Single-cell transcriptomics reveals a role for pancreatic duct cells as potential mediators of inflammation in diabetes mellitus.

Introduction: Inflammation of the pancreas contributes to the development of diabetes mellitus. Although it is well-accepted that local inflammation leads to a progressive loss of functional beta cell mass that eventually causes the onset of the disease, the development of islet inflammation remains unclear. Methods: Here, we used single-cell RNA sequencing to explore the cell type-specific molecular response of primary human pancreatic cells exposed to an inflammatory environment. Results: We identified a duct subpopulation presenting a unique proinflammatory signature among all pancreatic cell types. Discussion: Overall, the findings of this study point towards a role for duct cells in the propagation of islet inflammation, and in immune cell recruitment and activation, which are key steps in the pathophysiology of diabetes mellitus.

Neutral sphingomyelinase blockade enhances hematopoietic stem cell fitness through an integrated stress response.

Hematopoietic stem and progenitor cell (HSPC) transplantation serves as a curative therapy for many benign and malignant hematopoietic disorders and as a platform for gene therapy. However, growing needs for ex vivo manipulation of HSPC-graft products are limited by barriers in maintaining critical self-renewal and quiescence properties. The role of sphingolipid metabolism in safeguarding these essential cellular properties has been recently recognized, but not yet widely explored. Here, we demonstrate that pharmacologic and genetic inhibition of neutral sphingomyelinase 2 (nSMase-2) leads to sustained improvements in long-term competitive transplantation efficiency after ex vivo culture. Mechanistically, nSMase-2 blockade activates a canonical integrated stress response (ISR) and promotes metabolic quiescence in human and murine HSPCs. These adaptations result in part from disruption in sphingolipid metabolism that impairs the release of nSMase-2-dependent extracellular vesicles (EVs). The aggregate findings link EV trafficking and the ISR as a regulatory dyad guarding HSPC homeostasis and long-term fitness. Translationally, transient nSMase-2 inhibition enables ex vivo graft manipulation with enhanced HSPC potency.

A single-cell RNA-seq analysis unravels the heterogeneity of primary cultured human corneal endothelial cells.

The cornea is a transparent and avascular tissue located in front of the eye. Its inner surface is lined by a monolayer of corneal endothelial cells (CECs), which maintain the cornea transparency. CECs remain arrested in a non-proliferative state and damage to these cells can compromise their function leading to corneal opacity. The primary culture of donor-derived CECs is a promising cell therapy. It confers the potential to treat multiple patients from a single donor, alleviating the global donor shortage. Nevertheless, this approach has limitations preventing its adoption, particularly culture protocols allow limited expansion of CECs and there is a lack of clear parameters to identify therapy-grade CECs. To address this limitation, a better understanding of the molecular changes arising from the primary culture of CECs is required. Using single-cell RNA sequencing on primary cultured CECs, we identify their variable transcriptomic fingerprint at the single cell level, provide a pseudo-temporal reconstruction of the changes arising from primary culture, and suggest markers to assess the quality of primary CEC cultures. This research depicts a deep transcriptomic understanding of the cellular heterogeneity arising from the primary expansion of CECs and sets the basis for further improvement of culture protocols and therapies.

Single-Cell Transcriptomics Links Loss of Human Pancreatic ß-Cell Identity to ER Stress.

The maintenance of pancreatic islet architecture is crucial for proper ß-cell function. We previously reported that disruption of human islet integrity could result in altered ß-cell identity. Here we combine ß-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of ß-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and ß-cell phenotype, and strengthen the concept of altered ß-cell identity as a mechanism underlying the loss of functional ß-cell mass.

Single cell transcriptomics reveals the heterogeneity of the human cornea to identify novel markers of the limbus and stroma.

The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman's layer, stroma, Descemet's membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified expression of CAV1, HOMER3 and CPVL in the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, CXCL14 was expressed exclusively in the suprabasal/superficial limbus, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.

Oxidative Stress Leads to ß-Cell Dysfunction Through Loss of ß-Cell Identity.

Pancreatic ß-cell failure is a critical event in the onset of both main types of diabetes mellitus but underlying mechanisms are not fully understood. ß-cells have low anti-oxidant capacity, making them more susceptible to oxidative stress. In type 1 diabetes (T1D), reactive oxygen species (ROS) are associated with pro-inflammatory conditions at the onset of the disease. Here, we investigated the effects of hydrogen peroxide-induced oxidative stress on human ß-cells. We show that primary human ß-cell function is decreased. This reduced function is associated with an ER stress response and the shuttling of FOXO1 to the nucleus. Furthermore, oxidative stress leads to loss of ß-cell maturity genes MAFA and PDX1, and to a concomitant increase in progenitor marker expression of SOX9 and HES1. Overall, we propose that oxidative stress-induced ß-cell failure may result from partial dedifferentiation. Targeting antioxidant mechanisms may preserve functional ß-cell mass in early stages of development of T1D.

An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.

Tacrolimus-Induced BMP/SMAD Signaling Associates With Metabolic Stress-Activated FOXO1 to Trigger ß-Cell Failure.

Active maintenance of ß-cell identity through fine-tuned regulation of key transcription factors ensures ß-cell function. Tacrolimus, a widely used immunosuppressant, accelerates onset of diabetes after organ transplantation, but underlying molecular mechanisms are unclear. Here we show that tacrolimus induces loss of human ß-cell maturity and ß-cell failure through activation of the BMP/SMAD signaling pathway when administered under mild metabolic stress conditions. Tacrolimus-induced phosphorylated SMAD1/5 acts in synergy with metabolic stress-activated FOXO1 through formation of a complex. This interaction is associated with reduced expression of the key ß-cell transcription factor MAFA and abolished insulin secretion, both in vitro in primary human islets and in vivo in human islets transplanted into high-fat diet-fed mice. Pharmacological inhibition of BMP signaling protects human ß-cells from tacrolimus-induced ß-cell dysfunction in vitro. Furthermore, we confirm that BMP/SMAD signaling is activated in protocol pancreas allograft biopsies from recipients on tacrolimus. To conclude, we propose a novel mechanism underlying the diabetogenicity of tacrolimus in primary human ß-cells. This insight could lead to new treatment strategies for new-onset diabetes and may have implications for other forms of diabetes.

Dynamic adaptation of mesenchymal stem cell physiology upon exposure to surface micropatterns.

Human mesenchymal stem (hMSCs) are defined as multi-potent colony-forming cells expressing a specific subset of plasma membrane markers when grown on flat tissue culture polystyrene. However, as soon as hMSCs are used for transplantation, they are exposed to a 3D environment, which can strongly impact cell physiology and influence proliferation, differentiation and metabolism. Strategies to control in vivo hMSC behavior, for instance in stem cell transplantation or cancer treatment, are skewed by the un-physiological flatness of the standard well plates. Even though it is common knowledge that cells behave differently in vitro compared to in vivo, only little is known about the underlying adaptation processes. Here, we used micrometer-scale defined surface topographies as a model to describe the phenotype of hMSCs during this adaptation to their new environment. We used well established techniques to compare hMSCs cultured on flat and topographically enhanced polystyreneand observed dramatically changed cell morphologies accompanied by shrinkage of cytoplasm and nucleus, a decreased overall cellular metabolism, and slower cell cycle progression resulting in a lower proliferation rate in cells exposed to surface topographies. We hypothesized that this reduction in proliferation rate effects their sensitivity to certain cancer drugs, which was confirmed by higher survival rate of hMSCs cultured on topographies exposed to paclitaxel. Thus, micro-topographies can be used as a model system to mimic the natural cell micro-environment, and be a powerful tool to optimize cell treatment in vitro.

Muscle-Secreted Factors Improve Anterior Cruciate Ligament Graft Healing: An In Vitro and In Vivo Analysis.

One of the ligaments most often damaged during sports-the anterior cruciate ligament (ACL)-has poor healing capacity. On damage, reconstructive surgery is performed to restore the mechanical stability of the knee and to reduce the inflammatory milieu otherwise present in the joint. A return to normal activities, however, takes between 9 and 12 months. Thus, strategies capable of improving ACL graft healing are needed. Embryonic development of tendon and ligament (T/L) is regulated by a crosstalk between different cell types. We hypothesized that terminally differentiated skeletal-derived cells such as osteoblasts, chondrocytes, and myoblasts modulate T/L healing. Using an indirect coculture system, we discovered that myoblast-secreted signals-but not osteoblasts, chondrocytes, or stromal-secreted signals-are capable of upregulating classical T/L markers such as scleraxis and tenomodulin on human hamstring tendon-derived cells (hTC), which contribute to ACL graft healing. Transcriptome analysis showed that coculturing hTC with myoblasts led to an upregulation of extracellular matrix (ECM) genes and resulted in enhanced ECM deposition. In vivo, using a rat model of ACL reconstruction showed that conditioned media derived from human muscle tissue accelerated femoral tunnel closure, a key step for autograft integration. Collectively, these results indicate that muscle-secreted signals can be used to improve ACL graft healing in a clinical setting where muscle remnants are often discarded.

Linking the Transcriptional Landscape of Bone Induction to Biomaterial Design Parameters.

New engineering possibilities allow biomaterials to serve as active orchestrators of the molecular and cellular events of tissue regeneration. Here, the molecular control of tissue regeneration for calcium phosphate (CaP)-based materials is established by defining the parameters critical for tissue induction and those are linked to the molecular circuitry controlling cell physiology. The material properties (microporosity, ion composition, protein adsorption) of a set of synthesized osteoinductive and noninductive CaP ceramics are parameterized and these properties are correlated to a transcriptomics profile of osteogenic cells grown on the materials in vitro. Using these data, a genetic network controlling biomaterial-induced bone formation is built. By isolating the complex material properties into single-parameter test conditions, it is verified that a subset of these genes is indeed controlled by surface topography and ions released from the ceramics, respectively. The gene network points to a decisive role for extracellular matrix deposition in osteoinduction by genes such as tenascin C and hyaluronic acid synthase 2, which are controlled by calcium and phosphate ions as well as surface topography. This work provides insight into the biomaterial composition and material engineering aspects of bone void filling and can be used as a strategy to explore the interface between biomaterials and tissue regeneration.

A Single-Cell Transcriptome Atlas of the Human Pancreas.

To understand organ function, it is important to have an inventory of its cell types and of their corresponding marker genes. This is a particularly challenging task for human tissues like the pancreas, because reliable markers are limited. Hence, transcriptome-wide studies are typically done on pooled islets of Langerhans, obscuring contributions from rare cell types and of potential subpopulations. To overcome this challenge, we developed an automated platform that uses FACS, robotics, and the CEL-Seq2 protocol to obtain the transcriptomes of thousands of single pancreatic cells from deceased organ donors, allowing in silico purification of all main pancreatic cell types. We identify cell type-specific transcription factors and a subpopulation of REG3A-positive acinar cells. We also show that CD24 and TM4SF4 expression can be used to sort live alpha and beta cells with high purity. This resource will be useful for developing a deeper understanding of pancreatic biology and pathophysiology of diabetes mellitus.

Stepping into the omics era: Opportunities and challenges for biomaterials science and engineering.

The research paradigm in biomaterials science and engineering is evolving from using low-throughput and iterative experimental designs towards high-throughput experimental designs for materials optimization and the evaluation of materials properties. Computational science plays an important role in this transition. With the emergence of the omics approach in the biomaterials field, referred to as materiomics, high-throughput approaches hold the promise of tackling the complexity of materials and understanding correlations between material properties and their effects on complex biological systems. The intrinsic complexity of biological systems is an important factor that is often oversimplified when characterizing biological responses to materials and establishing property-activity relationships. Indeed, in vitro tests designed to predict in vivo performance of a given biomaterial are largely lacking as we are not able to capture the biological complexity of whole tissues in an in vitro model. In this opinion paper, we explain how we reached our opinion that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field. STATEMENT OF SIGNIFICANCE: In this opinion paper, we postulate that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field.

Exploring the Material-Induced Transcriptional Landscape of Osteoblasts on Bone Graft Materials.

During the past decades, there have been major advances in the field of biomaterials, thereby generating a vast variety of materials for a broad range of tissue engineering and regeneration applications. Although gene expression profiling has been used occasionally in biomaterial research, its usefulness for understanding cell-biomaterial interactions should be further explored for it to fulfill its promise as a tool to assess and improve material properties. Here, the transcriptional landscape induced by 23 materials is explored with a variety of properties within the scope of bone regeneration. An osteoblast cell line is used to identify the gene expression profiles that can be adopted in response to biophysical and chemical cues. It is shown that TGF-ß and WNT signaling may be involved in the cellular response to osteoinductive materials along with differential cell adhesion kinetics via attenuated FAK signaling. The previously reported effect of calcium and phosphate on BMP2 and TGF-ß signaling is confirmed and the biological effect of the addition of nanohydroxyapatite in poly (d,l-lactic acid) polymer particles is studied. Together with future applications, this approach will help researchers understand cellular responses in relation to material properties, which will promote the development of more effective biomaterials for applications in tissue regeneration.

MicroRNA Levels as Prognostic Markers for the Differentiation Potential of Human Mesenchymal Stromal Cell Donors.

The ability of human mesenchymal stromal/stem cells (hMSCs) to differentiate into various mesenchymal cell lineages makes them a promising cell source for the use in tissue repair strategies. Since the differentiation potential of hMSCs differs between donors, it is necessary to establish biomarkers for the identification of donors with high differentiation potential. In this study, we show that microRNA (miRNA) expression levels are effective for distinguishing donors with high differentiation potential from low differentiation potential. Twenty hMSC donors were initially tested for marker expression and differentiation potential. In particular, the chondrogenic differentiation potential was evaluated on the basis of histological matrix formation, mRNA expression levels of chondrogenic marker genes, and quantitative glycosaminoglycan deposition. Three donors out of twenty were identified as donors with high chondrogenic potential, whereas nine showed moderate and eight showed low chondrogenic potential. Expression profiles of miRNAs involved in chondrogenesis and cartilage homeostasis were used for the distinction between high-performance hMSCs and low-performance hMSCs. Global mRNA expression profiles of the donors before the onset of chondrogenic differentiation revealed minor differences in gene expression between low and high chondrogenic performers. However, analysis of miRNA expression during a 7-day differentiation period identified miR-210 and miR-630 as positive regulators of chondrogenesis. In contrast, miR-181 and miR-34a, which are negative regulators of chondrogenesis, were upregulated during differentiation in low-performing donors. In conclusion, profiling of hMSC donors for a specific panel of miRNAs may have a prognostic value for selecting donors with high differentiation potential to improve hMSC-based strategies for tissue regeneration.

Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: the effect of silanol groups.

Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si-OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL-Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL-Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL-Si scaffolds occur for up to 2weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL-Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies.

The effect of donor variation and senescence on endothelial differentiation of human mesenchymal stromal cells.

Application of autologous cells is considered for a broad range of regenerative therapies because it is not surrounded by the immunological and ethical issues of allo- or xenogenic cells. However, isolation, expansion, and application of autologous cells do suffer from variability in therapeutic efficacy due to donor to donor differences and due to prolonged culture. One important source of autologous cells is mesenchymal stromal cells (MSCs), which can differentiate toward endothelial-like cells, thus making them an ideal candidate as cell source for tissue vascularization. Here we screened MSCs from 20 donors for their endothelial differentiation capacity and correlated it with the gene expression profile of the whole genome in the undifferentiated state. Cells of all donors were able to form tubes on Matrigel and induced the expression of endothelial genes, although with quantitative differences. In addition, we analyzed the effect of prolonged in vitro expansion on the multipotency of human MSCs and found that endothelial differentiation is only mildly sensitive to expansion-induced loss of differentiation as compared to osteogenic and adipogenic differentiation. Our results show the robustness of the endothelial differentiation protocol and the gene expression data give insight in the differences in endothelial differentiation between donors.

Bioinformatics-based selection of a model cell type for in vitro biomaterial testing.

Biomaterial properties can be tailored for specific applications via systematic and high-throughput screening of biomaterial-cell interactions. However, progress in material development is often hampered by the lack of adequate in vitro testing methods, frequently due to incomplete understanding of involved in vivo mechanisms. In line with drug discovery in pharmacology, a crucial step in assay development for biomaterial screening is the identification of a target to direct the screen against. Herein, the cell type to be used for screening is of essential importance and has to be carefully chosen. So far, few attention has been put on selecting a cell type specifically suitable for in vitro testing of materials for predefined applications. In this manuscript, we describe an approach to define a suitable cell type for screening assays, for which biomaterials for bone regeneration served as example. Using a bioinformatics methodology, different cell lines are compared on three well-characterized model materials. The transcriptional profiles of MG63, iMSC, SV-HFO, hPPCT, hBPCT and SW480 cells are assessed on 3 calcium phosphate-based materials to evaluate their potential application in a screening assay. We show that MG63 is the most suitable cell line to evaluate biomaterials for bone regeneration applications, evidenced by their robust gene expression differences between the 3 model materials. The gene expression differences between the cell lines were assessed based on the overall gene expression profiles and specific subsets of genes and pathways related to osteogenesis and bone homeostasis in response to the 3 materials tested. In the next phase, this cell line will be used to identify a target correlating with in vivo biomaterial performance and henceforth to develop an in vitro screening system.

Predicting the therapeutic efficacy of MSC in bone tissue engineering using the molecular marker CADM1.

Mesenchymal stromal cells (hMSCs) are advancing into the clinic but the therapeutic efficacy of hMSCs faces the problem of donor variability. In bone tissue engineering, no reliable markers have been identified which are able to predict the bone-forming capacity of hMSCs prior to implantation. To this end, we isolated hMSCs from 62 donors and characterized systematically their in vitro lineage differentiation capacity, gene expression signature and in vivo capacity for ectopic bone formation. Our data confirms the large variability of in vitro differentiation capacity which did not correlate with in vivo ectopic bone formation. Using DNA microarray analysis of early passage hMSCs we identified a diagnostic bone-forming classifier. In fact, a single gene, CADM1, strongly correlated with the bone-forming capacity of hMSCs and could be used as a reliable in vitro diagnostic marker. Furthermore, data mining of genes expressed correlating with in vivo bone formation represented involvement in neurogenic processes and Wnt signaling. We will apply our data set to predict therapeutic efficacy of hMSCs and to gain novel insight in the process of bone regeneration. Our bio-informatics driven approach may be used in other fields of cell therapy to establish diagnostic markers for clinical efficacy.

Insulin-like growth factor-I enhances proliferation and differentiation of human mesenchymal stromal cells in vitro.

Human mesenchymal stromal cells (hMSCs) offer great potential for bone tissue engineering applications, but their in vivo performance remains limited. Preconditioning of these cells with small molecules to improve their differentiation before implantation, or incorporation of growth factors are possible solutions. Insulin-like growth factor-1 (IGF-1) is one of the most abundant growth factors in bone, involved in growth, development, and metabolism, but its effects on hMSCs are still subject of debate. Here we examined the effects of IGF-1 on proliferation and differentiation of hMSCs in vitro and we found that serum abolished the effects of IGF-1. Only in the absence of serum, IGF-1 increased proliferation, alkaline phosphatase expression, and osteogenic gene expression of hMSCs. Furthermore, we examined synergistic effects of bone morphogenetic protein-2 (BMP-2) and IGF-1 and, although IGF-1 enhanced BMP-2-induced mineralization, IGF-1 only slightly affected in vivo bone formation.