Development of a novel beta-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures.
Recently, it has been reported that islet transplantation into patients with Type 1 diabetes may achieve insulin independence for a year or longer [Shapiro et al., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med. 343 (2000) 230-238]. However, the amount of donor islet tissue is limited, therefore, multiple approaches are being explored to generate insulin-producing cells in vitro. Some promising results have been obtained using mouse and human stem cells and progenitor cells [Soria et al., From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus, Diabetologia. 4 (2001) 407-415; Lechner et al., Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus, Am J Physiol Endocrinol Metab. 284 (2003) 259-266; Bonner-Weir et al., In vitro cultivation of human islets from expanded ductal tissue, Proc Natl Acad Sci U S A, 97 (2000) 7999-8004; Assady et al., Insulin production by human embryonic stem cells, 50 (2001) Diabetes 1691-1697]. However, the efficiency of obtaining populations with high numbers of differentiated cells has been poor. In order to improve the efficiency of producing and selecting insulin-producing cells from undifferentiated cells, we have designed a novel beta-cell specific and glucose responsive promoter system designated pGL3.hINS-363 3x. This artificial promoter system exhibits significant luciferase activity not only in insulin-producing MIN6 m9 cells but also in isolated human islets. The pGL3.hINS-363 3x construct shows no activity in non-insulin-producing cells in low glucose conditions (2 mM glucose) but demonstrates significant activity and beta-cell specificity in high glucose conditions (16 mM glucose). Furthermore, pGL3.hINS-363 3x shows significant promoter activity in differentiated AR42J cells that can produce insulin after activin A and betacellulin treatment. Here, we describe a novel beta-cell specific and glucose responsive artificial promoter system designed for analyzing and sorting beta-like insulin-producing cells that have differentiated from stem cells or other progenitor cells.
Fukazawa
Takuya
T
First Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho 2-5-1, Okayama 700-8558, Japan. fukazawa@aol.com
Matsuoka
Junji
J
Naomoto
Yoshio
Y
Nakai
Toru
T
Durbin
Mary L
ML
Kojima
Itaru
I
Lakey
Jonathan R T
JR
Tanaka
Noriaki
N
D000818:Animals; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology*; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D007328:Insulin; D049452:Green Fluorescent Proteins; D005947:Glucose