ERK and GSK3 Signaling Pathway in cipsc

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1 ERK and GSK3 Signaling Pathway in cipsc (Experiment Designs & Results) Yangqing Lu, 2/21/ Background In naïve state ESC, MEK/ERK signaling pathway is dispensable for maintenance of the pluripotence and proliferation [1]. MEK/ERK signals differentiation in mouse ESC [2] and inhibition of this pathway by using facilitate the derivation of naïve state ESC and ipsc [3]. On the contrary, human ESC, which is considered resembling the mouse primed state ESC, requires the FGF/ERK signaling for maintenance, whereas inhibition of this pathway indirectly caused differentiation [4]. FGF has been supplemented in the culture and promote the growth of chicken ESC, EGC and PGC in vitro [5-7]. Two recent reports revealed that FGF/ERK signaling was activated in chicken PGC {Macdonald, 2010 #461}{Choi, 2010 #2831} and blocking of the MEK/ERK signaling by using significantly affected the cell growth in chicken PGC cultured in vitro [8, 9], indicating the necessity of the this pathway in chicken pluripotent cells. Glycogen synthase kinase 3 (GSK-3) mediate the phosphorylation of serine and threonine amino acid residues and plays its functions in cell proliferation, migration and many others. Blocking of GSK-3 signaling has reported to help ESC proliferate and inhibit non-neural differentiation {Sato, 2004 #3117}. Additionally, ground state ESCs and ipscs have been efficiently derived by simultaneous inhibition of GSK3 and ERK signaling {Ying, 2008 #2995}{Silva, 2008 #2993}. Up to date, little is known about the role of GSK-3 signaling in avian cells. Chicken pluripotent cells (including ESC and PGC) and the derived chimeras have high promise for research in developmental biology, pharmaceutical industry and disease resistant animals, but maintenance of the pluripotency in long term culture still remains notoriously difficulty in these avian pluripotent cells. Recently, we demonstrated the possibility of generating chicken ipsc (cipsc) by using human reprogramming factors. These cipsc are proliferative, growing in dome-shape and strongly positive for SSEA1 after serial passages. Moreover, a germ cell gene DDX4 (or chicken VASA homologue) is highly expressed in these cells. These findings indicate the germ line potential of these cipsc but also leave the state of these cipsc obscure, ESC-, EpiSC- or PGC-like? Unravel of the ERK and GSK3 signaling pathway in cipsc would be helpful to further understand the nature of cipsc, thereby facilitate the long term culture and chimera production using these cells. 1. Ying, Q.L., et al., The ground state of embryonic stem cell self-renewal. Nature, (7194): p Lanner, F. and J. Rossant, The role of FGF/Erk signaling in pluripotent cells. Development, (20): p Silva, J., et al., Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol, (10): p. e Bendall, S.C., et al., IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature, (7157): p Park, T.S. and J.Y. Han, Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod Dev, (4): p Pain, B., et al., Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities. Development, (8): p van de Lavoir, M.C., et al., Germline transmission of genetically modified primordial germ

2 cells. Nature, (7094): p Macdonald, J., et al., Characterisation and germline transmission of cultured avian primordial germ cells. PLoS One, (11): p. e Choi, J.W., et al., Basic fibroblast growth factor activates MEK/ERK cell signaling pathway and stimulates the proliferation of chicken primordial germ cells. PLoS One, (9): p. e Hypothesis ERK and GSK3 signaling is required in cipsc to maintain the proliferation and pluripotency. 2 Aim i. To determine whether ERK and GSK3 signaling is required in cipsc to maintain the proliferation and pluripotency. ii. To determine whether ERK and GSK3 signaling is required in cipsc to maintain the proliferation and pluripotency.. 3 Materials i. R-ciPSC BA4 P16.15A cultured in cksr on MEF (Subjected to re-reprogramming and SSEA1 positive) ii. MEK/ERK inhibitor () iii. GSK3 Inhibitor (CHIR99021) iv. (20% KSR medium conditioned on MEF) v. medium (PGC medium, 10% Knockout DMDEM conditioned with BRL) 4 Experiment I (Priliminary): i. On MEF cipscs cultured in cksr-mef will be tripsinized and plated on 1x6-well MEF plates at a density of 2.0X10^4 cells per plate. 3 wells are supplemented with ERK inhibitor (, 1.0 μm) and the other 3 wells with vehicle (DMSO). Cells will be cultured for 7 days and AP staining will be done at the end of culture. Number of colonies positive and negative for AP will be counted respectively. Results: all the colonies form each treatment are AP positive! ii. On Matrigel cipscs cultured in cksr-mef will be tripsinized and plated on 2x12-well Matrigel plates at a density of 3.0X10^5 cells per well. 1 plate will be fed with cksr and the other plate fed with cko medium. In each plate, 6 wells are supplemented with ERK inhibitor (, 1.0μM) and the other 6 wells with vehicle (DMSO). Cells will be cultured for 3 days. At the end of the culture, 3 wells of treated or non-treated cells will be counted for total cell number and the other 3 wells will stained and counted for SSEA1 positive cell using flow cytometry.

3 Table 1. Counting assay for the effect of ERK inhibitor on the proliferation of R-ciPSC BA4 cultured on Matrigel Total live cell count Treatment Plate x10^6 Vehicle AVE S.E AVE S.E Figure 1. Counting assay for the effect of ERK inhibitor on the proliferation of R-ciPSC BA4 cultured on Matrigel Efect of on proliferation of R-ciPS BA4 Live cell Count (x10^6) a b a c a,b,c: P<

4 Table 2. Flow cytometry assay for the effect of ERK inhibitor on the pluripotency of R-ciPSC BA4 cultured on Matrigel Treatment Vehicle Well % of SSEA Ave S.E Ave S.E Figure 2. Flow cytometry assay for the effect of ERK inhibitor on the pluripotency of R-ciPSC BA4 cultured on Matrigel Efect of on pluripotency of R-ciPS BA a a,c a,d SSEA1% b a,b,c,d: P<

5 5 Experiment II (Proliferation): cipscs cultured in cksr-mef will be tripsinized and plated on 2x12-well Matrigel plates at a density of 2.0X10^5 cells per well. One plate feed with cksr and the other with cko. 1) A (3 wells): control 2) B (3 wells): Supplemented with ERK inhibitor (, 1.0μM) 3) C (3 wells): Supplemented with GSK3 inhibitor (CHIR99021, 3.0μM) 4) D (3 wells): Supplemented with 2i (, 1.0μM; CHIR99021, 3.0μM) Cells will be cultured for 4 days. At the end of the culture, cells will be dissociated with 0.2mL accutase and inactivated by 0.2mL full medium. Mix 200mL of cells suspension with 200mL Trypan Blue and count the cells number by using Cellometer. Table 3. Counting assay for the effect of 2i (ERK and GSK3 inhibitors) on the proliferation of R-ciPSC BA4 cultured on Matrigel A B C D Treatment CHIR i Well Cell count X10^ AVE S.E AVE S.E AVE S.E AVE S.E

6 Fig 3. Efect of 2i on proliferation of R-ciPS BA4 in cksr Cell Count (x10^6) a b c d 0.7 CHIR i Fig 4. Efect of 2i on proliferation of R-ciPS BA4 in cko Cell Count (x10^6) CHIR i Fig 5. Efect of cksr and cko medium on proliferation of R-ciPS BA4 Cell Count (x10^6) * * * # *p<0.01 #p<0.05 N=3 0.5 CHIR i

7 6 Experiment III (Pluripotentcy): cipscs cultured in cksr-mef will be tripsinized and plated on 8x35mm Matrigel coated plates at a density of 5.0X10^5 cells per plate. Four plates feed with cksr and the other 4 with cko. And the treatment for each plate from cksr or cko is: 1) A: control 2) B: Supplemented with ERK inhibitor (, 1.0μM) 3) C: Supplemented with GSK3 inhibitor (CHIR99021, 3.0μM) 4) D: Supplemented with 2i (, 1.0μM; CHIR99021, 3.0μM) Cells will be subcultured for 3 passages. At the end of the culture, cells from each plate will be expanded to 3 wells in 6-well plate and, upon confluency, cells will be dissociated with accutase and fixed with 4% PFA and then subjected to flow cytometry (Neg Ctrl: CEF, Pos Ctrl:R-ciPSC on MEF) to determine the percentage of SSEA1+ cells. Table 4. Flow cytometry assay for the effect of 2i (ERK and GSK3 inhibitors) on the pluripotency of R-ciPSC BA4 cultured on Matrigel SSEA1% A B C D Treatment CHIR i Well Ave SS population FS population N/A N/A AVE S.E. N/A AVE S.E N/A N/A AVE S.E. N/A AVE S.E

8 Fig 6. Effect of 2i on R-ciPSC BA4 (After 2 passages) CHIR i

9 Fig 7. Effect of cksr and cko on pluripotency of R-ciPSC # # SSEA1% #P<0.05 N= CHIR i Note: only one replicate was run in and in Fig 8. Effect inhibitor on pluripotency of R-ciPSC BA4 cultured in cko (a,b,b: P<0.05, N=3) c c SSEA1% 75.0 b 70.0 a 65.0 CHIR i

10 Fig 9. cipscs culture in present in SS and FS population, different size and granularity demonstrate different rate of SSEA1+ SS FS Rate of SSEA1+ cipsc in SS and FS population cultured in cko SSEA1% SS FS CHIR i

11 Summary 1. Proliferation cipsc cultured in cko are more proliferative thant in cksr. In cksr medium, cipscs are more proliferative in supplement of ERK inhibitor () alone or both ERK inhibitor () and GSK3 inhibitor (CHIR99021), but not in GSK3 inhibitor (CHIR99021) alone. In cko medium, no significant difference of proliferation of cipscs was seen in supplement of ERK inhibitor () and GSK3 inhibitor (CHIR99021) alone or both. 2. Pluripotency Statistically, cipscs cultured in cko are less positive for SSEA1 than in cksr. In cksr medium, no difference in rate of SSEA1+ cipscs was seen in supplement of ERK inhibitor () and GSK3 inhibitor (CHIR99021) alone or both. In cko medium, cipscs are more positive for SSEA1 in supplement of ERK inhibitor () and GSK3 inhibitor (CHIR99021) alone or both, but cells in control maintained the best morphology. Conclusion ERK and GSK3 signaling is dispensable to maintain the proliferation and pluripotency of cipsc cultured in cko, but critical for proliferation while cultured in cksr. SSEA1+ cipscs behave more like naïve ESCs (dependent of ERK and possible required LIF secreted by BRL cells in cko medium).