Skip to main content
SpringerLink
Log in

Investigating neural differentiation of mouse P19 embryonic stem cells in a time-dependent manner by bioinformatic, microscopic and transcriptional analyses

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

As an available cell line, mouse pluripotent P19 has been widely employed for neuronal differentiation studies. In this research, by applying the in vitro differentiation of this cell line into neuron-like cells through retinoic acid (RA) treatment, the roles of some genes including DNMT3B, ICAM1, IRX3, JAK2, LHX1, SOX9, TBX3 and THY1 in neural differentiation was investigated.

Methods and results

Bioinformatics, microscopic, and transcriptional studies were conducted in a time-dependent manner after RA-induced neural differentiation. According to bioinformatics studies, we determined the engagement of the metabolic and developmental super-pathways and pathways in neural cell differentiation, particularly focusing on the considered genes. According to our qRT-PCR analyses, JAK2, SOX9, TBX3, LHX1 and IRX3 genes were found to be significantly overexpressed in a time-dependent manner (p < 0.05). In addition, the significant downregulation of THY1, DNMT3B and ICAM1 genes was observed during the experiment (p < 0.05). The optical microscopic investigation showed that the specialized extensions of the neuron-like cells were revealed on day 8 after RA treatment.

Conclusion

Accordingly, the neural differentiation of P19 cell line and the role of the considered genes during the differentiation were proved. However, our results warrant further in vivo studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data Availability

All data in this study can be obtained from the authors.

References

  1. Yang L, Jurczak KM, Ge L, van Rijn P (2020) High-throughput screening and hierarchical topography-mediated neural differentiation of mesenchymal stem cells. Adv Healthc Mater 9(11):2000117. https://doi.org/10.1002/adhm.202000117

    Article  CAS  Google Scholar 

  2. Jedari B, Rahmani A, Naderi M, Nadri S (2020) MicroRNA-7 promotes neural differentiation of trabecular meshwork mesenchymal stem cell on nanofibrous scaffold. J Cell Biochem 121(4):2818–2827. https://doi.org/10.1002/jcb.29513

    Article  CAS  PubMed  Google Scholar 

  3. Zakrzewski W, Dobrzyński M, Szymonowicz M et al (2019) Stem cells: past, present, and future. Stem Cell Res Ther 10:68. https://doi.org/10.1186/s13287-019-1165-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. van der Heyden MA, Defize LH (2003) Twenty one years of P19 cells: what an embryonal carcinoma cell line taught us about cardiomyocyte differentiation. Cardiovasc Res 58(2):292–302. https://doi.org/10.1016/S0008-6363(02)00771-X

    Article  CAS  PubMed  Google Scholar 

  5. Lee JH, Park SJ, Nakai K (2017) Differential landscape of non-CpG methylation in embryonic stem cells and neurons caused by DNMT3s. Sci Rep 7(1):1–1. https://doi.org/10.1038/s41598-017-11800-1

    Article  CAS  Google Scholar 

  6. Nguyen LS, Fregeac J, Bole-Feysot C, Cagnard N, Iyer A, Anink J, Aronica E, Alibeu O, Nitschke P, Colleaux L (2018) Role of miR-146a in neural stem cell differentiation and neural lineage determination: relevance for neurodevelopmental disorders. Mol Autism 9(1):1–2. https://doi.org/10.1186/s13229-018-0219-3

    Article  CAS  Google Scholar 

  7. de Araujo TM, Razolli DS, Correa-da-Silva F, de Lima-Junior JC, Gaspar RS, Sidarta-Oliveira D, Victorio SC, Donato JrJ, Kim YB, Velloso LA (2019) The partial inhibition of hypothalamic IRX3 exacerbates obesity. EBioMedicine 39:448–460. https://doi.org/10.1016/j.ebiom.2018.11.048

    Article  PubMed  Google Scholar 

  8. Kong X, Gong Z, Zhang L, Sun X, Ou Z, Xu B, Huang J, Long D, He X, Lin X et al (2019) JAK2/STAT3 signaling mediates IL-6-inhibited neurogenesis of neural stem cells through DNA demethylation/methylation. Brain Behav Immun 79:159–173. https://doi.org/10.1016/j.bbi.2019.01.027

    Article  CAS  PubMed  Google Scholar 

  9. Symmank J, Bayer C, Reichard J, Pensold D, Zimmer-Bensch G (2020) Neuronal Lhx1 expression is regulated by DNMT1-dependent modulation of histone marks, vol 15. Epigenetics, pp 1259–1274. 11https://doi.org/10.1080/15592294.2020.1767372

  10. Mokabber H, Najafzadeh N, Mohammadzadeh Vardin M (2019) miR-124 promotes neural differentiation in mouse bulge stem cells by repressing Ptbp1 and Sox9. J Cell Physiol 234(6):8941–8950. https://doi.org/10.1002/jcp.27563

    Article  CAS  PubMed  Google Scholar 

  11. Lee YY, Choi HJ, Lee SY, Park SY, Kang MJ, Han J, Han JS (2020) Bcl-2 overexpression induces neurite outgrowth via the Bmp4/Tbx3/NeuroD1 cascade in H19-7 cells. Cell Mol Neurobiol 40(1):153–166. https://doi.org/10.1007/s10571-019-00732-1

    Article  CAS  PubMed  Google Scholar 

  12. Stoltz JF, de Isla N, Li YP, Bensoussan D, Zhang L, Huselstein C, Chen Y, Decot V, Magdalou J, Li N et al (2015) Stem cells and regenerative medicine: myth or reality of the 21th century. Stem Cells Int 15. https://doi.org/10.1155/2015/734731

  13. Alvarez CV, Garcia-Lavandeira M, Garcia-Rendueles ME, Diaz-Rodriguez E, Garcia-Rendueles AR, Perez-Romero S, Vila TV, Rodrigues JS, Lear PV, Bravo SB (2012) Defining stem cell types: understanding the therapeutic potential of ESCs, ASCs, and iPS cells. J Mol Endocrinol 49(2):R89–111. https://doi.org/10.1530/jme-12-0072

    Article  CAS  PubMed  Google Scholar 

  14. Wongtrakoongate P, Li J, Andrews PW (2014) DNMT3B inhibits the re-expression of genes associated with induced pluripotency. Exp Cell Res 321(2):231–239. https://doi.org/10.1016/j.yexcr.2013.11.024

    Article  CAS  PubMed  Google Scholar 

  15. Feng J, Chang H, Li E, Fan G (2005) Dynamic expression of de novo DNA methyltransferases Dnmt3a and Dnmt3b in the central nervous system. J Neurosci Res 79(6):734–746. https://doi.org/10.1002/jnr.20404

    Article  CAS  PubMed  Google Scholar 

  16. Watanabe D, Uchiyama K, Hanaoka K (2006) Transition of mouse de novo methyltransferases expression from Dnmt3b to Dnmt3a during neural progenitor cell development. Neuroscience 142(3):727–737. https://doi.org/10.1016/j.neuroscience.2006.07.053

    Article  CAS  PubMed  Google Scholar 

  17. Magli A, Incitti T, Kiley J, Swanson SA, Darabi R, Rinaldi F, Selvaraj S, Yamamoto A, Tolar J, Yuan C et al (2017) PAX7 targets, CD54, integrin α9β1, and SDC2, allow isolation of human ESC/iPSC-derived myogenic progenitors. Cell Rep 19(13):2867–2877. https://doi.org/10.1016/j.celrep.2017.06.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lee KS, Cha SH, Kang HW, Song JY, Lee KW, Ko KB, Lee HT (2013) Effects of serial passage on the characteristics and chondrogenic differentiation of canine umbilical cord matrix derived mesenchymal stem cells. Asian-Australas J Anim Sci 26(4):588. https://doi.org/10.5713/ajas.2012.12488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Xing F, Li L, Zhou C, Long C, Wu L, Lei H, Kong Q, Fan Y, Xiang Z, Zhang X (2019) Regulation and directing stem cell fate by tissue engineering functional microenvironments: scaffold physical and chemical cues. Stem cells Int. 2019. https://doi.org/10.1155/2019/2180925

  20. Li D, Sakuma R, Vakili NA, Mo R, Puviindran V, Deimling S, Zhang X, Hopyan S, Hui CC (2014) Formation of proximal and anterior limb skeleton requires early function of Irx3 and Irx5 and is negatively regulated by shh signaling. Dev Cell 29(2):233–240. https://doi.org/10.1016/j.devcel.2014.03.001

    Article  CAS  PubMed  Google Scholar 

  21. Houweling AC, Dildrop R, Peters T, Mummenhoff J, Moorman AF, Rüther U, Christoffels VM (2001) Gene and cluster-specific expression of the Iroquois family members during mouse development. Mech Dev 107(1–2):169–174. https://doi.org/10.1016/S0925-4773(01)00451-8

    Article  CAS  PubMed  Google Scholar 

  22. Holmquist ML, Lindell-Munther S, Yasui H, Jansson C, Esfandyari J, Karlsson J, Lau K, Hui CC, Bexell D, Hopyan S et al (2019) The Iroquois homeobox proteins IRX3 and IRX5 have distinct roles in Wilms tumour development and human nephrogenesis. J Pathol 247(1):86–98. https://doi.org/10.1002/path.5171

    Article  CAS  Google Scholar 

  23. Tan Z, Kong M, Wen S, Tsang KY, Niu B, Hartmann C, Chan D, Hui CC, Cheah KS (2020) IRX3 and IRX5 inhibit adipogenic differentiation of hypertrophic chondrocytes and promote osteogenesis. J Bone Miner Res 35(12):2444–2457. https://doi.org/10.1002/jbmr.4132

    Article  CAS  PubMed  Google Scholar 

  24. Somerville TD, Simeoni F, Chadwick JA, Williams EL, Spencer GJ, Boros K, Wirth C, Tholouli E, Byers RJ, Somervaille TC (2018) Derepression of the iroquois homeodomain transcription factor gene IRX3 confers differentiation block in acute leukemia. Cell Rep 22(3):638–652. https://doi.org/10.1016/j.celrep.2017.12.063

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sankar S, Yellajoshyula D, Zhang B, Teets B, Rockweiler N, Kroll KL (2016) Gene regulatory networks in neural cell fate acquisition from genome-wide chromatin association of Geminin and Zic1. Sci Rep 6(1):1–6. https://doi.org/10.1038/srep37412

    Article  CAS  Google Scholar 

  26. Kim YH, Chung JI, Woo HG, Jung YS, Lee SH, Moon CH, Suh-Kim H, Baik EJ (2010) Differential regulation of proliferation and differentiation in neural precursor cells by the Jak pathway. Stem Cells 28(10):1816–1828. https://doi.org/10.1002/stem.511

    Article  CAS  PubMed  Google Scholar 

  27. Sun F, Park KK, Belin S, Wang D, Lu T, Chen G, Zhang K, Yeung C, Feng G, Yankner BA et al (2011) Sustained axon regeneration induced by co-deletion of PTEN and SOCS3. Nature 480(7377):372–375. https://doi.org/10.1038/nature10594

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cheng X, Yeung PK, Zhong K, Zilundu PL, Zhou L, Chung SK (2019) Astrocytic endothelin-1 overexpression promotes neural progenitor cells proliferation and differentiation into astrocytes via the Jak2/Stat3 pathway after stroke. J Neuroinflammation 16(1):1–6. https://doi.org/10.1186/s12974-019-1597-y

    Article  CAS  Google Scholar 

  29. Inoue J, Ueda Y, Bando T, Mito T, Noji S, Ohuchi H (2013) The expression of LIM-homeobox genes, Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation. Dev Growth Differ 55(7):668–675. https://doi.org/10.1111/dgd.12074

    Article  CAS  PubMed  Google Scholar 

  30. Zhao Y, Kwan KM, Mailloux CM, Lee WK, Grinberg A, Wurst W, Behringer RR, Westphal H (2007) LIM-homeodomain proteins Lhx1 and Lhx5, and their cofactor Ldb1, control Purkinje cell differentiation in the developing cerebellum. Proc. Natl. Acad. Sci. U.S.A. 104(32):13182-6. https://doi.org/10.1073/pnas.0705464104

  31. Cepeda-Nieto AC, Pfaff SL, Varela-Echavarría A (2005) Homeodomain transcription factors in the development of subsets of hindbrain reticulospinal neurons. Mol Cell Neurosci 28(1):30–41. https://doi.org/10.1016/j.mcn.2004.06.016

    Article  CAS  PubMed  Google Scholar 

  32. Bröhl D, Strehle M, Wende H, Hori K, Bormuth I, Nave KA, Müller T, Birchmeier C (2008) A transcriptional network coordinately determines transmitter and peptidergic fate in the dorsal spinal cord. Dev Biol 322(2):381–393. https://doi.org/10.1016/j.ydbio.2008.08.002

    Article  CAS  PubMed  Google Scholar 

  33. Cheungm M, Briscoe J (2003) Neural crest development is regulated by the transcription factor Sox9. Development 130(23):5681-93. https://doi.org/10.1242/dev.00808

  34. Vogel JK, Wegner M (2021) Sox9 in the developing central nervous system: a jack of all trades? Neural Regen Res 16(4):676. https://doi.org/10.4103/1673-5374.295327

    Article  CAS  PubMed  Google Scholar 

  35. Scott CE, Wynn SL, Sesay A, Cruz C, Cheung M, Gaviro MV, Booth S, Gao B, Cheah KS, Lovell-Badge R et al (2010) SOX9 induces and maintains neural stem cells. Nat Neurosci 13(10):1181. https://doi.org/10.1038/nn.2646

    Article  CAS  PubMed  Google Scholar 

  36. Esmailpour T, Huang T (2012) TBX3 promotes human embryonic stem cell proliferation and neuroepithelial differentiation in a differentiation stage-dependent manner. Stem Cells 30(10):2152–2163. https://doi.org/10.1002/stem.1187

    Article  CAS  PubMed  Google Scholar 

  37. Kartikasari AE, Zhou JX, Kanji MS, Chan DN, Sinha A, Grapin-Botton A, Magnuson MA, Lowry WE, Bhushan A (2013) The histone demethylase Jmjd3 sequentially associates with the transcription factors Tbx3 and eomes to drive endoderm differentiation. EMBO J 32(10):1393–1408. https://doi.org/10.1038/emboj.2013.78

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, Schafer X, Lun Y, Lemischka IR (2006) Dissecting self-renewal in stem cells with RNA interference. Nature 442(7102):533–538. https://doi.org/10.1038/nature04915

    Article  CAS  PubMed  Google Scholar 

  39. Han J, Yuan P, Yang H, Zhang J, Soh BS, Li P, Lim SL, Cao S, Tay J, Orlov YL et al (2010) Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature 463(7284):1096–1100. https://doi.org/10.1038/nature08735

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Niwa H, Ogawa K, Shimosato D, Adachi K (2009) A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 460(7251):118–122. https://doi.org/10.1038/nature08113

    Article  CAS  PubMed  Google Scholar 

  41. Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A (2003) Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113(5):643–655. https://doi.org/10.1016/S0092-8674(03)00392-1

    Article  CAS  PubMed  Google Scholar 

  42. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S (2003) The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631–642. https://doi.org/10.1016/S0092-8674(03)00393-3

    Article  CAS  PubMed  Google Scholar 

  43. Khan SF, Damerell V, Omar R, Du Toit M, Khan M, Maranyane HM, Mlaza M, Bleloch J, Bellis C, Sahm BD, Moraes DA, Sibov TT, Pavon LF, Alvim PQ, Bonadio RS, Da Silva JR, Pic-Taylor A, Toledo OA, Marti LC, Azevedo RB, Oliveira DM et al (2020) (2016). A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells. Stem Cell Res. Ther. 7(1):1–4. https://doi.org/10.1186/s13287-016-0359-3

  44. Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, Jones SB, Brooks JD, Andrews PW, Brown PO, Thomson JA (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc. Natl. Acad. Sci. U.S.A. 100:13350–13355. https://doi.org/10.1073/pnas.2235735100

  45. Gallardo TD, Hammer RE, Garry DJ (2003) RNA amplification and transcriptional profiling for analysis of stem cell populations, vol 37. Genesis, pp 57–63. https://doi.org/10.1002/gene.10223

  46. Wei CL, Miura T, Robson P, Lim SK, Xu XQ, Lee MY, Gupta S, Stanton L, Luo Y, Schmitt J et al (2005) Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 23:166–185. https://doi.org/10.1634/stemcells.2004-0162

    Article  CAS  PubMed  Google Scholar 

  47. Sundberg M, Jansson L, Ketolainen J, Pihlajamäki H, Suuronen R, Skottman H, Inzunza J, Hovatta O, Narkilahti S (2009) CD marker expression profiles of human embryonic stem cells and their neural derivatives, determined using flow-cytometric analysis, reveal a novel CD marker for exclusion of pluripotent stem cells. Stem Cell Res 2(2):113–124. https://doi.org/10.1016/j.scr.2008.08.001

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are thankful to Graduate Office of the University of Isfahan for financially supporting the project.

Funding

This study was performed at the University of Isfahan (Isfahan, Iran) and financially supported by the Graduate.

Studies Office at this university (Grant No. 2206).

Author information

Authors and Affiliations

  1. Division of Genetics, Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Science and Technology, University of Isfahan, 81746-73441, Isfahan, Iran

    Marzieh Moazeny, Moein Dehbashi & Zohreh Hojati

  2. Division of Animal Sciences, Department of Plant and Animal Biology, Faculty of Biological Science and Technology, University of Isfahan, 81746-73441, Isfahan, Iran

    Fariba Esmaeili

Authors
  1. Marzieh Moazeny
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Moein Dehbashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zohreh Hojati
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fariba Esmaeili
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.M., M.D., Z.H. and F.E. designed the research; M.M. and M.D. carried out the research; M.D. and Z.H. analyzed the data; M.D. wrote the manuscript; and Z.H. edited the manuscript. All authors have reviewed the manuscript too.

Corresponding author

Correspondence to Zohreh Hojati.

Ethics declarations

Conflict of interest

The authors declare that no conflicts of interest exist.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Moazeny, M., Dehbashi, M., Hojati, Z. et al. Investigating neural differentiation of mouse P19 embryonic stem cells in a time-dependent manner by bioinformatic, microscopic and transcriptional analyses. Mol Biol Rep 50, 2183–2194 (2023). https://doi.org/10.1007/s11033-022-08166-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-022-08166-7

Keywords

Search

Navigation