Development of a reliable automated screening system to identify small molecules and biologics that promote human β-cell regeneration.
Numerous compounds stimulate rodent β-cell proliferation; however, translating these findings to human β-cells remains a challenge. To examine human β-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human β-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled β-cells with high specificity using both nuclear and cytoplasmic markers. β-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1β signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+β-cells, whereas treatment with other compounds had limited to no effect on human β-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human β-cell proliferation, thus allowing for increased testing of candidate human β-cell mitogens.
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee.
Kristie I
KI
Aamodt
Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.
Radhika
R
Aramandla
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee.
Judy J
JJ
Brown
Division of Endocrinology, Diabetes, and Bone Disease, Department of Medicine, Mount Sinai Medical Center, New York, New York; and.
Nathalie
N
Fiaschi-Taesch
Division of Endocrinology, Diabetes, and Bone Disease, Department of Medicine, Mount Sinai Medical Center, New York, New York; and.
Peng
P
Wang
Division of Endocrinology, Diabetes, and Bone Disease, Department of Medicine, Mount Sinai Medical Center, New York, New York; and.
Andrew F
AF
Stewart
Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee; marcela.brissova@vanderbilt.edu.
Marcela
M
Brissova
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee.|Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.|Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee.
Alvin C
AC
Powers
D028341:Activins / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D058908:Adenosine A2 Receptor Agonists / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000328:Adult; D001331:Automation; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects*; D004353:Drug Evaluation, Preclinical; D004921:Erythropoietin / Q000494:pharmacology; D000077270:Exenatide; D005260:Female; D018682:GABA Agents / Q000494:pharmacology; D006247:Harmine / Q000494:pharmacology; D006801:Humans; D054795:Incretins / Q000494:pharmacology; D050417:Insulin-Secreting Cells / Q000187:drug effects*; D008297:Male; D008875:Middle Aged; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D055435:Myostatin / Q000494:pharmacology; D009705:Nucleosides / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011388:Prolactin / Q000494:pharmacology; D012038:Regeneration / Q000187:drug effects; D012701:Serotonin / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D014665:Vasodilator Agents / Q000494:pharmacology; D014688:Venoms / Q000494:pharmacology; D055815:Young Adult; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
C422646:A 134974; D058908:Adenosine A2 Receptor Agonists; D018682:GABA Agents; D054795:Incretins; D008996:Monoamine Oxidase Inhibitors; D055435:Myostatin; D009705:Nucleosides; D010455:Peptides; D010982:Platelet-Derived Growth Factor; D017366:Serotonin Receptor Agonists; C557757:UK-432097; D014665:Vasodilator Agents; D014688:Venoms; C422405:activin A; D028341:Activins; D004921:Erythropoietin; D012701:Serotonin; D019830:Adenosine-5'-(N-ethylcarboxamide); D006247:Harmine; D005680:gamma-Aminobutyric Acid; D011388:Prolactin; D000077270:Exenatide; D000241:Adenosine
human islet; β-cell proliferation