BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells

PLOS ONE, Jun 2010

Background Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells. Methodology/Principal Findings We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP-6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation. Conclusions/Significance Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance.

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BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells

Choe S (2010) BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells. PLoS ONE 5(6): e11167. doi:10.1371/journal.pone.0011167 BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells Elvira Valera 0 Michael J. Isaacs 0 Yasuhiko Kawakami 0 Juan Carlos Izpisu a Belmonte 0 Senyon 0 Catherine M. Verfaillie, Katholieke Universiteit Leuven, Belgium 0 1 Structural Biology Laboratory, Salk Institute, La Jolla, California, United States of America, 2 Joint Center for Biosciences , Incheon , Korea , 3 Gene Expression Laboratory, Salk Institute , La Jolla, California , United States of America Background: Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells. Methodology/Principal Findings: We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation. Conclusions/Significance: Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance. - Funding: This work was partly supported by grants from the National Institutes of Health HD013527 (SC) and HD042167 (JCIB), CIRM (SC), Korea Stem Cell Research Center and WCU (SC), and the G. Harold and Leila Y. Mathers Charitable Foundation (JCIB). EV was the recipient of a fellowship from the Ministerio de Ciencia e Innovacio n, Instituto de Salud Carlos III (Spain) in collaboration with the Salk Institute for Biological Studies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Embryonic stem (ES) cell lines derived from the epiblast tissue of the inner cell mass of a blastocyst or earlier morula stage embryos are pluripotent and can develop to the three primary germ layers: ectoderm, endoderm and mesoderm. For that reason, human embryonic stem (hES) cells represent an excellent clinical source of precursor cells for cell-based therapy if they can be guided to differentiate into a certain cell type needed to treat damaged tissue in chronic diseases or injuries [1]. In addition to tissue regeneration, hES cell derivatives can be used in studies of cellular development, tumorigenesis, and in the discovery or cytotoxicity screening of new drug candidates [2]. Different members of the Transforming Growth Factor b (TGFb) family have been implicated in various developmental stages and processes. One subfamily, the Bone Morphogenetic Proteins (BMPs), have been traditionally studied with regard to bone and cartilage development, but recently their effects have been studied in mouse and human stem cell cultures [3]. Murine and human stem cells display differences in their behavior upon incubation with BMPs. BMP signaling promotes self-renewal in mouse stem cells when incubated together with Leukemia Inhibitory Factor in the absence of serum replacement or conditioned medium [4]. In contrast, inhibition of BMP signaling is a requirement for long term maintenance of hES cells [5], while incubation with BMPs is a potent inductor of differentiation of these cells in conditions that would otherwise support self-renewal [6]. The diverse members of the BMP subfamily exert different effects on hES cells. BMP-2 and BMP-6 have been involved in osteogenesis of human mesenchymal stem cells [7,8] and BMP-2 is also known to induce extraembryonic endoderm differentiation of hES ce (...truncated)


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Elvira Valera, Michael J. Isaacs, Yasuhiko Kawakami, Juan Carlos Izpisúa Belmonte, Senyon Choe. BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells, PLOS ONE, 2010, Volume 5, Issue 6, DOI: 10.1371/journal.pone.0011167