Quantitative 3D imaging of the cranial microvascular environment at single-cell resolution.

Vascularization is critical for skull development, maintenance, and healing. Yet, there remains a significant knowledge gap in the relationship of blood vessels to cranial skeletal progenitors during these processes. Here, we introduce a quantitative 3D imaging platform to enable the visualization and analysis of high-resolution data sets (>100 GB) throughout the entire murine calvarium. Using this technique, we provide single-cell resolution 3D maps of vessel phenotypes and skeletal progenitors in the frontoparietal cranial bones. Through these high-resolution data sets, we demonstrate that CD31hiEmcnhi vessels are spatially correlated with both Osterix+ and Gli1+ skeletal progenitors during postnatal growth, healing, and stimulated remodeling, and are concentrated at transcortical canals and osteogenic fronts. Interestingly, we find that this relationship is weakened in mice with a conditional knockout of PDGF-BB in TRAP+ osteoclasts, suggesting a potential role for osteoclasts in maintaining the native cranial microvascular environment. Our findings provide a foundational framework for understanding how blood vessels and skeletal progenitors spatially interact in cranial bone, and will enable more targeted studies into the mechanisms of skull disease pathologies and treatments. Additionally, our technique can be readily adapted to study numerous cell types and investigate other elusive phenomena in cranial bone biology.

Multicolor 3D Confocal Imaging of Thick Tissue Sections.

In multicellular organisms, most physiological and pathological processes involve an interplay between various cells and molecules that act both locally and systemically. To understand how these complex and dynamic processes occur in time and space, imaging techniques are key. Advances in tissue processing techniques and microscopy now allow us to probe these processes at a large scale and at the same time at a level of detail previously unachievable. Indeed, it is now possible to reliably quantify multiple protein expression levels at single-cell resolution in whole organs using three-dimensional fluorescence imaging techniques. Here we describe a method to prepare adult mouse bone tissue for multiplexed confocal imaging of thick tissue sections. Up to eight different fluorophores can be multiplexed using this technique and spectrally resolved using standard confocal microscopy. The optical clearing method described allows detection of these fluorophores up to a depth of >700 µm in the far-red. Although the method was initially developed for bone tissue imaging, we have successfully applied it to several other tissue types.

Development of Humanized Ossicles: Bridging the Hematopoietic Gap.

Ectopic 'humanized ossicles' (hOss) are miniaturized, engineered human bone organs in mice displaying a similar structure and function to native mouse bones. However, they are composed of human mesenchymal derived cells forming a humanized bone marrow niche. This in vivo reconstitution of human skeletal and hematopoietic compartments provides an opportunity to investigate the cellular and molecular processes involved in their establishment and functions in a human setting. However, current hOs strategies vary in their engineering methods and their downstream applications, undermining comprehensive exploitation of their potential. This review describes the specificities of the hOs models and highlights their potential and limits. Ultimately, we propose directions for the development of hOss as a technological platform for human hematopoietic studies.

Fate Distribution and Regulatory Role of Human Mesenchymal Stromal Cells in Engineered Hematopoietic Bone Organs.

The generation of humanized ectopic ossicles (hOss) in mice has been proposed as an advanced translational and fundamental model to study the human hematopoietic system. The approach relies on the presence of human bone marrow-derived mesenchymal stromal cells (hMSCs) supporting the engraftment of transplanted human hematopoietic stem and progenitor cells (HSPCs). However, the functional distribution of hMSCs within the humanized microenvironment remains to be investigated. Here, we combined genetic tools and quantitative confocal microscopy to engineer and subsequently analyze hMSCs' fate and distribution in hOss. Implanted hMSCs reconstituted a humanized environment including osteocytes, osteoblasts, adipocytes, and stromal cells associated with vessels. By imaging full hOss, we identified rare physical interactions between hMSCs and human CD45+/CD34+/CD90+ cells, supporting a functional contact-triggered regulatory role of hMSCs. Our study highlights the importance of compiling quantitative information from humanized organs, to decode the interactions between the hematopoietic and the stromal compartments.

In vitro biomimetic engineering of a human hematopoietic niche with functional properties.

In adults, human hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment. Our understanding of human hematopoiesis and the associated niche biology remains limited, due to human material accessibility and limits of existing in vitro culture models. The establishment of an in vitro BM system would offer an experimentally accessible and tunable platform to study human hematopoiesis. Here, we develop a 3D engineered human BM analog by recapitulating some of the hematopoietic niche elements. This includes a bone-like scaffold, functionalized by human stromal and osteoblastic cells and by the extracellular matrix they deposited during perfusion culture in bioreactors. The resulting tissue exhibited compositional and structural features of human BM while supporting the maintenance of HSPCs. This was associated with a compartmentalization of phenotypes in the bioreactor system, where committed blood cells are released into the liquid phase and HSPCs preferentially reside within the engineered BM tissue, establishing physical interactions with the stromal compartment. Finally, we demonstrate the possibility to perturb HSPCs' behavior within our 3D niches by molecular customization or injury simulation. The developed system enables the design of advanced, tunable in vitro BM proxies for the study of human hematopoiesis.

Inductive and Selective Effects of GSK3 and MEK Inhibition on Nanog Heterogeneity in Embryonic Stem Cells.

Embryonic stem cells (ESCs) display heterogeneous expression of pluripotency factors such as Nanog when cultured with serum and leukemia inhibitory factor (LIF). In contrast, dual inhibition of the signaling kinases GSK3 and MEK (2i) converts ESC cultures into a state with more uniform and high Nanog expression. However, it is so far unclear whether 2i acts through an inductive or selective mechanism. Here, we use continuous time-lapse imaging to quantify the dynamics of death, proliferation, and Nanog expression in mouse ESCs after 2i addition. We show that 2i has a dual effect: it both leads to increased cell death of Nanog low ESCs (selective effect) and induces and maintains high Nanog levels (inductive effect) in single ESCs. Genetic manipulation further showed that presence of NANOG protein is important for cell viability in 2i medium. This demonstrates complex Nanog-dependent effects of 2i treatment on ESC cultures.

Multicolor quantitative confocal imaging cytometry.

Multicolor 3D quantitative imaging of large tissue volumes is necessary to understand tissue development and organization as well as interactions between distinct cell types in situ. However, tissue imaging remains technically challenging, particularly imaging of bone and marrow. Here, we describe a pipeline to reproducibly generate high-dimensional quantitative data from bone and bone marrow that may be extended to any tissue. We generate thick bone sections from adult mouse femurs with preserved tissue microarchitecture and demonstrate eight-color imaging using confocal microscopy without linear unmixing. We introduce XiT, an open-access software for fast and easy data curation, exploration and quantification of large imaging data sets with single-cell resolution. We describe how XiT can be used to correct for potential artifacts in quantitative 3D imaging, and we use the pipeline to measure the spatial relationship between hematopoietic cells, bone matrix and marrow Schwann cells.

Three-dimensional map of nonhematopoietic bone and bone-marrow cells and molecules.

The bone marrow (BM) microenvironment contains many types of cells and molecules with roles in hematopoiesis, osteogenesis, angiogenesis and metabolism. The spatial distribution of the different bone and BM cell types remains elusive, owing to technical challenges associated with bone imaging. To map nonhematopoietic cells and structures in bone and BM, we performed multicolor 3D imaging of osteoblastic, vascular, perivascular, neuronal and marrow stromal cells, and extracellular-matrix proteins in whole mouse femurs. We analyzed potential interactions between cells and molecules on the basis of colocalization of markers. Our results shed light on the markers expressed by different osteolineage cell types; the heterogeneity of vascular and perivascular cells; the neural subtypes innervating marrow and bone; the diversity of stromal cells; and the distribution of extracellular-matrix components. Our complete imaging data set is available for download and can be used in research in bone biology, hematology, vascular biology, neuroscience and extracellular-matrix biology.

Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios.

The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.

Tissue engineering of rat bladder using marrow-derived mesenchymal stem cells and bladder acellular matrix.

Bladder replacement or augmentation is required in congenital malformations or following trauma or cancer. The current surgical solution involves enterocystoplasty but is associated with high complication rates. Strategies for bladder tissue engineering are thus actively sought to address this unmet clinical need. Because of the poor efficacy of synthetic polymers, the use of bladder acellular matrix (BAM) has been proposed. Indeed when cellular components are removed from xenogenic or allogeneic bladders, the extracellular matrix scaffold thus obtained can be used alone or in combination with stem cells. In this study, we propose the use of BAM seeded with marrow-derived mesenchymal stem cells (MSCs) for bladder tissue engineering. We optimized a protocol for decellularization of bladder tissue from different species including rat, rabbit and swine. We demonstrate the use of non-ionic detergents followed by nuclease digestion results in efficient decellularization while preserving the extracellular matrix. When MSCs were seeded on acellular matrix scaffold, they remained viable and proliferative while adopting a cellular phenotype consistent with their microenvironment. Upon transplantation in rats after partial cystectomy, MSC-seeded BAM proved superior to unseeded BAM with animals recovering nearly 100% normal bladder capacity for up to six months. Histological analyses also demonstrated increased muscle regeneration.

Single-cell technologies sharpen up mammalian stem cell research.

Analysis of the mechanisms underlying cell fates requires the molecular quantification of cellular features. Classical techniques use population average readouts at single time points. However, these approaches mask cellular heterogeneity and dynamics and are limited for studying rare and heterogeneous cell populations like stem cells. Techniques for single-cell analyses, ideally allowing non-invasive quantification of molecular dynamics and cellular behaviour over time, are required for studying stem cells. Here, we review the development and application of these techniques to stem cell research.

Loss of the osteogenic differentiation potential during senescence is limited to bone progenitor cells and is dependent on p53.

DNA damage can lead to the induction of cellular senescence. In particular, we showed that exposure to ionizing radiation (IR) leads to the senescence of bone marrow-derived multipotent stromal cells (MSC) and osteoblast-like stromal cells (OB-SC), a phenotype associated with bone loss. The mechanism by which IR leads to bone dysfunction is not fully understood. One possibility involves that DNA damage-induced senescence limits the regeneration of bone progenitor cells. Another possibility entails that bone dysfunction arises from the inability of accumulating senescent cells to fulfill their physiological function. Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB-SC could still form mineralize nodules. This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes. Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors. These results are the first to demonstrate that senescence impacts osteogenesis in a cell type dependent manner and suggest that the accumulation of senescent osteoblasts is unlikely to significantly contribute to bone dysfunction in a cell autonomous manner.

Probing cellular processes by long-term live imaging--historic problems and current solutions.

Living organisms, tissues, cells and molecules are highly dynamic. The importance of their continuous and long-term observation has been recognized for over a century but has been limited by technological hurdles. Improvements in imaging technologies, genetics, protein engineering and data analysis have more recently allowed us to answer long-standing questions in biology using quantitative continuous long-term imaging. This requires a multidisciplinary collaboration between scientists of various backgrounds: biologists asking relevant questions, imaging specialists and engineers developing hardware, and informaticians and mathematicians developing software for data acquisition, analysis and computational modeling. Despite recent improvements, there are still obstacles to be addressed before this technology can achieve its full potential. This Commentary aims at providing an overview of currently available technologies for quantitative continuous long-term single-cell imaging, their limitations and what is required to bring this field to the next level. We provide an historical perspective on the development of this technology and discuss key issues in time-lapse imaging: keeping cells alive, using labels, reporters and biosensors, and hardware and software requirements. We highlight crucial and often non-obvious problems for researchers venturing into the field and hope to inspire experts in the field and from related disciplines to contribute to future solutions.

The immune plasticity of mesenchymal stromal cells from mice and men: concordances and discrepancies.

During the last decade, mesenchymal stromal cells (MSCs) have generated numbers of clinical trials to address inflammatory diseases such as GVHD, Crohn's disease and lupus. Animal models and therapeutic protocols in patients have demonstrated their anti-inflammatory and immunosuppressive properties towards adaptive immune cells. However, the basis of their immune suppression remains hotly debated. In the present review, we discuss the comparative isolation of human and rodent MSCs, their respective immune properties, whether constitutive or licensed by inflammatory or immune reactions, as well as differential efficacy as observed in GVHD clinical trials and related mouse models. Rodent MSCs display a number of immune differences with human MSCs regarding to ease of isolation, licensing pathways resulting in immunosuppression, and expression of immune mediators. These observations urge for caution when translating results generated in murine models into clinical settings.

Roles of FGF signaling in stem cell self-renewal, senescence and aging.

The aging process decreases tissue function and regenerative capacity, which has been associated with cellular senescence and a decline in adult or somatic stem cell numbers and self-renewal within multiple tissues. The potential therapeutic application of stem cells to reduce the burden of aging and stimulate tissue regeneration after trauma is very promising. Much research is currently ongoing to identify the factors and molecular mediators of stem cell self-renewal to reach these goals. Over the last two decades, fibroblast growth factors (FGFs) and their receptors (FGFRs) have stood up as major players in both embryonic development and tissue repair. Moreover, many studies point to somatic stem cells as major targets of FGF signaling in both tissue homeostasis and repair. FGFs appear to promote self-renewing proliferation and inhibit cellular senescence in nearly all tissues tested to date. Here we review the role of FGFs and FGFRs in stem cell self-renewal, cellular senescence, and aging.

Inhibition of cellular senescence by developmentally regulated FGF receptors in mesenchymal stem cells.

Bone-derived mesenchymal stem cells (MSCs) are important cells for use in cell therapy, tissue engineering, and regenerative medicine, but also to study bone development, homeostasis, and repair. However, little is known about their developmental ontology and in vivo identity. Because fibroblast growth factors (FGFs) play key roles in bone development and their receptors are developmentally regulated in bones, we hypothesized that MSCs should express FGF receptors (FGFRs), reflecting their developmental origin and potential. We show here that FGFR1/2 are expressed by rare mesenchymal progenitors in putative MSC niches in vivo, including the perichondrium, periosteum, and trabecular marrow. FGFR1⁺ cells often appeared as pericytes. These cells display a characteristic MSC phenotype in vitro when expanded with FGF-2, which appears to maintain MSC stemness by inhibiting cellular senescence through a PI3K/AKT-MDM2 pathway and by promoting proliferation. FGFRs may therefore be involved in MSC self-renewal. In summary, FGFR1/2 are developmentally regulated markers of MSCs in vivo and in vitro and are important in maintaining MSC stemness.

Hierarchical scaffold design for mesenchymal stem cell-based gene therapy of hemophilia B.

Gene therapy for hemophilia B and other hereditary plasma protein deficiencies showed great promise in pre-clinical and early clinical trials. However, safety concerns about in vivo delivery of viral vectors and poor post-transplant survival of ex vivo modified cells remain key hurdles for clinical translation of gene therapy. We here describe a 3D scaffold system based on porous hydroxyapatite-PLGA composites coated with biomineralized collagen 1. When combined with autologous gene-engineered factor IX (hFIX) positive mesenchymal stem cells (MSCs) and implanted in hemophilic mice, these scaffolds supported long-term engraftment and systemic protein delivery by MSCs in vivo. Optimization of the scaffolds at the macro-, micro- and nanoscales provided efficient cell delivery capacity, MSC self-renewal and osteogenesis respectively, concurrent with sustained delivery of hFIX. In conclusion, the use of gene-enhanced MSC-seeded scaffolds may be of practical use for treatment of hemophilia B and other plasma protein deficiencies.

Circadian rhythms and clock genes in psychotic disorders.

Numerous lines of evidence suggest that a disordered circadian system contributes to the etiology and symptomatology of major psychiatric disorders. Sleep disturbances, particularly rapid eye movement (REM) sleep, have been observed in bipolar affective disorder (BPD) and schizophrenia. Therapies aimed at altering the timing and duration of sleep and realigning circadian rhythms, including sleep scheduling, wake extension, light therapy and drug therapies that alter sleep and circadian rhythms appear beneficial for affective disorders. Interventional studies aiming to correct sleep and circadian disturbances in schizophrenia are scarce, although exogenous melatonin has been shown to improve both sleep structure and psychotic symptoms. The study of molecular clock mechanisms in psychiatric disorders is also gaining interest. Genetics studies have found associations with CLOCK, PERIOD1, PERIOD3, and TIMELESS in schizophrenia. Most research on BPD has focused on polymorphisms of CLOCK, but the lithium target GSK-3 may also be significant. New research examining the role of circadian rhythms and clock genes in major mental illness is likely to produce rapid advances in circadian-based therapeutics.

Three-dimensional porous scaffolds at the crossroads of tissue engineering and cell-based gene therapy.

In the last 20 years, more than 1,500 gene therapy clinical trials have been approved worldwide targeting a variety of indications, from inherited monogenic diseases to acquired conditions such as cancer, cardiovascular and infectious diseases. However, concerns about the safety and efficacy of gene therapy pharmaceuticals justify the development of alternative strategies to ensure the clinical translation of this still promising field. In particular, ex vivo gene therapy strategies using autologous adult stem cells coupled to three-dimensional (3D) porous scaffolds show great promises in preclinical studies. Developments in the fields of biomaterial sciences and tissue engineering have already helped understanding how we can harness to regenerative potential of many cell types to create artificial tissues and organs and vastly improve the engraftment of ex vivo manipulated adult stem cells. In this article, we will review the current state of the art in tissue engineering by exploring the various types of clinically available biomaterials and the methods used to process them into complex 3D scaffolds. We will then review how these technologies are applied in cell-based gene therapy and identify novel avenues of research that may benefit patients in the near future.

Improved autograft survival of mesenchymal stromal cells by plasminogen activator inhibitor 1 inhibition.

Mesenchymal stromal cells (MSCs) display robust reparative properties through their ability to limit apoptosis, enhance angiogenesis, and direct positive tissue remodeling. However, low in vivo survival of transplanted cells limits their overall effectiveness and significantly affects their clinical usage. Consequently, identifying strategies to improve cell survival in vivo are a priority. One explanation for their low survival is that MSCs are often transplanted into ischemic tissue, such as infarcted myocardium, where there is poor blood supply and low oxygen tension. Therefore, we examined how MSCs respond to a hypoxic, nutrient-poor stress environment to identify trophic factors that could be manipulated in advance of MSC transplantation. Combining microarray and proteomic screens we identified plasminogen activator inhibitor 1 (PAI-1) as one factor consistently upregulated in our in vitro ischemia-mimicking conditions. Subsequent genetic and chemical manipulation studies define PAI-1 as a negative regulator of MSC survival in vivo. Mechanistically, MSC-derived PAI-1 does not alter MSC survival through a plasmin-dependent mechanism but rather directly impacts on the adhesiveness of MSCs to their surrounding matrices. Thus we can conclude that post-transplantation, PAI-1 negatively impacts MSC survival by promoting anoikis via matrix detachment.