Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential.
OBJECTIVE
The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-L-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue.


METHODS
Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions.


RESULTS
Human pancreatic cell (HPC) cultures coexpressed alpha-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein.


CONCLUSIONS
These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.
Linning
Katrina D
KD
Department of Physiology, Michigan State University, East Lansing, Michigan 48824, USA.
Tai
Mei-Hui
MH
Madhukar
Burra V
BV
Chang
C C
CC
Reed
Donald N
DN
Ferber
Sarah
S
Trosko
James E
JE
Olson
L Karl
LK
D000111:Acetylcysteine / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D000328:Adult; D000418:Albumins / Q000096:biosynthesis / Q000235:genetics; D002096:C-Peptide / Q000096:biosynthesis / Q000235:genetics; D002449:Cell Aggregation; D002454:Cell Differentiation / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured / Q000166:cytology; D002867:Chromones / Q000494:pharmacology; D003470:Culture Media / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D000077270:Exenatide; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics / Q000494:pharmacology; D005934:Glucagon / Q000096:biosynthesis / Q000235:genetics; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000096:biosynthesis / Q000235:genetics; D007381:Intermediate Filament Proteins / Q000096:biosynthesis / Q000235:genetics; D007424:Intracellular Fluid / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology*; D009025:Morpholines / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D064231:Nestin; D009536:Niacinamide / Q000494:pharmacology; D010084:Oxidation-Reduction; D010455:Peptides / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013004:Somatostatin / Q000096:biosynthesis / Q000235:genetics; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014688:Venoms / Q000494:pharmacology; D014746:Vimentin / Q000096:biosynthesis / Q000235:genetics; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics
D000418:Albumins; D002096:C-Peptide; D002867:Chromones; D003470:Culture Media; D016895:Culture Media, Serum-Free; D018398:Homeodomain Proteins; D007328:Insulin; D007381:Intermediate Filament Proteins; D009025:Morpholines; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; D010455:Peptides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014688:Venoms; D014746:Vimentin; C084010:pancreatic and duodenal homeobox 1 protein; D009536:Niacinamide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D013004:Somatostatin; D005934:Glucagon; D000077270:Exenatide; D000516:alpha-Amylases; D000111:Acetylcysteine