Deciphering Metabolic Regulation of Hematopoietic Stem Cell Fate

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. This unique characteristic makes them an ideal candidate for cell-based therapies to treat various hematological malignancies. Their extensive use in the clinic is often hampered due to insufficient number of cells obtained from donors. Countless attempts to expand HSCs in vitro have failed, primarily due to our inability to recapitulate key features of the native bone marrow microenvironment, termed niche, in a dish. The absence of important niche signals in vitro results in rapid proliferation of HSCs with a concomitant loss of their long-term multi lineage blood reconstitution potential. The niche in the bone marrow involves a highly complex network of physical and biochemical signals that, in concert with cell-intrinsic mechanisms, is believed to control HSC fate choices. Moreover, the hypoxic conditions in the niche presents an extreme metabolic environment, imposing HSCs to attain a distinct metabolic identity as compared to their differentiated progeny. However, despite decades of research it is currently very poorly understood how HSCs take the decision to either undergo self-renewal or differentiation. Insights into the mechanisms regulating HSC fate choices are key to design better strategies for HSC maintenance and expansion in vitro for use in clinical transplantations. The overall goal of this thesis is to employ innovative experimental tools to explore the role of metabolism in regulating HSC fate choices. In the first part of this thesis a versatile cell-tracking assay was developed to follow HSC divisions in vitro. A combination of cell tracking and immunostaining was used to systematically map phenotypic changes in HSCs up to four divisions, under defined culture conditions imposing specific fates. We found that the proportion of cells maintaining an HSC phenotype decreased with increasing number of cell divisions, supporting the notion that faster cycling results in HSC exhaustion. In the second part of this thesis, we for the first time identify a link between mitochondrial metabolism and HSC fate decision. Using flow cytometry and long-term blood reconstitution assays low mitochondrial activity was established as a reliable marker of functional HSCs, independent of their cell cycle state. Consequently, we could use this marker to reliable identify self-renewing HSCs from heterogeneous in vitro cultures. Strikingly, we found that HSC fate could be altered by artificially modulating their mitochondrial activity in vitro. These results suggest that mitochondrial activity is a determinant of HSC fate. The last part of this thesis describes an experimental paradigm to analyze in vivo niche-instructed fate choices in paired HSC daughter cells. Live single cell imaging revealed a significant increase in asynchronous divisions in niche activated HSCs compared to control cells, suggesting a possible involvement of niche-instructed asymmetric cell division program. Indeed, a significantly higher level of asymmetric gene expression was found in paired daughter cells arising from niche-instructed HSCs. This analysis led to the identification of 12 asymmetrically expressed genes, among them were key enzymes belonging to glycolytic and mitochondrial TCA cycle metabolic pathways. Altogether, this thesis successfully employed unique experimental strategies to provide an intriguing link between metabolism and HSC fate choices

Specification of haematopoietic stem cell fate via modulation of mitochondrial activity

Haematopoietic stem cells (HSCs) differ from their committed progeny by relying primarily on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production. However, whether this change in the metabolic program is the cause or the consequence of the unique function of HSCs remains unknown. Here we show that enforced modulation of energy metabolism impacts HSC self-renewal. Lowering the mitochondrial activity of HSCs by chemically uncoupling the electron transport chain drives self-renewal under culture conditions that normally induce rapid differentiation. We demonstrate that this metabolic specification of HSC fate occurs through the reversible decrease of mitochondrial mass by autophagy. Our data thus reveal a causal relationship between mitochondrial metabolism and fate choice of HSCs and also provide a valuable tool to expand HSCs outside of their native bone marrow niches

The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance

It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD(+)-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD(+)-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.Peer reviewe

In Vivo Pre-Instructed HSCs Robustly Execute Asymmetric Cell Divisions In Vitro

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. Under homeostasis, HSCs in their native bone marrow niches are believed to undergo asymmetric cell divisions (ACDs), with one daughter cell maintaining HSC identity and the other committing to differentiate into various mature blood cell types. Due to the lack of key niche signals, in vitro HSCs differentiate rapidly, making it challenging to capture and study ACD. To overcome this bottleneck, in this study, we used interferon alpha (IFN alpha) treatment to "pre-instruct" HSC fate directly in their native niche, and then systematically studied the fate of dividing HSCs in vitro at the single cell level via time-lapse analysis, as well as multigene and protein expression analysis. Triggering HSCs' exit from dormancy via IFN alpha was found to significantly increase the frequency of asynchronous divisions in paired daughter cells (PDCs). Using single-cell gene expression analyses, we identified 12 asymmetrically expressed genes in PDCs. Subsequent immunocytochemistry analysis showed that at least three of the candidates, i.e., Glut1, JAM3 and HK2, were asymmetrically distributed in PDCs. Functional validation of these observations by colony formation assays highlighted the implication of asymmetric distribution of these markers as hallmarks of HSCs, for example, to reliably discriminate committed and self-renewing daughter cells in dividing HSCs. Our data provided evidence for the importance of in vivo instructions in guiding HSC fate, especially ACD, and shed light on putative molecular players involved in this process. Understanding the mechanisms of cell fate decision making should enable the development of improved HSC expansion protocols for therapeutic applications

EXTENSIVE RADICULAR CYST OF THE MANDIBLE: A RARE CASE REPORT

The radicular cyst is the most common inflammatory odontogenic cystic lesion of the jaws. It usually originates as a sequel to a periapical inflammatory process, following chemical, physical or bacterial injury. Due to its chronic etiology, the cyst usually appears towards the later stage of life. It has a male sex predilection, with the maxillary anterior region as the most common site of involvement. This article reports a rare case of a large radicular cyst in the mandible, its management and follow up along one year

Automated analysis of single stem cells in microfluidic traps

We report a reliable strategy to perform automated image cytometry of single (non-adherent) stem cells captured in microfluidic traps. The method rapidly segments images of an entire microfluidic chip based on the detection of horizontal edges of microfluidic channels, from where the position of the trapped cells can be derived and the trapped cells identified with very high precision (>97%). We used this method to successfully quantify the efficiency and spatial distribution of single-cell loading of a microfluidic chip comprised of 2048 single-cell traps. Furthermore, cytometric analysis of trapped primary hematopoietic stem cells (HSC) faithfully recapitulated the distribution of cells in the G1 and S/G2-M phase of the cell cycle that was measured by flow cytometry. This approach should be applicable to automatically track single live cells in a wealth of microfluidic systems

Cross sectional analysis of mandibular anthropometric points using CBCT to derive biometric measurements for a safer approach to mandible osteotomies

Purpose: This study aims to derive a series of biometric measurements using cone-beam computed tomography (CBCT) from a cross sectional group of population to help the surgeon accurately locate the mandibular foramen and the mental foramen during mandibular osteotomies. Methods: CBCT images of 800 subjects were evaluated. Various measurements were noted and compared between the two sides of the mandible in and between the sexes. Result: Statistically significant values were noted between the right and left sides of Line X to Point A in female subjects, Line Z & Line B only in male subjects and Line X’ in both male and female subjects. However, Line Y was found to be significant when comparing both sides in both males and females and also on correlation between the genders. Conclusion: Although the identification of the mandibular lingula and anatomical landmarks is an important step during mandibular osteotomies, the position of one side, however, cannot be blindly extrapolated to the contra lateral side. Also, pre operative CBCT is a useful tool to derive measurements which when transferred clinically during the surgery gives an accurate and safe approach for localisation of lingula, thus reducing the incidence of post operative neurologic morbidities

Kinesin-2 transports Orco into the olfactory cilium of Drosophila melanogaster at specific developmental stages

The cilium, the sensing centre for the cell, displays an extensive repertoire of receptors for various cell signalling processes. The dynamic nature of ciliary signalling indicates that the ciliary entry of receptors and associated proteins must be regulated and conditional. To understand this process, we studied the ciliary localisation of the odour-receptor coreceptor (Orco), a seven-pass transmembrane protein essential for insect olfaction. Little is known about when and how Orco gets into the cilia. Here, using Drosophila melanogaster, we show that the bulk of Orco selectively enters the cilia on adult olfactory sensory neurons in two discrete, one-hour intervals after eclosion. A conditional loss of heterotrimeric kinesin-2 during this period reduces the electrophysiological response to odours and affects olfactory behaviour. We further show that Orco binds to the C-terminal tail fragments of the heterotrimeric kinesin-2 motor, which is required to transfer Orco from the ciliary base to the outer segment and maintain within an approximately four-micron stretch at the distal portion of the ciliary outer-segment. The Orco transport was not affected by the loss of critical intraflagellar transport components, IFT172/Oseg2 and IFT88/NompB, respectively, during the adult stage. These results highlight a novel developmental regulation of seven-pass transmembrane receptor transport into the cilia and indicate that ciliary signalling is both developmentally and temporally regulated.ISSN:1553-7390ISSN:1553-740