Stem Cells. 2017 Jan;35(1):181-196.

Quiescence Preconditioned Human Multipotent Stromal Cells Adopt a Metabolic Profile Favorable for Enhanced Survival under Ischemia.

Moya A1, Larochette N1, Paquet J1, Deschepper M1, Bensidhoum M1, Izzo V2,3,4,5, Kroemer G2,3,4,5,6,7,8, Petite H1, Logeart-Avramoglou D1.

1 Laboratory of Bioengineering and Bioimaging for Osteo-Articular tissues, UMR 7052, CNRS, Paris Diderot University, Sorbonne Paris Cité, Paris, France.
2 Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.
3 Cell Biology and Metabolomics platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France.
4 INSERM, U1138, Paris, France.
5 Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
6 Université Pierre et Marie Curie, Paris, France.
7 Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France.
8 Department of Women’s and Children’s Health, Karolinska Institute, Karolinska University Hospital Q2:07, Stockholm, Sweden.

 

Abstract

A major impediment to the development of therapies with mesenchymal stem cells/multipotent stromal cells (MSC) is the poor survival and engraftment of MSCs at the site of injury. We hypothesized that lowering the energetic demand of MSCs by driving them into a quiescent state would enhance their survival under ischemic conditions. Human MSCs (hMSCs) were induced into quiescence by serum deprivation (SD) for 48 hours. Such preconditioned cells (SD-hMSCs) exhibited reduced nucleotide and protein syntheses compared to unpreconditioned hMSCs. SD-hMSCs sustained their viability and their ATP levels upon exposure to severe, continuous, near-anoxia (0.1% O2 ) and total glucose depletion for up to 14 consecutive days in vitro, as they maintained their hMSC multipotential capabilities upon reperfusion. Most importantly, SD-hMSCs showed enhanced viability in vivo for the first week postimplantation in mice. Quiescence preconditioning modified the energy-metabolic profile of hMSCs: it suppressed energy-sensing mTOR signaling, stimulated autophagy, promoted a shift in bioenergetic metabolism from oxidative phosphorylation to glycolysis and upregulated the expression of gluconeogenic enzymes, such as PEPCK. Since the presence of pyruvate in cell culture media was critical for SD-hMSC survival under ischemic conditions, we speculate that these cells may utilize some steps of gluconeogenesis to overcome metabolic stress. These findings support that SD preconditioning causes a protective metabolic adaptation that might be taken advantage of to improve hMSC survival in ischemic environments. © 2016 AlphaMed Press.

KEYWORDS:

Autophagy; Cell metabolism; Cell survival; Human mesenchymal stromal cells; Ischemia; Quiescence

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Supplemental information

Multipotent Stromal Cells (MSCs) are ideal candidates for applications in the fields of tissue engineering (TE) and regenerative medicine due to their capability to (i) proliferate, (ii) secrete various growth factors and cytokines pertinent to new tissue formation, and (iii) differentiate into cells, such as osteoblasts, chondrocytes and adipocytes from various tissues (Figure 1). These adult stem cells are relatively easy to harvest from patients, in particular, those derived from bone marrow or adipose tissue. For these reasons, MSCs and MSCs-based therapies are widely investigated with more than 20 000 scientific studies published (PubMed) and close to 5000 clinical trials indexed on https://clinicaltrials.gov.

In the cases of bone TE several research groups, including our own, have demonstrated the beneficial effect of MSCs associated with ceramics and adequate biological factors to repair large bone defects [1] [2]. However, the clinical outcomes of these composite materials remain inferior to current gold standard treatment which is the autologous bone graft. One of the major impediments in TE and in stem cell based therapies concern the poor cell survival and engraftment upon implantation (80-90% cell-death within 2 weeks) that drastically reduces the potential benefits of such therapies [3]. While this massive cell death is multifactorial, the harsh ischemic microenvironment (lack of both nutrients and oxygen supplies) cells experience after implantation is the prime cause of this cell death in situ [4]. In fact, MSCs fail to survive in such post-implantation milieu because they face a situation where their energy demands far exceed their available cellular resources and cannot adapt their metabolism to ensure their survival (Figure 2).

In the cases of bone TE applications, aiming at repairing bone defect of a critical size, the larger the defect is the more ischemic the environment will be; due to the short range of oxygen diffusion from blood capillaries (100-200 µm). As mentioned above, this type of deleterious environment not only induces a massive cell death, but also reduces the functionalities of surviving cells. When lacking of energy substrate cells need to rewire their energy route to ensure their survival and as a result diminish their contribution to tissue repair. For example, without the presence of exogenous glucose, MSCs are not capable to secrete the adequate growth factors and cytokines (chemo-attractive, pro-angiogenic and immunomodulatory) that are pertinent to tissue repair (Paquet, unpublished data).

Most studies focuses solely on the role of oxygen because it modulates several critical cellular processes (e.g., cell adhesion, metabolism, proliferation, differentiation) as well as their paracrine function [5]. Recently, studies from our group, in sheep[6], and humans [7] MSCs (hMSCs) challenged the pivotal role of oxygen in MSC survival and established that MSCs can adapt to near-anoxia conditions (0.1 O2) in vitro and increased their in vivo survival rate as long as they have a sufficient glucose supply.

In the study presented here, rather than providing the necessary glucose supply to ensure long term MSC survival; we tried a preconditioning aiming at reducing MSC energy demands and provide them with an adequate metabolism to face the ischemic conditions. We used a 48h serum deprivation to induce cellular quiescence in hMSCs. Quiescence is characterized by exit from the cell cycle and profound reductions of the metabolic rate that cells, particularly stem cells, adopt in their niche to preserve their key features upon activation (Figure 1). Moreover, the quiescent state is a reversible and the physiological state of adult stem cells in their niche and as a result suited for environments with low oxygen tension (pO2:  1-8%).

As mentioned in the abstract above, were able to demonstrate that the quiescent hMSCs reduces their energy demands and adopt a metabolic profile favorable for enhanced survival in vitro and in vivo (Figure 3). Interestingly, our data provided new insights on metabolism of quiescent stem cells, suggesting that these cells utilize some steps of gluconeogenesis and highlighting the pivotal role of pyruvate which was essential to their survival under ischemic conditions (Figure 4). Moreover this cellular quiescence preconditioning is a simple, safe, cost-effective, yet very efficient solution for enhancing cell survival in ischemia that may be applicable not only for adult stem cells (hMSC) but also other cell types. While this study does not demonstrate an enhancement of tissue regeneration correlated with the enhanced cell survival; a recent study has shown that an increase in murine periosteum-derived cell survival after 3 days post-implantations would enhance bone formation after 8 weeks [8]. Therefore, it is reasonable to speculate that the quiescence preconditioning could also enhance de novo tissue formation in vivo. Another limitation regarding TE and stem-cell therapies concern the number of cells needed to achieve the therapeutic effect desired; in most cases, these numbers can reach 10 to 100 million of cells. As a result, stem cells need to be amplified ex vivo prior to their implantation. However, adult stem cell differentiation and proliferation potentials are reduced when these cells exceed a number of cellular divisions, which diminish their therapeutic potential. Improving stem cell survival could then, help reduce the total number of cells needed for TE applications thus reducing the ex vivo amplification phase and ultimately enhancing their efficacy.

 

 

Figure 1: Illustration of MSCs role in tissue homeostasis.

 

 

Figure 2: Schematic representation of the ischemic milieu post-implantation

 

 

Figure 3: Metabolic profile of hMSCs in either (A) « normoxia » (21% O2), (B) ischemia, or (C) quiescence; metabolic pathways are inhibited (white), activated (black) or not affected (grey). Red color indicates an inhibitory pathway.

 

 

Figure 4: Proposed illustration of the quiescent hMSCs metabolic « shwitch » obtained by analysis of 84 genes implicated in the cellular metabolism. Enzymes level of expressions are represented in green (>2-fold upregulated), red (< 2-fold downregulated), black (stable) and violet (not studied). Reversible (dual heads) and non-reversible reactions (single head) are represented by arrows. Phosphoenolpyruvate (PEP), Acetyl coenzyme A (Acetyl-CoA), Tricarboxylic acid cycle (TCA cycle), Oxidative phosphorylation (OXPHOS), Pyruvate kinase Liver and RBC (PKLR), Phosphoenolpyruvate carboxylase 1 & 2 (PEPCK1, PEPCK2), Lactate Dehydrogenase (LDH), Pyruvate carboxylase (PC), Malate dehydrogenase 1 & 1B (MDH1, MDH1B), Malic enzyme 1 (ME1), Pyruvate dehydrogenase phosphatase regulatory subunit (PDPR), Pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), Pyruvate dehydrogenase A1 & B (PDHA1, PDHB), Dihydrolipoamide S-acetyltransferase (DLAT), Phosphoinositide dependent protein kinase 1, 2,3 & 4 (PDK1, PDK2, PDK3, PDK 4).

 

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