Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates an in vitro model of bone marrow adipocyte subtypes.

Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step.

MarrowQuant Across Aging and Aplasia: A Digital Pathology Workflow for Quantification of Bone Marrow Compartments in Histological Sections.

The bone marrow (BM) exists heterogeneously as hematopoietic/red or adipocytic/yellow marrow depending on skeletal location, age, and physiological condition. Mouse models and patients undergoing radio/chemotherapy or suffering acute BM failure endure rapid adipocytic conversion of the marrow microenvironment, the so-called "red-to-yellow" transition. Following hematopoietic recovery, such as upon BM transplantation, a "yellow-to-red" transition occurs and functional hematopoiesis is restored. Gold Standards to estimate BM cellular composition are pathologists' assessment of hematopoietic cellularity in hematoxylin and eosin (H&E) stained histological sections as well as volumetric measurements of marrow adiposity with contrast-enhanced micro-computerized tomography (CE-µCT) upon osmium-tetroxide lipid staining. Due to user-dependent variables, reproducibility in longitudinal studies is a challenge for both methods. Here we report the development of a semi-automated image analysis plug-in, MarrowQuant, which employs the open-source software QuPath, to systematically quantify multiple bone components in H&E sections in an unbiased manner. MarrowQuant discerns and quantifies the areas occupied by bone, adipocyte ghosts, hematopoietic cells, and the interstitial/microvascular compartment. A separate feature, AdipoQuant, fragments adipocyte ghosts in H&E-stained sections of extramedullary adipose tissue to render adipocyte area and size distribution. Quantification of BM hematopoietic cellularity with MarrowQuant lies within the range of scoring by four independent pathologists, while quantification of the total adipocyte area in whole bone sections compares with volumetric measurements. Employing our tool, we were able to develop a standardized map of BM hematopoietic cellularity and adiposity in mid-sections of murine C57BL/6 bones in homeostatic conditions, including quantification of the highly predictable red-to-yellow transitions in the proximal section of the caudal tail and in the proximal-to-distal tibia. Additionally, we present a comparative skeletal map induced by lethal irradiation, with longitudinal quantification of the "red-to-yellow-to-red" transition over 2 months in C57BL/6 femurs and tibiae. We find that, following BM transplantation, BM adiposity inversely correlates with kinetics of hematopoietic recovery and that a proximal to distal gradient is conserved. Analysis of in vivo recovery through magnetic resonance imaging (MRI) reveals comparable kinetics. On human trephine biopsies MarrowQuant successfully recognizes the BM compartments, opening avenues for its application in experimental, or clinical contexts that require standardized human BM evaluation.

Injectable, scalable 3D tissue-engineered model of marrow hematopoiesis.

Modeling the interaction between the supportive stroma and the hematopoietic stem and progenitor cells (HSPC) is of high interest in the regeneration of the bone marrow niche in blood disorders. In this work, we present an injectable co-culture system to study this interaction in a coherent in vitro culture and in vivo transplantation model. We assemble a 3D hematopoietic niche in vitro by co-culture of supportive OP9 mesenchymal cells and HSPCs in porous, chemically defined collagen-coated carboxymethylcellulose microscaffolds (CCMs). Flow cytometry and hematopoietic colony forming assays demonstrate the stromal supportive capacity for in vitro hematopoiesis in the absence of exogenous cytokines. After in vitro culture, we recover a paste-like living injectable niche biomaterial from CCM co-cultures by controlled, partial dehydration. Cell viability and the association between stroma and HSPCs are maintained in this process. After subcutaneous injection of this living artificial niche in vivo, we find maintenance of stromal and hematopoietic populations over 12 weeks in immunodeficient mice. Indeed, vascularization is enhanced in the presence of HSPCs. Our approach provides a minimalistic, scalable, biomimetic in vitro model of hematopoiesis in a microcarrier format that preserves the HSPC progenitor function, while being injectable in vivo without disrupting the cell-cell interactions established in vitro.

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.

High-throughput, nonperturbing quantification of lipid droplets with digital holographic microscopy.

In vitro differentiating adipocytes are sensitive to liquid manipulations and have the tendency to float. Assessing adipocyte differentiation using current microscopy techniques involves cell staining and washing, while using flow cytometry involves cell retrieval in suspension. These methods induce biases, are difficult to reproduce, and involve tedious optimizations. In this study, we present digital holographic microscopy (DHM) as a label-free, nonperturbing means to quantify lipid droplets in differentiating adipocytes in a robust medium- to high-throughput manner. Taking advantage of the high refractive index of lipid droplets, DHM can assess the production of intracellular lipid droplets by differences in phase shift in a quantitative manner. Adipocytic differentiation, combined with other morphological features including cell confluence and cell death, was tracked over 6 days in live OP9 mesenchymal stromal cells. We compared DHM with other currently available methods of lipid droplet quantification and demonstrated its robustness with modulators of adipocytic differentiation in a dose-responsive manner. This study suggests DHM as a novel marker-free nonperturbing method to study lipid droplet accumulation and may be envisioned for drug screens and mechanistic studies on adipocytic differentiation.

Single-cell analyses identify bioengineered niches for enhanced maintenance of hematopoietic stem cells.

The in vitro expansion of long-term hematopoietic stem cells (HSCs) remains a substantial challenge, largely because of our limited understanding of the mechanisms that control HSC fate choices. Using single-cell multigene expression analysis and time-lapse microscopy, here we define gene expression signatures and cell cycle hallmarks of murine HSCs and the earliest multipotent progenitors (MPPs), and analyze systematically single HSC fate choices in culture. Our analysis revealed twelve differentially expressed genes marking the quiescent HSC state, including four genes encoding cell-cell interaction signals in the niche. Under basal culture conditions, most HSCs rapidly commit to become early MPPs. In contrast, when we present ligands of the identified niche components such as JamC or Esam within artificial niches, HSC cycling is reduced and long-term multipotency in vivo is maintained. Our approach to bioengineer artificial niches should be useful in other stem cell systems.Haematopoietic stem cell (HSC) self-renewal is not sufficiently understood to recapitulate in vitro. Here, the authors generate gene signature and cell cycle hallmarks of single murine HSCs, and use identified endothelial receptors Esam and JamC as substrates to enhance HSC growth in engineered niches.

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.