Skip to main content
Log in

Evaluating differentiation propensity of in-house derived human embryonic stem cell lines KIND-1 and KIND-2

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

Human embryonic stem (hES) cells possess the ability to self-renew indefinitely and provide a potential source of differentiated progeny representing all three embryonic germ layers. Although hES cell lines share the expression of typical pluripotency markers, limited data is available regarding their differentiation capabilities. We have earlier reported the in-house derivation of two hES cell lines, KIND-1 and KIND-2 on human feeders. Here, we describe a comparative study carried out on both these cell lines to better understand the differentiation potential of KIND-1 and KIND-2 by gene expression analysis of representative gene transcripts reflecting pluripotency and the three germ layers viz. ectoderm, mesoderm, and endoderm. Gene expression analysis and immunolocalization studies were undertaken on (a) 7- and 14-d old embryoid bodies (EBs) (b) spontaneously differentiated cells from EBs, (c) cells derived from EBs under the influence of various growth factor treatments and (d) KIND-1 and KIND-2 cells co-cultured on mouse embryonic visceral endoderm-like feeder (END-2). Despite both the cell lines being XX, derived, passaged, and cultured similarly, KIND-1 exhibits preferential differentiation towards endodermal lineage whereas KIND-2 spontaneously forms beating cardiomyocytes. Perhaps the occurrence of discrete epigenetic profile in both the cell lines predisposes them to encompass different developmental potential in vitro. Our data provide evidence for existence of distinct differentiation propensity among hES cell lines and emphasizes the need to derive more hES cell lines for future regenerative medicine.

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

Access this article

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
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Abeyta M. J.; Clark A. T.; Rodriguez R. T.; Bodnar M. S.; Pera R. A.; Firpo M. T. Unique gene expression signatures of independently-derived human embryonic stem cell lines. Hum. Mol. Genet. 13: 601–608; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Arai A.; Yamamoto K.; Toyama J. Murine cardiac progenitor cells require visceral embryonic endoderm and primitive streak for terminal differentiation. Dev. Dyn. 210: 344–353; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Atlasi Y.; Mowla S. J.; Ziaee S. A.; Gokhale P. J.; Andrews P. W. OCT4 spliced variants are differentially expressed in human pluripotent and non-pluripotent cells. Stem Cells 26: 3068–3074; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Bain G.; Kitchens D.; Yao M.; Huettner J. E.; Gottlieb D. Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168: 342–357; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Beqqali A.; Kloots J.; Ward-van Oostwaard D.; Mummery C.; Passier R. Genome-wide transcriptional profiling of human embryonic stem cells differentiating to cardiomyocytes. Stem Cells 24: 1956–1967; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Bhattacharya B.; Cai J.; Luo Y.; Miura T.; Mejido J.; Brimble S. N.; Zeng X.; Schulz T. C.; Rao M. S.; Puri R. K. Comparison of the gene expression profile of undifferentiated human embryonic stem cell lines and differentiating embryoid bodies. BMC Dev. Biol. 5: 22; 2005.

    Article  PubMed  Google Scholar 

  • Bhattacharya B.; Puri S.; Puri R. K. A review of gene expression profiling of human embryonic stem cell lines and their differentiated progeny. Curr. Stem Cell Res. Ther. 4: 98–106; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Bibikova M.; Chudin E.; Wu B.; Zhou L.; Garcia E. W.; Liu Y.; Shin S.; Plaia T. W.; Auerbach J. M.; Arking D. E.; Gonzalez R.; Crook J.; Davidson B.; Schulz T. C.; Robins A.; Khanna A.; Sartipy P.; Hyllner J.; Vanguri P.; Savant-Bhonsale S.; Smith A. K.; Chakravarti A.; Maitra A.; Rao M.; Barker D. L.; Loring J. F.; Fan J. B. Human embryonic stem cells have a unique epigenetic signature. Genome Res. 16: 1075–1083; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Burridge P. W.; Anderson D.; Priddle H.; Barbadillo Munoz M. D.; Chamberlain S.; Allegrucci C.; Young L. E.; Denning C. Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells 25: 929–938; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Chen H. F.; Kuo H. C.; Chien C. L.; Shun C. T.; Yao Y. L.; Ip P. L.; Chuang C. Y.; Wang C. C.; Yang Y. S.; Ho H. N. Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum. Reprod. 22: 567–577; 2007.

    Article  PubMed  Google Scholar 

  • D’Amour K. A.; Bang A. G.; Eliazer S.; Kelly O. G.; Agulnick A. D.; Smart N. G.; Moorman M. A.; Kronn E.; Carpenter M. K.; Baetge E. E. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 24: 1392–1401; 2006.

    Article  PubMed  Google Scholar 

  • Denning C.; Allegrucci C.; Priddle H.; Barbadillo-Munoz M. D.; Anderson D.; Self T.; Smith N. M.; Parkin C. T.; Young L. E. Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7. Int. J. Dev. Biol. 50: 27–37; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Feki A.; Hovatta O.; Jaconi M. Letter to the Editor: Derivation of human embryonic stem cell lines from single cells of 4-cell stage embryos: be aware of the risks. Hum. Reprod. 23: 2874; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Gan Q.; Yoshida T.; McDonald O. G.; Owens G. K. Concise review: epigenetic mechanisms contribute to pluripotency and cell lineage determination of embryonic stem cells. Stem Cells 25: 2–9; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Geens M.; Mateizel I.; Sermon K.; De Rycke M.; Spits C.; Cauffman G.; Devroey P.; Tournaye H.; Liebaers I.; Van de Velde H. Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos. Hum. Reprod. 24: 2709–2717; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Hart A. H.; Hartley L.; Ibrahim M.; Robb L. Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human. Dev. Dyn. 230: 187–198; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Hoffman L. M.; Carpenter M. K. Human embryonic stem cell stability. Stem Cell Rev. 1: 139–144; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Hoffman L. M.; Hall L.; Batten J. L.; Young H.; Pardasani D.; Baetge E. E.; Lawrence J.; Carpenter M. K. X-inactivation status varies in human embryonic stem cell lines. Stem Cells 23: 1468–1478; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Hyslop L.; Stojkovic M.; Armstrong L.; Walter T.; Stojkovic P.; Przyborski S.; Herbert M.; Murdoch A.; Strachan T.; Lako M. Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages. Stem Cells 23: 1035–1043; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Inamdar M. S.; Venu P.; Srinivas M. S.; Rao K.; VijayRaghavan K. Derivation and characterization of two sibling human embryonic stem cell lines from discarded grade III embryos. Stem Cells Dev. 18: 423–433; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Kim S. E.; Kim B. K.; Gil J. E.; Kim S. K.; Kim J. H. Comparative analysis of the developmental competence of three human embryonic stem cell lines in vitro. Mol. Cells 23: 49–56; 2007.

    PubMed  CAS  Google Scholar 

  • Klimanskaya I.; Chung Y.; Becker S.; Lu S. J.; Lanza R. Derivation of human embryonic stem cells from single blastomeres. Nat. Protoc. 2: 1963–1972; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Kumar N.; Hinduja I.; Nagvenkar P.; Pillai L.; Zaveri K.; Mukadam L.; Telang J.; Desai S.; Mangoli V.; Mangoli R.; Padgaonkar S.; Kaur G.; Puri C.; Bhartiya D. Derivation and characterization of two genetically unique human embryonic stem cell Lines on in-house-derived human feeders. Stem Cells Dev. 18: 67–77; 2009.

    Article  Google Scholar 

  • Lagarkova M. A.; Volchkov P. Y.; Lyakisheva A. V.; Philonenko E. S.; Kiselev S. L. Diverse epigenetic profile of novel human embryonic stem cell lines. Cell Cycle 5: 416–420; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Lakshmipathy U.; Love B.; Adams C.; Thyagarajan B.; Chesnut J. D. Micro RNA profiling: an easy and rapid method to screen and characterize stem cell populations. Meth. Mol. Biol. 407: 97–114; 2007a.

    Article  CAS  Google Scholar 

  • Lakshmipathy U.; Love B.; Goff L. A.; Jornsten R.; Graichen R.; Hart R. P.; Chestnut J. D. MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells. Stem Cells Dev. 16: 1003–1016; 2007b.

    Article  PubMed  CAS  Google Scholar 

  • Li J. Y.; Pu M. T.; Hirasawa R.; Li B. Z.; Huang Y. N.; Zeng R.; Jing N. H.; Chen T.; Li E.; Sasaki H.; Xu G. L. Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog. Mol. Cell. Biol. 27: 8748–8759; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Loser P.; Schirm J.; Guhr A.; Wobus A. M.; Kurtz A. Human embryonic stem cell lines and their use in international research. Stem Cells 28: 240–246; 2010.

    PubMed  Google Scholar 

  • Lough J.; Sugi Y. Endoderm and heart development. Dev. Dyn. 217: 327–342; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Mandal A.; Bhowmik S.; Patki A.; Viswanathan C.; Majumdar A. S. Derivation, characterization, and gene expression profile of two new human ES cell lines from India. Stem Cell Res. 5: 173–187; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Mandal A.; Tipnis S.; Pal R.; Ravindran G.; Bose B.; Patki A.; Rao M. S.; Khanna A. Characterization and in vitro differentiation potential of a new human embryonic stem cell line, ReliCellhES1. Differentiation 74: 81–90; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Mehta A.; Mathew S.; Viswanathan C.; Sen Majumdar A. Intrinsic properties and external factors determine differentiation bias of human embryonic stem cell lines. Cell Biol. Int. 34: 1021–1031; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Mikkola M.; Olsson C.; Palgi J.; Ustinov J.; Palomaki; Horelli-Kuitunen N.; Knuutila S.; Lundin K.; Otonkoski T.; Tuuri T. Distinct differentiation characteristics of individual human embryonic stem cell lines. BMC Dev. Biol. 6: 40; 2006.

    Article  PubMed  Google Scholar 

  • Mummery C.; Ward D.; Passier R. Differentiation of human embryonic stem cells to cardiomyocytes by co-culture with endoderm in serum-free medium. Curr. Protoc. Stem Cell Biol. Chap 1: Unit 1F.2; 2007.

  • Mummery C.; Ward-van Oostwaard D.; Doevendans P.; Spijker P.; van den Brink S.; Hassink R.; van der Heyden M.; Opthof T.; Pera M.; de la Riviere A. B.; Passier R.; Tertoolen L. Differentiation of human embryonic stem cells to cardiomyocytes: role of co-culture with visceral endoderm-like cells. Circulation 107: 2733–2740; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Nascone N.; Mercola M. An inductive role for the endoderm in Xenopus cardiogenesis. Development 121: 515–523; 1995.

    PubMed  CAS  Google Scholar 

  • Niwa H.; Miyazaki J.; Smith A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24: 372–376; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Oh S. K.; Kim H. S.; Ahn H. J.; Seol H. W.; Kim Y. Y.; Park Y. B.; Yoon C. J.; Kim D. W.; Kim S. H.; Moon S. Y. Derivation and characterization of new human embryonic stem cell lines: SNUhES1, SNUhES2, and SNUhES3. Stem Cells 23: 211–219; 2005.

    Article  PubMed  Google Scholar 

  • Osafune K.; Caron L.; Borowiak M.; Martinez R. J.; Fitz-Gerald C. S.; Sato Y.; Cowan C. A.; Chien K. R.; Melton D. A. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26: 313–315; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Pal R.; Totey S.; Mamidi M. K.; Bhat V. S.; Totey S. Propensity of human embryonic stem cell lines during early stage of lineage specification controls their terminal differentiation into mature cell types. Exp. Biol. Med. 234: 1230–1243; 2009.

    Article  CAS  Google Scholar 

  • Park Y. B.; Kim Y. Y.; Oh S. K.; Chung S. G.; Ku S. Y.; Kim S. H.; Choi Y. M.; Moon S. Y. Alterations of proliferative and differentiation potentials of human embryonic stem cells during long-term culture. Exp. Mol. Med. 40: 98–108; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Pekkanen-Mattila M.; Kerkela E.; Tanskanen J. M.; Pietila M.; Pelto-Huikko M.; Hyttinen J.; Skottman H.; Suuronen R.; Aalto-Setala K. Substantial variation in the cardiac differentiation of human embryonic stem cell lines derived and propagated under the same conditions—a comparison of multiple cell lines. Ann. Med. 41: 360–370; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Pevny L. H.; Sockanathan S.; Placzek M.; Lovell-Badge R. A role of SOX1 in neural determination. Development 125: 1967–1978; 1998.

    PubMed  CAS  Google Scholar 

  • Rohwedel J.; Maltsev V.; Bober E.; Arnold H. H.; Hescheler J.; Wobus A. M. Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev. Biol. 164: 87–101; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Saga Y.; Miyagawa-Tomita S.; Takagi A.; Kitajima S.; Miyazaki J.; Inoue I. MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Development 126: 3437–3447; 1999.

    PubMed  CAS  Google Scholar 

  • Schuldiner M.; Yanuka O.; Itskovitz-Eldor J.; Melton D. A.; Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. PNAS 97: 11307–11312; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Schultheiss T. M.; Xydas S.; Lassar A. B. Induction of avian cardiac myogenesis by anterior endoderm. Development 121: 4203–4214; 1995.

    PubMed  CAS  Google Scholar 

  • Skottman H.; Mikkola M.; Lundin K.; Olsson C.; Stromberg A. M.; Tuuri T.; Otonkoski T.; Hovatta O.; Lahesmaa R. Gene expression signatures of seven individual human embryonic stem cell lines. Stem Cells 23: 1343–1356; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Slager H. G.; Van Inzen W.; Freund E.; Van der Eijnden-Van Raaij A. J.; Mummery C. L. Transforming growth factor-beta in the early mouse embryo: implications for the regulation of muscle formation and implantation. Dev. Genet. 14: 212–224; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Strelchenko N.; Verlinsky O.; Kukharenko V.; Verlisnky Y. Morula-derived embryonic stem cells. Reprod. Biomed. Online 9: 623–629; 2004.

    Article  PubMed  Google Scholar 

  • Tavakoli T.; Xu X.; Derby E.; Serebryakova Y.; Reid Y.; Rao M. S.; Mattson M. P.; Ma W. Self-renewal and differentiation capabilities are variable between human embryonic stem cell lines I3, I6 and BG01V. BMC Cell Biol. 10: 44; 2009.

    Article  PubMed  Google Scholar 

  • Thomson J. A.; Itskovitz-Eldor J.; Shapiro S. S.; Waknitz M. A.; Swiergiel J. J.; Marshall V. S.; Jones J. M. Embryonic stem cell lines derived from human blastocysts. Science 282: 1145–1147; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Tropepe V.; Hitoshi S.; Sirard C.; Mak T. W.; Rossant J.; van der Kooy D. Direct neural fate specification from embryonic stem cells: A primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 30: 65–78; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Venu P.; Chakraborty S.; Inamdar M. S. Analysis of long-term culture properties and pluripotent character of two sibling human embryonic stem cell lines derived from discarded embryos. In Vitro Cell. Dev. Biol. Anim. 46: 200–205; 2010.

    Article  PubMed  Google Scholar 

  • Weitzer W. Embryonic stem cell-derived embryoid bodies: An in vitro model of eutherian pregastrulation development and early gastrulation. Handb. Exp. Pharmacol. 174: 21–51; 2006.

    Article  PubMed  Google Scholar 

  • Yao S.; Chen S.; Clark J.; Hao E.; Beattie G. M.; Hayek A.; Ding S. Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions. PNAS 103: 6907–6912; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Zeineddine D.; Papadimou E.; Chebli K.; Gineste M.; Liu J.; Grey C.; Thurig S.; Behfar A.; Wallace V. A.; Skerjanc I. S.; Puceat M. Oct3/4 dose dependently regulates specification of embryonic stem cells towards a cardiac lineage and early heart development. Dev. Cell 11: 535–546; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Zhang X.; Stojkovic P.; Przyborski S.; Cooke M.; Armstrong L.; Lako M.; Stojkovic M. Derivation of human embryonic stem cells from developing and arrested embryos. Stem Cells 24: 2669–2676; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Zhou J.; Ou-Yang Q.; Li J.; Zhou X. Y.; Lin G.; Lu G. X. Human feeder cells support establishment and definitive endoderm differentiation of human embryonic stem cells. Stem Cells Dev. 17: 737–749; 2008.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Professor Christine Mummery for providing the END-2 cells. We are grateful to Dr E Baetge for providing RNA sample (a pool of various fetal human endoderm tissues and differentiated CyT49 cells). This study was financially supported by the Department of Biotechnology, Govt. of India.

Conflict of interest

The authors declare that they have no competing financial interests.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Deepa Bhartiya.

Additional information

Editor: T. Okamoto

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nagvenkar, P., Pethe, P., Pawani, H. et al. Evaluating differentiation propensity of in-house derived human embryonic stem cell lines KIND-1 and KIND-2. In Vitro Cell.Dev.Biol.-Animal 47, 406–419 (2011). https://doi.org/10.1007/s11626-011-9420-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-011-9420-9

Keywords

Navigation