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Pharmacological Inhibition of p38 MAPK Rejuvenates Bone Marrow Derived-Mesenchymal Stromal Cells and Boosts their Hematopoietic Stem Cell-Supportive Ability

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Abstract

The therapeutic value of mesenchymal stromal cells (MSCs) for various regenerative medicine applications, including hematopoietic stem cell transplantations (HSCT), has been well-established. Owing to their small numbers in vivo, it becomes necessary to expand them in vitro, which leads to a gradual loss of their regenerative capacity. Stress-induced mitogen-activated protein kinase p38 (p38 MAPK) signaling has been shown to compromise the MSC functions. Therefore, we investigated whether pharmacological inhibition of p38 MAPK signaling rejuvenates the cultured MSCs and boosts their functionality. Indeed, we found that the ex vivo expanded MSCs show activated p38 MAPK signaling and exhibit increased oxidative stress. These MSCs show a decreased ability to secrete salutary niche factors, thereby compromising their ability to support hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation. We, therefore, attempted to rejuvenate the cultured MSCs by pharmacological inhibition of p38 MAPK – a strategy broadly known as “priming of MSCs”. We demonstrate that priming of MSCs with a p-38 MAPK inhibitor, PD169316, boosts their niche-supportive functions via upregulation of various HSC-supportive transcription factors. These primed MSCs expand multipotent HSCs having superior homing and long-term reconstitution ability. These findings shed light on the significance of non-cell-autonomous mechanisms operative in the hematopoietic niche and point towards the possible use of pharmacological compounds for rejuvenation of ex vivo cultured MSCs. Such approaches could improve the outcome of regenerative therapies involving in vitro cultured MSCs.

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Acknowledgements

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

Funding

This study was funded by Department of Biotechnology (DBT), Ministry of Science & Technology, New Delhi, Government of India. P.B. was provided with Junior Research Fellowship (JRF) by DBT under this project (No.BT/PR23620/MED/31/368/2017).

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  1. Anuradha Vaidya

    Present address: Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Gram: Lavale, Taluka: Mulshi, Pune, 412115, India

Authors and Affiliations

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

    Pallavi Budgude, Vaijayanti Kale & Anuradha Vaidya

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

    Pallavi Budgude

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Contributions

Pallavi Budgude: Investigation, Validation, Formal Analysis, Writing – original draft preparation; Vaijayanti Kale: Conceptualization, Methodology, Formal Analysis, Reviewing & Editing; Anuradha Vaidya: Conceptualization, Visualization, Methodology, Formal Analysis, Resources, Writing—Reviewing & Editing, Supervision, Project Administration, Funding Acquisition.

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Correspondence to Anuradha Vaidya.

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All the animal experiments were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of the Symbiosis School of Biological Sciences (SSBS) (Approval Number: SSBS/IAEC/09–2016).

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12015_2021_10240_MOESM1_ESM.pdf

Supplementary file1 (PDF 1853 KB) Fig. S1: (A) The graph depicts the quantification of native p38 MAPK and p-p38 MAPK in naïve and primed MSCs as determined by densitometry of bands obtained in the blots. (B) The bar represents the ratio of phosphorylated p38 MAPK to native p38 MAPK protein. (C) The graph depicts percent cell viability of MSCs after treatment with DMSO and 10µM PD169316 for 24 and 48h, respectively (n=3). (D) A representative phase contrast image showing the morphology of ex vivo expanded MSCs when treated with DMSO and 10µM PD169316 for 24 and 48h. (10X magnification, scale = 20µm). (E) The frequency of DCF+ cells and (F) Mean Fluorescence Intensity (MFI) of DCF was measured using flow cytometry to determine the levels of intracellular ROS in naïve and primed MSCs. The data are represented as mean ± SD. ***p≤0.001

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Supplementary file2 (PDF 2173 KB) Fig. S2: Phase contrast images showing the ex vivo expansion of HSCs when they were co-cultured with (A) naïve and (B) primed MSCs (10X magnification), respectively. (C) The graph represents the total number of nucleated cells (TNCs) (n=25). (D-F) Flow panels depicting the frequency of HSC subsets such as (D) Lin- (upper panel), (E) LSK (middle panel), (F) LT-HSC and ST-HSC (lower panel) in HSCs co-cultured with naïve and primed MSCs. (G-I) Graphs depict the absolute numbers of (G) Lin-, (H) LSK, (I) LT-HSCs and ST-HSCs subsets in HSCs co-cultured with naïve and primed MSCs. The absolute number of each HSC subset was calculated from the total hematopoietic output and the percentage obtained in flow cytometry analysis (n=4). The data are represented as mean ± SD. * p≤0.05, ** p≤0.01, ***p≤0.001

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Supplementary file3 (PDF 329 KB) Fig. S3: Gating strategy used to determine the different HSC progenitors by flow cytometry is shown

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Supplementary file4 (PDF 541 KB) Fig. S4: (A-D) Flow panels depicting the frequency of HSC progenitors such as (A-B) CLPs (left panels) and (C-D) CMP, GMP and MEP (middle panels) in HSCs co-cultured with naïve and primed MSCs. (E-F) Flow panels depicting the frequency of CXCR4+ cells in naïve and primed LSK HSCs (right panels).

Supplementary file5 (XLSX 15 KB)

Supplementary file6 (DOCX 14 KB)

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Budgude, P., Kale, V. & Vaidya, A. Pharmacological Inhibition of p38 MAPK Rejuvenates Bone Marrow Derived-Mesenchymal Stromal Cells and Boosts their Hematopoietic Stem Cell-Supportive Ability. Stem Cell Rev and Rep 17, 2210–2222 (2021). https://doi.org/10.1007/s12015-021-10240-9

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