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The Intercellular Communication Between Mesenchymal Stromal Cells and Hematopoietic Stem Cells Critically Depends on NF-κB Signalling in the Mesenchymal Stromal Cells

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Abstract

Mesenchymal stromal cells (MSCs) regulate the fate of the hematopoietic stem cells (HSCs) through both cell-cell interactions and paracrine mechanisms involving multiple signalling pathways. We have previously shown that co-culturing of HSCs with CoCl2-treated MSCs expands functional HSCs. While performing these experiments, we had observed that the growth of CoCl2-treated MSCs was significantly stunted. Here, we show that CoCl2-treated MSCs possess activated NF-κB signalling pathway, and its pharmacological inhibition significantly relieves their growth arrest. Most interestingly, we found that pharmacological inhibition of NF-κB pathway in both control and CoCl2-treated MSCs completely blocks their intercellular communication with the co-cultured hematopoietic stem and progenitor cells (HSPCs), resulting in an extremely poor output of hematopoietic cells. Mechanistically, we show that this is due to the down-regulation of adhesion molecules and various HSC-supportive factors in the MSCs. This loss of physical interaction with HSPCs could be partially restored by treating the MSCs with calcium ionophore or calmodulin, suggesting that NF-κB regulates intracellular calcium flux in the MSCs. Importantly, the HSPCs co-cultured with NF-κB-inhibited-MSCs were in a quiescent state, which could be rescued by re-culturing them with untreated MSCs. Our data underscore a critical requirement of NF-κB signalling in the MSCs in intercellular communication between HSCs and MSCs for effective hematopoiesis to occur ex vivo. Our data raises a cautionary note against excessive use of anti-inflammatory drugs targeting NF-κB.

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Acknowledgements

The authors wish to thank Symbiosis Centre for Research & Innovation, Symbiosis International (Deemed University) (SIU) for providing infrastructural support.

Funding

This study was funded by SIU. SP was provided with a Senior Research Fellowship (SRF) by SIU.

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  1. Symbiosis Centre for Stem Cell Research, Symbiosis International (Deemed University), Gram: Lavale, Taluka: Mulshi, Pune, 412115, Maharashtra, India

    Shalmali Pendse, Vaijayanti Kale & Anuradha Vaidya

  2. Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Gram: Lavale, Taluka: Mulshi, Pune, 412115, Maharashtra, India

    Shalmali Pendse, Vaijayanti Kale & Anuradha Vaidya

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Contributions

Investigation, Interpretation and Data Analysis, Validation, Writing – original draft preparation: SP, VK, AV; Conceptualization, Methodology, Writing –Editing; VK, AV; Visualization, Resources and Funding acquisition, Writing—Reviewing, Supervision: AV.

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Correspondence to Anuradha Vaidya.

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Supplementary Information

Supplementary Fig. 1

Flow cytometry panels showing MSCs (A) positive for MSC specific markers (CD44, CD105, CD73 and CD90) and (B) negative for HSC specific markers (CD45 and CD34). (PNG 985 kb)

High Resolution Image (TIF 3110 kb)

Supplementary Fig. 2

(A) The graph represents the growth curve of MSCs treated or not with either CoCl2 or hypoxia (n=3). (*Comparison with control; #Comparison with hypoxia). (B and D) Representative images show immunofluorescence staining of MSCs treated with CoCl2 or hypoxia using antibodies against Ki67 (Scale 50μm; Magnification 20X) (B) or NF-κB1 p105/p50 (D) (Scale 20μm; Magnification 40X). (C) Graph represents the percentage of Ki67 positive (+ve) MSCs treated with various concentrations of NF-κB inhibitor. The data represent mean ± SD. * p≤0.05, ** p≤0.01, ***p≤0.001. (PNG 5081 kb)

High Resolution Image (TIF 7100 kb)

Supplementary Fig. 3

(A) Flow cytometry panel depicting the gating strategy used for phenotypic analysis of the output cells harvested from MSC-HSC co-cultures. (B) The graph represents the number of viable TNCs harvested after co-culture of HSCs with control MSCs and CoCl2-MSCs treated or not with various concentrations of NF-κB inhibitor (n=3). (C-E) The graphical representation shows the frequencies of Lin- cells (C), LSK HSCs (D), and LSK CXCR4+ cells (E) (n=3) obtained in the above mentioned co-cultures. The comparisons between the groups that are statistically not significant have not been shown in the graphs. The data represents mean ± SD. ** p≤0.01, ***p≤0.001. (PNG 2136 kb)

High Resolution Image (TIF 7086 kb)

Supplementary Fig. 4

(A-C) The mean fluorescence intensity (MFI) of (A) N-cadherin (B) integrin-α4 and (C) integrin-α5 after Calmodulin (CaM) treatment is represented graphically (n=6). (D-F) The graphs represent the mean fluorescence intensity (MFI) of N-cadherin (D), integrin-α4 (E), and integrin-α5 (F) after calcium ionophore (Cal Ion) treatment. (A.U. Arbitrary units). The comparisons between the groups that are statistically not significant have not been shown in the graphs. The data represents mean ± SD. * p≤0.05, ** p≤0.01, ***p≤0.001. *comparison with control, #comparison with NKI, @comparison between Cal Ion or CaM groups. (PNG 2430 kb)

High Resolution Image (TIF 7801 kb)

Supplementary Fig. 5

The absolute numbers of (A) Lin- cells, (B) LSK-HSCs, and (C) LT-HSCs and ST-HSCs obtained in the serial transfer experiments are graphically depicted (n=3). (D) The graph shows the frequency of Sca-1+ c-kit- and Sca-1- c-kit+ cells in the harvested cells after serial co-culture (n=3). The data represent mean ± SD. * p≤0.05, ***p≤0.001. (PNG 1016 kb)

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Pendse, S., Kale, V. & Vaidya, A. The Intercellular Communication Between Mesenchymal Stromal Cells and Hematopoietic Stem Cells Critically Depends on NF-κB Signalling in the Mesenchymal Stromal Cells. Stem Cell Rev and Rep 18, 2458–2473 (2022). https://doi.org/10.1007/s12015-022-10364-6

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