Proliferation of sorted human and rat beta cells.
OBJECTIVE
The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors.


METHODS
Human beta cells were purified by FACS by virtue of their high zinc content using Newport Green, and excluding ductal and dead cells. Rat beta cells were sorted by autofluorescence or using the same method developed for human cells. Cells were plated on poly-L-lysine or ECMs from rat or human bladder carcinoma cells or bovine corneal ECM and incubated in the presence of BrdU with or without growth factors.


RESULTS
The newly developed method for sorting human beta cells yields a population containing 91.4 +/- 2.8% insulin-positive cells with a low level of spontaneous apoptosis and a robust secretory response to glucose. Beta cells from 8-week-old rats proliferated in culture and this was increased by ECM. Among growth factors, only human growth hormone (hGH) and the glucagon-like peptide-1 analogue liraglutide enhanced proliferation of rat beta cells, with a significant increase on both poly-L-lysine and ECM. By contrast, sorted adult human beta cells from 16 donors aged 48.9 +/- 14.3 years (range 16-64 years) failed to replicate demonstrably in vitro regardless of the substratum or growth factors used.


CONCLUSIONS
These findings indicate that, in our conditions, the fully differentiated human adult insulin-producing beta cell was unable to proliferate in vitro. This has important implications for any attempt to expand cells from pancreases of donors of this age group. By contrast, the rat beta cells used here were able to divide in vitro, and this was enhanced by ECM, hGH and liraglutide.
Parnaud
G
G
Department of Genetic Medicine and Development, University Medical Center, 1 rue Michel-Servet, 1211, Geneva-4, Switzerland. geraldine.parnaud@medecine.unige.ch
Bosco
D
D
Berney
T
T
Pattou
F
F
Kerr-Conte
J
J
Donath
M Y
MY
Bruun
C
C
Mandrup-Poulsen
T
T
Billestrup
N
N
Halban
P A
PA
D000293:Adolescent; D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002469:Cell Separation; D003920:Diabetes Mellitus / Q000628:therapy*; D005109:Extracellular Matrix; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000502:physiology*; D016381:Islets of Langerhans Transplantation / Q000379:methods; D008875:Middle Aged; D051381:Rats; D013045:Species Specificity