least to show the presence of 
Neurog3+ cells in their condition. Therefore, what is presented is most likely the conversion of 
non-beta to beta cells that can be referred to general term of regeneration or cell conversion but 
not neogenesis. Furthermore, no high resolution data is shown to indicate that the yellow cells are 
derived from the ductal epithelium. For instance, in FigureC, lower part a yellow cell population is 
shown that is far away from the ductal region. If such cells are derived from duct is it possible that 
they move that far in a short period within the slice culture? For me the presented data are of low 
quality and to show that there is cell conversion in this system the authors need to provide high 
resolution picture with high magnification involving different pancreatic markers. This might 
provide more information about the origin of the converted cells. If they cannot provide any data 
regarding the presence of Neurog3+ cells they need to revisit using the neogenesis term. 
 
-The same concept also applies to the figure 5 on human slices. I do understand the limitations 
with lineage tracing in human systems and I appreciate the design of this system in figure 5. 
However, the conclusion claimed by the authors here might not truly reflect the presented data. 
Again, there is no data showing the presence of NEUROG3+ progenitors. The low quality and 
magnification of the presented data makes it very difficult to monitor the actual process of cell 
conversion in this system. Most importantly, the authors failed to show that treatment of BMP7 
result in increase in yellow population statistically. For me increased levels of EGFP is not enough 
to claim increase in total beta cells. First, the data presented in fig5 c and h showing the % of slice 
expressing EGFP but do not show if there is increase in beta cell number. Second, there is no data 
to rule out that treatment of BMP7 does not increase insulin expression and levels. 
 
Other points 
-introduction: “…are more likely to reflect the native biology of the organ” not completely true. 
They are the closest system to the in vivo condition so far, but till which degree they reflect native 
biology of the organ is not clear. 
 
-Fig1b-d. Could this analysis be extended to 10 days? This would help to monitor the system 
better over 10 day experimental course presented in later figures. 
-Page 7: “…. in transwells have less-defined boundaries (Fig. 2j) and with less defined 
boundaries.” Repeated wording. 
 
-Some figures miss scale bar. 
-Fig2K. Are all these cell alive? Is there any analysis that can show the live vs dead cells in 
endocrine compartments? 
-Fig3g,I, What is 3G, please indicate it in the legend. 
-Fig3i, where is the reference or control sample at day 1? 
-Fig3K-m, here only shows that the experiment technically work. I found the actual data regarding 
the PFC and TW cultures in the supplementary data. I would suggest to put those data in the main 
figure. 
-The experiment on mouse slices is done using pancreas from 6-8-week-old mice. This is relatively 
young age for mice. Therefore, the pancreatic lineage might still retain higher degree of plasticity. 
I am curious if old animal also present the formation of new beta cells when treated with BMP-7 in 
the slice culture. This would be more relevant for diabetes condition in older population. 
-Page13: “… due to the treatment of slices with BMP7.” They mean “alloxan”? 
-Fig4c, is the lower picture cropped from the upper part? For me it doesn’t seem to be. 
-Fig4i. The green signal is barely detectable. The same for Fig4f. 
-Page16: “We could also detect several regions of non-β-cells transitioning to EGFP-expressing 
cells, mirroring previous observations using mouse slices (Fig. 5a).” I don’t see this transitioning in 
the picture. High magnification and resolution is required. 

 
-Fig5d, is not mentioned within the text. 
 
-The conclusion section especially on neogenesis is too strong. It needs to be written more based 
on the actual data. 
 
-Figure S5, the control and MBP-7-treated samples present similar levels of yellow cells. While the 
EGFP signal is different between the two condition. This might indicate the possible effect of BMP-7 
on insulin levels rather than increase in cell conversion. Similar data are also presented in Fig5 c 
and h. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
In this manuscript Qadir et al set out to develop novel methodology for long term culture and 
subsequent examination of function and islet cell regeneration using human pancreatic slices. 
Authors report successful development of enhanced pancreatic slice culture system that allows for 
improved oxygenation, slice viability and endocrine functionality (i.e. glucose and KCl-stimulated 
insulin secretion). Furthermore, investigators utilized adenoviral vectors to perform longitudinal 
lineage tracing tracking of potential beta cell neogenesis in response to the treatment with BMP7, 
a compound previously shown by this group to induce human beta cell neogenesis from exocrine 
cells in the pancreas (i.e. purported ductal cell progenitors). 
 
Critique: 
 
1. Figure 1: Authors state that parameters used to model oxygen consumption are estimated 
based on values reported in references 15. The data in reference 15 however reports data from 
isolated/cultured porcine acinar cells and islets kept in culture from 0 to 8 days. It’s not clear why 
this data set was chosen to approximate/model oxygen consumption in intact human slices given 
clear differences in tissue handling and also reported differences between porcine and human 
islets/exocrine tissue. In addition, lactate production (panel e) data is presented (in hours) which 
is inconsistent with data presentation in other graphs (i.e. in days). Furthermore, authors need to 
include units for Fig 1e and 1f (i.e. lactate production and glucose consumption). Finally, the claim 
that ‘enhanced oxygenation” preserves oxidative phosphorylation is not supported by presented 
data. Decreased “glucose consumption” in PFC group can be attributed to numerous factors and 
cellular changes and for example may reflect attenuated metabolic rate or changes in cellular 
proliferation/survival. Authors need to perform additional experiments to directly demonstrate 
enhanced mitochondrial function/oxygen consumption/oxidative metabolism or use glucose carbon 
isotope techniques to demonstrate enhanced glucose oxidation under PFC condition. 
 
2. Figure 2. Given focus of the manuscript on endocrine pancreas biology, authors should perform 
additional studies of endocrine pancreas viability/expression in PFC vs. TW culture conditions. This 
should include examination of cell turnover (proliferation and apoptosis) in beta and alpha cells. 
This will be particularly important given the focus on beta cell regeneration and turnover. In 
addition, author should examine expression of key maturation markers for beta (e.g. MAFA, 
NKX6.1, PDX-1 etc) and alpha (e.g. ARX) cells. This is important given dramatic increases in 
insulin secretion reported in Fig. 3 under PFC conditions. Finally, since authors suggest enhanced 
oxygen delivery and improved mitochondrial oxidation in PFC group, staining for hypoxia-regulated 
transcription factors (e.g. HIF1, c-JUN etc) and/or markers of mitochondrial integrity will provide 
supporting evidence for the stated hypothesis. 
 
3. Figure 3. Authors report enhanced glucose-stimulated insulin secretion under perifusion 
conditions with 10 day culture using PFC dishes. Although this data is impressive, there are 
number of questions that arise from these studies. Firstly, contrary to what authors state, none of 

the groups display “classic biphasic pattern” of glucose-stimulated insulin release. In PFC group, 
insulin concentrations continuously rise during 16mM glucose period without clear 1st/2nd phase 
distinctions. The physiological significance of pancreas slice perifusion will be reinforced by data 
demonstrating physiological release of other important endocrine hormones such as glucagon and 
somatostatin. Finally, PFC group has a significant increase in glucose-independent insulin release 
to KCl. KCl is commonly used as a measure of maximal insulin output and used as a marker of 
total beta cell mass/numbers. Authors should comment on the reason for increased KCl-stimulated 
insulin release in PFC group despite comparable levels of insulin positive cells reported in sections 
from PFC vs. TW slices. 
 
4. Figures 4 and 5. Data on longitudinal tracking of purported beta cell regeneration in 
mouse/human slices is intriguing, but is largely preliminary and not supportive of main conclusions 
of the study. Firstly, to prove generation of “newly” formed beta cells (with BMP-7) authors need 
to show clear increase in overall beta cell numbers (or other measures of insulin + area). These 
data need to show that newly formed cells express key beta cell maturation transcription factors 
(e.g. NKX6.1, PDX1 etc) as well as other markers of functionality. Demonstrating that treatment 
with BMP-7 also positively alters insulin secretion in perifusion setting (especially KCl-mediated 
insulin release) will also significantly strengthen the argument that beta cell neogenesis has 
occurred in this model. In addition, the concept of beta cell regeneration implies replacement of 
lost beta cells, however current study in human slices examines response of otherwise “healthy’ 
beta cells to a supra-physiological stimuli of BMP-7. Authors need to clearly acknowledge that 
there are limitations of this approach in human tissue and the study of human beta cell 
physiological neogenesis is not possible using this model. Indeed, repeated studies in mice and 
humans have demonstrated lack of any evidence for physiological occurrence of beta cell 
neogenesis.