Comparison of Emotional Processing Between Patients with Substance Use Disorder and Those with Dual Diagnosis: Relationship with Severity of Dependence and Use During Treatment.

This study analyzed, in a Spanish sample, the differences in emotional processing in patients diagnosed with substance use disorder (SUD) and patients with a dual diagnosis (DD), and tested whether alterations in emotional regulation were related to the severity of dependence and consumption during treatment. A descriptive follow-up study was conducted with 88 adult outpatients (83% men) who were receiving treatment for alcohol and cocaine SUD. Of the sample, 43.2% presented dual diagnosis according to DSM-IV-TR criteria. Emotional processing was assessed with the IAPS, and dependence severity with the SDSS. Consumption was determined with self-reports and toxicological tests. Regression analyses revealed that the DD group had more difficulties in identifying the valence and arousal of the images than patients with SUD. Patients with DD presented more difficulty in identifying images in which valence was manipulated, but not in those in which arousal was manipulated. Cocaine use during treatment was associated with difficulties in identifying unpleasant (U = 734.0; p < .05) and arousing (U = 723.5; p < .05) images. Although these results are preliminary, findings suggest that impaired emotional processing is aggravated in dual patients, although it may be a common transdiagnostic factor in SUD and other comorbid mental disorders. Findings highlight the importance of evaluating emotional regulation to better understand its possible role in the maintenance of substance use.

Harnessing human adipose-derived stromal cell chondrogenesis in vitro for enhanced endochondral ossification.

Endochondral ossification (ECO), the major ossification process during embryogenesis and bone repair, involves the formation of a cartilaginous template remodelled into a functional bone organ. Adipose-derived stromal cells (ASC), non-skeletal multipotent progenitors from the stromal vascular fraction (SVF) of human adipose tissue, were shown to recapitulate ECO and generate bone organs in vivo when primed into a hypertrophic cartilage tissue (HCT) in vitro. However, the reproducibility of ECO was limited and the major triggers remain unknown. We studied the effect of the expansion of cells and maturation of HCT on the induction of the ECO process. SVF cells or expanded ASC were seeded onto collagen sponges, cultured in chondrogenic medium for 3-6 weeks and implanted ectopically in nude mice to evaluate their bone-forming capacities. SVF cells from all tested donors formed mature HCT in 3 weeks whereas ASC needed 4-5 weeks. A longer induction increased the degree of maturation of the HCT, with a gradually denser cartilaginous matrix and increased mineralization. This degree of maturation was highly predictive of their bone-forming capacity in vivo, with ECO achieved only for an intermediate maturation degree. In parallel, expanding ASC also resulted in an enrichment of the stromal fraction characterized by a rapid change of their proteomic profile from a quiescent to a proliferative state. Inducing quiescence rescued their chondrogenic potential. Our findings emphasize the role of monolayer expansion and chondrogenic maturation degree of ASC on ECO and provides a simple, yet reproducible and effective approach for bone formation to be tested in specific clinical models.

Different niches for stem cells carrying the same oncogenic driver affect pathogenesis and therapy response in myeloproliferative neoplasms.

Aging facilitates the expansion of hematopoietic stem cells (HSCs) carrying clonal hematopoiesis-related somatic mutations and the development of myeloid malignancies, such as myeloproliferative neoplasms (MPNs). While cooperating mutations can cause transformation, it is unclear whether distinct bone marrow (BM) HSC-niches can influence the growth and therapy response of HSCs carrying the same oncogenic driver. Here we found different BM niches for HSCs in MPN subtypes. JAK-STAT signaling differentially regulates CDC42-dependent HSC polarity, niche interaction and mutant cell expansion. Asymmetric HSC distribution causes differential BM niche remodeling: sinusoidal dilation in polycythemia vera and endosteal niche expansion in essential thrombocythemia. MPN development accelerates in a prematurely aged BM microenvironment, suggesting that the specialized niche can modulate mutant cell expansion. Finally, dissimilar HSC-niche interactions underpin variable clinical response to JAK inhibitor. Therefore, HSC-niche interactions influence the expansion rate and therapy response of cells carrying the same clonal hematopoiesis oncogenic driver.

Engineering of immunoinstructive extracellular matrices for enhanced osteoinductivity.

The increasing recognition of the contribution of the immune system to activate and prime regeneration implies that tissue engineering strategies and biomaterials design should target regulation of early immunological processes. We previously proposed the cell-based engineering and devitalization of extracellular matrices (ECMs) as a strategy to generate implant materials delivering custom-defined signals. Here, in the context of bone regeneration, we aimed at enhancing the osteoinductivity of such ECMs by enriching their immunomodulatory factors repertoire. Priming with IL1ß a cell line overexpressing BMP-2 enabled engineering of ECMs preserving osteoinductive signals and containing larger amounts of angiogenic (VEGF) and pro-inflammatory molecules (IL6, IL8 and MCP1). Upon implantation, these IL1ß-induced materials enhanced processes typical of the inflammatory phase (e.g., vascular invasion, osteoclast recruitment and differentiation), leading to 'regenerative' events (e.g., M2 macrophage polarization) and ultimately resulting in faster and more efficient bone formation. These results bear relevance towards the manufacturing of potent off-the-shelf osteoinductive materials and outline the broader paradigm of engineering immunoinstructive implants to enhance tissue regeneration.

Toward 3D Bioprinting of Osseous Tissue of Predefined Shape Using Single-Matrix Cell-Bioink Constructs.

Engineering living bone tissue of defined shape on-demand has remained a challenge. 3D bioprinting (3DBP), a biofabrication process capable of yielding cell constructs of defined shape, when combined with developmental engineering can provide a possible path forward. Through the development of a bioink possessing appropriate rheological properties to carry a high cell load and concurrently yield physically stable structures, printing of stable, cell-laden, single-matrix constructs of anatomical shapes is realized without the need for fugitive or support phases. Using this bioink system, constructs of hypertrophic cartilage of predesigned geometry are engineered in vitro by printing human mesenchymal stromal cells at a high density to drive spontaneous condensation and implanted in nude mice to evoke endochondral ossification. The implanted constructs retain their prescribed shape over a 12-week period and undergo remodeling to yield ossicles of the designed shape with neovascularization. Microcomputed tomography, histological, and immunohistochemistry assessments confirm bone tissue characteristics and the presence of human cells. These results demonstrate the potential of 3DBP to fabricate complex bone tissue for clinical application.

Mini- and macro-scale direct perfusion bioreactors with optimized flow for engineering 3D tissues.

Bioreactors enabling direct perfusion of cell suspensions or culture media through the pores of 3D scaffolds have long been used in tissue engineering to improve cell seeding efficiency as well as uniformity of cell distribution and tissue development. A macro-scale U-shaped bioreactor for cell culture under perfusion (U-CUP) has been previously developed. In that system, the geometry of the perfusion chamber results in rather uniform flow through most of the scaffold volume, but not in the peripheral regions. Here, the design of the perfusion chamber has been optimized to provide a more homogenous perfusion flow through the scaffold. Then, the design of this macro-scale flow-optimized perfusion bioreactor (macro-Flopper) has been miniaturized to create a mini-scale device (mini-Flopper) compatible with medium-throughput assays. Computational fluid dynamic (CFD) modeling of the new chamber design, including a porous scaffold structure, revealed that Flopper bioreactors provide highly homogenous flow speed, pressure, and shear stress. Finally, a proof-of-principle of the functionality of the Flopper systems by engineering endothelialized stromal tissues using human adipose tissue-derived stromal vascular fraction (SVF) cells has been offered. Preliminary evidence showing that flow optimization improves cell maintenance in the engineered tissues will have to be confirmed in future studies. In summary, two bioreactor models with optimized perfusion flow and complementary sizes have been proposed that might be exploited to engineer homogenous tissues and, in the case of the mini-Flopper, for drug testing assays with a limited amount of biological material.

A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise.

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects.

Cholinergic signals preserve haematopoietic stem cell quiescence during regenerative haematopoiesis.

The sympathetic nervous system has been evolutionary selected to respond to stress and activates haematopoietic stem cells via noradrenergic signals. However, the pathways preserving haematopoietic stem cell quiescence and maintenance under proliferative stress remain largely unknown. Here we found that cholinergic signals preserve haematopoietic stem cell quiescence in bone-associated (endosteal) bone marrow niches. Bone marrow cholinergic neural signals increase during stress haematopoiesis and are amplified through cholinergic osteoprogenitors. Lack of cholinergic innervation impairs balanced responses to chemotherapy or irradiation and reduces haematopoietic stem cell quiescence and self-renewal. Cholinergic signals activate α7 nicotinic receptor in bone marrow mesenchymal stromal cells leading to increased CXCL12 expression and haematopoietic stem cell quiescence. Consequently, nicotine exposure increases endosteal haematopoietic stem cell quiescence in vivo and impairs hematopoietic regeneration after haematopoietic stem cell transplantation in mice. In humans, smoking history is associated with delayed normalisation of platelet counts after allogeneic haematopoietic stem cell transplantation. These results suggest that cholinergic signals preserve stem cell quiescence under proliferative stress.

Engineering of fully humanized and vascularized 3D bone marrow niches sustaining undifferentiated human cord blood hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are frequently located around the bone marrow (BM) vasculature. These so-called perivascular niches regulate HSC function both in health and disease, but they have been poorly studied in humans due to the scarcity of models integrating complete human vascular structures. Herein, we propose the stromal vascular fraction (SVF) derived from human adipose tissue as a cell source to vascularize 3D osteoblastic BM niches engineered in perfusion bioreactors. We show that SVF cells form self-assembled capillary structures, composed by endothelial and perivascular cells, that add to the osteogenic matrix secreted by BM mesenchymal stromal cells in these engineered niches. In comparison to avascular osteoblastic niches, vascularized BM niches better maintain immunophenotypically-defined cord blood (CB) HSCs without affecting cell proliferation. In contrast, HSPCs cultured in vascularized BM niches showed increased CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) numbers. The vascularization also contributed to better preserve osteogenic gene expression in the niche, demonstrating that niche vascularization has an influence on both hematopoietic and stromal compartments. In summary, we have engineered a fully humanized and vascularized 3D BM tissue to model native human endosteal perivascular niches and revealed functional implications of this vascularization in sustaining undifferentiated CB HSPCs. This system provides a unique modular platform to explore hemato-vascular interactions in human healthy/pathological hematopoiesis.

Manufacturing of Human Tissues as off-the-Shelf Grafts Programmed to Induce Regeneration.

Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, a conceptual shift based on the manufacturing of devitalized human hypertrophic cartilage (HyC), as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification, is reported. The strategy relies on a customized human mesenchymal line expressing bone morphogenetic protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization, and storage, the resulting off-the-shelf cartilage tissue exhibits unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy are demonstrated by bioreactor-based production and subsequent orthotopic assessment. The findings exemplify the broader paradigm of programming human cell lines as biological factory units to engineer customized ECMs, designed to activate specific regenerative processes.

Culturing patient-derived malignant hematopoietic stem cells in engineered and fully humanized 3D niches.

Human malignant hematopoietic stem and progenitor cells (HSPCs) reside in bone marrow (BM) niches, which remain challenging to explore due to limited in vivo accessibility and constraints with humanized animal models. Several in vitro systems have been established to culture patient-derived HSPCs in specific microenvironments, but they do not fully recapitulate the complex features of native bone marrow. Our group previously reported that human osteoblastic BM niches (O-N), engineered by culturing mesenchymal stromal cells within three-dimensional (3D) porous scaffolds under perfusion flow in a bioreactor system, are capable of maintaining, expanding, and functionally regulating healthy human cord blood-derived HSPCs. Here, we first demonstrate that this 3D O-N can sustain malignant CD34+ cells from acute myeloid leukemia (AML) and myeloproliferative neoplasm patients for up to 3 wk. Human malignant cells distributed in the bioreactor system mimicking the spatial distribution found in native BM tissue, where most HSPCs remain linked to the niches and mature cells are released to the circulation. Using human adipose tissue-derived stromal vascular fraction cells, we then generated a stromal-vascular niche and demonstrated that O-N and stromal-vascular niche differentially regulate leukemic UCSD-AML1 cell expansion, immunophenotype, and response to chemotherapy. The developed system offers a unique platform to investigate human leukemogenesis and response to drugs in customized environments, mimicking defined features of native hematopoietic niches and compatible with the establishment of personalized settings.

Evolutionary Perspective and Expression Analysis of Intronless Genes Highlight the Conservation of Their Regulatory Role.

The structure of eukaryotic genes is generally a combination of exons interrupted by intragenic non-coding DNA regions (introns) removed by RNA splicing to generate the mature mRNA. A fraction of genes, however, comprise a single coding exon with introns in their untranslated regions or are intronless genes (IGs), lacking introns entirely. The latter code for essential proteins involved in development, growth, and cell proliferation and their expression has been proposed to be highly specialized for neuro-specific functions and linked to cancer, neuropathies, and developmental disorders. The abundant presence of introns in eukaryotic genomes is pivotal for the precise control of gene expression. Notwithstanding, IGs exempting splicing events entail a higher transcriptional fidelity, making them even more valuable for regulatory roles. This work aimed to infer the functional role and evolutionary history of IGs centered on the mouse genome. IGs consist of a subgroup of genes with one exon including coding genes, non-coding genes, and pseudogenes, which conform approximately 6% of a total of 21,527 genes. To understand their prevalence, biological relevance, and evolution, we identified and studied 1,116 IG functional proteins validating their differential expression in transcriptomic data of embryonic mouse telencephalon. Our results showed that overall expression levels of IGs are lower than those of MEGs. However, strongly up-regulated IGs include transcription factors (TFs) such as the class 3 of POU (HMG Box), Neurog1, Olig1, and BHLHe22, BHLHe23, among other essential genes including the ß-cluster of protocadherins. Most striking was the finding that IG-encoded BHLH TFs fit the criteria to be classified as microproteins. Finally, predicted protein orthologs in other six genomes confirmed high conservation of IGs associated with regulating neural processes and with chromatin organization and epigenetic regulation in Vertebrata. Moreover, this study highlights that IGs are essential modulators of regulatory processes, such as the Wnt signaling pathway and biological processes as pivotal as sensory organ developing at a transcriptional and post-translational level. Overall, our results suggest that IG proteins have specialized, prevalent, and unique biological roles and that functional divergence between IGs and MEGs is likely to be the result of specific evolutionary constraints.

Biomimetic human bone marrow tissues: models to study hematopoiesis and platforms for drug testing.

We propose an in vitro 3D culture system combining perfusion bioreactors, scaffolds and human primary cells to engineer fully-humanized, biomimetic and customizable bone marrow tissues. This system could serve as a model to investigate human hematopoietic stem cell niches, but also as a drug testing platform for pharmaceutical research and patient-personalized medicine.

The Autonomic Nervous System Pulls the Strings to Coordinate Circadian HSC Functions.

As for many other adult stem cells, the behavior of hematopoietic stem and progenitor cells (HSPCs) is subjected to circadian regulatory patterns. Multiple HSPC functions, such as proliferation, differentiation or trafficking exhibit time-dependent patterns that require a tight coordination to ensure daily blood cell production. The autonomic nervous system, together with circulating hormones, relay circadian signals from the central clock-the suprachiasmatic nucleus in the brain-to synchronize HSC niche physiology according to light/darkness cycles. Research over the last 20 years has revealed how specific neural signals modulate certain aspects of circadian HSC biology. However, only recently some studies have started to decipher the cellular and molecular mechanisms that orchestrate this complex regulation in a time-dependent fashion. Here we firstly review some of the recent key findings illustrating how different neural signals (catecholaminergic or cholinergic) regulate circadian HSC egress, homing, maintenance, proliferation, and differentiation. In particular, we highlight the critical role of different neurotransmitter receptors in the bone marrow microenvironment to channel these neural signals and regulate antagonistic processes according to circadian cues and organismal demands. Then, we discuss the potential biological meaning of HSC circadian regulation and its possible utility for clinical purposes. Finally, we offer our perspective on emerging concepts in HSC chronobiology.

Extracellular Matrices to Modulate the Innate Immune Response and Enhance Bone Healing.

Extracellular matrices (ECMs) have emerged as promising off-the-shelf products to induce bone regeneration, with the capacity not only to activate osteoprogenitors, but also to influence the immune response. ECMs generated starting from living cells such as mesenchymal stromal cells (MSCs) have the potential to combine advantages of native tissue-derived ECMs (e.g., physiological presentation of multiple regulatory factors) with those of synthetic ECMs (e.g., customization and reproducibility of composition). MSC-derived ECMs could be tailored by enrichment not only in osteogenic cytokines, but also in immunomodulatory factors, to skew the innate immune response toward regenerative processes. After reviewing the different immunoregulatory properties of ECM components, here we propose different approaches to engineer ECMs enriched in factors capable to regulate macrophage polarization, recruit host immune and mesenchymal cells, and stimulate the synthesis of other immunoinstructive cytokines. Finally, we offer a perspective on the possible evolution of the paradigm based on biological and chemico-physical design considerations, and the use of gene editing approaches.

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes ß2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced ß3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with ß3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.

Dual cholinergic signals regulate daily migration of hematopoietic stem cells and leukocytes.

Hematopoietic stem and progenitor cells (HSPCs) and leukocytes circulate between the bone marrow (BM) and peripheral blood following circadian oscillations. Autonomic sympathetic noradrenergic signals have been shown to regulate HSPC and leukocyte trafficking, but the role of the cholinergic branch has remained unexplored. We have investigated the role of the cholinergic nervous system in the regulation of day/night traffic of HSPCs and leukocytes in mice. We show here that the autonomic cholinergic nervous system (including parasympathetic and sympathetic) dually regulates daily migration of HSPCs and leukocytes. At night, central parasympathetic cholinergic signals dampen sympathetic noradrenergic tone and decrease BM egress of HSPCs and leukocytes. However, during the daytime, derepressed sympathetic noradrenergic activity causes predominant BM egress of HSPCs and leukocytes via ß3-adrenergic receptor. This egress is locally supported by light-triggered sympathetic cholinergic activity, which inhibits BM vascular cell adhesion and homing. In summary, central (parasympathetic) and local (sympathetic) cholinergic signals regulate day/night oscillations of circulating HSPCs and leukocytes. This study shows how both branches of the autonomic nervous system cooperate to orchestrate daily traffic of HSPCs and leukocytes.

Daily Onset of Light and Darkness Differentially Controls Hematopoietic Stem Cell Differentiation and Maintenance.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

A Linked List-Based Algorithm for Blob Detection on Embedded Vision-Based Sensors.

Blob detection is a common task in vision-based applications. Most existing algorithms are aimed at execution on general purpose computers; while very few can be adapted to the computing restrictions present in embedded platforms. This paper focuses on the design of an algorithm capable of real-time blob detection that minimizes system memory consumption. The proposed algorithm detects objects in one image scan; it is based on a linked-list data structure tree used to label blobs depending on their shape and node information. An example application showing the results of a blob detection co-processor has been built on a low-powered field programmable gate array hardware as a step towards developing a smart video surveillance system. The detection method is intended for general purpose application. As such, several test cases focused on character recognition are also examined. The results obtained present a fair trade-off between accuracy and memory requirements; and prove the validity of the proposed approach for real-time implementation on resource-constrained computing platforms.

BMSCs and hematopoiesis.

Recent discoveries have significantly expanded our previous knowledge about the role of bone marrow mesenchymal stem cells (BMSCs) in hematopoiesis. BMSCs and their derivatives modulate blood production and immunity at different levels but a prominent role has emerged for BMSCs in the regulation of hematopoietic stem and progenitor cells (HSPCs). Additionally, BMSC-like cells regulate B and T cell lymphopoiesis and also probably myelopoiesis. Furthermore, BMSCs might also exhibit key regulatory properties in non-physiological conditions. BMSCs in extramedullary sites might provide a permissive microenvironment to allow for transient hematopoiesis. BMSCs might be also involved in the manifestation and/or the development of hematopoietic diseases, as stemming from their emerging roles in the progression of hematological malignancies. Here we review some key molecular pathways, adhesion molecules and ligand/receptor interactions that mediate the crosstalk between BMSCs and hematopoietic stem cells (HSCs) in health and disease. The development of novel markers to visualize and isolate individual cells will help to dissect the stromal-hematopoietic interplay.