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.

Cryogel-based Injectable 3D Microcarrier Co-culture for Support of Hematopoietic Progenitor Niches.

Although hematopoietic stem cell (HSC) transplantation can restore functional hematopoiesis upon immune or chemotherapy-induced bone marrow failure, complications often arise during recovery, leading to up to 25% transplant-related mortality in treated patients. In hematopoietic homeostasis and regeneration, HSCs in the bone marrow give rise to the entirety of cellular blood components. One of the challenges in studying hematopoiesis is the ability to successfully mimic the relationship between the stroma and hematopoietic stem and progenitor cells (HSPCs). This study and the described protocols propose an advantageous method for culturing and assessing stromal hematopoietic support in three dimensions, representing a simplified in vitro model of the bone marrow niche that can be transplanted in vivo by injection. By co-culturing OP9 bone marrow-derived stromal cells (BMSCs) and cKit+ Sca-1+ Lin- (KLS+ ) HSPCs on collagen-coated carboxymethylcellulose scaffolds for 2 weeks in the absence of cytokines, we established a methodology for in vivo subcutaneous transplantation. With this model we were able to detect early signs of extramedullary hematopoiesis. This work can be useful for studying various stromal cell populations in co-culture, as well as simple transfer by injection of these scaffolds in vivo for heterotopic regeneration of the marrow microenvironment. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of HSPCs from mice Basic Protocol 2: Co-seeding of HSPCs and BMSCs on collagen-coated CCMs Basic Protocol 3: Maintenance, real-time imaging, and analysis of co-seeded scaffolds Basic Protocol 4: End-point analysis of co-seeded scaffolds using flow cytometry and CFU assays Basic Protocol 5: Transplantation of scaffolds by subcutaneous injection Support Protocol: Preparation of custom scaffold drying device.

Bone marrow adiposity and the hematopoietic niche: A historical perspective of reciprocity, heterogeneity, and lineage commitment.

PURPOSE: Here we review the current knowledge on bone marrow adipocytes (BMAds) as active contributors to the regulation of the hematopoietic niche, and as potentially pivotal players in the progression of hematological malignancies. We highlight the hierarchical and functional heterogeneity of the adipocyte lineage within the bone marrow, and how potentially different contexts dictate their interactions with hematopoietic populations. RECENT FINDINGS: Growing evidence associates the adipocyte lineage with important functions in hematopoietic regulation within the BM niche. Initially proposed to serve as negative regulators of the hematopoietic microenvironment, studies have also demonstrated that BMAds positively influence the survival and maintenance of hematopoietic stem cells (HSCs). These seemingly incongruous findings may at least be partially explained by stage-specificity across the adipocytic differentiation axis and by BMAds subtypes, suggesting that the heterogeneity of these populations allows for differential context-based interactions. One such distinction relies on the location of adipocytes. Constitutive bone marrow adipose tissue (cBMAT) historically associates to the "yellow" marrow containing so-called "stable" BMAs larger in size, less responsive to stimuli, and linked to HSC quiescence. On the other hand, regulated bone marrow adipose tissue (rBMAT)-associated adipocytes, also referred to as "labile" are smaller, more responsive to hematopoietic demand and strategically situated in hematopoietically active regions of the skeleton. Here we propose a model where the effect of distinct BM stromal cell populations (BMSC) in hematopoiesis is structured along the BMSC-BMAd differentiation axis, and where the effects on HSC maintenance versus hematopoietic proliferation are segregated. In doing so, it is possible to explain how recently identified, adipocyte-primed leptin receptor-expressing, CXCL12-high adventitial reticular cells (AdipoCARs) and marrow adipose lineage precursor cells (MALPs) best support active hematopoietic cell proliferation, while adipose progenitor cells (APCs) and maturing BMAd gradually lose the capacity to support active hematopoiesis, favoring HSC quiescence. Implicated soluble mediators include MCP-1, PAI-1, NRP1, possibly DPP4 and limiting availability of CXCL12 and SCF. How remodeling occurs within the BMSC-BMAd differentiation axis is yet to be elucidated and will likely unravel a three-way regulation of the hematopoietic, bone, and adipocytic compartments orchestrated by vascular elements. The interaction of malignant hematopoietic cells with BMAds is precisely contributing to unravel specific mechanisms of remodeling. SUMMARY: BMAds are important operative components of the hematopoietic microenvironment. Their heterogeneity directs their ability to exert a range of regulatory capacities in a manner dependent on their hierarchical, spatial, and biological context. This complexity highlights the importance of (i) developing experimental tools and nomenclature adapted to address stage-specificity and heterogeneity across the BMSC-BMAd differentiation axis when reporting effects in hematopoiesis, (ii) interpreting gene reporter studies within this framework, and (iii) quantifying changes in all three compartments (hematopoiesis, adiposity and bone) when addressing interdependency.

An Injectable Meta-Biomaterial: From Design and Simulation to In Vivo Shaping and Tissue Induction.

A novel type of injectable biomaterial with an elastic softening transition is described. The material enables in vivo shaping, followed by induction of 3D stable vascularized tissue. The synthesis of the injectable meta-biomaterial is instructed by extensive numerical simulation as a suspension of irregularly fragmented, highly porous sponge-like microgels. The irregular particle shape dramatically enhances yield strain for in vivo stability against deformation. Porosity of the particles, along with friction between internal surfaces, provides the elastic softening transition. This emergent metamaterial property enables the material to reversibly change stiffness during deformation, allowing native tissue properties to be matched over a wide range of deformation amplitudes. After subcutaneous injection in mice, predetermined shapes can be sculpted manually. The 3D shape is maintained during excellent host tissue integration, with induction of vascular connective tissue that persists to the end of one-year follow-up. The geometrical design is compatible with many hydrogel materials, including cell-adhesion motives for cell transplantation. The injectable meta-biomaterial therefore provides new perspectives in soft tissue engineering and regenerative medicine.

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.

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society.

The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced µCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt-/-, KitW/W-v) and development of specific BMA deletion models would be highly desirable for this purpose.

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.

Brief Report From the 3rd International Meeting on Bone Marrow Adiposity (BMA 2017).

The 3rd International Meeting on Bone Marrow Adiposity (BMA) was held at the Olympic Museum in Lausanne, Switzerland, on August 31st and September 1st, 2017. This brief monograph summarizes the scientific contents of the meeting and highlights the birth of the International Bone Marrow Adiposity Society (BMAS).

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.

Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue.

BACKGROUND: The utility of mesenchymal stromal cells (MSCs) in therapeutic applications for regenerative medicine has gained much attention. Clinical translation of MSC-based approaches requires in vitro culture-expansion to achieve a sufficient number of cells. The ideal cell culture medium should be devoid of any animal derived components. We have evaluated whether human Platelet Lysate (hPL) could be an attractive alternative to animal supplements. METHODS: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three different commercially available hPL fulfilling good manufacturing practice criteria for clinical use. BMSCs and ASCs cultured in Minimum Essential Medium Eagle-alpha supplemented with 5% PLT-Max (Mill Creek), Stemulate™ PL-S and Stemulate™ PL-SP (COOK General Biotechnology) were compared to standard culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. RESULTS: Regardless of manufacturer, BMSCs and ASCs cultured in hPL media showed a significant increase in proliferation capacity compared to FBS medium. In general, the immunophenotype of both BMSCs and ASCs fulfilled International Society for Cellular Therapy (ISCT) criteria after hPL media expansion. Comparative genomic hybridization measurements demonstrated no unbalanced chromosomal rearrangements for BMSCs or ASCs cultured in hPL media or FBS medium. The BMSCs and ASCs could differentiate into osteogenic, adipogenic, or chondrogenic lineages in all four culture conditions. CONCLUSION: All three clinically approved commercial human platelet lysates accelerated proliferation of BMSCs and ASCs and the cells meet the ISCT mesenchymal phenotypic requirements without exhibiting chromosomal aberrations.

Influence of vascular endothelial growth factor stimulation and serum deprivation on gene activation patterns of human adipose tissue-derived stromal cells.

INTRODUCTION: Stimulation of mesenchymal stromal cells and adipose tissue-derived stromal cells (ASCs) with vascular endothelial growth factor (VEGF) has been used in multiple animal studies and clinical trials for regenerative purposes. VEGF stimulation is believed to promote angiogenesis and VEGF stimulation is usually performed under serum deprivation. Potential regenerative molecular mechanisms are numerous and the role of contributing factors is uncertain. The aim of the current study was to investigate the effect of in vitro serum deprivation and VEGF stimulation on gene expression patterns of ASCs. METHODS: Gene expressions of ASCs cultured in complete medium, ASCs cultured in serum-deprived medium and ASCs stimulated with VEGF in serum-deprived medium were compared. ASC characteristics according to criteria set by the International Society of Cellular Therapy were confirmed by flow cytometry. Microarray gene expressions were obtained using the Affymetrix HT HG-U133+ GeneChip®. Gene set enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes and gene ontology terms. Transcription of selected genes of interest was confirmed by quantitative PCR. RESULTS: Compared to ASCs in complete medium, 190 and 108 genes were significantly altered by serum deprivation and serum deprivation combined with VEGF, respectively. No significant differences in gene expression patterns between serum-deprived ASCs and serum-deprived ASCs combined with VEGF stimulation were found. Genes most prominently and significantly upregulated by both conditions were growth factors (IGF1, BMP6, PDGFD, FGF9), adhesion molecule CLSTN2, extracellular matrix-related proteins such as matricellular proteins SMOC2, SPON1 and ADAMTS12, and inhibitors of proliferation (JAG1). The most significantly downregulated genes included matrix metalloproteinases (MMP3, MMP1), and proliferation markers (CDKN3) and GREM2 (a BMP6 antagonist). CONCLUSION: The decisive factor for the observed change in ASC gene expression proves to be serum starvation rather than VEGF stimulation. Changes in expression of growth factors, matricellular proteins and matrix metalloproteinases in concert, diverge from direct pro-angiogenic paracrine mechanisms as a primary consequence of the used protocol. In vitro serum starvation (with or without VEGF present) appears to favour cardioprotection, extracellular matrix remodelling and blood vessel maturation relevant for the late maturation phase in infarct healing.

Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF.

BACKGROUND: Human mesenchymal stromal cells from the bone marrow (BMSCs) are widely used as experimental regenerative treatment of ischemic heart disease, and the first clinical trials using adipose-derived stromal cells (ASCs) are currently being conducted. Regenerative mechanisms of BMSCs and ASCs are manifold and in vitro pretreatment of the cells with growth factors has been applied to potentially enhance these properties. When characterizing the transcriptional activity of these cellular mechanisms in vitro it is important to consider the effect of the growth factor treatment on reference genes (RGs) for the normalization of qPCR data. RESULTS: BMSCs and ASCs were stimulated with vascular endothelial growth factor A-165 (VEGF) for one week, and compared with un-stimulated cells from the same donor. The stability of nine RGs through VEGF treatment as well as the donor variation was assessed using the GenEx software with the subprograms geNorm and Normfinder.The procedure of stepwise elimination was validated by poor performance of eliminated RGs in a normalization experiment using vWF as target gene. Normfinder found the TATA box binding protein (TBP) to be the most stable single RG for both BMSCs and ASCs. The optimal number of RGs for ASCs was two, and the lowest variance for vWF normalization was found using TBP and YWHAZ. For BMSCs, the optimal number of RGs was four, while the two-RG combination producing the most similar results was TBP and YWHAZ. CONCLUSIONS: A common reference gene, TBP, was found to be the most stable standalone gene, while TBP and YWHAZ were found to be the best two-RG combination for qPCR analyses for both BMSCs and ASCs through the VEGF stimulation. The presented stepwise elimination procedure was validated, while we found the final normalization experiment to be essential.

Identical effects of VEGF and serum-deprivation on phenotype and function of adipose-derived stromal cells from healthy donors and patients with ischemic heart disease.

BACKGROUND: Adipose-derived stromal cells (ASCs) stimulated with vascular endothelial growth factor (VEGF) and serum-deprived, are applied in the first in-man double-blind placebo-controlled MyStromalCell Trial, as a novel therapeutic option for treatment of ischemic heart disease (IHD). This in vitro study explored the effect of VEGF and serum deprivation on endothelial differentiation capacity of ASCs from healthy donors and IHD patients. METHODS: ASCs stimulated with rhVEGF(A165) in serum-deprived medium for one to three weeks were compared with ASCs in serum-deprived (2% fetal bovine serum) or complete medium (10% fetal bovine serum). Expression of VEGF receptors, endothelial and stem cell markers was measured using qPCR, flow cytometry and immunocytochemistry. In vitro tube formation and proliferation was also measured. RESULTS: ASCs from VEGF-stimulated and serum-deprived medium significantly increased transcription of transcription factor FOXF1, endothelial marker vWF and receptor VEGFR1 compared with ASCs from complete medium. ASCs maintained stem cell characteristics in all conditions. Tube formation of ASCs occurred in VEGF-stimulated and serum-deprived medium. The only difference between healthy and patient ASCs was a variation in proliferation rate. CONCLUSIONS: ASCs from IHD patients and healthy donors proved equally inclined to differentiate in endothelial direction by serum-deprivation, however with no visible additive effect of VEGF stimulation. The treatment did not result in complete endothelial differentiation, but priming towards endothelial lineage.