Embed Size (px)
Citation preview
ORIGINAL ARTICLE Reproductive biology
Suppression of transforming growthfactorb signaling promotesground statepluripotency from single blastomeresSeyedeh-Nafiseh Hassani1,2,†, Mohammad Pakzad1,†, Behrouz Asgari1,Adeleh Taei1, and Hossein Baharvand1,2,*1Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology andTechnology, ACECR, PO Box 19395-4644, Tehran, Iran 2Department of Developmental Biology, University of Science and Culture, ACECR,Tehran, Iran
*Correspondence address. Tel: +98-21-22306485; Fax: +98-21-23562507; E-mail: [email protected]
Submitted on January 27, 2014; resubmitted on May 1, 2014; accepted on May 12, 2014
study question: Can transforming growth factorb (TGFb) inhibition promote ground state pluripotency of embryonic stem cells (ESCs)from single blastomeres (SBs) of cleavage embryos in different mouse stains?
summary answer: Small molecule suppression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and TGFb signaling(designated as R2i) can enhance the generation of mouse ESCs from SBs of different cleavage stage embryos compared with the dual suppressionof ERK1/2 and glycogen synthase kinase 3 (GSK3), designated as 2i, regardless of the strain of mouce.
what is known already: It is known that chemical inhibition of TGFb promotes ground state pluripotency in the generation and sus-tenance of naı̈ve ES cells from mouse blastocysts compared with the well-known 2i condition. However, the positive effect of this inhibition onmouse ESCs from early embryonic SBs remains obscure.
study design, size, duration: We used 155 cleavage-stage mouse embryos to optimize the culture conditions for blastocyst de-velopment. Then, to assess the effects of R2i and 2i on ESC generation from SBs, we cultured isolated SBs in 2i and R2i for 10 days. SBs werereplated under the same conditions to produce ESCs. In total, 46 embryos and 321 SBs from two- to eight-cell stages were recovered fromNMRI and BALB/c mouse strains and used in this study.
participants/materials, setting, methods: Blastomeres from 2- to 8-cell stage mouse embryos were dispersed and indi-vidually seeded into a 96-well plates that included mitotically inactivated feeder cells. ESCs were generated in B27N2 defined medium supplemen-ted with R2i or 2i. Randomly selected ESC lines, generated from SBs of each stage, were assessed for pluripotency and germ-line transmission.
main results and the role of chance: We demonstrated that dual inhibition of ERK1/2 and TGFb (R2i) enhanced efficientblastocyst development and efficient establishment of ESCs from SB of 2- to 8-cell stage mouse embryos compared with the dual inhibition ofERK1/2 and GSK3 (2i) regardless of the embryonic stage and strain of mice. The proportions of SBs that produced ESC were 50–60 versus20–30%.
limitations, reasons for caution: This study was done with mouse embryos, it is not known whether these findings are trans-ferable to humans.
wider implications of the findings: These findings resulted in an increased efficiency of ESC generation from one biopsiedblastomere for autogeneic or allogeneic matched pluripotent cells without the need to destroy viable embryos. The results also provided infor-mation about the developmental capacity of early embryonic blastomeres.
study funding/competing interest(s): This study was funded by grants provided from Royan Institute, the Iranian Council ofStem Cell Research and Technology and the Iran National Science Foundation. The authors have no conflict of interest to declare.
Key words: TGFb inhibition / ERK inhibition / single blastomeres / ground state pluripotency / mouse embryonic stem cell derivation
† These authors contributed equally in this work.
& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: [email protected]
Human Reproduction, Vol.0, No.0 pp. 1–10, 2014
doi:10.1093/humrep/deu134
Hum. Reprod. Advance Access published June 24, 2014 by guest on June 25, 2014
http://humrep.oxfordjournals.org/
Dow
nloaded from
IntroductionEmbryonic stem cells (ESCs) are pluripotent cells conventionally derivedfrom the inner cell mass (ICM) of blastocyst-stage embryos cultured onmitotically inactivated mouse embryonic fibroblasts (MEFs) as feedercells with fetal calf serum (FCS) (Baharvand and Matthaei, 2004). Thegrowth factors leukemia inhibitory factor (LIF) and bone morphogeneticprotein 4 can substitute for MEF and FCS, respectively, enabling ESC pro-duction indefined culture medium(Smithetal., 1988; Williams et al., 1988;Ying et al., 2003). However, these conditions restrict the production ofESCs to the Sv129 mouse strain (Batlle-Morera et al., 2008).
In recent years, major improvements in the development of culturemedium have dramatically increased the success rates for generationand maintenance of mouse ESCs. The application of chemical inhibitorsof differentiation-inducing signaling pathways has allowed for the efficientgeneration and optimum propagation of mouse ESCs from differentmouse strains in a chemically defined medium. The combination of smallmolecules PD0325901 and CHIR99021, known as 2i, that inhibit extracel-lular signal-regulated protein kinases 1 and 2 (ERK1/2) and glycogen syn-thase kinase 3 (GSK3), respectively, made a breakthrough in establishingmouse ESCs with enhanced ESC clonal growth and viability (Ying et al.,2008; Wray et al., 2010). The chemical inhibitor-based culture mediumhas obviated the need for growth factors and it is presumed that ESCsare at a ground state pluripotency, although 2i/LIF has been introducedto optimize results (Blair et al., 2011). Recently, we have shown that sup-pression of the transforming growth factorb (TGFb) signaling pathway incombination with ERK1/2 inhibitor can also promote ground state pluri-potency (Hassani et al., 2014a,b). The small molecules SB431542 thatinhibit TGFb type I receptors [activin receptor-like kinase (ALK)-4, -5and -7] and PD0325901—the combination has been named as R2i—allow for highlyefficient, reproducible derivation of ESCs from mouse blas-tocysts of strains previously considered refractory and non-permissive(Hassani et al., 2012, 2014a,b). We have observed that mouse ESCs gen-erated with R2i (R2i cells) exhibit enhanced clonal growth and homogen-ous expression of pluripotency-related transcription factor proteins suchas Nanog and Stella. R2i cells have less differentiation leakage (expressionof genes such as Lefty 1, Lefty 2, Brachyury) in comparison with serum- andeven 2i-cultured ESCs. In comparison with the 2i compound, R2i protectsa more stable karyotype in mouse ESCs during long-term passaging(Hassani et al., 2014a,b). More recently, we also found that the derivationand expansion of pluripotent embryonic germ cells from primordial germcells of embryos of various ages can be performed under the R2i conditionwith greater efficiency compared with 2i (Attari et al., 2014).
In the present study, we attempt to improve the generation of mouseESCs from early embryonic blastomeres using the TGFb signalingpathway inhibitor, SB431542. We provide evidence that dual inhibitionof ERK1/2 and TGFb (R2i condition) leads to efficient blastocyst devel-opment and more effective (2-fold increase) generation of ESCs fromsingle blastomeres (SBs) of preimplantation embryos in comparisonwith the dual inhibition of ERK1/2 and GSK3 (2i condition), regardlessof the embryonic stage and strain of mice.
Materials and Methods
Animals, embryos and blastomeresAll animal experiments in this study were conducted in accordance with theapproval of the Royan Institutional Review Board and Institutional Ethical
Committee (Royan Institute, Tehran, Iran). Mouse strains (obtained fromPasteur Institute, Tehran, Iran) were maintained on a 12-h light/darkregimen. Sexually mature NMRI and BALB/c female mice were eachinduced to superovulate by an injection with 7.5 IU pregnant mare serum go-nadotrophin (Intervet, Brussels) followed by an injection of 7.5 IU humanchorionic gonadotrophin (hCG, Tehran, Iran) 48 h later. Females werethen paired with stud NMRI or BALB/c males, respectively. Detection of acopulation plug the next morning was considered to be 0.5 day postcoitum(dpc). All early cleavage embryos were flushed from the oviducts withmedium used to culture the SBs (Supplementary data, Table SI). Cleavageembryos at two-, four- and eight-cell stages were retrieved at 44, 54 and68 h, respectively, after hCG injection. MEF was obtained from NMRIstrain E13.5 fetuses by a standard protocol.
Establishment of mouse ESCs from SBsThe zona pellucida of each cleavage embryo was removed with acid Tyrode’ssolution. Denuded two- to eight-cell embryos were transferred to Ca2+/Mg2+-free phosphate-buffered saline (PBS, Invitrogen, Carlsbad, CA, USA)after which they were gently pipetted several times in the relevant culturemedium in order to dissociate the blastomeres. The media used in thisstudy included knockout serum replacement (KSOM, Invitrogen), serum/LIF, N2B27/LIF and N2B27/small molecules/LIF.
Serum/LIF medium contained knockout Dulbecco’s modified eaglemedium (DMEM) (Invitrogen), 15% fetal bovine serum (FBS) (HyClone,Logan, UT), 2 mM L-glutamine (Invitrogen), 1% non-essential amino acids(Invitrogen), penicillin (100 U/ml) streptomycin (100 mg/ml, Invitrogen),0.1 mM b-mercaptoethanol (Sigma-Aldrich, Saint Louis, MO, USA) and1000 U/ml mouse LIF (Millipore, Billerica, MA, USA). N2B27/LIF mediumcontained DMEM/F12 (Invitrogen) and neurobasal (Invitrogen) at a 1:1
Figure 1 Effects of various culture media on early embryo develop-ment. Development of two-cell whole embryos from the outbredmouse strain NMRI to intact hatched blastocysts in different culturemedia. All media were supplemented with leukemia inhibitory factor(LIF), with the exception of knockout serum replacement (KSOM).The basal medium for 2i (extracellular signal-regulated protein kinases1 and 2 (ERK1/2) and glycogen synthase kinase 3 (GSK3) inhibitors)and R2i was N2B27. Bars and whiskers represent the mean+ SD offive independent experiments. *Significant differences compared withKSOM (***P , 0.0001; **P , 0.001). †Significant differences comparedwith N2B27/2i/LIF (††P , 0.001). #Significant differences comparedwith N2B27/R2i/LIF (###P , 0.0001; ##P , 0.001; #P , 0.05).
2 Hassani et al.
by guest on June 25, 2014http://hum
rep.oxfordjournals.org/D
ownloaded from
.............................................................................................................................................................................................
.............................................................................................................................................................................................
............................................................................................ .............................................................................................
.............................................................................................................................................................................................
.............................................................................................................................................................................................
.............................................................................................................................................................................................
............................................................................................ .............................................................................................
.............................................................................................................................................................................................
Table I Derivation efficiency of ESCs from SBs of two-cell embryos.
NMRI Strain
Addition to N2B27/LIF Medium
R2i 2i
Serial no. ofembryo
Number of dissociatedblastomeres
Number ofestablished ESC lines
Serial no. ofembryo
Number of dissociatedblastomeres
Number of establishedESC lines
1 2 0 1 2 0
2 2 2 2 2 2
3 2 2 3 2 0
4 2 2 4 2 0
5 2 0 5 2 0
6 2 2 6 2 2
7 2 1 7 2 0
8 2 2 8 2 1
9 2 2 9 2 1
10 2 0 10 2 1
11 2 2 11 2 0
12 2 0 12 2 0
13 2 2 13 2 0
14 2 1 14 2 0
15 2 0 15 2 1
16 2 0 16 2 0
17 2 2 17 2 0
Total 34 20 Total 34 8
Efficiency of establishing ESC lines (%)
From embryos From dissociatedblastomeres
From embryos From dissociatedblastomeres
65 59 35 24
BALB/c Strain
Addition to N2B27/LIF Medium
R2i 2i
Serial no. ofembryo
Number of dissociatedblastomeres
Number of establishedESC lines
Serial no. ofembryo
Number of dissociatedblastomeres
Number ofestablished ESC lines
1 2 2 1 2 0
2 2 0 2 2 2
3 2 0 3 2 0
4 2 0 4 2 0
5 2 2 5 2 2
6 2 1 6 2 1
7 2 1 7 2 0
8 2 2 8 2 0
Total 16 8 Total 16 5
Efficiency of establishing ESC lines (%)
From embryos From dissociatedblastomeres
From embryos From dissociatedblastomeres
63 50 38 31
LIF, leukemia inhibitory factor.
Gain of pluripotency from isolated blastomeres 3
by guest on June 25, 2014http://hum
rep.oxfordjournals.org/D
ownloaded from
ratio, 1% N2 supplement (Invitrogen), 1% B27 supplement (Invitrogen),2 mM L-glutamine (Invitrogen), 1% non-essential amino acids, penicillin/streptomycin, 0.1 mM b-mercaptoethanol, 5 mg/ml bovine serumalbumin (Sigma-Aldrich) and 1000 U/ml mouse LIF. Small molecules usedin this study were the 2i compound that consisted of PD0325901 (1 mM,Sigma-Aldrich) and CHIR99021 (3 mM, Stemgent, San Diego, CA, USA),and the R2i compound that consisted of PD0325901 (1 mM) andSB431542 (10 mM, Sigma-Aldrich). In one experiment adrenocorticotropichormone (ACTH, Sigma-Aldrich) was added to N2B24/R2i/LIF. Eachblastomere was seeded into a well of an MEF-coated 96-well plates that con-tained N2B24/2i/LIF or N2B24/R2i/LIF. Usually after 10 days, the formedoutgrowths were dissociated by treatment with EGTA (500 mM) for3–5 min followed by 2.5X trypsin (0.625% w/v, Invitrogen) for 3 min.Trypsin was neutralized by serum medium and the dissociated cell solution
was centrifuged to remove the serum. Dissociated cells were then platedon gelatin-coated plates that contained the relevant medium. The establishedESC lines were routinely passaged every 2 days.
In vitro and in vivo differentiation of mouseESCsIn vitro spontaneous differentiation of ESCs was performed via embryoidbody (EB) formation. ESCs were dissociated by trypsin and cultured as sus-pensions in bacterial dishes that contained serum media without LIF. Seven-day old EBs were plated onto gelatin-coated plates and evaluated 7 days laterfor spontaneous differentiation. For induced differentiation into neuronallineages, 3-day EBs were transferred to medium that contained 2 mM retinoicacid for 1 week, then plated onto gelatinized plates and assayed 3–4 days
.............................................................................................................................................................................................
.............................................................................................................................................................................................
............................................................................................ .............................................................................................
.............................................................................................................................................................................................
.............................................................................................................................................................................................
.............................................................................................................................................................................................
............................................................................................ .............................................................................................
.............................................................................................................................................................................................
Table II Derivation efficiency of ESCs from SBs of four-cell embryos.
NMRI Strain
Addition to N2B27/LIF Medium
R2i 2i
Serial no. ofembryo
Number of dissociatedblastomeres*
Number ofestablished ESC lines
Serial no. ofembryo
Number of dissociatedblastomeres*
Number of establishedESC lines
1 3 2 1 3 1
2 3 1 2 3 2
3 3 2 3 3 0
4 3 2 4 3 0
5 3 0 5 3 1
Total 15 7 Total 15 4
Efficiency of establishing ESC lines (%)
From embryos From dissociatedblastomeres
From embryos From dissociatedblastomeres
80 47 60 27
BALB/c Strain
Addition to N2B27/LIF Medium
R2i 2i
Serial no. ofembryo
Number of dissociatedblastomeres*
Number ofestablished ESC lines
Serial no. ofembryo
Number of dissociatedblastomeres*
Number ofestablished ESC lines
1 4 2 1 3 2
2 3 1 2 3 0
3 3 3 3 3 1
4 3 1 4 4 2
5 4 2 5 3 2
6 4 3 6 3 0
7 3 3 7 4 0
Total 24 15 Total 23 7
Efficiency of establishing ESC lines (%)
From embryos From dissociatedblastomeres
From embryos From dissociatedblastomeres
100 63 57 31
* Early stage 4-cell embryos were used in this study. They were difficult to disperse and usually only 3 blastomere per embryo were obtained.
4 Hassani et al.
by guest on June 25, 2014http://hum
rep.oxfordjournals.org/D
ownloaded from
later. For cardiomyocyte differentiation, ESCs were trypsinized and culturedat a density of 800 cells/20 ml hanging drop for 2 days in serum mediumwithout LIF supplemented with 0.1 mM ascorbic acid. Subsequently, cellswere transferred to the bacterial dishes for an additional 3 days then rinsedonto gelatinized plates. Beating cells were usually observed 1–5 days afterplating. For endoderm lineage differentiation, EB formation was performedin N2B27 medium for 3–4 days, after which 50 nM activin A was addedfor 5 days. The EBs were plated onto gelatinized cell culture dishes andassayed 72 h later.
Chimeric mice were generated by injection of single-cell mouse ESCs into2.5 dpc embryos from the C57BL/6 strain. Usually 7–9 injected blastocystswere transferred into the uterine horns of pseudo-pregnant foster mice.Chimeric mice, identified by coat color, were crossed with mice from theinjected strain of the ESC lines to test for germ-line transmission.
Alkaline phosphatase activity andimmunocytochemistryAlkaline phosphatase (ALP) activity was determined by a manufacturer’s kit(Sigma-Aldrich, 86R). For immunofluorescence staining, mouse ESCs were
fixed in 4% paraformaldehyde for 20 min then permeabilized with 0.2%Triton X-100 for 30 min. Cells were washed twice in PBS-Tween (0.2%Tween-20), then placed in blocking buffer (PBS-Tween supplemented with10% goat serum) for 1 h at 378C. Cells were incubated with the primary anti-bodies overnight at48C, washedand incubated with secondaryantibodies for1 h at 378C. The primary antibodies were: Oct4 (1:100, sc-5279, Santa CruzBiotechnology, Inc., Santa Cruz, CA, USA), Nanog (1:100, 560259, BD Phar-mingen, San Diego, CA, USA) and SSEA1 (1:100, MAB2155, R&D, USA).The secondary antibodies were Alexa Fluor 488 goat anti-mouse immuno-globulin G (Invitrogen, A-11001, 1:500) and Alexa Fluor 488 goat anti-mouseimmunoglobulin M (Invitrogen, A-21042, 1:500). Nuclei were counter-stained with 1 mg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich,D9564). An Olympus fluorescent microscope (Olympus, Japan) was used forvisualizing cells.
Statistical analysisStatistical analyses were performed by one-way ANOVA to determinethe statistical significance of the data, using SPSS version 16.0 software.Data are presented as mean+ SD from a minimum of three independentexperiments.
.............................................................................................................................................................................................
.............................................................................................................................................................................................
............................................................................................ .............................................................................................
.............................................................................................................................................................................................
.............................................................................................................................................................................................
.............................................................................................................................................................................................
............................................................................................ .............................................................................................
.............................................................................................................................................................................................
Table III Derivation efficiency of ESCs from SBs of eight-cell embryos.
NMRI Strain
Addition to N2B27/LIF Medium
R2i 2i
Serial no. ofembryo
Number of dissociatedblastomeres
Number ofestablished ESC lines
Serial no. ofembryo
Number of dissociatedblastomeres
Number of establishedESC lines
1 8 3 1 8 0
2 8 5 2 8 2
3 8 4 3 8 4
Total 24 12 Total 24 6
Efficiency of establishing ESC lines (%)
From embryos From dissociatedblastomeres
From embryos From dissociatedblastomeres
100 50 67 25
BALB/c Strain
Addition to N2B27/LIF Medium
R2i 2i
Serial no. ofembryo
Number of dissociatedblastomeres
Number of establishedESC lines
Serial no. ofembryo
Number of dissociatedblastomeres
Number ofestablished ESC lines
1 8 3 1 8 2
2 8 2 2 8 0
3 8 3 3 8 4
4 8 5 4 8 0
5 8 4 5 8 4
6 8 5 6 8 1
Total 48 22 Total 48 11
Efficiency of establishing ESC lines (%)
From embryos From dissociatedblastomeres
From embryos From dissociatedblastomeres
100 46 67 23
Gain of pluripotency from isolated blastomeres 5
by guest on June 25, 2014http://hum
rep.oxfordjournals.org/D
ownloaded from
Results
R2i increased the efficiency of blastocystdevelopment from two-cell whole embryosOur intent was to investigate the capability of R2i to induce pluripotencyfrom SBs of cleavage-stage mouse embryos therefore, we initially soughtto select an appropriate basal culture medium that supported earlyembryo development. We assessed the development of two-cellwhole embryos from an outbred NMRI mouse strain toward intacthatched blastocysts in droplets of different media that included KSOM(as conventional cleavage medium), serum/LIF, N2B27/LIF, N2B27/2i/LIF and N2B27/R2i/LIF (Supplementary data, Table SI). Weobserved the presence of hatched blastocysts by Days 4–5 in all ofthe above-mentioned media. Experimental results demonstrated that21.6% of the two-cell embryos developed into intact hatched blasto-cysts when cultured in KSOM medium. However, only 8.3% of thetwo-cell embryos developed into intact hatched blastocysts inserum/LIF medium, which showed its minimal support of early embry-onic development. In comparison with KSOM, N2B27/LIF mediumhad a similar result with 23.3% two-cell embryos developing to blasto-cysts. The addition of small molecules to N2B27/LIF considerablyincreased blastocyst development efficiency. N2B27/2i/LIF with an ef-ficiency of 41.6% and N2B27/R2i/LIF with an efficiency of 79.9%showed �2- and 3.7-fold increases in intact hatched blastocyst devel-opment from the two-cell stage, respectively, compared with KSOM(Fig. 1). However, only the R2i compound generated statistically signifi-cantly more intact hatched blastocysts compared with KSOM (P ,
0.0001; Fig. 1).Previous studies have shown the efficient derivation of mouse ESCs
from SBs by the addition of ACTH to KOSR-containing medium(Wakayama et al., 2007; Lorthongpanich et al., 2008). It seems thatACTH by inhibition of adenylyl cyclase activity can stimulate survival andproliferation of mouse ESCs (Ogawa et al., 2004). Furthermore, it has
been reported that 2i and ACTH supplemented KOSR-containingmedium synergistically enhanced the establishment of mouse ESCs fromSBs of two- to eight-cell embryos (Lee et al., 2012). We added ACTHto N2B27/R2i/LIF medium in order to determine optimal culture condi-tions for early embryo development. Our results showed that althoughN2B27/R2i/ACTH/LIF supported blastocyst development fromtwo-cell embryos significantly more than KSOM, serum/LIF andN2B27/LIF (P , 0.05), it could not enhance the effect of N2B27/R2i/LIF medium (Fig. 1). Therefore, for establishment of mouse ESCs fromSBs of two- to eight-cell embryos we used chemically defined N2B27/R2i/LIF medium and compared it with N2B27/2i/LIF medium as thecontrol to establish ground state pluripotency (Ying et al., 2008).
R2i enhances the establishment of mouseESCs from SBsAs we reported the highest efficiency of ICM-derived ESCsestablishment by N2B27/R2i/LIF medium in feeder-free condition(Hassani et al., 2014a,b), the derivation of ESCs from SBs was initially per-formed on gelatin-coated plates. SBs from 2-, 4- and 8-cell embryos ofoutbred NMRI mouse strains were plated on gelatin-coated 96-wellplates in N2B27/R2i/LIF or N2B27/2i/LIF media. However, itbecame apparent that gelatin could not support the proliferation of dis-sociated blastomeres (Supplementary data, Table SII). This observationwas consistent with previous reports that the generation of ESC from SBsrequired feedercells, which were more likely to provide a properphysicalsubstrate (Chung et al., 2006; Lorthongpanich et al., 2008; Lee et al.,2012). Therefore, SB of 2- to 8-cell embryos from outbred NMRIstrain was cultured on MEF-coated 96-well plates in N2B27/R2i/LIFand N2B27/2i/LIF media (Tables I– III, upper panels). Our resultsshowed that R2i supported the generation of SB-derived ESCs with effi-ciencies of 59% (two-cell), 47% (four-cell) and 50% (eight-cell). In con-trast, when SBs were cultured in 2i the efficiency with which ESCswere established was 24% (two-cell), 27% (four-cell) and 25% (eight-cell). To confirm our results further, we recapitulated ESCs generationfrom SBs of inbred BALB/c strain mouse embryos (Tables I– III,bottom panels). We observed that with R2i, the efficacy of derivingESCs was 50, 62.5 and 46% from SB of BALB/c two-, four- and eight-cellembryos, respectively. In 2i, however, 31, 30.5 and 23% of SBs fromBALB/c two-, four- and eight-cell embryos, respectively, perpetuatedas mouse ESCs. Overall, our results indicated that R2i with �50–60%and 2i with �20–30% efficacy of deriving ESCs from SB of two- to eight-cell embryonic stages could support the generation of SB-derived ESCs.This efficiency in R2i was nearly 2-fold more than 2i under similar condi-tions regardless of the embryonic stage and strain of mice (Fig. 2). Afterestablishment, all single SB-derived ESCs were propagated in feeder-freegelatin-coated plates in N2B27/2i/LIF or N2B27/R2i/LIF medium andevaluated for pluripotency criteria.
Characterization of SB-derived R2i mouseESCsThe established SB-derived ESCs by R2i (SB-R2i cells) demonstratedtheir capability to undergo long-term passaging for at least 10 timesand indicated high nuclear–cytoplasmic ratios in the cells. Werandomly selected three SB-R2i cell lines—C50S2-1, C69S4-1 andC60S8-1, which were derived from SBs of two-, four- and eight-cellmouse embryos, respectively, for additional characterization. These
Figure 2 R2i elevates the efficiency of single blastomere (SB)-derivedembryonic stem cell (ESC) generation. The graph shows the foldchanges of ESC derivation efficiency from SBs of two-, four- and eight-cell embryos in R2i (ERK1/2 and transforming growth factor b inhibi-tors) versus 2i (ERK1/2 and GSK3 inhibitors). Two strains of mice,outbred NMRI and inbredBALB/c, were evaluated for ESCs generation.The results are shown in greater detail in Tables I– III.
6 Hassani et al.
by guest on June 25, 2014http://hum
rep.oxfordjournals.org/D
ownloaded from
SB-R2i cells showed expressions of archetype ESC markers by immunos-taining for ALP, Oct4, Nanog and SSEA1 (Fig. 3). In addition, theyshowedpluripotent potency during in vitro and in vivo differentiation. Throughoutspontaneous differentiation by EB formation, the randomly selectedSB-R2i ESCs from two-, four- and eight-cell stages showed the expres-sion of marker genes from derivatives of three embryonic germ layers
(Fig. 4A). Induced differentiation toward neuronal-, cardiomyocyte-and endodermal-lineages verified the pluripotency of SB-R2i ESCs(Fig. 4B). The in vivopotencyof SB-R2i ESCs to differentiate toward three-germ layers and germ-line was assessed by contribution to chimeric mice.After microinjection of SB-R2i ESCs into 2.5 dpc host embryos, theembryos were transferred to uterine horns of the foster mothers.
Figure 3 Characteristics of the SB-derived ESCs by R2i (SB-R2i cells). Phase contrast, alkaline phosphatase staining and immunofluorescence labeling forOct4, Nanog and SSEA-1, counterstained for DAPI, are shown for three SB-R2i cell lines (C50S2-1, C69S4-1 and C60S8-1) which were derived from SB oftwo-, four- and eight-cell mouse embryos, respectively.
Gain of pluripotency from isolated blastomeres 7
by guest on June 25, 2014http://hum
rep.oxfordjournals.org/D
ownloaded from
Pups that were born alive showed a high level of chimerism as indicatedby their black and white coat colors. Germ-line transmission from chi-meras was demonstrated after backcross breeding and indicated bycoat color of pups (Table IV and Fig. 4C).
DiscussionWe have described a highly efficient and reproducible method for thegeneration of mouse ESCs from SBs of two- to eight-cell stage
Figure 4 In vitro and in vivo evaluation of SB-R2i cell pluripotency. (A) RT-PCR analysis for various markers of the three embryonic germ layers at Day 14(7-day embryoid bodies and 7-day plating). (B) Phase contrast microscopy of differentiated neuronal cells (top left picture) and immunostaining for thederivatives of three embryonic germ layers of SB-R2i cells after induced differentiation. Expressions of Nestin as a neuronal marker, Mhc as a cardiomyocytemarker and Foxa2 as an endodermal marker are shown. (C) Chimera formation and germ-line transmission of SB-R2i cells after blastocyst injection ofdifferent lines.
.............................................................................................................................................................................................
Table IV Chimera and germ-line competency of SBs-derived R2i ESCs (BALB/c strain).
Developmentalstage of SB
Line name Embryos injected Live births Chimeras Confirmation ofgerm-line transmission
Two-cell RC50S2-1 34 8 8 Yes
Four-cell RC69S4-1 32 5 5 Ongoing
Eight-cell RC60S8-1 18 7 6 Yes
8 Hassani et al.
by guest on June 25, 2014http://hum
rep.oxfordjournals.org/D
ownloaded from
embryos using a combination of small molecule inhibitors of TGFb andmitogen-activated protein kinase (MAPK) signal transduction. Inthe chemically defined N2B27/LIF medium supplemented with chemi-cals SB431542 and PD0325901, or R2i (Hassani et al., 2014a,b),�50–60% of SBs gained the ability to produce ESCs, regardless of theembryonic stages and strain of mice. This may be related to technicallosses as well as to the occasional cessation of cell division of somefresh isolated SBs. In culture, in the course of conventional methodsfor ESCs generation, SBs usually tend to grow into small blastocyststhat lack ICM cells, or into cells with trophoblast-like shapes (Chunget al., 2006; Lee et al., 2012). Compared with earlier methods, the appli-cation of R2i increased the rate of establishment of SB-derived ESCs(Chung et al., 2006; Wakayama et al., 2007; Lorthongpanich et al.,2008; Lee et al., 2012). In comparison with 2i, the compound thatincluded chemical inhibitors for GSK3 and ERK1/2 signaling pathways(Ying et al., 2008), R2i has shown an �2-fold higher efficiency forSB-derived ESCs generation. This is especially important since it is pre-sumed that the application of CHIR99021 as a GSK3 inhibitor in 2i canenhance SB proliferation and so boost establishment of ESCs (Yinget al., 2008; Lee et al., 2012). It appeared that R2i was more effectivefor SB-derived ESC generation when compared with other reportedmethods (Chung et al., 2006; Wakayama et al., 2007; Lorthongpanichet al., 2008; Gonzalez et al., 2010; Lee et al., 2012).
Although ICM is the prevailing source for ESC generation, the use ofSBs can relieve ethical concerns about the destruction of earlyembryos. However, the efficacy of deriving ESCs from SB is usuallylower than from ICM. Derivation of mouse ESCs from SB was firstreported in 2006. The researchers utilized the aggregation of SBs withan established ESC line to take advantage of its secretome to suppressdifferentiation (Chung et al., 2006). The success rate for this reportwas only 4% in a conventional ESC culture condition that included MEFand medium that contained serum. Subsequently, a more successfulresult was reported by Wakayama et al. (2007) by cultivation of SB inKOSR-based medium that contained ACTH 1–24 on 96-well MEF-coated plates. This report indicated that whenever all blastomeres of asingle embryo were dispersed SB-derived ESCs could be obtainedfrom two-cell, late four-cell and eight-cell embryos with efficiencies of33, 8 and 8%, respectively. The authors implied that only one blastomerefrom either four- or eight-cell embryos could give rise to ESCs. In con-trast, our study demonstrated that R2i caused the derivation of threeESCs from all three SBs of four-cell embryos (Table II). As well, two tofive SBs from single eight-cell embryos perpetuated as ESCs (Table III).The other value of this study was the use of the refractory mousestrains to generate ESCs. Most previous studies have used the hybridof a refractory mouse strain, such as C57BL/6, and a highly permissive129/Sv imprinting control region (ICR) strain for SB-derived ESCs estab-lishment (Chung et al., 2006; Wakayama et al., 2007; Lee et al., 2012).However, we utilized the purebred NMRI and BALB/c mouseembryos, which are considered to be recalcitrant strains for ESC gener-ation (Brook and Gardner, 1997; Baharvand et al., 2004). In a previousstudy, we were unable to derive ESCs from outbred NMRI blastocystsby using conventional ESC culture condition and only F1 of theNMRI × BALB/c were able to generate ESCs (Baharvand et al., 2004).In addition, although the proliferation of SB in this study was dependenton MEF, the application of chemically defined N2B27 supplementedmedium has provided defined culture conditions for facilitating the evalu-ation of the ESCs derivation procedure.
Although it is shown that the derivation efficiency of SB-derived ESCscould be increased in the lower stages of embryo development(Wakayama et al., 2007), in this study R2i has demonstrated a high gen-eration rate of ESCs from SBs of both the eight-cell and lower stages. Thisis especially important if we extend this result for deriving human ESCs byremoving only one blastomere from eight-cell embryos, which is consist-ent with PGD experience. It is presumed that the generation of humanESCs from PGD blastomeres can circumvent the ethical concern regard-ing destruction of human embryos. Recently, we have successfullyderived human ESCs from SB of six- to eight-cell embryos by use ofthe chemical inhibitors of GSK3 and Rho-associated kinase signal trans-duction with an efficiency of up to 31% of the outgrowths (Taei et al.,2013). Presumptively, the inhibition of TGFb signaling might adverselyimpact establishment of human ESCs (Hassani et al., 2014a,b). Wewere unable to generate human ESCs from SBs using SB431542 (Taeiet al., 2013). In contrast, a recent study showed that the treatment ofhuman embryos with SB431542 led to an increase in the number ofNANOG-positive ICM cells and permitted the generation of ESCsfrom human blastocysts (Van der Jeught et al., 2014). However, thenumber of human blastocysts used for this study was insufficient to indi-cate if SB affected the efficacy of human ESC establishment.
In summary, these findings have revealed the pivotal role of TGFb sig-naling in ESC derivation from SBs of cleavage-stage embryos regardless ofthe embryonic stages and strainof mice. These findings maycontribute tothe answers to the fundamental questions in developmental capacity ofearly embryonic blastomeres. Further experiments, by modulatingextrinsic signaling, may facilitate the extension of the present study tosuccessful generation of one biopsied SB-derived ESC for autogeneicor allogeneic matched pluripotent cells without the destruction ofviable human embryos.
Supplementary dataSupplementary data areavailable athttp://humrep.oxfordjournals.org/.
AcknowledgementsWe thank the members of the Department of Stem Cells and Develop-mental Biology labs for their helpful suggestions and critical reading of thismanuscript.
Authors’ rolesS.-N.H., M.P. and H.B. designed all experiments. S.-N.H. and H.B.interpreted data and wrote the manuscript. A.T. performed immu-nostaining and ALK staining. B.A. prepared embryos and generatedchimeric mice.
FundingThis study was funded by grants provided from Royan Institute, theIranian Council of Stem Cell Research and Technology and the IranNational Science Foundation (INSF).
Gain of pluripotency from isolated blastomeres 9
by guest on June 25, 2014http://hum
rep.oxfordjournals.org/D
ownloaded from
Conflict of interestNone declared.
ReferencesAttari F, Sepehri H, Ansari H, Hassani SN, Esfandiari F, Asgari B, Shahverdi A,
Baharvand H. Efficient induction of pluripotency in primordial germ cells bydual inhibition of TGFbeta and ERK signaling pathways. Stem Cells Dev2014;23:1050–1061.
Baharvand H, Matthaei KI. Culture condition difference for establishment ofnew embryonic stem cell lines from the C57BL/6 and BALB/c mousestrains. In Vitro Cell Dev Biol Anim 2004;40:76–81.
Baharvand H, Taee A, Moulamohammadi S, Masumi M, Jafari H, Hatami M,Zarei-Moradi SH. Effects of mouse strain on establishment of embryonicstem cell lines. J Iran Anat Sci 2004;2:21–31.
Batlle-Morera L, Smith A, Nichols J. Parameters influencing derivationof embryonic stem cells from murine embryos. Genesis 2008;46:758–767.
Blair K, Wray J, Smith A. The liberation of embryonic stem cells. PLoS Genet2011;7:e1002019.
Brook FA, Gardner RL. The origin and efficient derivation of embryonic stemcells in the mouse. Proc Natl Acad Sci USA 1997;94:5709–5712.
Chung Y, Klimanskaya I, Becker S, Marh J, Lu SJ, Johnson J, Meisner L, Lanza R.Embryonic and extraembryonic stem cell lines derived from single mouseblastomeres. Nature 2006;439:216–219.
Gonzalez S, Ibanez E, Santalo J. Establishment of mouse embryonic stem cellsfrom isolated blastomeres and whole embryos using three derivationmethods. J Assist Reprod Genet 2010;27:671–682.
Hassani SN, Totonchi M, Farrokhi A, Taei A, Larijani MR, Gourabi H,Baharvand H. Simultaneous suppression of TGF-beta and ERK signalingcontributes to the highly efficient and reproducible generation of mouseembryonic stem cells from previously considered refractory andnon-permissive strains. Stem Cell Rev 2012;8:472–481.
Hassani SN, Totonchi M, Gourabi H, Scholer HR, Baharvand H. Signalingroadmap modulating naive and primed pluripotency. Stem Cells Dev2014a;23:193–208.
Hassani SN, Totonchi M, Sharifi-Zarchi A, Mollamohammadi S, Pakzad M,Moradi S, Samadian A, Masoudi N, Mirshahvaladi S, Farrokhi A et al.
Inhibition of TGFbeta signaling promotes ground state pluripotency.Stem Cell Rev 2014b;10:16–30.
Lee KH, Chuang CK, Guo SF, Tu CF. Simple and efficient derivation of mouseembryonic stem cell lines using differentiation inhibitors or proliferationstimulators. Stem Cells Dev 2012;21:373–383.
Lorthongpanich C, Yang SH, Piotrowska-Nitsche K, Parnpai R, Chan AW.Chemical enhancement in embryo development and stem cellderivation from single blastomeres. Cloning Stem Cells 2008;10:503–512.
Ogawa K, Matsui H, Ohtsuka S, Niwa H. A novel mechanism for regulatingclonal propagation of mouse ES cells. Genes Cells 2004;9:471–477.
Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M,Rogers D. Inhibition of pluripotential embryonic stem cell differentiationby purified polypeptides. Nature 1988;336:688–690.
Taei A, Hassani SN, Eftekhari-Yazdi P, Rezazadeh Valojerdi M,Nokhbatolfoghahai M, Masoudi NS, Pakzad M, Gourabi H, Baharvand H.Enhanced generation of human embryonic stem cells from singleblastomeres of fair and poor-quality cleavage embryos via inhibition ofglycogen synthase kinase beta and Rho-associated kinase signaling. HumReprod 2013;28:2661–2671.
Van der Jeught M, Heindryckx B, O’Leary T, Duggal G, Ghimire S, Lierman S,Van Roy N, Chuva de Sousa Lopes SM, Deroo T, Deforce D et al.Treatment of human embryos with the TGFbeta inhibitor SB431542increases epiblast proliferation and permits successful human embryonicstem cell derivation. Hum Reprod 2014;29:41–48.
Wakayama S, Hikichi T, Suetsugu R, Sakaide Y, Bui HT, Mizutani E,Wakayama T. Efficient establishment of mouse embryonic stem cell linesfrom single blastomeres and polar bodies. Stem Cells 2007;25:986–993.
Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP,Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukaemiainhibitory factor maintains the developmental potential of embryonicstem cells. Nature 1988;336:684–687.
Wray J, Kalkan T, Smith AG. The ground state of pluripotency. Biochem SocTrans 2010;38:1027–1032.
Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteinssuppresses differentiation and sustains embryonic stem cell self-renewalin collaboration with STAT3. Cell 2003;115:281–292.
Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P,Smith A. The ground state of embryonic stem cell self-renewal. Nature2008;453:519–523.
10 Hassani et al.
by guest on June 25, 2014http://hum
rep.oxfordjournals.org/D
ownloaded from
