Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the

Capacity for Endocrine Cell FateGomez DL, O’Driscoll M, Sheets TP, Hruban RH, Oberholzer J, McGarrigle JJ, et

al. (2015) PLoS ONE

Presented by Stacey N. Van Pelt

The human pancreasThe human pancreas has exocrine and endocrine

functions

Exocrine pancreas: duct and acinar cells

Digestive juices, secreted by acinar cells, flow through pancreatic ducts to the small intestine

Endocrine pancreas: islets

Structures called pancreatic islets are dispersed throughout the organ and contain hormone producing cells that regulate blood glucose levels

Beta cells, located in pancreatic islets, produce insulin

Diabetes mellitus type 1 is a disease resulting from the autoimmune destruction of insulin-producing beta cells in the pancreas

Patients with type 1 diabetes have to take insulin injections to regulate their blood glucose levels

Diabetes treatments/cureType 1 diabetes is a 2 prong disease that currently has no cure

Autoimmune

Pancreatic function

Focus on how to restore pancreatic function and leave the rest to immunologists

One potential treatment option is Islet transplantation

Islets from two donor organs are purified and implanted into the patient’s liver

Problem: donor/host compatibility → rejection

Solution: use a patient’s own cells

Dedifferentiate exocrine cells and turn them into insulin-secreting endocrine cells

Can NGN3 be used to reprogram a patient’s own cells?

Notch signaling in the developing pancreasCanonical Notch signaling pathwayIn the developing human pancreas, Notch ligand (JAG1) binds to the Notch receptor, and the intracellular domain (NICD) is cleaved and travels to the nucleus where it activates the transcription of HES1, a neurogenin 3 (NGN3) repressor.

Neurogenin 3 (NGN3) in the developing pancreasA transcription factor that upregulates NEUROD1 (another TF, activation

commits cells to endocrine fate) Necessary and sufficient for endocrine differentiation during pancreatic

developmentExpressed by a population of progenitor cells that give rise exclusively to

hormone-secreting cells within isletsNGN3 expression in the developing pancreas leads to endocrine cell fate (via

NEUROD1)

Does NGN3 have a role in the adult human pancreas? Does Notch negatively regulate NGN3 in the adult pancreas?

Expression of NGN3 in the adult human pancreas

Experiment #1:Aim: Determine if NGN3 plays a role in the duct cells of

the adult human pancreas

Sections of histologically normal pancreatic tissue from living subjects were stained for:

NGN3, CK19 (duct cell), nuclei

Figure 1A shows colocalization of NGN3 and CK19 (duct cell marker) in the same cells

These results show that NGN3 is present in the adult human pancreatic duct cells

What about acinar cells?

CK19

NGN3

Expression of NGN3 in the adult human pancreasExperiment #2:

Aim: Determine if NGN3 plays a role in the acinar cells of the adult human pancreas

Sections of histologically normal pancreatic tissue from living subjects (same as experiment #1) were stained for :

NGN3, amylase (acinar marker), nuclei

Figure 1E shows colocalization of NGN3 and amylase in the same cells

These results show that NGN3 is present in the adult human pancreatic acinar cells

Taken together, experiments 1 and 2 show that NGN3 is present in the adult human EXOCRINE pancreas

CD133/NGN3 Co-expression CD133 is a cell surface glycoprotein that’s been shown to be coexpressed with NGN3 in human fetal pancreas.

What is the relationship between CD133 and NGN3 in the adult human pancreas? *Important implications for cell sorting

Experiment #3:IHC: Islet depleted pancreatic tissue was stained for CD133, NGN3,

and nuclei

Figure D shows colocalization of NGN3 and CD133 in the same cells

FACS analysis confirmed ~94% CD133+ cells were NGN3+

CD133 NGN3

Regulation by Notch in adult pancreasExperiment #4: Western blot analysis of Notch signaling

Left side:

Sort cells into CD133+/CD133D (depleted)

Run whole cell lysates on a gel

Stain for HES1, NICD

Results showed the expression of Notch pathway genes in CD133+ cells (NGN3+)

Right side:

Use CD133+ cells only

Nuclear extracts vs. cytoplasmic extracts

Both Notch signaling proteins are localized in the nucleus

Surprise!

Why are genes that negatively regulate NGN3 in NGN3+ cells?

Notch signalingInhibitor of DNA binding proteins (ID1-4) can form heterodimers with HES1 to block transcriptional regulation

Experiment #5: Culture cells for 4 days → whole cell lysates

CoIP for HES1

Western blot

Stain for ID1, ID2, ID4

Gel shows that ID proteins are interacting with HES1, preventing it from downregulating NGN3

Other activating/repressing experiments showed that Notch signaling was regulating NGN3 at both a transcriptional and translational level in the same way that it occurs in the developing pancreas.

Pancosphere (PS) formationWhen CD133+cells are suspended in media conditioned

with human SDEC cells, formation of spherical aggregates called pancospheres (Fig A) occurs

There are 2 phases to PS formation - 21 days total, and these phases are characterized by different expression patterns of endocrine related proteins

CD133D cells do not form PS

Significance: Islets are aggregates of different cell types that express different sets of hormones. PS formation mimics the structure of pancreatic islets.

Fig B: PDX1 expression in PS nuclei

Maturing beta cells co-express PDX1, NKX6, CHGA

Images show PS on day 6 of formation

PS characterizationPhase I (day 4-13)

Proliferation (detected by EdU incorporation to DNA)

Increased PS diameter

High expression of KI67 compared to CD133+ starting population

Phase II (day 15-21)

Decreased expression of KI67

Increased expression of NEUROD1 (regulates terminal differentiation and mature function of islet cells) and NGN3 (positively regulates NEUROD1)

MAFA/MAFB peak at final step of differentiation (required for beta cell function)

PS show signs of endocrine differentiationExperiment #9:

Aim: To show if PS are expressing proteins associated with endocrine pancreatic cells

IHC/confocal microscopy: PS were stained for CPEP, glucagon, and PDX1 and nuclei on day 21 of formation (phase II)

Figures C and D show cells that co-express PDX1 and C-peptide (insulin), which are proteins produced by maturing/mature beta cells, and other cells producing glucagon (an alpha cell marker)

Figures E and F show pancospheres expressing a variety of endocrine related proteins, including glucagon, chromogranin A, C-peptide, and PDX1

What next?Exocrine cells can acquire endocrine fate in vitro, but what about in

vivo?

NGN3+ cells may be a future treatment for type 1 diabetes

Other treatments might include pharmacologically targeting the negative regulation of NGN3, leading to islet neogenesis

Immunologists have to do their part to prevent future damage to any new islets created