Research Article Reprogramming of Mice Primary …downloads.hindawi.com/journals/jdr/2014/716163.pdfResearch Article Reprogramming of Mice Primary Hepatocytes into Insulin-Producing

Research ArticleReprogramming of Mice Primary Hepatocytesinto Insulin-Producing Cells by Transfection withMulticistronic Vectors

Haizhao Luo12 Rongping Chen1 Rui Yang1 Yan Liu1

Youping Chen2 Yi Shu2 and Hong Chen1

1 Department of Endocrinology Zhujiang Hospital Southern Medical University No 253 Gong Ye Road Guangzhou 510282 China2Department of Endocrinology Nanhai Hospital Southern Medical University No 40 Foping Road Foshan 528200 China

Correspondence should be addressed to Hong Chen rubychq163com

Received 28 December 2013 Accepted 30 April 2014 Published 19 May 2014

Academic Editor Dong-qi Tang

Copyright copy 2014 Haizhao Luo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The neogenesis of insulin-producing cells (IPCs) from non-beta-cells has emerged as a potential method for treating diabetesmellitus (DM) Many groups have documented that activation of pancreatic transcription factor(s) in hepatocytes can improvethe hyperglycemia in diabetic mice In the present study we explored a novel protocol that reprogrammed primary hepatocytesinto functional IPCs by using multicistronic vectors carrying pancreatic and duodenal homeobox-1 (Pdx1) neurogenin 3 (Ngn3)and v-musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) These triple-transfected cells activated multiple beta-cellgenes synthesized and stored considerable amounts of insulin and released the hormone in a glucose-regulated manner in vitroFurthermore when transplanted into streptozotocin-induced diabetic mice the cells markedly ameliorated glucose tolerance Ourresults indicated that ectopic expression of Pdx1 Ngn3 and MafA facilitated hepatocytes-to-IPCs reprogramming This approachmay offer opportunities for treatment of DM

1 Introduction

According to data from International Diabetes Federation(IDF) there were about 371 million people suffering fromdiabetes mellitus (DM) in 2012 [1] Islet transplantationhas been considered as a promising strategy for curingDM whereas it is limited by both scarcity of donor cellsand immunologic rejection It is widely believed that cellreplacement for curing DM will be applied on a large scaleonly when new sources of insulin-producing cells (IPCs) arediscovered

On the phase of organogenesis the same as pancreaticislet beta-cells hepatocytes also are derived from endodermand both of them share many of their epigenomes such asglucose transporter-2 and glucokinase (Gk) [2] Conversionbetween hepatocytes and pancreatic islet beta-cells maytherefore require fewer epigenetic changes Hepatocytes canserve as the potential source of IPCs

Transcription factors are the elements that regulate dif-ferentiation and development of cells It was documentedthat hepatocytes could be reprogrammed into IPCs byintroducing certain transcription factor(s) Pancreatic andduodenal homeobox-1 (Pdx1) was proved to play a crucialrole on pancreatic morphogenesis and function in postnatalislet [3] Ferber et al reported that ectopic expression ofPdx1 could induce the conversion from hepatocytes to IPCsand improve the hyperglycemia in streptozotocin- (STZ-)treated diabetic mouse [4] Thereafter several studies haveconfirmed that many other factors involved in pancreasdevelopment including neurogenin 3 (Ngn3) [5] beta-cellulin [6] neurogenic differentiation (NeuroD) [7] andv-musculoaponeurotic fibrosarcoma oncogene homolog A(MafA) [8] could also turn on endocrine program in hep-atocytes but not to generate functional beta-cells Recentlyan exciting and interesting breakthrough in islet regenerationwas found by Melton and his colleagues Among more than

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2014 Article ID 716163 7 pageshttpdxdoiorg1011552014716163

2 Journal of Diabetes Research

1100 pancreas-associated transcription factors they estab-lished a specific combination (Pdx1 Ngn3 and MafA) whichwas amost efficient precept in reprogramming nonpancreaticbeta-cells into IPCs that closely resemble endogenous 120573-cells[9]

Many groups have attempted to set up a practicalinduction of islet regeneration by virus-mediated protocolHowever safety concerns have been the main bottleneck tothe studies of viral gene delivery In 1999 the death of avolunteer due to gene therapy in a clinical trial was causedby administering adenovirus vectors within 98 hours [10]The autopsy report revealed that the patient succumbedto multiorgan failure owing to the fatal immune responsetriggered by the administered adenovirus [11] In order toallow clinical use of reprogramming it is urgent to explorea feasible nonviral strategy

Here we showed that coexpression of Pdx1 Ngn3 andMafA in primary hepatocytes induced hepatocytes-to-IPCsreprogramming and reversal of hyperglycemia in diabeticanimals by using multicistronic vectors via liposome

2 Materials and Methods

21 Plasmid Construction The transcription factors ofmousePdx1 Ngn3 andMafA (gene ID 0088143 0097196 1943501)were PCR-amplified from total RNA and ligated with anopen reading frame (ORF) or 31015840-untranslated regions (UTR)subsequently cloned into a shuttle vector pcDNA31 (+)(Clontech USA) respectively Electrophoretic analysis andgene sequencing were conducted to make sure that all therecombinant plasmids were correct

22 Cell Culture 4-week-old male C57BL6J mice (Lab-oratory Animal Center of Southern Medical UniversityGuangzhou China) were kept in a controlled-temperature(22ndash25∘C) animal room with a 12 h light and 12 h dark cyclePelleted commercial chow (Laboratory Animal Center ofSouthern Medical University) and purified water were avail-able The experimental procedures performed in this studywere in accordance with the guidelines of the InstitutionalAnimal Ethics Committee for the Care andUse of LaboratoryAnimals Hepatocytes were isolated and purified from theanimals by in situ collagenase perfusion as mentionedpreviously [12] The hepatocytes were plated at 6-well platesprecoated with collagen (Corning USA) and incubated at37∘C with 5 humidified CO

2

23 Transfection Primary hepatocytes were transfected byLipofectamine 2000 (Invitrogen USA) according to themanufacturerrsquos protocol In brief we prepared DNA-reagentcomplexes at ratios of 1 25 (total volume = 500 120583L) Twentyminutes later the complexes were added to each well Toinvestigate transfection efficiency a green fluorescent pro-tein (GFP) plasmid was introduced 48 hours after trans-fection flow cytometry analysis was conducted The cellsthat displayed green fluorescence were examined throughFL 1 channel (excitation 488 nm emission 520 plusmn 10 nmFACSCalibur BD USA) The efficiency was defined as the

percentage of GFP-positive cells within all viable cells in 3independent experiments Hepatocytes treated without GFPserved as control

24 Reverse Transcription Polymerase Chain Reaction TotalRNA was extracted using TRIzol (Takara Japan) from two-day cultured hepatocytes RT-PCR was performed accordingto the manufacturerrsquos instructions (PrimeScript RT-PCR KitTakara) The following gene-specific oligonucleotide primerswere used for amplification Pax6 (314 bp) CAG TCA CAGCGG AGT GAA TCA GC (forward) and GCC ATC TTGCGT AGG TTG CCC TG (reverse) Nkx61 (381 bp) GTTCCT CCT CCT CCT CTT CCT C (forward) and AAG ATCTGC TGT CCG GAA AAA G (reverse) and Isl1 (268 bp)GTG CGG AGT GTA ATC AGT ATT TGG (forward)and GTC ATC TCT ACC AGT TGC TCC TTC (reverse)Amplification conditions were initial denaturation at 94∘Cfor 10min followed by 35 cycles of denaturation at 94∘C for30 sec annealing at 60∘C for 30 sec and extension at 72∘C for30 sec and at last an extension step of 10min at 72∘C

25 Real-Time Fluorescence Relative Quantitative PCR(qPCR) Total RNA was obtained as above The primerswere the following Gk (islet type) CAG AGA CAC AACAAC CTT TTC CC (forward) and GCT GTC TCA CTGGCT GAC TT (reverse) Ins1 TTG GTG CAC TTC CTACCC CT (forward) and CAC ACA CCA GGT AGA GAGCC (reverse) and Ins2 CCA TCA GCA AGC AGG AAGGTT A (forward) and CAG GTG GGA ACC ACA AAG GT(reverse) The SYBR-Green real-time PCR reaction solutionwas prepared and the PCR conditions were set accordingto the manufacturerrsquos protocol (ABI USA) The data wereanalyzed for target gene expression by the 2minusΔΔCt methodNormal C57BL6J mice islet was used as a control

26 Western Blot Assay Five days after transfection proteinswere isolated The protein concentrations were determinedby protein assay (KeyGEN China) Electrophoresis on a 12SDS polyacrylamide gel was performed and the protein wastransferred to a PVDFmembrane (Millipore Germany) Pro-tein bands were detected using an enhanced chemilumines-cence system (Pierce Biotechnology USA) with antibodies(Santa Cruz USA)

27 Insulin and C-Peptide Detection 5-day cultured hepato-cytes were treated with acid-ethanol at 4∘C overnight Thesupernatants were collected and the intracellular C-peptidelevels were measured by an enzyme-linked immunosorbentassay (C-peptide-ELISA kit Millipore) Insulin secretion wasdetermined by an ELISA kit (Insulin-ELISA kit Millipore)after exposure toKrebs-Ringer bicarbonate (KRB) bufferwith01 BSA containing various concentrations of glucose (0 5or 25mM) as described [13] The values of insulin and C-peptide were normalized relative to the total protein contentwhich was detected by protein assay (KeyGEN China)

Journal of Diabetes Research 3Ev

ents

0

101 102 103

FL 1 Log

293 plusmn 11

100

104

Lipofectamine 2000Lipofectamine 2000 + GFP

Figure 1 Flow cytometry quantitation of GFP-positive cells demon-strates that treatment with GFP results in a 16-fold higher numbercompared with those without GFP 119899 = 3

28 Streptozotocin-Induced Diabetic Mice and Cell Transplan-tation 6-week-old male C57BL6J mice (Laboratory Ani-mal Center of Southern Medical University) were injectedintraperitoneally with streptozotocin (STZ Merck Ger-many) at a dose of 180mgkg body weight Diabetes wasdiagnosed by blood glucose levels gt300mgdL (167mM)on two consecutive measurements Recombinant plasmidswhich encode Pdx1 Ngn3 and MafA (MNP) as well as nullvectors (NV) were delivered into hepatocytes 3 days beforetransplantation 6ndash8 times 106 cells suspended in 02mL PBSwere transplanted into the liver parenchyma of diabetic micethrough portal injection

29 Glucose Tolerance Test Mice were injected intraperi-toneally with glucose at a dose of 1 gkg body weight after6 h fast Blood samples were collected from the tail vein tomonitor glucose levels at the indicated time points

210 Statistical Analysis All values are expressed as means plusmnSDThe differences were analyzed with two-sample Studentrsquos119905-test and 119875 lt 005 was considered to be significant

3 Results

31 Transfection Efficiency in Our Experiment Transfectionefficiency was recognized as the proportion of GFP-positivecells among all viable cells at 48 h after transfection Ourdata indicated that transfection efficiency was 293 plusmn 11(Figure 1) which was close to previous study [14]

MaFA

Ngn3

Pdx1

Pax6

Nkx 61

P N M NP

MP

MN

MN

P

NV

120573-Actin

Isl1

Figure 2 Gene expression profiles in transfected hepatocytesTotal RNA was isolated from hepatocytes treated with pPdx1 (P)pNgn3 (N) pMafA (M) pNgn3+Pdx1 (NP) pMafA+Pdx1 (MP)pMafA+Ngn3 (MN) pMafA+Ngn3+Pdx1 (MNP) and null vector(NV)

0

01

02

03

04

05

06

07

08

09

1

M N P MN NP MP MNP NV

GkIns1Ins2

lowastlowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowastlowastlowastRelat

ive l

evels

to is

let o

f pan

crea

tic m

arke

rs

Figure 3 Relative levels to mice islet of endogenous pancreaticmarkers in 5-day cultured hepatocytes evaluated by real-time PCR119899 = 3 lowast119875 lt 005 versus MNP

GAPDH

P N M NP

MP

MN

MN

P

NV

Ins1

Ins2

Isle

t

Figure 4 Protein levels of Ins1 and Ins2 in hepatocytes on day 5after transfection evaluated by Western blotting with mice islet aspositive control


lowast lowastlowast

lowastlowast

lowast

lowast

1200

1000

800

600

400

200

0M N P MN NP MN MNP NV

C-pe

ptid

e con

tent

(pg120583

g pr

otei

n)

(a)

0

50

100

150

200

250

300

NV MNP

KRB

Insu

lin se

cret

ion

( o

f bas

al)

lowast

lowast

KRB + 25mM glucoseKRB + 5mM glucose

(b)

Figure 5 Evaluation of insulin synthesis and secretary ability in transfected cells (a)Measurements of intracellular C-peptidewere conductedby an ELISA kit 119899 = 3 (b) Glucose-induced insulin secretion from transfected hepatocytes Values are mean plusmn SD of insulin in 3 differentexperiments relative to 0mM glucose lowast119875 lt 005

32 Ectopic Coexpression of Pdx1 Ngn3 and MafA MarkedlyPromotes Hepatocytes-to-IPCs Reprogramming Using RT-PCRwe investigated the expression profiles of the transfectedtranscription factors and islet-related genes As expected thetransfected transcription factor(s) was (were) detectable incorresponding group Interestingly some factors were foundin their transfection-default group For example exogenousMafA could induce the expression of the endogenous Pdx1On the other hand the mRNA expression of pancreatictranscription factors including Pax6 Nkx61 and Isl1 inMNPgroup was higher than the other groups However the genesabove were not detectable in control NV group (Figure 2)

A better illustration for the extent of the transdifferentia-tion process is to quantify the expression of the endogenouspancreatic markers in transfected hepatocytes Compared toMNP group differences in the expression of islet-type Gk inbicistronic groups were not significant It is documented thatIns2 is detectable in yolk sac and developing brain as well asin islet cells while Ins1 appears in islet cells only In thesehepatocytes the relative values of Ins2 and Ins1 inMNPgroupwere 082 plusmn 004 and 076 plusmn 004 respectively which werehigher than that in the rest groups (Figure 3)

To confirm that transfected hepatocytes can express thetarget protein encodes by Ins1 and Ins2 we performedWestern blot analysis As shown in Figure 4 among allgroups level of both Ins1 and Ins2 protein was the highestin MNP group except positive control group These datasuggested that transfection with Pdx1 Ngn3 and MafA acti-vated an endocrine developmental shift in fully differentiatedhepatocytes

33 Triple Transfection Enables Hepatocytes to Reprograminto IPCs In order to examine insulin biosynthesizing intransfected cells intracellular C-peptide contents were mea-sured The figure of C-peptide in MNP group which reached1200 pg120583g protein was significantly higher (2ndash120-fold) thanthe other groups (Figure 5(a)) Glucose-sensing ability andthe coupling between glucose sensing and insulin secretionplay an important role in pancreatic beta-cell functionThere-fore insulin secretion was evaluated after static incubationwith glucose using a commercial ELISAkit FromFigure 5(b)we can see that the hormone was secreted in a glucose-responsive manner in MNP group These results togetherwith data from C-peptide secretion implied that IPCs repro-grammed from hepatocytes with triple transfection closelyresembles normal pancreatic beta-cells

34 IPCs Ameliorate STZ-Induced Hyperglycemia In Vivo Tofurther assess effectiveness of IPCs in maintaining glucosehomeostasis they were transplanted into diabetic micewhich were treated with STZ to specifically damage islet120573-cells Meanwhile hepatocytes transfected with NV weretransplanted as a negative control A glucose tolerance testwas conducted daily until day 19 after transplantation Asshown in Figure 6(a) 2 days after transplantation fastingblood glucose levels were reduced by IPCs FurthermoreIPCs-transplanted mice exhibited a complete reversal ofhyperglycemia on day 7 It is noted that hypoglycemia wasnot observed (Figure 6(b)) These data suggested that trans-fection with Pdx1 Ngn3 andMafAmulticistronic expressionvector through Lipofectamine 2000 was a sufficient nonviral

Journal of Diabetes Research 5

0

5

10

15

20

25

30

0 3 7 11 15 19

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L) lowast

Time (days)

lowast

Nondiabetes (n = 10)

(a)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 9)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 7


(b)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 19

lowast

lowast


(c)

Figure 6 Effects on blood glucose levels by transplanting IPCs into diabetic mice at different times A glucose tolerance test was performedand glucose levels were determined from blood drawn from the tail vein (a) Fasting blood glucose of mice until day 19 ((b) and (c)) Glucoselevels during a glucose tolerance test of mice on days 7 and 19 Data are presented as mean plusmn SD lowast119875 gt 005 versus NV

119875 gt 005 versusnondiabetes

approach to trigger hepatocytes-to-IPCs reprogrammingHowever profound effect of improving hyperglycemia wasnot obtained at day 19 Differences at the time point of 0and 30min during a glucose tolerance test between miceimplanted with IPCs and NV-treated hepatocytes were notsignificant (Figure 6(c))

4 Discussion

It has been confirmed that one kind of adult somatic cellscan transdifferentiate into another one without undergoingthe process of dedifferentiation [9 15 16]This was termed ascell direct reprogramming technology It was demonstratedthat the coexpression of Pdx1 Ngn3 and MafA in pancreatic

exocrine cells was themost effective way for IPCs reprogram-ming [9] In this study we examined the potential of IPCsreprogramming by using multicistronic vectors-mediatedcoexpression of the three transcription factors in hepatocytes

As promoters of hepatocytes-to-IPCs reprogrammingwe focused on the three transcription factors Pdx1 Ngn3and MafA Pdx1 is a ldquoswitchrdquo of pancreas development andfunction and Ngn3 is expressed in endocrine progenitorsand is in charge of islet differentiation [17] With respectto MafA it is a key calibrator of glucose-responsive insulinsecretion [18] Despite that all the three factors could triggerthe process none of them could achieve the reprogrammingalone while the specific combination does Similar resultswere obtained in our study Substantial increase in insulingene expression (Figures 3 and 4) andC-peptide (Figure 5(a))


was found in MNP group Furthermore IPCs respondedto glucose challenge both in vitro (Figure 5(b)) and in vivo(Figure 6(b)) These results suggested that IPCs acquired thecapacity of insulin synthesis storing secretion and glucose-sensing The exact mechanisms underlying the processes arestill elusive at present However the following possibilitiesmay be important in explaining it Firstly as mentionedabove embryological homology between hepatocytes andislet-cells may facilitate the transdifferentiation Secondlyit could be attributed to the powerful synergistic effect ofpancreatic transcriptional network including Pax6 Nkx61and Isl1 which were elicited by triple overexpression pro-moting [19] RT-PCR analysis revealed large induction ofexpression of Pax6 Nkx61 and Isl1 inMNP group (Figure 2)The powerful synergistic effect of pancreatic transcrip-tional network was proved to promote insulin expression[20 21]

Besides embryological homology there is another advan-tage when using hepatocytes as a source of IPCs FromGreek myth about Prometheus ancient Greeks had real-ized the regenerative capacity of the liver In moderntimes it has been proved by many studies that injuriesincluding chemical insult and surgical remove can triggerliver regeneration [22] The regenerative procedure willfinish within one week in rodents even there are onlyapproximately 30 of liver left [23] More importantly thestructure and function of the liver maintained during therepopulation [24 25] Additionally laparoscopic approachhas been considered as a safe and effective therapeuticoption in hepatectomy Thus the acquirement of hepato-cytes by laparoscopic approach makes autotransplantationpossible allowing diabetic patients to be the donors ofthemselves

Viral vectors are attractive tools for gene delivery becauseof their high efficiency Nevertheless questions regardingtoxicity immunogenicity and a probable risk for insertionalmutagenesis following viral vectors are hurdles that maypreclude their widespread use [26] The success of genetherapy requires the development of the gene delivery vectorOwing to its ability to mediate stable transgene efficiencylarge cloning capacity devoid of eliciting a major humoralimmune response and lower cost multicistronic vector hasentered the realm of the current therapeutic gene transferarena recently [27] By using it we achieved the hepatocytes-to-IPCs reprogramming

In conclusion our study reveals that multicistronicvectors-mediated expression of Pdx1 together with Ngn3 andMafA in hepatocytes was a feasible regimen for inducingislet neogenesis in vitro and resulting in correction ofdiabetic state in vivo Additional investigations are neededto prolong duration of euglycemia in animals We believethat this information is valuable for cell replacement ofdiabetes

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Haizhao Luo and Rongping Chen contributed equally to thiswork

Funding

This work was supported by grants from the Natural ScienceFoundation of Guangdong Province (BE2010768)

Acknowledgments

The authors would like to thank Songhao Cai Ruyi ZhangShuangshuang Zhang and Eva Xu for their valuable sugges-tions

References

[1] L Guariguata ldquoBy the numbers new estimates from the IDFDiabetes Atlas Update for 2012rdquo Diabetes Research and ClinicalPractice vol 98 no 3 pp 524ndash525 2012

[2] K S Zaret and M Grompe ldquoGeneration and regeneration ofcells of the liver and pancreasrdquo Science vol 322 no 5907 pp1490ndash1494 2008

[3] K Fujimoto and K S Polonsky ldquoPdx1 and other factorsthat regulate pancreatic 120573-cell survivalrdquo Diabetes Obesity andMetabolism vol 11 no 4 pp 30ndash37 2009

[4] S Ferber A Halkin H Cohen et al ldquoPancreatic and duode-nal homeobox gene 1 induces expression of insulin genes inliver and ameliorates streptozotocin-induced hyperglycemiardquoNature Medicine vol 6 no 5 pp 568ndash572 2000

[5] A Y Wang A Ehrhardt H Xu and M A Kay ldquoAdenovirustransduction is required for the correction of diabetes usingPdx-1 or neurogenin-3 in the liverrdquo Molecular Therapy vol 15no 2 pp 255ndash263 2007

[6] H Kojima M Fujimiya K Matsumura et al ldquoNeuroD-betacellulin gene therapy induces islet neogenesis in the liverand reverses diabetes in micerdquo Nature Medicine vol 9 no 5pp 596ndash603 2003

[7] H Kaneto Y Nakatani TMiyatsuka et al ldquoPDX-1VP16 fusionprotein together with neurod or Ngn3 markedly inducesinsulin gene transcription and ameliorates glucose tolerancerdquoDiabetes vol 54 no 4 pp 1009ndash1022 2005

[8] H Kaneto T-A Matsuoka Y Nakatani et al ldquoA crucial roleof MafA as a novel therapeutic target for diabetesrdquo Journal ofBiological Chemistry vol 280 no 15 pp 15047ndash15052 2005

[9] Q Zhou J Brown A Kanarek J Rajagopal and D A MeltonldquoIn vivo reprogramming of adult pancreatic exocrine cells to 120573-cellsrdquo Nature vol 455 no 7213 pp 627ndash632 2008

[10] E Marshall ldquoGene therapy death prompts review of adenovirusvectorrdquo Science vol 286 no 5448 pp 2244ndash2245 1999

[11] S E Raper N Chirmule F S Lee et al ldquoFatal systemic inflam-matory response syndrome in a ornithine transcarbamylasedeficient patient following adenoviral gene transferrdquoMolecularGenetics and Metabolism vol 80 no 1-2 pp 148ndash158 2003

[12] M Morita Y Watanabe and T Akaike ldquoProtective effectof hepatocyte growth factor on interferon-gamma-inducedcytotoxicity in mouse hepatocytesrdquo Hepatology vol 21 no 6pp 1585ndash1593 1995


[13] L Le Brigand A Virsolvy D Manechez et al ldquoIn vitro mech-anism of action on insulin release of S-22068 a new putativeantidiabetic compoundrdquo British Journal of Pharmacology vol128 no 5 pp 1021ndash1026 1999

[14] S Gao E Seker M Casali et al ldquoEx vivo gene delivery to hep-atocytes techniques challenges and underlying mechanismsrdquoAnnals of Biomedical Engineering vol 40 no 9 pp 1851ndash18612012

[15] T Vierbuchen A Ostermeier Z P Pang Y Kokubu T CSudhof and M Wernig ldquoDirect conversion of fibroblasts tofunctional neurons by defined factorsrdquo Nature vol 463 no7284 pp 1035ndash1041 2010

[16] M Ieda J-D Fu P Delgado-Olguin et al ldquoDirect reprogram-ming of fibroblasts into functional cardiomyocytes by definedfactorsrdquo Cell vol 142 no 3 pp 375ndash386 2010

[17] K S Zaret ldquoGenetic programming of liver and pancreas pro-genitors lessons for stem-cell differentiationrdquo Nature ReviewsGenetics vol 9 no 5 pp 329ndash340 2008

[18] C Zhang T Moriguchi M Kajihara et al ldquoMafA is a keyregulator of glucose-stimulated insulin secretionrdquo Molecularand Cellular Biology vol 25 no 12 pp 4969ndash4976 2005

[19] A M Ackermann and M Gannon ldquoMolecular regulation ofpancreatic 120573-cell mass development maintenance and expan-sionrdquo Journal of Molecular Endocrinology vol 38 no 1-2 pp193ndash206 2007

[20] J M Rukstalis and J F Habener ldquoNeurogenin3 a masterregulator of pancreatic islet differentiation and regenerationrdquoIslets vol 1 no 3 pp 177ndash184 2009

[21] H Kaneto T Miyatsuka Y Fujitani et al ldquoRole of PDX-1 andMafA as a potential therapeutic target for diabetesrdquo DiabetesResearch and Clinical Practice vol 77 no 3 pp S127ndashS137 2007

[22] I Uriarte M G Fernandez-Barrena M J Monte et alldquoIdentification of fibroblast growth factor 15 as a novel mediatorof liver regeneration and its application in the prevention ofpost-resection liver failure in micerdquoGut vol 62 no 6 pp 899ndash910 2013

[23] T Sakamoto Z Liu N Murase et al ldquoMitosis and apoptosisin the liver of interleukin-6-deficient mice after partial hepate-ctomyrdquo Hepatology vol 29 no 2 pp 403ndash411 1999

[24] R Taub ldquoLiver regeneration frommyth tomechanismrdquoNatureReviewsMolecular Cell Biology vol 5 no 10 pp 836ndash847 2004

[25] S Hoehme M Brulport A Bauer et al ldquoPrediction and vali-dation of cell alignment alongmicrovessels as order principle torestore tissue architecture in liver regenerationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 107 no 23 pp 10371ndash10376 2010

[26] N Somia and I M Verma ldquoGene therapy trials and tribula-tionsrdquo Nature Reviews Genetics vol 1 no 2 pp 91ndash99 2000

[27] T Niidome and L Huang ldquoGene therapy progress andprospects nonviral vectorsrdquo Gene Therapy vol 9 no 24 pp1647ndash1652 2002

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


1100 pancreas-associated transcription factors they estab-lished a specific combination (Pdx1 Ngn3 and MafA) whichwas amost efficient precept in reprogramming nonpancreaticbeta-cells into IPCs that closely resemble endogenous 120573-cells[9]

Many groups have attempted to set up a practicalinduction of islet regeneration by virus-mediated protocolHowever safety concerns have been the main bottleneck tothe studies of viral gene delivery In 1999 the death of avolunteer due to gene therapy in a clinical trial was causedby administering adenovirus vectors within 98 hours [10]The autopsy report revealed that the patient succumbedto multiorgan failure owing to the fatal immune responsetriggered by the administered adenovirus [11] In order toallow clinical use of reprogramming it is urgent to explorea feasible nonviral strategy

Here we showed that coexpression of Pdx1 Ngn3 andMafA in primary hepatocytes induced hepatocytes-to-IPCsreprogramming and reversal of hyperglycemia in diabeticanimals by using multicistronic vectors via liposome

2 Materials and Methods

21 Plasmid Construction The transcription factors ofmousePdx1 Ngn3 andMafA (gene ID 0088143 0097196 1943501)were PCR-amplified from total RNA and ligated with anopen reading frame (ORF) or 31015840-untranslated regions (UTR)subsequently cloned into a shuttle vector pcDNA31 (+)(Clontech USA) respectively Electrophoretic analysis andgene sequencing were conducted to make sure that all therecombinant plasmids were correct

22 Cell Culture 4-week-old male C57BL6J mice (Lab-oratory Animal Center of Southern Medical UniversityGuangzhou China) were kept in a controlled-temperature(22ndash25∘C) animal room with a 12 h light and 12 h dark cyclePelleted commercial chow (Laboratory Animal Center ofSouthern Medical University) and purified water were avail-able The experimental procedures performed in this studywere in accordance with the guidelines of the InstitutionalAnimal Ethics Committee for the Care andUse of LaboratoryAnimals Hepatocytes were isolated and purified from theanimals by in situ collagenase perfusion as mentionedpreviously [12] The hepatocytes were plated at 6-well platesprecoated with collagen (Corning USA) and incubated at37∘C with 5 humidified CO

2

23 Transfection Primary hepatocytes were transfected byLipofectamine 2000 (Invitrogen USA) according to themanufacturerrsquos protocol In brief we prepared DNA-reagentcomplexes at ratios of 1 25 (total volume = 500 120583L) Twentyminutes later the complexes were added to each well Toinvestigate transfection efficiency a green fluorescent pro-tein (GFP) plasmid was introduced 48 hours after trans-fection flow cytometry analysis was conducted The cellsthat displayed green fluorescence were examined throughFL 1 channel (excitation 488 nm emission 520 plusmn 10 nmFACSCalibur BD USA) The efficiency was defined as the

percentage of GFP-positive cells within all viable cells in 3independent experiments Hepatocytes treated without GFPserved as control

24 Reverse Transcription Polymerase Chain Reaction TotalRNA was extracted using TRIzol (Takara Japan) from two-day cultured hepatocytes RT-PCR was performed accordingto the manufacturerrsquos instructions (PrimeScript RT-PCR KitTakara) The following gene-specific oligonucleotide primerswere used for amplification Pax6 (314 bp) CAG TCA CAGCGG AGT GAA TCA GC (forward) and GCC ATC TTGCGT AGG TTG CCC TG (reverse) Nkx61 (381 bp) GTTCCT CCT CCT CCT CTT CCT C (forward) and AAG ATCTGC TGT CCG GAA AAA G (reverse) and Isl1 (268 bp)GTG CGG AGT GTA ATC AGT ATT TGG (forward)and GTC ATC TCT ACC AGT TGC TCC TTC (reverse)Amplification conditions were initial denaturation at 94∘Cfor 10min followed by 35 cycles of denaturation at 94∘C for30 sec annealing at 60∘C for 30 sec and extension at 72∘C for30 sec and at last an extension step of 10min at 72∘C

25 Real-Time Fluorescence Relative Quantitative PCR(qPCR) Total RNA was obtained as above The primerswere the following Gk (islet type) CAG AGA CAC AACAAC CTT TTC CC (forward) and GCT GTC TCA CTGGCT GAC TT (reverse) Ins1 TTG GTG CAC TTC CTACCC CT (forward) and CAC ACA CCA GGT AGA GAGCC (reverse) and Ins2 CCA TCA GCA AGC AGG AAGGTT A (forward) and CAG GTG GGA ACC ACA AAG GT(reverse) The SYBR-Green real-time PCR reaction solutionwas prepared and the PCR conditions were set accordingto the manufacturerrsquos protocol (ABI USA) The data wereanalyzed for target gene expression by the 2minusΔΔCt methodNormal C57BL6J mice islet was used as a control

26 Western Blot Assay Five days after transfection proteinswere isolated The protein concentrations were determinedby protein assay (KeyGEN China) Electrophoresis on a 12SDS polyacrylamide gel was performed and the protein wastransferred to a PVDFmembrane (Millipore Germany) Pro-tein bands were detected using an enhanced chemilumines-cence system (Pierce Biotechnology USA) with antibodies(Santa Cruz USA)

27 Insulin and C-Peptide Detection 5-day cultured hepato-cytes were treated with acid-ethanol at 4∘C overnight Thesupernatants were collected and the intracellular C-peptidelevels were measured by an enzyme-linked immunosorbentassay (C-peptide-ELISA kit Millipore) Insulin secretion wasdetermined by an ELISA kit (Insulin-ELISA kit Millipore)after exposure toKrebs-Ringer bicarbonate (KRB) bufferwith01 BSA containing various concentrations of glucose (0 5or 25mM) as described [13] The values of insulin and C-peptide were normalized relative to the total protein contentwhich was detected by protein assay (KeyGEN China)


ents

0

101 102 103

FL 1 Log

293 plusmn 11

100

104






3 Results


MaFA

Ngn3

Pdx1

Pax6

Nkx 61

P N M NP

MP

MN

MN

P

NV

120573-Actin

Isl1


0

01

02

03

04

05

06

07

08

09

1


GkIns1Ins2

lowastlowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast


ive l

evels

to is

let o

f pan

crea

tic m

arke

rs


GAPDH

P N M NP

MP

MN

MN

P

NV

Ins1

Ins2

Isle

t



lowast lowastlowast

lowastlowast

lowast

lowast

1200

1000

800

600

400

200


C-pe

ptid

e con

tent

(pg120583

g pr

otei

n)

(a)

0

50

100

150

200

250

300

NV MNP

KRB

Insu

lin se

cret

ion

( o

f bas

al)

lowast

lowast


(b)








0

5

10

15

20

25

30

0 3 7 11 15 19

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L) lowast

Time (days)

lowast


(a)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 9)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 7


(b)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 19

lowast

lowast


(c)




4 Discussion













Funding


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ents

0

101 102 103

FL 1 Log

293 plusmn 11

100

104






3 Results


MaFA

Ngn3

Pdx1

Pax6

Nkx 61

P N M NP

MP

MN

MN

P

NV

120573-Actin

Isl1


0

01

02

03

04

05

06

07

08

09

1


GkIns1Ins2

lowastlowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast


ive l

evels

to is

let o

f pan

crea

tic m

arke

rs


GAPDH

P N M NP

MP

MN

MN

P

NV

Ins1

Ins2

Isle

t



lowast lowastlowast

lowastlowast

lowast

lowast

1200

1000

800

600

400

200


C-pe

ptid

e con

tent

(pg120583

g pr

otei

n)

(a)

0

50

100

150

200

250

300

NV MNP

KRB

Insu

lin se

cret

ion

( o

f bas

al)

lowast

lowast


(b)








0

5

10

15

20

25

30

0 3 7 11 15 19

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L) lowast

Time (days)

lowast


(a)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 9)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 7


(b)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 19

lowast

lowast


(c)




4 Discussion













Funding


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast lowastlowast

lowastlowast

lowast

lowast

1200

1000

800

600

400

200


C-pe

ptid

e con

tent

(pg120583

g pr

otei

n)

(a)

0

50

100

150

200

250

300

NV MNP

KRB

Insu

lin se

cret

ion

( o

f bas

al)

lowast

lowast


(b)








0

5

10

15

20

25

30

0 3 7 11 15 19

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L) lowast

Time (days)

lowast


(a)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 9)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 7


(b)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 19

lowast

lowast


(c)




4 Discussion













Funding


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

30

0 3 7 11 15 19

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L) lowast

Time (days)

lowast


(a)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 9)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 7


(b)

0

5

10

15

20

25

30

35

40

0 30 60 120

NV (n = 6)

IPCs (n = 8)

Bloo

d gl

ucos

e lev

els (m

mol

L)

Time (min)

Day 19

lowast

lowast


(c)




4 Discussion













Funding


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Funding


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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