Cellular reprogramming of human amniotic fluid cells to express insulin.
Islet transplantation represents a potential cure for type 1 diabetes; however, a lack of sufficient donor material limits its clinical use. To address the shortfall of islet availability, surrogate insulin-producing cells are sought. Studies suggest that human amniotic fluid (hAF) contains multipotent progenitor cells capable of differentiating to all three germ layers. Here, we used high-content, live-cell imaging to assess the ability to reprogram hAF cells towards a beta cell phenotype. A fluorescent reporter system was developed where DsRed express (DSRE) expression is driven by the human insulin promoter. Using integrative lentiviral technology, we created stable reporter hAF cells that could be routinely monitored for insulin promoter activation. These cells were subjected to combinatorial high-content screening using adenoviral-mediated expression of up to six transcription factors important for beta cell development. Cells were monitored for DSRE expression which revealed an optimal combination of the transcription factors required to induce insulin gene expression in hAF cells. These optimally induced cells were examined for expression of additional beta cell transcription factors and proteins involved in glucose sensing and insulin processing. RT-qPCR revealed very low level expression of insulin that was ultimately insufficient to reverse streptozotocin-induced diabetes following sub-capsular kidney transplantation. High-content, live-cell imaging using fluorescent reporter cells provides a convenient method for repeated assessment of cellular reprogramming. hAF cells could be reprogrammed to express key beta cell proteins, however insulin gene expression was insufficient to reverse hyperglycemia in diabetic animals.
Gage
Blair K
BK
Laboratory of Molecular and Cellular Medicine, Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, Canada.
Riedel
Michael J
MJ
Karanu
Francis
F
Rezania
Alireza
A
Fujita
Yukihiro
Y
Webber
Travis D
TD
Baker
Robert K
RK
Wideman
Rhonda D
RD
Kieffer
Timothy J
TJ
D000256:Adenoviridae / Q000235:genetics; D000653:Amniotic Fluid / Q000166:cytology*; D000818:Animals; D002478:Cells, Cultured; D005434:Flow Cytometry; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D008297:Male; D051379:Mice; D011401:Promoter Regions, Genetic; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism
D007328:Insulin; D014157:Transcription Factors