We have recently identified hormone negative endocrine cells (Chromogranin A positive hormone negative [CPHN]) cells in human pancreas ( in non-diabetic and diabetic subjects) and in HIP rats.
http://www.ncbi.nlm.nih.gov/pubmed/26700560
http://www.ncbi.nlm.nih.gov/pubmed/27300574
http://www.ncbi.nlm.nih.gov/pubmed/27472443
Now I am planning to characterize those cells. Given that the frequency of those cells are very low, I want to FACS sort out those cells from all the endocrine cells. One approach could be isolating all the endocrine cells first and then sort Chromogranin(+ve)/endocrine cell(-ve) cells. It seems a great challenge to me, however, I am hoping expert opinions from the researchgate community.
Thanks a lot.
I am curious about the reason behind your criteria of defining islets versus cell clusters, 4 versus 3 positive cells, respectively.
No answers but a few, hopefully useful, comments:
One has to be careful about making assessment based on immunofluorescence signal - threshold of detection can make cell appear negative, whereas the reality may be different. In case of sections of islets from rodents, I have noted that under severe challenge (insulin resistance or continuous infusion of 40% glucose solution at a rate of 1-2ml/hr for several days), many beta cells seem to be negative for insulin. But then when you look for insulin mRNA, the picture is quite opposite - mRNA production is increased 2-4 fold compared to normal controls.
All of the above and more was published well over a couple of decades ago. We also often did serial sections and included much larger sample size.
Thanks a lot Dr. Alam for your concern and comments. Regarding our research criteria of considering clusters of endocrine cells Vs islets, in human pancreas (very rare in adult rodent pancreas) hormone positive endocrine cells are frequently seen in single cells or clustered in 2-3 cells (increased in T1D and T2D). we decided not to consider 2-3 cells as an islet based on the possibility that those cells might be originated from non-islet origin (for example from acinar or ductal origin). Moreover we are very interested in those clustered cells. Since our lab pioneered studying T2D in human pancreas first (http://www.ncbi.nlm.nih.gov/pubmed/12502499), we set our criteria to emphasize those clustered cells in human pancreas.
Your findings of insulin negative beta cells is very interesting and quite relevant to ongoing hypothesis of beta cell dedifferentiation in diabetes. (http://www.ncbi.nlm.nih.gov/pubmed/22980982).
Our data also indicated that pancreatic beta cells lose their identity in response to stress and that occurs even before onset of diabetes. We also very curious about those hormone negative (in your case insulin negative) endocrine cells. But still in human, we are not be able to say whether those cells are dedifferntiated or newly forming endocrine cells. It's only possible by Lineage Tracing and in human we cannot do that.
However I am curious about your protocol of isolating mRNAs to those specific insulin negative beta cells. Maybe that can also help me to find my solution.
Thanks again.
We did not isolate RNA from single cells. In fact, there is no need for it. A more convenient and adequately informative way is to perform in situ hybridization for mRNA of your interest. When deemed necessary, we performed in situ hybridization for insulin, glucagon, somatostatin, amylin, Glut2 and glucokinase on serial sections of pancreas.
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