Download Generation of Insulin-Secreting Glucose-Responsive ß

Generation of insulin-secreting glucose-responsive
-cells from human stem cells
Adam Ross, Nan Sook Lee, Robert H. Chow
METHODS
ABOUT ME
RESULTS
Stem-cell-derived insulin-positive cells
Five stages of differentiation
My name is Adam Douglas Jackson Ross. I am originally
from Pasadena, California, and
currently attend the
University of San Francisco where I am a rising junior. At the
University of San Francisco, I am a Biology Major with a
Chemistry and a Business Minor.
A. Endpoint RT-PCR with MEF
B. Immunocyto Staining
(Cell Staining)
Betacellulin+ Nicotinamide
CONCLUSION
ABSTRACT
Human embryonic stem cells offer an unlimited but unrealized
source of cells to treat Type 1 diabetes. Proper gene
expression is critical for normal development of human
embryonic stem cells. Protocols to direct differentiation of
stem cells to express insulin (INS) already exist. However, the
yield of INS positive cells is low, difficult to reproduce, and the
cells do not secrete when stimulated by glucose. We have
modified existing protocols to give more reliable production of
differentiated stem cells expressing INS. The new five-stage
protocol yields roughly 10% INS cells, which means any
analysis of the whole population is confounded by the
presence of the predominant non-INS cells. To identify and
purify the INS-producing cells, Dr. Nan Sook Lee has created
a reporter construct that encodes Green Fluorescent Protein
(GFP) and Red Fluorescent Protein (mCherry). The construct
is designed so that when introduced into human embryonic
stem cells, they will shine green if the INS gene is present, or
red if the INS gene is not present. The cells will be processed
by a fluorescence-activated cell sorter (FACS). Unlike normal
insulin-secreting beta cells, the differentiated stem cells
containing the INS gene do not respond to glucose. To
address this problem, we will compare the genetic information
for these two cell types. We hypothesize that expression of
the proteins that facilitate glucose responsiveness is regulated
by micro RNAs (miRNAs) acting on messenger RNAs
(mRNAs). Once we identify the relevant miRNAs, we will be
able to turn on the glucose-response genes to produce fully
glucose-responsive beta-like cells in vitro.
• We have made a few major findings, thus far:
2
6
(Differentiation 18 days)
H1
INS
E
Figure 3.
A. Endpoint RT-PCR of insulin transcripts in stage-5 (ST5) cells using the modified protocol
and mouse embryonic feeder cells (MEF) showing INS production in mRNA at stage-5.
B. The fluorescent imaging distinguishes insulin (INS) and glucagon (GCG) immunofluorescence of stage-5 cells using antibodies to report the presence of the INS and GCG
proteins.
Endpoint RT-PCR with M-15
A novel fluorescent reporter that identifies
insulin-producing single cells and also
reports insulin secretion
ST1
ST5
IS
(Islet)
INS--
Reporter: Dual-color design
Green--insulin+ cells
Red--non-insulin+ cell
pCMV-L-mCherry-pA-L
• In order to reliably distinguish the insulin-producing
cells from those that are non-insulin-producing, we
have constructed a fluorescent reporter system to
facilitate in the sorting of the cells
• The next steps will be:
• Compare miRNAs and mRNAs of glucoseresponse genes for stage-5 stem-cell-derived cells
(glucose nonresponsive) and normal human beta
cells (glucose responsive)
• Demonstrate that glucose response genes can be
turned on by changing specific miRNA levels
GAPDH--
pA-Cre-phINS
• Differentiation of human embryonic stem cells to
insulin-producing cells can be reliably induced
using a protocol we have optimized by adding
betacellulin and nicotinamide at stages 4 and 5
Program Benefits
-GFP-pA
Figure 4. This Gel Electrophoresis picture displays the results of an Endpoint PCR of
insulin transcripts in stage-5 cells using the mouse mesoderm cell line (M-15). Compared
are the amount of INS produced by the specified cell types, thus, if the gene is expressed,
it will be shown on the gel.
This program has provided me with a wealth of
experiences that will help me reach my future goals. It
has provided me with an understanding of how basic
research is conducted. I now really appreciate how
much hard work goes into making the scientific
advances needed to create new medical treatments.
The insights I have gained have inspired me to work
harder and to consider research and medicine as
future career goals.
PILOT RESULTS
HYPOTHESIS
CLINICAL RELEVANCE
We hypothesize that miRNAs are responsible for the turning on
of glucose-response genes. Furthermore, we hypothesize that
we can make fully glucose-responsive beta-like cells by intentionally controlling the levels of the critical miRNAs.
Insulin Production
Figure 1. Illustration showing how micro RNAs (miRNAs) act
as molecular switches to turn-off gene expression.
Figure 2. The diagram illustrates the expression of Green Florescent Protein
(GFP) in hamster insulinoma cell line (HIT-T15) containing the gene segment
CMV-LoxP-mCherry-LoxP-GFP-phIns-Cre-pA. The dual-color reporter enables
researchers to reliably separate cells that are insulin-producing from cells that are
not.
Figure 5. Pilot results showing comparison of miRNA level in
the heterogeneous populations of stage-5 H1 and NT2 cells
versus human beta cells prior to creation of the reporter.
Figure 6. This illustration displays the potential this process has for diabetes therapy in the
future.