Optogenetic Regulation of intracellular cyclic adenosine monophosphate and glucose-stimulated insulin secretion in pancreatic beta cells.
Zhang, Fan.
2019
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Diabetes affects
millions of people worldwide. Islet or whole pancreas transplantation is a promising
method for the treatment of Type I diabetes (T1D), which results from autoimmune damage
or dysfunction of pancreatic beta cells, which producing insulin. While transplantation
is limited by the shortage of healthy donor tissues, cell therapy is a promising
modality for the treatment or even ... read morecure of diabetes. Sources of insulin-producing cells
include pancreatic beta-cell lines, adult cells trans-differentiated into beta-cell-like
cells and human embryonic or induced pluripotent stem cells converted into pancreatic
beta cells. These cells should also maintain physiologically relevant glucose stimulated
insulin secretion (GSIS) ability. In this work, mouse insulin-producing pancreatic
beta-cell lines were used because of the ease in maintaining these cells compared with
cells from human embryonic stem cell differentiation. These cell lines were further
modified using optogenetic methods, which were developed in recent years to precisely
and timely control cellular behavior free of the side effects associated with the use of
chemical agents. MIN6 cells were engineered with the incorporation of a gene encoding a
photoactivated adenylyl cyclase (PAC) from the soil bacterium Beggiatoa (bPAC). In
pancreatic beta cells, the glucose metabolism increases the level of cyclic adenosine
monophosphate (cAMP), which in turn mediates insulin secretion and acts as an amplifier
of GSIS. A bPAC expressing plasmid was designed and a recombinant adenovirus was
constructed for gene delivery to animal cells. including beta cells. Transduction of
MIN6 cells led to a 3- to 5-fold increase of cAMP with blue light illumination for 10
minutes. After a 30-minute stimulation with blue light a 2- to 3-fold increase of
insulin secretion was observed. The effect of photoactivation on insulin secretion was
comparable to the amplification resulting after treatment with known secretagogues like
forskolin or IBMX. Cultured MIN6 cells in spinner flask formed 3D aggregates (termed
pseudoislets or PIs). The transduction of PIs as well as of native islets isolated from
mice with the bPAC adenovirus resulted in increased insulin secretion. Subsequently, a
MIN6 cell line (MIN6-bPAC) with stable bPAC expression was generated through
transduction with a bPAC lentivirus. Further testing on this cell line showed that there
was no significant difference on cell growth rate compared with regular MIN6 cells. The
continuous light illumination also caused no significant apoptosis. MIN6-bPAC cells were
cultured in spinner flask to form PIs, which were further encapsulated in
calcium-alginate beads. The PIs/beads were transplanted into STZ induced diabetic mice.
Mice transplanted with cells and stimulated with blue light showed improved glucose
tolerance. In summary, this dissertation describes the engineering pancreatic beta cells
for on demand increase of insulin secretion using light as the stimulus. Engineering
beta cells amenable to optogenetic control of their function paves a novel way for of
cell replacement therapy for diabetes. The tools generated in this research will also be
useful in the study of the role of cAMP in beta-cell function under normal and disease
conditions.
Thesis (Ph.D.)--Tufts University, 2019.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisor: Emmanuel Tzanakakis.
Committee: Qiaobing Xu, James Van Deventer, and Athanassios Sambanis.
Keyword: Chemical engineering.read less - ID:
- vd66wc393
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