Novel tools to dissect stem cell metabolism at single cell level

Hematopoietic stem cells (HSC) are responsible for the life-long maintenance of our blood system. Their long-term capacity to both self-renew and differentiate and the ability to efficiently âhomeâ to their bone marrow niches when injected in the blood stream, makes these rare cells ideal candidates for transplantation for the treatment of various blood cancers. However, countless attempts to expand HSC in vitro without losing their stem cell potential have failed, which is, to a large extent, linked to our poor understanding of the mechanisms that control HSC fate. Recent discoveries have revealed a crucial role of metabolism in controlling HSC function in vivo. However, because of major technical difficulties, we do not yet know the underlying mechanisms behind the metabolic control of HSC fate. Traditional, population-level experimental approaches link the fate of a stem cell to a cell surface protein expression phenotype. This method does not take into account the large heterogeneity of metabolic states in HSC. Consequently, to get mechanistic insights on HSC fate decision-making, metabolism must be studied at single cell level. Therefore, the overall goal of this thesis is to establish novel tools to study metabolic regulation of HSC at single cell level. Here we specifically focused on two functional metabolic read-outs, protein turnover and mitochondrial pH. Mitochondrial matrix pH is one of the most important functional parameters of mitochondria, as it is directly related to the efficiency of ATP production and thus reflects the metabolic state of the mitochondria. Here, a new ratiometric fluorescent probe, 5FA-PIP-Cy5-DA, able to reliably quantify mitochondrial pH, has been developed. It selectively accumulates in mitochondria of live cells and can be used to sensitively detect mitochondrial pH fluctuations upon various external stimuli. Spectral properties of 5FA-PIP-Cy5-DA allow it to be combined with the total mitochondrial membrane potential-dependent probes, enabling simultaneous imaging of total mitochondrial potential and mitochondrial pH. To complement the data on mitochondrial pH in live cells with an additional independent parameter of mitochondrial metabolism, a method of correlated TEM and nanoscale secondary ion mass-spectrometry (NanoSIMS) imaging was developed, that for the first time allows to study the subcellular protein turnover in single stem cells. Application of these novel tools to study HSC metabolism revealed a striking correlation between the levels of mitochondrial protein turnover and mitochondrial pH. This correlation reflects the burst in mitochondrial metabolism upon HSC activation. Intriguingly, an extreme burst in both mitochondrial pH alkalinization and protein turnover rate was observed for the most potent long-term HSC (LT-HSC) upon their activation and induction of differentiation. A new model is proposed to explain the link between the observed activation of mitochondrial protein structure reorganization and increase in mitochondrial pH level of LT-HSC undergoing differentiation. Finally, to demonstrate the broad range of applicability of the new mitochondrial pH probe, we applied it to measure the impact of anticancer treatment on mitochondrial metabolism in ovarian cancer cells exposed to two anticancer drugs, RAPTA-T and cis-platin. Distinctly different mitochondrial pH responses were measured for these two drugs, revealing alternative mechanisms behind their impact on mitochondria.

Metabolic and microenvironmental strategies to accelerate hematopoietic recovery post-aplasia

Vasco Ledebur de Antas de Campos

The procedure-associated mortality rate of bone marrow transplantations (BMT) remains close to 25%. The BMT is therefore only indicated for life-threatening diseases, with no replacement of this technology to be expected in the foreseeable future. Modest improvements such as the use of G-CSF to accelerate hematopoietic recovery significantly reduce mortality. Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and are mainly quiescent, dividing only to self-renew. Via extracellular signals and interactions with other cells of the BM (microenvironment), a stable HSC pool is maintained, while hematopoietic progenitor cells (HPC) proliferate and differentiate to mature blood cell types. Marrow adipocytes (BMA) have recently been recognized as a hematopoietic regulatory entity, although the mechanisms by which it interacts with HSCs and HPCs (HSPC) in vivo, remain unknown. During the hematopoietic recovery period post-BMT, HSPCs increase their metabolism as a response to hematopoietic stress, partly modulated by the microenvironment. In this work we identified novel pharmacological approaches to accelerate hematopoietic recovery in the post-BMT period, addressed via two strategies: (i) by directly modulating HSC metabolism, and (ii) by regulating the differentiation of BMA. Increasing oxidative phosphorylation, combined with an impaired mitochondrial stress response, can severely compromise the regenerative capacity of HSCs. Here we demonstrate that the NAD+-boosting agent Nicotinamide Riboside (NR) reduces mitochondrial activity within HSCs through increased mitophagy, the recycling mechanism of damaged mitochondria. We observed in vitro NR treatment of HSCs to lead to increased asymmetric HSC divisions, which resulted in a significantly enlarged pool of progenitor cells, without HSC exhaustion. Dietary supplementation of NR to mice undergoing BMT accelerated blood recovery and improved survival. We therefore established a novel link between HSC mitochondrial stress, mitophagy and stem cell fate decision. Recent studies have suggested BMA maintain HSCs quiescent, which, may be detrimental to the increased expansion of HPCs in the recovery phase post-BMT. BM stromal cells (BMSC) are the cellular precursors of both adipocytes and osteoblasts, and they have been strongly implicated in supporting hematopoiesis by secreting cytokines such as SCF and Cxcl12. We observed that adipocytes quickly accumulate the marrow space of mice undergoing BMT, followed by an expansion hematopoiesis-supportive multipotent Sca1+/CD24+ stromal cells, coinciding with hematopoietic recovery. We developed a novel screening platform using Digital Holographic Microscopy (DHM), quantifying in vitro adipocytic differentiation non-invasively, allowing for follow-up co-cultures with HSPCs. Using the OP9 BMSC-line, we modulated adipocyte differentiation prior to co-culture using a pharmacological approach and observed that high adipocytic content was associated to a decrease in HPC expansion. We perfomed a screening of over 4000 FDA-approved drugs and natural compounds and identified one to efficiently prevent adipocytic differentiation and maintain the hematopoietic supportive capacity of OP9 stroma. Altogether, this work opens the door to alternative strategies accelerating hematopoietic reconstitution and unveils the potential to improve recovery of patients undergoing BMT.

EPFL2019

Role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells

Isabelle Chareyron

Glucose sensing and regulation of insulin secretion are the two main functions performed by the pancreatic beta-cell. Together these processes contribute to the tight control of blood glucose in our body. Compromising the ability of the beta-cells to properly secrete insulin can lead to the development of Type 2 diabetes. Mitochondria play an important role in beta-cell function, by integrating nutrients signals and modulating insulin secretion. In this thesis, we aimed at better understanding the role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells. To that end, we designed three studies looking at different aspects of beta-cell mitochondrial function. In the first one, we developed an in vitro model of glucose overload in human islet. We report an over activation of the mitochondria when returned to basal conditions after a 4 day culture in elevated glucose. Additionally, these mitochondria only poorly responded to nutrient stimulation. Impaired metabolism secretion coupling and insulin secretion indicated a partial loss of beta-cell function, despite the increased mitochondrial activity. After 4 days of elevated glucose treatment, unusually high levels of glycerol-3-phosphate and pyruvate, even in sub-stimulatory extracellular glucose concentrations, were observed. We propose that the accumulation of these metabolites renders beta-cells blind to subsequent glucose stimulation. After removing the glucose stress for 24 hours the mitochondrial energy metabolism was fully restored and metabolism-secretion coupling was normalized. In the second approach, we measured for the first time the matrix pH in primary human pancreatic beta-cells. We showed that the mitochondrial pH in beta-cell is more acidic than in other cell types. Furthermore, in response to glucose, the beta-cell mitochondrial matrix slightly acidifies, possibly due to uptake and metabolism of pyruvate. Lastly, we observed that adenoviruses used to deliver the genetically encoded pH sensor can have an intrinsic impact on mitochondrial pH and calcium signaling. It must be used at low infection units per cell in the study of primary beta-cells. In the third project, we tried to modify the mitochondrial energy metabolism in beta-cells by inhibiting mitochondrial protein synthesis. We used different antibiotics to achieve this, in particular chloramphenicol, known to interfere with the activity of the mitochondrial ribosome. Surprisingly, we observe that when mitochondrial protein synthesis is inhibited, the respiration of insulin-secreting cells (INS-1E cells, human islets) is not affected. Expression of mtDNA encoded respiratory chain subunits is reduced and a mitochondrial unfolded protein response initiated without reducing respiratory rates. This raises the possibility that limited activation of unfolded protein response has a positive effect on the cells. This doctoral thesis provides novel insights into various aspects of mitochondrial behavior in pancreatic beta-cells, both during physiological and pathophysiological conditions. Additional work is needed to better understand the molecular mechanisms underlying the mitochondrial adaptation to nutrient overload and the associated beta-cell dysfunction leading to the accelerated impairment of glucose homeostasis during the development of Type 2 diabetes.

EPFL2019

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

George Coukos, Mukul Girotra, Alexandre Harari, Olaia Maria Naveiras Torres-Quiroga, Nicola Vannini

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

2019

Role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells

Isabelle Chareyron

EPFL2019

Metabolic and microenvironmental strategies to accelerate hematopoietic recovery post-aplasia

Vasco Ledebur de Antas de Campos

EPFL2019