Supplementary Material Current Pharmaceutical Biotechnology, 2016, Vol. 17, No.

4 i

SUPPLEMENTARY MATERIAL

Beta Cell Formation in vivo Through Cellular Networking, Integration and

Processing (CNIP) in Wild Type Adult Mice

Bruno Doiron*, Wenchao Hu and Ralph A DeFronzo

Diabetes Division, University of Texas Health Science Center at San Antonio, Mail Code 7886, 7703 Floyd Curl Drive,
San Antonio, TX 78229, USA

In vivo method for targeted gene delivery to adult pancreas.

To study beta cell formation in adult animals, we employed a novel in vivo approach to target the adult pancreas using a
lentiviral vector (2). We have validated this technique (2) and employed it in our CNIP approach to generate pancreatic beta
cells in vivo. Quantitative morphometric analysis of pancreatic transduction by the Lentivirus vector, based on GFP expression,
showed that 60% of the tissue expressed GFP (2). This level of transduction efficiency is similar to that previously published
(2). With each viral construct individually and with the triple cocktail, the expression of each transgene in the pancreas was
greater than 60%. Importantly, expression was detected in the pancreas even after four weeks (Fig. 1D and 1E). The Lentivirus
vector expressed green fluorescent protein was not found in any other tissue in the body including heart, lung, liver, brain, leg
muscle and kidney by histology and PCR (data not shown). Pancreatic tissue was stained with H & E to look for evidence of
inflammation (pancreatitis) at day 2 and day 14 post-injection of Lentivirus. No evidence of inflammation was observed (2).
Following Lentiviral vector injection containing the experimental cocktail or control placebo cocktail, daily food intake was
monitored for four weeks post-injection (CNIP-treated mice ingested 5.4 0.3 grams/day [n=4] and control cocktail mice in-
gested 5.9 0.5 grams/day [n=6], p=NS). Weight gain was similar in CNIP and control groups. No diarrhea was observed in
either the cocktail placebo control or experimental cocktail groups after Lentivirus injection. Pancreatic (lipase) and hepatic
(AST, ALT) enzymes were normal, consistent with previous observations (2). These results demonstrate that the Lentivirus
injection technique does not cause adverse gastrointestinal, pancreatic, or hepatic effects.

Male C57BL/6 mice (Charles River, Wilmington, MA, USA) 8 weeks of age were maintained on an ad libitum diet of water
and normal chow for all experiments. At day 1 post-injection with lentiviral vector, mice were injected i.p. daily with BrdU (50
g/g body weight) (Sigma-Aldrich, St Louis, MO, USA) in PBS for 12 days to quantitate beta cell proliferation. At 4-weeks
post-lentiviral injection, the entire pancreas was removed for histological analysis in blinded fashion to the examiner. Partial
pancreatectomy was performed through mid-incision (0.5 cm) in the right side of the mouse. The tail of the pancreas was ex-
posed and propped up with forceps to expose the body of the pancreas. A Bovie cautery (Bovie Medical Cor. Clearwater, FL)
was used to cauterize the lineal artery and lineal veins, lieno-pancreatic veins, lieno-pancreatic artery and pancreatic duct. A
transversal cut with the Bovie cautery was made to remove ~ 70-80 % of the pancreas. Within 5-7 days the mice were eating
and growing normally and the white blood cell count was normal. Animal protocols were approved by the University of Texas
Health Science Center Animal Care Committee. We randomly allocated mice to the experimental and control group.
ii Current Pharmaceutical Biotechnology, 2016, Vol. 17, No. 4 Supplementary Material

RNA Extraction and Real-time PCR

Total RNA was extracted from frozen pancreatic tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) with Qiagen
RNeasy (Qiagen, Valencia, CA). Integrity of each RNA sample was confirmed post-extraction using denaturing (glyoxal) aga-
rose gel electrophoresis. Reverse transcription was carried out with 0.5 g total RNA using ImProm II reverse transcription
system (Promega, Madison, WI) according to manufacturers instruction. Real-time quantitative PCR was then preformed using
2 l cDNA with primer and 5-terminal 6-carboxyfluorescein (FAM)-labelled TaqMan probe mix from Applied Biosystems
(assay ID Hs00193409; Foster City, CA). Relative expression values were calculated from a standard curve and sample of each
cDNA and were normalized to actin RNA.

Statistical Analysis

Results are presented as mean SE. Statistical comparisons were performed with Students t test or one-way ANOVA, as
appropriate. Results were considered to be statistically when p < 0.05.

WILD-TYPE AND PARTIALLY PANCREATECTOMIZED DIABETIC MOUSE MODEL

We selected a wild-type partially pancreatectomized diabetic mouse model because all other rodent diabetic models are as-
sociated with long-lasting conditions that are not present in human diabetes and that facilitate cell proliferation. Briefly, strep-
tozotocin (STZ) and alloxan are transported into the cell via the GLUT2 transporter and generate reactive oxygen species and
free radicals leading to cell injury, inflammation, and apoptosis (3). Thus, the remaining pancreatic beta cells, as well as any
other cells (liver, brain, gut, kidney) expressing the glucose transporter GLUT2, are subject to injury (3, 4, 5). STZ also pos-
sesses alkylating properties, which can lead to DNA fragmentation (6) and cause cell proliferation, independent of the therapeu-
tic intervention employed to induce beta cell formation. STZ also is carcinogenic and can induce tumors in rat kidney, liver and
pancreas when given in vivo (6, 7, 8, 9). In vitro, STZ is a potent carcinogen when added to cultured rat pancreatic duct epithe-
lial cells (10). While STZ-induced diabetes has proven to be a useful model to study the pathophysiology of diabetes, investiga-
tors have cautioned that its genotoxic and tumor-initiating potential can complicate the interpretation of studies when cell
growth/proliferation and tumor induction are unwanted (11, 12, 13). Because STZ can cause DNA damage and elicit a cell-
proliferative response, we chose the partially pancreatectomized diabetic mouse model to study our CNIP effect on beta cell
proliferation. If given to STZ-treated mice, the cocktail of agents used to promote beta cell formation will be enhanced, making
it difficult to attribute any observed beta cell proliferative effect to the cocktail itself.

The use of immune-deficient diabetic mouse models to study the effect of agents on beta cell proliferation also is associated
with interpretive problems. Severe-combined immunodeficiency (SCID) mice lack a functionally active subunit of the DNA-
dependent protein kinase enzyme that is required to generate rearranged antigen receptor in lymphocytes (14). Thus, they are
unable to generate an antigen-specific immune response. SCID mice RAG-2 -/- develop spontaneous intestinal neoplasia (15)
and the RAG-2 -/- x STAT -/- mice develop spontaneous intestinal and mammary tumors (15). The relation between immu-
nologic defects in SCID mice and increased incidence of spontaneous cancer creates a confounding variable whereby any
molecule(s) used to induce pancreatic beta cell formation or proliferation will be unable to untangle the effect of the molecule
cocktail per se from the natural spontaneous cell proliferative effect of the model. NOD mice develop thyroiditis, Sjogrens
Syndrome and other immunological disorders and are characterized by altered thyroid hormone secretion, increased activity of
the interleukin 1 system, and inflammation (3, 16), all of which create a confounding effect when evaluating any intervention
that promotes beta cell formation in vivo.
Supplementary Material Current Pharmaceutical Biotechnology, 2016, Vol. 17, No. 4 iii

Genetically modified animals (transgenic or knock-outs) do not represent good models to study complex human diseases
such as diabetes, both type 1 and type 2. Cre toxicity (17) is an important potential problem in conditional gene-disruption ex-
periments using Cre recombinase. While most Cre-transgenic mice lines appear to develop normally, some studies have dem-
onstrated that Cre can be toxic to cells by damaging genomic DNA as a result the recombinase activity of Cre (17). The authors
(17) suggest that many Cre-expressing mouse lines are not completely normal, but overcome Cre toxicity through developmen-
tal selection and adaptation processes. If Cre toxicity produces a DNA mutation that causes activation of an oncogene that in-
creases the potential for cell proliferation, this would complicate the interpretation of any cocktail of molecules that were used
to induce beta cell proliferation.

THE QUEST TO REPLACE INSULIN INJECTION TREATMENT

The quest to replace insulin injection treatment has focused on two strategies: in vivo islet transplantation and in vitro nu-
clear reprogramming to produce differentiated beta cells. Ultimately, both strategies rely on encapsulation to implant islets or
cultured cells into the human body (18). The many hurdles involved with islet transplantation have yet to be overcome and,
even if successful, the paucity of pancreatic donors limits this approach. An alternative approach has attempted to recapitulate
the embryonic development of pancreatic beta cell in vitro using stem cells (19, 20). However, our knowledge about embryonic
beta cell development remains fragmentary and the ability to reprogram stem cells into mature beta cells that are responsive to
changes in plasma glucose concentration remains problematic (19, 20, 21, Viacyte Inc. Clinicaltrial.gov # NCT02239354).
Even if stem cells can be made to differentiate into beta cells, all of the problems associated with transplantation and immune
rejection remain. Further, there is no assurance that a cell that has been reprogrammed in vitro will function as expected when
transplanted in vivo, where both the local and systemic environment differs markedly from the in vitro situation. The CNIP in
vivo post-development approach was developed to circumvent the problems associated with the in vitro cell culture approach
based upon use of transcriptional factors implicated in embryonic development to form beta cells. The reductionism approach
focuses only on one molecular step, i.e. transcription at nuclear level, and ignores other key cellular steps that are essential for
induction of beta cell formation (22). Therefore, a strategy that simply overexpresses a cohort of transcriptional factors in cell
culture to mimic the in vivo development is likely to be unsuccessful (19, 23, 24, 25). Pancreatic organogenesis in vivo develops
by stages though gut-tube endoderm to pancreatic endoderm and eventually to endocrine precursor cells that express endocrine
hormones (19). While there exists a considerable body of literature regarding the in vitro differentiation of stem cells that can
guide strategies, many aspects of beta cell development remain poorly understood (19, 23, 24, 25). Also, cells in vitro may not
behave like cells which develop in the embryonic environment (19). Transdifferentiation of hepatocytes to produce insulin has
been attempted by overexpressing pancreatic and duodenal homeobox 1 (PDX-1) with an adenovirus vector in the liver (26,
27). RT-PCR (55 cycles) and histology were used to provide evidence that hepatocytes produce insulin after overexpressing
PDX-1. When > 35 cycles are used in RT-PCR, one begins to pick up every gene present in the genome, and this is independent
of the cell phenotype (28). This artifact is referred to as illegitimate transcription (28). A second complicating feature relates to
the fact that hepatocytes have large amounts of insulin bound to their plasma membrane, and the insulin antibody cannot distin-
guish between insulin which is secreted by pancreatic beta cells and then binds to hepatocytes versus ectopic insulin expression
in hepatocytes by transdifferention. Third, the authors do not present any physiological data that their approach restores en-
dogenous insulin secretion or improves glucose tolerance. Fourth, use of adenovirus as the transfection vector is problematic
since transfected DNA will not be incorporated into the cells genome after cell division. Further, because of its inflammatory
properties, adenovirus vector cannot be used for repeat administration. Fifth, even if hepatocytes could be targeted to transdif-
ferentiate and secrete insulin, this would result in marked peripheral hyperinsulinemia.
iv Current Pharmaceutical Biotechnology, 2016, Vol. 17, No. 4 Supplementary Material

Use of stem cells as a source of beta cells has received interest (21). However, this approach requires an in vitro culture sys-
tem with subsequent islet cell transplantation (18, 29). CNIP bypasses the problems associated with in vitro cell culture, i.e.
good manufacturing practice, and the problems associated with islet transplantation that have limited this approach for more
than two decades (18). Stem cells are highly proliferative and readily can form teratocarcinomas (20). Further, many of the
transcription factors employed in stem cell transformation include oncogenes like MafA (30). Further, the in vitro stem cell
differentiation protocols do not produce a consistent level of beta cell-like differentiation (20), and the beta cell-like differenti-
ated stem cells can continue to differentiate into other cell types after injection in vivo, resulting in cells with a very different
profile than present in the petri dish (31, 32). Moreover, after stem cells are injected in vivo, one still has to deal with all of the
hurdles encountered in islet transplantation, including rejection (18). The in vivo CNIP approach obviates all of these hurdles.
Because only endogenous pancreatic cells are transfected with CNIP, e.g. cells normally present in the pancreas and that have
not been rejected, rejection should not occur.

The fundamental principle underlying CNIP is to target simultaneously and integrate three key levels of cellular physiology
that have been implicated in beta cell formation. This is in contrast to the non-integrated model which targets only nuclear re-
programming.

REFERENCES
[1] Zufferey R,; Donello JE,; Trono D,; Hope TJ. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes
delivered by retroviral vectors. J. Viol. 1999, 73, 2886-2892.
[2] Doiron B,; Hu W,; Norton L,; DeFronzo RA. Lentivirus shRNA Grb10 targeting the pancreas induces apoptosis and improved glucose tolerance due
to decreased plasma glucagon levels. Diabetologia 2012, 55, 719-729.
[3] Chatzigeorgiou A,; Halapas A,; Kalafatakis K,; Kamper E. The use of animal models in the study of diabetes mellitus. In Vivo 2009, 23, 245-258.
[4] Cerf ME,; Williams K,; van Rooyen J,; Esterhuyse AJ.; Muller CJ,; Louw J. Gestational 30% and 40% fat diets increase brain GLUT2 and neuropep-
tide Y immunoreactivity in neonatal Wistar rats. Int J Dev. Neurosci. 2010, 28, 625-630.
[5] Scheepers A,; Joost HG,; Schurmann A. The glucose transporter families SGLT and GLUT: molecular basic on normal and aberrant function. JPEN J
Parent Enteral Nutr. 2004, 28, 364-371.
[6] Bolzan AD; and Bianchi MS. Genotoxicity of streptozotocin Mutat Res. 2002, 512, 121-134.
[7] Iwase M,; Nunoi K,; Sadoshima S,; Kikuchi M,; Fujishima M.; Tohoku. Liver, Kidney and islet cell tumors is spontaneously hypertensive and nor-
motensive rats treated neonatally with streptozotocin. J Exp Med. 1989, 159, 83-90.
[8] Steiner H,; Oelz O,; Zahnd G,; Froesch ER. Studies on islet cell regeneration, hyperplasia and intrainsular cellular interrelations in long lasting strepto-
zotocin diabetes in rats. Diabetologia 1970, 6, 558-564.
[9] Yamagami T,; Miwa A,; Takasawa S,; Yamamoto H. Induction of rat pancreatic B-cell tumors by the combined administration of streptozotocin or
alloxan and poly(adenosine diphosphate ribose) synthetase inhibitors. Cancer Res 1985, 45, 1845-1849.
[10] Shepherd JG,; Chen JR,; Tsao MS,; Duguid WP. Neoplastic transformation of propagable cultured rat pancreatic duct epithelial cells by azaserine and
streptozotocin. Carcinogenesis 1993, 14, 1027-1033.
[11] Bennet RA,; Pegg AE. Alkylation of DNA in rat tissues following administration of streptozotocin. Cancer Res. 1981, 41, 2786-2790.
[12] Iwase M, Nnunoi K, Wakisaka M, Kikichi M, Maki Y, Sadoshima S, Fujishima M. Spontaneous recovery from non-insulin-dependent diabetes melli-
tus induced by neonatal streptozotocin treatment in spontaneously hypertensive rats. Metabolism 1991, 40, 10-14.
[13] Kraynak AR et al. Extent and persistence of streptozotocin-induced DNA damage and cell proliferation in rat kidney as determined by in vivo alkaline
elution and BrdU labeling assays. Toxicol Appl Pharmacol. 1995, 135, 279-286.
[14] Dune GP,; Bruce AT,; Ikeda H,; Old LJ,; Schreiber RD. Cancer immunoediting: from immunosurveillance to tumor escape. Nature Immunol 2002, 3,
991-998.
[15] Shankaran V, et al. IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 2001, 410, 1107-
1111.
[16] Noorchashm H,; Noorchashm N,; Kern J,; Rostami SY,; Barker CF,; Naji A. B-cells are required for the initiation of insulitis and sialitis in nonobese
diabetic mice. Diabetes 1997, 46, 941-946.
[17] Schimidt-Supprian M,; Rajewsky K. Vagaries of conditional gene targeting. Nature Immunol. 2007, 8, 665-668.
[18] Krishnan R,; Alexander M,; Robles L,; Forster CE,; Lakey JR. Islet and stem cell encapsulation for clinical transplantation. Rev.Diabet Stud. 2014, 11,
84-101.
[19] DAmour KA et al., Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nature Biotechnology 2006, 24,
1392-1401.
[20] Hayden EC. Stem Cells: The growing pains of pluripotency. Nature 2011, 473, 272-274.
[21] Pagliuca FW, et al., Generation of Functional Human Pancreatic Cells In Vitro. Cell 2014, 159, 428-439.
[22] Zhou Q,; Brown J,; Kanarek A,; Rajagopal J,; Melton DA. In vivo reprogramming of adult pancreatic exocrine cell to beta-cells. Nature 2008, 455,
627-632.
[23] Jensen J. Gene regulatory factors in pancreatic development. Dev. Dyn. 2004, 229, 176-200.
[24] Murtaugh LC,; Melton DA. Genes, signals, and lineages in pancreas development. Annu. Rev. Cell. Dev. Biol. 2003, 19, 71-89.
Supplementary Material Current Pharmaceutical Biotechnology, 2016, Vol. 17, No. 4 v

[25] Wilson ME,; Scheel D,; German MS. Gene expression cascades in pancreatic development. Mech.Dev. 2003, 120, 65-80.
[26] Aviv V,; Meivar-Levy I,; Rachmut IH,; Rubinek T,; Mor E,; Ferber S. Exendin-4 promoter liver cell proliferation and enchances the PDX-1 induced
liver to pancreas transdifferentiation process. J.Biol.Chem. 2009, 284, 33509-33520.
[27] Ber I, et al., Functional, persistent, and extended liver to pancreas transdifferentiation. J.Biol.Chem. 2003, 278, 31950-31957.
[28] Chelly J,; Concordet JP,; Kaplan JC,; Kahn A. Illegitimate transcription: transcription of any gene in any cell type. Proc Nalt Acad Sci USA. 1989, 86,
2617-2621.
[29] Bradley JA,; Bolton EM,; Pedersen R.A. Stem cell medicine encounters the immune system. Nat Rev Immunol. 2002, 2, 859-871.
[30] Jaenisch R,; Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 2008, 132, 567-582.
[31] Dlouhy BJ, Awe O, Rao RC, Kirby PA, Hitchon W. Autograft-derived spinal cord mass following olfactory mucosal cell transplantation in a spinal
cord injury patient. Case report. Journal of Neurosurgery: Spine 2014, 21, 618-622.
[32] Ideguchi M, Palmer TD, Recht LD, Weimann JM. Murine embryonic stem cell-derived pyramidal neurons integrate into the cerebral cortex and ap-
propriately project axons to subcortical targets. J. Neurosci 2011, 30, 894-904.