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Divergent Levels of LBP and TGFβ1 in Murine MSCs Lead to Heterogenic Response to TLR and Proinflammatory Cytokine Activation

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Abstract

The outstanding heterogeneity of stem cell populations is a major obstacle on the way to their clinical application. It is therefore paramount to identify the molecular mechanisms that underlay this heterogeneity. Individually derived bone marrow mesenchymal stromal cells (MSCs) preparations, studied here, diverged markedly in various properties, despite of being all tripotent in their differentiation potential. Microarray analysis showed that MSC diversity is evident also in highly variable gene expression patterns. Differentially expressed genes were significantly enriched in toll-like receptors (TLRs) and differentiation pathways. Marked differences were observed in LPS binding protein (LBP) and transforming growth factor (TGF)β1 expression. These differences correlated with MSC functionality. Therefore, the possible contribution of these molecules to MSC diversity was examined. In the TLR signaling pathway, LBP levels predicted the ability of specific MSCs to secrete interleukin (IL)-6 in response to LPS. A relatively higher expression of TGFβ1 endowed MSCs with a capacity to respond to IL-1β by reduced osteogenic differentiation. This study thus demonstrates major diversity within MSC isolates, which appears early on following derivation and persists following long–term culture. MSC heterogeneity results from highly variable transcriptome. Differential expression of LBP and TGFβ1, along with other genes, in different MSC preparations, produces the variable responses to external stimuli.

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Acknowledgments

This study was supported by a research grant from the Leona M. and Harry B. Helmsley Charitable Trust and by Roberto and Renata Ruhman, Brazil. D.Z. is the incumbent of the Joe and Celia Weinstein Professorial Chair.

Conflict of Interest Disclosures

The authors declare no conflict of interest.

Author Contributions

S.L and M.P.F designed, performed experiments and wrote the paper, S.K designed and performed experiments. H. L, D.G and A.W performed experiments. G.F analyzed data. D.Z supervised the project.

Author information

Authors and Affiliations

  1. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 76100

    Sarit Levin, Meirav Pevsner-Fischer, Sivan Kagan, Hila Lifshitz, Ada Weinstock, Diana Gataulin & Dov Zipori

  2. The Biological Services Unit, Weizmann Institute of Science, Rehovot, Israel, 76100

    Gilgi Friedlander

  3. Department of Molecular Cell Biology, Weizmann Institute of Science, 234 Herzel St., Rehovot, 7632700, Israel

    Dov Zipori

Authors
  1. Sarit Levin
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  2. Meirav Pevsner-Fischer
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  3. Sivan Kagan
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  4. Hila Lifshitz
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  5. Ada Weinstock
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  6. Diana Gataulin
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  7. Gilgi Friedlander
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  8. Dov Zipori
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Corresponding author

Correspondence to Dov Zipori.

Additional information

Sarit Levin and Meirav Pevsner-Fischer contributed equally to this work.

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Figure 1S

Tripotent MSCs express functional TLRs. (A) MSCs I-V were induced to differentiate into osteocytes, adipocytes or chondrocytes. After up to 3 weeks, cell cultures were fixed and stained with Alizarin red, Oil red O or Alcian Blue, respectively. (B) MSCs I-V were analyzed by flow cytometry for expression of surface antigens presented by percentage of positive cells. (C) Total RNA from MSC I - V was subjected to quantitative PCR amplification with TLR1 to TLR9 specific primers. Presented are expression levels relative to HPRT. (JPEG 205 kb)

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Figure 2S

Early passage MSCs respond differently to TLR activation. MSCs of passages 4-7 were induced to differentiate into osteocytes without or with 20μg/ml of the TLR ligands Pam3Cys, PG, LPS and Poly(I:C) or 1ng/ml TNFα and IL-1β. (A) MSCs were fixed and stained with Alizarin red. Original magnifications: x10. Summary of osteogenic differentiation: No change (), augmentation () or inhibition (). (B) Alizarin red stain was extracted and quantified ((*) p < 0.05). A single repeat was conducted. (JPEG 352 kb)

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Figure 3S

Pam3Cys generally promotes MSC I-V proliferation and inhibits their basal migration. (A) MSCs proliferation was measured without or with 20μg/ml Pam3Cys, Poly(I:C), PG or LPS. (B) For migration, in vitro "wound healing" assay was performed. MSC were allowed to migrate into the “wound” without or with 20μg/mL of Pam3Cys, PG, LPS or Poly(I:C). Three to five days later, cells were fixed and stained. Original magnifications: x12.5. (C) The diameters of the circles were measured and quantified using Adobe Photoshop 7.0 software. The results represent the means ± SD of a total of 8 “wounds” per treatment ((*) p < 0.05). One representative experiment out of least 3 repeats is presented. (JPEG 207 kb)

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Figure 4S

MSC clones within an individual mouse respond differently to TLR activation. MSC clones were induced to differentiate into osteocytes without or with 20μg/ml of the TLR ligands Pam3Cys, PG, LPS and Poly(I:C). (A) Clones from mouse #2 were fixed and stained with Alizarin red. Original magnifications: x10. Summary of osteogenic differentiation: No change (), augmentation () or inhibition (). (B) Alizarin red stain was extracted and quantified. Shown quantifications of mouse #2 clones ((*) p < 0.05). Representative experiment out of at least 6 repeats are shown. (JPEG 422 kb)

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Figure 5S

Pam3cys and Poly(I:C) induce NFkB translocation to the nucleus in MSCs I-V. MSCs were incubated with 1μg/ml Pam3Cys (A) or Poly(I:C) (B) for the indicated times. Nuclear extracts were blotted with anti-NFkB or anti-nucleolin antibodies. One representative experiment out of 2 repeats is presented. (JPEG 171 kb)

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Figure 6S

MSCs I-V exhibit similar phosphorylation kinetics of ERK, JNK and p38 in response to TLR activation. MSCs were incubated with 1μg/ml Pam3Cys or Poly(I:C) for the indicated times. MSCs were harvested and proteins were extracted. The extracts were quantified, run on SDS-PAGE gel and blotted with anti-phosphorylated or anti total ERK1/2, P38 or JNK antibodies. Representative results from 3 repeats are presented. (JPEG 264 kb)

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Figure 7S

MSC clones within an individual mouse respond heterogeneously to IL-1β and TNFα activation. MSC clones were induced to differentiate into osteocytes with or without 1ng/ml IL-1β or 1ng/ml TNFα. (A) Summary of osteogenic differentiation: No change (), augmentation () or inhibition (). (B) Cell cultures were fixed and stained with Alizarin red. Original magnifications: x10. (C) Alizarin red stain was extracted and quantified. ((*) p < 0.05). Representative experiment out of at least 6 repeats is presented. (JPEG 566 kb)

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Table 1

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Table 2

List of 100 most differentially expressed genes (JPEG 182 kb)

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ESM 1

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Levin, S., Pevsner-Fischer, M., Kagan, S. et al. Divergent Levels of LBP and TGFβ1 in Murine MSCs Lead to Heterogenic Response to TLR and Proinflammatory Cytokine Activation. Stem Cell Rev and Rep 10, 376–388 (2014). https://doi.org/10.1007/s12015-014-9498-z

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