mice , including nod / lt mice for tail - tip fibroblasts isolation , were maintained in a specific pathogen - free animal facility under monash university approved ethics ( mmca2007/34 ) .
an 1.5 cm length of tail - tip from 8-week - old male nod mice was washed with 70% ethanol and pbs , the superficial dermis was peeled away , and the remaining tissue was cut into 1-mm pieces using a scalpel .
five or six pieces were plated in one well of a six - well plate and cultured with 2 ml of nttf medium ( dulbecco s modied eagle s medium with 10% fbs and 50 units / ml penicillin / streptomycin ) for 57 days .
retrovirus production and ipsc induction were performed as described previously with minor modifications ( 16 ) .
briefly , pmx - based retroviral vectors ( addgene ) encoding mouse oct4 , sox2 , and klf4 sequences were independently transfected into plat - e cells . the next day ,
nttfs at passage 4 were seeded at a density of 1  10 cells per well in six - well plates overnight .
the virus - containing supernatants were collected 48 h after transfection , mixed , filtered , and added to the nttfs together with 4 g / ml polybrene ( sigma - aldrich , st .
louis , mo ) for 24 h. subsequently , nttfs were cultured in mouse esc medium supplemented with 2 mol / l valproic acid ( vpa ) , which was refreshed daily for 7 days .
mouse esc culture medium was composed of knockout - dulbecco s modied eagle s medium , 20% knockout serum replacement , 0.1 mmol / l nonessential amino acids , 1 mmol / l glutamax , 0.1 mmol / l -mercaptoethanol ( all from invitrogen , carlsbad , ca ) , and 1,000 units / ml leukemia inhibitory factor ( lif ) ( millipore , billerica , ma ) .
cells were cultured in a 37c , 5% co2 incubator . at day 14 after transduction
the colonies were dissociated into single cells and transferred to wells in 96-well plates with mouse embryonic fibroblast ( mef ) feeder cells and 200 l esc medium .
primary antibodies were oct4 ( 1:100 , santa cruz biotechnology , inc . , santa cruz , ca ) , nanog ( 1:100 , abcam , cambridge , u.k . ) , and ssea-1 ( 1:100 , millipore ) .
total rna was isolated using the rneasy kit ( qiagen , hilden , germany ) , and genomic dna contamination was removed using dna - free kit ( ambion , austin , tx ) .
one microgram of total rna was used for cdna synthesis using superscript iii reverse transcriptase and oligo ( dt ) primers ( invitrogen ) .
genomic dna was isolated from approximately 2  10 ipscs for each cell line using dneasy blood and tissue kit ( qiagen ) .
genomic dna from nod ips , es ( d3 line ) , and nttf cells were extracted and processed for bisulfite genomic sequencing analysis as described previously ( 17 ) . nested - pcr and sequencing was performed using primers listed in table 1 . to form embryoid bodies ( ebs ) in vitro , nod - ipscs
were dissociated into single cells and resuspended in mes medium without lif in bacteriological petri dishes ( falcon , bd biosciences ) for 1 week . to form teratomas in vivo ,
2  10 nod ipscs were injected into the hind leg muscle of nod / scid mice .
host embryos were collected at the blastocyst stage from c57bl/6 female mice at 3.5 days post coitum , and 1015 nod ipscs were injected into each . after a recovery period of 2 h , 10 to 15 injected blastocysts were transferred into one uterine horn of pseudopregnant cbac57bl/6 f1 female mice at 2.5 days post coitum .
chimeric mice were identified by coat color , and the male chimera was assessed for germline transmission by mating with nod female mice .
single cell suspensions were prepared from individual spleens and erythrocytes were lysed by treating the sample with tris - nh4cl followed by washing with facs buffer containing 2% fbs .
fc receptor binding was blocked by incubating cells with 2.4g2 ( bd biosciences pharmingen , san diego , ca ) .
the following monoclonal antibodies were used in multiparameter flow cytometric analysis : anti - cd19 ( 6d5 , alexa fluor 700 conjugated ) and anti - h-2k ( af688.5 , percp / cy5.5 conjugated ) from biolegend and anti - h-2k ( sf11.1 , fluorescein isothiocyanate conjugated ) , anti - i - a ( ox-6 , fluorescein isothiocyanate conjugated ) , and anti - i - a ( af6120.1 , phycoerythrin conjugated ) from bd pharmingen .
cells were analyzed with an lsr fortessa ( bd biosciences , franklin lakes , nj ) and flowjo ( tree star ) software using propidium iodide incorporation to exclude dead cells .
dna was extracted from the nod - ips#5 cell line and genotyped for 112 microsatellite markers across the mouse genome covering 19 autosomes and the x chromosome .
, using the applied biosystems ( carlsbad , ca ) 3730 dna analyzer and ab gene mapper software .
mice , including nod / lt mice for tail - tip fibroblasts isolation , were maintained in a specific pathogen - free animal facility under monash university approved ethics ( mmca2007/34 ) .
an 1.5 cm length of tail - tip from 8-week - old male nod mice was washed with 70% ethanol and pbs , the superficial dermis was peeled away , and the remaining tissue was cut into 1-mm pieces using a scalpel .
five or six pieces were plated in one well of a six - well plate and cultured with 2 ml of nttf medium ( dulbecco s modied eagle s medium with 10% fbs and 50 units / ml penicillin / streptomycin ) for 57 days .
retrovirus production and ipsc induction were performed as described previously with minor modifications ( 16 ) .
briefly , pmx - based retroviral vectors ( addgene ) encoding mouse oct4 , sox2 , and klf4 sequences were independently transfected into plat - e cells . the next day ,
nttfs at passage 4 were seeded at a density of 1  10 cells per well in six - well plates overnight .
the virus - containing supernatants were collected 48 h after transfection , mixed , filtered , and added to the nttfs together with 4 g / ml polybrene ( sigma - aldrich , st .
louis , mo ) for 24 h. subsequently , nttfs were cultured in mouse esc medium supplemented with 2 mol / l valproic acid ( vpa ) , which was refreshed daily for 7 days .
mouse esc culture medium was composed of knockout - dulbecco s modied eagle s medium , 20% knockout serum replacement , 0.1 mmol / l nonessential amino acids , 1 mmol / l glutamax , 0.1 mmol / l -mercaptoethanol ( all from invitrogen , carlsbad , ca ) , and 1,000 units / ml leukemia inhibitory factor ( lif ) ( millipore , billerica , ma ) .
cells were cultured in a 37c , 5% co2 incubator . at day 14 after transduction , colonies were picked individually and incubated with 20 l of 0.25% trypsin/1 mmol
the colonies were dissociated into single cells and transferred to wells in 96-well plates with mouse embryonic fibroblast ( mef ) feeder cells and 200 l esc medium .
primary antibodies were oct4 ( 1:100 , santa cruz biotechnology , inc . , santa cruz , ca ) , nanog ( 1:100 , abcam , cambridge , u.k . ) , and ssea-1 ( 1:100 , millipore ) .
total rna was isolated using the rneasy kit ( qiagen , hilden , germany ) , and genomic dna contamination was removed using dna - free kit ( ambion , austin , tx ) .
one microgram of total rna was used for cdna synthesis using superscript iii reverse transcriptase and oligo ( dt ) primers ( invitrogen ) .
genomic dna was isolated from approximately 2  10 ipscs for each cell line using dneasy blood and tissue kit ( qiagen ) .
genomic dna from nod ips , es ( d3 line ) , and nttf cells were extracted and processed for bisulfite genomic sequencing analysis as described previously ( 17 ) . nested - pcr and sequencing was performed using primers listed in table 1 .
to form embryoid bodies ( ebs ) in vitro , nod - ipscs were dissociated into single cells and resuspended in mes medium without lif in bacteriological petri dishes ( falcon , bd biosciences ) for 1 week . to form teratomas in vivo ,
2  10 nod ipscs were injected into the hind leg muscle of nod / scid mice .
host embryos were collected at the blastocyst stage from c57bl/6 female mice at 3.5 days post coitum , and 1015 nod ipscs were injected into each . after a recovery period of 2 h , 10 to 15 injected blastocysts were transferred into one uterine horn of pseudopregnant cbac57bl/6 f1 female mice at 2.5 days post coitum .
chimeric mice were identified by coat color , and the male chimera was assessed for germline transmission by mating with nod female mice .
single cell suspensions were prepared from individual spleens and erythrocytes were lysed by treating the sample with tris - nh4cl followed by washing with facs buffer containing 2% fbs .
fc receptor binding was blocked by incubating cells with 2.4g2 ( bd biosciences pharmingen , san diego , ca ) .
the following monoclonal antibodies were used in multiparameter flow cytometric analysis : anti - cd19 ( 6d5 , alexa fluor 700 conjugated ) and anti - h-2k ( af688.5 , percp / cy5.5 conjugated ) from biolegend and anti - h-2k ( sf11.1 , fluorescein isothiocyanate conjugated ) , anti - i - a ( ox-6 , fluorescein isothiocyanate conjugated ) , and anti - i - a ( af6120.1 , phycoerythrin conjugated ) from bd pharmingen .
cells were analyzed with an lsr fortessa ( bd biosciences , franklin lakes , nj ) and flowjo ( tree star ) software using propidium iodide incorporation to exclude dead cells .
dna was extracted from the nod - ips#5 cell line and genotyped for 112 microsatellite markers across the mouse genome covering 19 autosomes and the x chromosome .
genotyping was performed by the australian genome research facility ltd . , using the applied biosystems ( carlsbad , ca ) 3730 dna analyzer and ab gene mapper software .
1a ) , clusters of cells started to aggregate together . by day 14 postviral infection , es - like colonies
were individually picked , trypsinized , and transferred to mef feeder cells in 96-well plates for expansion ( fig .
a total of 41 putative colonies were picked in three replicated experiments , and 18 colonies were successfully expanded in six - well plates ( supplementary table 1 ) .
the cells formed compacted colonies showing a refractile halo effect when observed by phase - contrast microscopy ( fig .
next , we transfected one of the nod - ipsc lines with an mcherry expression cassette ( cmv - mcherry - hygro ) by lipofectamine ( invitrogen ) as described previously ( 18 ) . after selection with hygromycin and expansion ,
nttfs were plated at a density of 1  10 cells per well in six - well plates and infected with retroviruses encoding osk factors for 24 h. the infected nttfs were cultured in fibroblast medium for 2 days , switched to mouse esc medium supplemented with vpa for 1 week , and subsequently cultured in mouse esc medium .
b : representative images of colonies observed at different stages as indicated in the panels ( p , passage number ) and the representative colonies stably transfected with cmv - mcherry - hygro construct .
c : integration of oct4 , sox2 , and klf4 transgenes in the three osk - induced ipsc lines was confirmed by genomic pcr using transgene - specific primers , with h2o and nttf as negative controls and the construct plasmids as positive control .
sox2 , klf4 , nanog , and rex1 ; expression of transgenes oct4 , sox2 , and klf4 in the tail - tip fibroblasts ; and the three osk - induced ipsc lines were assessed by rt - pcr , with h2o as negative control and appropriate samples as corresponding positive controls .
e : immunofluorescence staining shows expression of pluripotency markers ( oct4 , nango , and ssea1 ) in the three osk - induced ipsc lines .
genomic dna from mouse escs ( esd3 ) , fibroblasts ( nttf ) , and the three osk - induced ipsc lines were processed for bisulfite sequencing .
( a high - quality digital representation of this figure is available in the online issue . ) of the 18 nod ipsc lines generated , three were selected for further characterization .
we confirmed by genomic dna pcr that the three reprogramming transgenes ( osk ) were integrated in the genome of the ipscs ( fig .
rt - pcr analysis confirmed that osk transgenes were efficiently silenced in the three nod ipsc lines ( fig .
in addition , reactivation of endogenous oct4 and sox2 genes , as well as other pluripotent - associated transcription factors such as nanog and rex1 ( also known as zfp42 ) , was observed in all nod ipsc lines tested in the study ( fig .
immunohistochemical analysis showed that the ipscs stained positively for oct4 , nanog , and ssea1 ( fig .
further , bisulfite sequencing analysis revealed that promoter regions examined for both oct4 and nanog were demethylated in nod - ipscs , in a manner similar to mouse escs ( esd3 cell line ) , compared with heavily methylated patterns observed for the same regions in the parental nttf cells ( fig .
karyotyping analysis showed a normal chromosome number ( 40 , xy ) in the three ipsc lines ( fig . 1 g ) , and genome - wide genotyping ( 112 microsatellite markers ) of nod - ips # 5 cell line confirmed the nod genetic background ( supplementary table 2 ) .
we next evaluated the differentiation potential of the nod ipscs in vitro by eb formation and in vivo by teratoma induction .
the ipscs readily formed ebs on culture in the absence of feeders and lif ( fig .
rt - pcr of the ebs showed strong suppression of pluripotency genes including oct4 and nanog , with coincident activation of lineage - specific genes representing the three germ layers : nestin ( ectoderm ) ; vimentin and brachyury ( mesoderm ) ; and gata6 , sox17 , and foxa2 ( endoderm ) ( fig .
all three nod - ipsc lines generated well - differentiated teratomas in the nod / scid mice .
hematoxylin - eosin staining of teratoma sections showed tissues representative of the germ layers , including keratinized - epithelium ( ectoderm ) , cartilage ( mesoderm ) , and secreting - gland epithelium ( endoderm ) ( fig . 2c ) .
a : in vitro differentiation of cmv - mcherry - hygro vector transfected nod ipscs to ebs ( bright field and fluorescence field ) .
b : rt - pcr analysis of total rna isolated from ebs generated from the three nod ipsc lines , one nod ipsc line ( no .
the expression of oct4 and nanog ( pluripotency markers ) ; vimentin and brachyury ( mesoderm ) ; gata6 , foxa2 , sox17 ( endoderm markers ) ; and nestin ( ectoderm marker ) were examined .
histologic analysis of teratomas indicates ipscs contribute to tissues from the three germ layers , including keratinized - epithelium ( ectoderm ) , cartilage ( mesoderm ) , and secreting - gland epithelium ( endoderm ) .
d : contribution of nod - ips#5 cells in chimeric mouse ( xa2401 ) as detected by agouti coat color .
e : hematopoietic chimerism assessment by flow cytometry analysis of spleen samples from a wild - type nod mouse , the chimeric mouse ( xa2401 ) , and a c57bl/6 mouse .
top : h-2k ( c57bl/6 ) and h-2k ( nod ) mhc class i expression on cd19 cells .
bottom : i - a ( c57bl/6 ) and i - a ( nod ) mhc class ii expression on cd19 cells .
( a high - quality digital representation of this figure is available in the online issue . ) a total of 24 c57bl/6 blastocysts injected with nod - ips#5 cells were transferred into the uteri of two pseudopregnant mice .
both recipients became pregnant and delivered a total of 17 pups , with one male pup ( designated as xa2401 ) showing 4050% coat color chimerism ( fig .
we crossed the chimeric male with wild - type nod female mice to examine germ - line transmission capability ; however , no albino offspring resulted from three litters examined ( 18 pups born ) .
last , we assessed the hematopoietic chimerism of male xa2401 by cell surface marker expressions of the major histocompatibility complex ( mhc ) .
nod mice express the mhc class i molecule k and class ii molecule i - a , whereas c57bl/6 mice express k and i - a molecules , respectively .
flow cytometric analysis of b - cell expression of mhc molecules in the spleen indicated that the xa2401 mouse showed a c57bl/6 hematopoietic system ( fig .
the major goal of our current research was to demonstrate that somatic fibroblasts from nod mice can be reprogrammed to generate ipscs .
we used three yamanaka factors , oct4 , sox2 , and klf4 , but not c - myc , to fulfill the reprogramming process of nttf in this study .
c - myc was excluded because previous studies have shown that potential reactivation of the c - myc transgene could result in tumor development in the chimeras and their progeny derived from the ipscs ( 9,19 ) . instead , the chromatin remodeling small - molecule vpa ( a histone deacetylase inhibitor ) was used because it has been shown to greatly increase the efficiency of reprogramming human and mouse fibroblasts ( 20,21 ) .
male ( xy ) donor cells were also chosen for induction to pluripotency for three reasons .
1 ) male pluripotent cell - derived differentiated cells are easier to track in vivo , based on y - chromosome specific probes , when used for transplantation studies into female recipients .
2 ) aberrant x - inactivation associated with female ( xx ) esc lines can result in xo cells , which compromises euploidy of cells , chimera generation , and germ - line transmission ( 22,23 ) .
3 ) male esc chimeras can be rapidly bred with more than one wild - type female to produce multiple litters . by using this modified approach ,
we show that nod ipscs are readily generated from nttfs by transduction with retroviral vectors encoding oct4 , sox2 , and klf4 and supplementation with vpa in the culture medium for 1 week .
all three transgenes were silenced in the nod ipscs , suggesting complete reprogramming of the somatic nttf .
the nod ipscs could be dissociated into single cells by trypsinization and maintained for > 50 passages .
the nod ipscs derived in this study maintained esc - like pluripotent characteristics and did not convert to an epiblast stem ( epis ) cell - like state , which is characterized by distinct colonies with flat morphology ( 24,25 ) .
( 4 ) , because their nod ipscs appeared to be metastable and acquired an alternative epis cell - like identity after removal of exogenous klf4 or cmyc factor expression .
the epis cell colonies not only were morphologically distinct from mouse escs but also lost the ability to be propagated after dissociation into single cells by trypsinization . in our study , the derivation and maintenance of nod ipscs were achieved without constitutive expression of klf4 transgene or addition of small molecules of gsk3/erk pathway inhibitors .
this difference may be due to the source of adult somatic tissue : we used nttf , whereas hanna et al .
nonetheless , our modified method provides a robust alternative strategy for the generation of esc - like pluripotent stem cells from a nonpermissive mouse strain without using embryos .
 objectivethe nod mouse strain has been widely used to investigate the pathology and genetic susceptibility for type 1 diabetes . induced pluripotent stem cells ( ipscs ) derived from this unique mouse strain would enable new strategies for investigating type 1 diabetes pathogenesis and potential therapeutic targets . 
 the objective of this study was to determine whether somatic fibroblasts from nod mice could be reprogrammed to become ipscs , providing an alternative source of stem cells for the production of genetically modified nod cells and mice.research design and methodsadult tail - tip fibroblasts from male nod mice were reprogrammed by retroviral transduction of the coding sequences of three transcription factors , oct4 , sox2 , and klf4 , in combination with a histone deacetylase inhibitor , valproic acid.resultseighteen nod ipsc lines were generated , and three of these cell lines were further characterized . 
 all three cell lines exhibited silencing of the three reprogramming transgenes and reactivation of endogenous pluripotent markers ( oct4 , sox2 , nanog , rex1 , and ssea1 ) . 
 these nod ipscs readily differentiated in vitro to form embryoid bodies and in vivo by teratoma formation in immunodeficient mice . 
 moreover , nod ipscs were successfully transfected with a reporter transgene and were capable of contributing to the inner cell mass of c57bl/6 blastocysts , leading to the generation of a chimeric mouse.conclusionsadult tail - tip fibroblasts from nod mice can be reprogrammed , without constitutive ectopic expression of transcription factors , to produce ipscs that exhibit classic mouse embryonic stem cell ( esc ) features . 
 these nod ipscs can be maintained and propagated under normal esc culture conditions to produce genetically altered cell lines , differentiated cells , and chimeric mice . 
RESEARCH DESIGN AND METHODS
Mice.
Tail-tip fibroblast isolation.
Retrovirus production and iPSC induction.
Cell culture and colony pick-up.
Immunocytochemistry.
RT-PCR and genomic DNA PCR.
Bisulfite genomic sequencing.
Differentiation of NOD-iPSCs.
Blastocyst injection and embryo transfer.
Flow cytometric analysis of splenic B cells.
Microsatellite genotyping of cell lines.
RESULTS
DISCUSSION